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Cadmium-Induced Autophagy and Autophagic Contribution to
Cell Death in Cadmium-Treated Mesangial Cells
by
Bilal Ahmadi
A thesis submitted in conformity with the requirements
for the degree of Master of Science
Department of Laboratory Medicine and Pathobiology
University of Toronto
© Copyright by Bilal Ahmadi 2016
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Cadmium-Induced Autophagy and Autophagic Contribution to Cell
Death in Cadmium-Treated Mesangial Cells
Bilal Ahmadi
Master of Science
Department of Laboratory Medicine and Pathobiology
University of Toronto
2016
Abstract
Cadmium (Cd) is a nephrotoxic metal that has a number of consequences for renal function.
Cells exposed to Cd may have methods to counteract toxic effects with processes like autophagy.
Autophagy is an intracellular degradation system that delivers cytoplasmic constituents to the
lysosome. This study investigated the mechanism of Cd-induced autophagy, and the relationship
between autophagy and cell survival. At 10 µM exposure for 24 hours, Cd-induced autophagy
was found to be reactive oxygen species-independent, and suppressed with thapsigargin
treatment, suggesting the necessity of intracellular Ca2+. Inhibition of the Jun-N-Terminal Kinase
resulted in the greatest suppression of Cd-induced autophagy. Autophagy was found to be
protective against cell death elicited by the toxic metal, and the absence of autophagy resulted in
increased apoptosis in mouse embryonic fibroblast cells. This study adds insight to the
mechanism and protective effects of Cd-induced autophagy, and provides the groundwork for
future experiments.
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Acknowledgments
I would like to thank Dr. Douglas Templeton first and foremost for his guidance and
support throughout my Master’s Degree. I am eternally grateful for the chance to work in your
laboratory, and you were always ready with an amusing anecdote or a thought-provoking
question to keep me entertained and enlightened. Your patience, wisdom and care has helped me
become a better scientist and person, and I will not easily forget the ‘relevance’ of cadmium
toxicity thanks to you!
To Dr. Ying Liu, I am enormously thankful that you were present in the lab during my
Master’s work. You are a wonderfully caring, thoughtful and intelligent person and I appreciate
all the help you provided in helping me succeed.
I would like to thank former members of the laboratory including Kathy Xiao, Grace
Choong, Bryce Chen and Jenny Nguyen for your assistance, friendship and kindness. To all my
close friends from the 6th floor of the Medical Science Building, you have kept me entertained
and eager to come to work, and I feel extremely lucky to have you in my life.
Most importantly, my love and gratitude to my family for helping me in all matters big
and small for the past two years. My three younger brothers never fail to make me smile, and are
always prepared to help me waste time. To my parents, you have been my most passionate
supporters and I will never be able to repay you back for all of your warmth, love and
understanding. Thank you.
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Table of Contents
Acknowledgments.......................................................................................................................... iii
Table of Contents ........................................................................................................................... iv
Abbreviations ................................................................................................................................ vii
List of Figures and Tables................................................................................................................x
1 Introduction .................................................................................................................................1
1.1 Cadmium ..............................................................................................................................1
1.1.1 Physicochemical Properties of Cadmium ................................................................1
1.1.2 Occurrence and Sources of Exposure ......................................................................2
1.2 Cadmium Toxicity ...............................................................................................................4
1.2.1 Routes of Exposure ..................................................................................................4
1.2.2 Cadmium Transport in the Body..............................................................................5
1.2.3 Organs Affected by Cadmium Toxicity ...................................................................7
1.2.4 Cadmium Nephrotoxicity.........................................................................................8
1.2.5 Environmental Exposure ........................................................................................10
1.3 Molecular Changes with Cd Exposure ..............................................................................13
1.3.1 Reactive Oxygen Species .......................................................................................13
1.3.2 Cd2+ Impact on Ca2+ Signalling .............................................................................16
1.3.3 Endoplasmic Reticulum Stress ..............................................................................18
1.3.4 Cancer, Proliferation and Survival in Response to Cd2+ ........................................18
1.3.5 Cell Death Mechanisms .........................................................................................20
1.4 Autophagy ..........................................................................................................................24
1.4.1 Definition and Purpose of Autophagy ...................................................................24
1.4.2 General Process and Molecular Machinery ...........................................................26
1.4.3 Three Types of Autophagy ....................................................................................30
v
1.4.4 Autophagy as a Cell Death Mechanism .................................................................31
1.4.5 Autophagy as a Homeostatic or Cell Survival Mechanism ...................................32
1.4.6 Agents that Modulate Autophagy ..........................................................................34
1.4.7 Metals and Induction of Autophagy ......................................................................37
1.4.8 Cadmium-induced Autophagy ...............................................................................39
1.5 The Mesangial Cell Model.................................................................................................40
1.5.1 Physiology of Mesangial Cells ..............................................................................40
1.5.2 Relevance to Cd Nephrotoxicity ............................................................................41
1.6 Hypotheses and Objectives ................................................................................................42
2 Materials and Methods ..............................................................................................................44
2.1 Materials ............................................................................................................................44
2.2 Cell Culture ........................................................................................................................44
2.3 Cell Treatments ..................................................................................................................45
2.4 1% NP-40 whole-cell lysate for detection of p62, β-actin and caspase-3 proteins............45
2.5 Triton X-100 whole cell lysate for detection of LC3-II proteins .......................................46
2.6 Western Blotting ................................................................................................................46
2.7 Immunofluorescence ..........................................................................................................46
2.8 Viability Assay...................................................................................................................47
2.9 Statistical Analyses ............................................................................................................47
3 Results .......................................................................................................................................48
3.1 Cadmium induces autophagy in RMC ...............................................................................48
3.1.1 Cadmium-induced autophagy is time and concentration dependent .....................48
3.1.2 At 24 hours of Cd exposure, autophagy is not the only cell fate ...........................50
3.2 Cadmium-induced autophagy is decreased by JNK inhibition ..........................................52
3.3 Cadmium-induced autophagy is suppressed with Tg treatment ........................................54
3.4 Cadmium-induced autophagy progresses independently of ROS accumulation ...............56
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3.5 Selective autophagy protein p62 expression declines with increasing Cd concentration ..58
3.6 Cells that cannot undergo autophagy are less viable .........................................................60
3.6.1 Rat mesangial cell viability after 24h of Cd exposure ...........................................60
3.6.2 Viability of autophagy-deficient mouse embryonic fibroblasts exposed to Cd .....61
3.7 MEF cells that cannot undergo autophagy experience greater apoptosis ..........................63
4 Discussion .................................................................................................................................66
4.1 Cadmium and autophagy ...................................................................................................66
4.2 JNK activation and autophagy ...........................................................................................68
4.3 Ca2+ and Cd-induced autophagy ........................................................................................70
4.4 Selective autophagy with Cd exposure ..............................................................................72
4.5 ROS and Cd-induced autophagy ........................................................................................73
4.6 Autophagy and apoptosis ...................................................................................................74
5 Summary and Significance .......................................................................................................76
Bibliography ..................................................................................................................................77
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Abbreviations
AIF Apoptosis-inducing factor
ATF-6 Activating transcription factor-6
ATG Autophagy related gene
BHA Butylated hydroxyanisole
Bif1 Bax-interacting factor 1
BSA Bovine serum albumin
CAMKII Ca2+/calmodulin-dependent protein kinase II
Cd Cadmium
CHO Chinese hamster ovary
CHOP CCAAT-enhancer-binding protein homologous protein
Cpt Campothecin
CQ Chloroquine
DMSO Dimethyl sulfoxide
DMT-1 Divalent metal transporter-1
DTT Dithiothreitol
EDTA Ethylenediaminetetraacetic acid
EGFRK Epidermal Growth Factor Receptor Kinase
EGTA Ethylene glycol tetraacetic acid
ER Endoplasmic reticulum
ERK Extracellular-signal-regulated kinase
ETC Electron transport chain
FBS Fetal bovine serum
GFR Glomerular filtration rate
GPR78 G-protein coupled receptor 78
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GPCR G-protein coupled receptor
GSH Glutathione, reduced
GSK- β Glycogen synthase kinase-3 β
HRP Horseradish peroxidase
IARC International Agency for the Research on Cancer
IL-1β Interleukin-1 beta
IP3 Inositol-1,4,5-triphosphate
IP3R1 Inositol 1,4,5-triphosphate receptor 1
IRE1 Inositol-requiring protein-1
JNK Jun-N-terminal kinase
LAMP Lysosome-associated protein
LC3 Micotubular-associated protein 1 light chain 3
MAPK Mitogen activated protein kinases
MEF Mouse embryonic fibroblasts
MT Metallothionein
mTOR Mammalian target of Rapamycin
MTT Thiozolyl blue tetrazolium bromide
NAG N-acetyl-β-d-glucosaminidase
NBR1 Neighbor of BRCA1 gene 1 protein
Nrf2 Nuclear factor (erythroid-derived 2)-like 2
p38 p38 mitogen-activated protein kinase
PBS Phosphate-buffered saline
PI3K Phosphatidylinositol-4,5-bisphosphate 3-kinase
PE Phosphatidylethanolamine
PERK Protein kinase RNA-like endoplasmic reticulum kinase
PLC Phospholipase C
ix
PMSF Phenylmethanesulfonyl fluoride
RIP Receptor-interacting protein
RMC Rat mesangial cells
RNAi Ribonucleic acid interference
ROS Reactive oxygen species
SDS PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis
SERCA Sarcoplasmic/endoplasmic reticulum Ca2+-ATPase
SF Serum free
SNARE Soluble NSF attachment protein receptor
SOD Superoxide dismutase
SP SP600125 (JNK inhibitor)
SQSTM1 Sequestosome 1 (also known as p62)
TBS Tris-buffered saline
Tg Thapsigargin
TNF- α Tumor necrosis factor alpha
ULK1 Unc-51-like kinase 1
UPR Unfolded protein response
VDCC Voltage dependant calcium channels
VPS34 Vacuolar protein sorting 34
WHO World Health Organization
x
List of Figures and Tables
Figure 1. Cadmium transport in the body. ...................................................................................... 6
Figure 2. Cadmium induction of oxidative stress. ........................................................................ 16
Figure 3. Pathways of Cd-induced apoptosis. ............................................................................... 23
Figure 4. Schematic diagram of the stages of autophagy. ............................................................ 27
Figure 5. The molecular execution of autophagy. ........................................................................ 29
Figure 6. Anatomy of the glomerulus. .......................................................................................... 41
Figure 7. Time- and concentration-dependent autophagy in Cd-exposed RMC. ......................... 49
Figure 8. Effects of Cd on autophagic vacuolization as visualized by fluorescent dye. ............... 51
Figure 9. Extent of autophagy when Cd is co-treated with various kinase inhibitors. ................. 53
Figure 10. Determining the extent of autophagy with co-treatment of Cd and Tg. ...................... 55
Figure 11. Extent of autophagy when cells are treated with a ROS scavenger. ........................... 57
Figure 12. Monitoring p62-assisted selective autophagy in Cd-treated mesangial cells. ............. 59
Figure 13. Rat mesangial cell viability measured by MTT assay after 24h of Cd treatment. ...... 60
Figure 14. MEFWT and MEFATG-/- viability measured by MTT. .............................................. 62
Figure 15. Monitoring apoptosis and autophagy in MEFWT and MEFATG-/- cells. ................... 64
Table 1. Exposure levels associated with kidney and bone defects in recent environmental studies
of Cd exposure. ............................................................................................................................. 12
Table 2. Compounds known to activate autophagy. ..................................................................... 35
Table 3. Compounds known to inhibit autophagy. ....................................................................... 36
1
1 Introduction
1.1 Cadmium
1.1.1 Physicochemical Properties of Cadmium
Cadmium (Cd) is a highly toxic metal located in group 12 of the periodic table of
elements with an atomic number of 48. Cadmium is found at concentrations ranging from 0.1 to
1 ppm in the earth’s crust, with anthropogenic activities contributing significantly to its increased
bioavailability in the environment (Joseph, 2009). Cadmium has several industrial applications
and is used in electroplating, in pigments, as a stabilizer in paint and in nickel-cadmium batteries.
In the United States, it is estimated that the average daily individual consumption of Cd is 30 μg,
with a substantially higher consumption in China and Japan (Templeton and Liu, 2010).
The major toxic form of Cd is the cadmium ion (Cd2+), which competes with essential metal ions
for entry into cells, disrupts cellular functions and can lead to disease (Thévenod and Lee,
2013a). The Cd2+ ion’s similarity to physiologically relevant metal ions Ca2+ and Zn2+ is of
particular note in mammalian toxicity. Zinc and Cd belong to the same group in the periodic
table with filled 3d and 4d orbitals, respectively, and they consequently share many properties.
The Cd2+ ion is the only state in which Cd is accessible in physiological conditions, and the large
and easily polarized Cd2+ cation is classified as a ‘soft’ ion, unlike zinc which is classified as
‘borderline’. The soft ion classification refers to ions that readily share their electron cloud and
more easily bind via covalent interactions to inorganic anions such as I-, or organic molecules
containing sulfur (sulfhydryl, disulfide, thioether) or nitrogen (amino, imidazole, histidine,
nucleotide base) (Thévenod, 2010). In metallothionein (MT), which will be discussed in section
1.2.2, Cd2+ being a soft ion needs only to be one thousandth of the concentration of Zn2+ in order
to effectively compete for binding (Thévenod and Lee, 2013b).
Cadmium and calcium share very similar physicochemical properties, allowing Cd2+ to
act as a Ca2+ mimetic. Numerous studies have shown that Cd disrupts calcium homeostasis,
leading to apoptosis in a variety of cells in vitro (Son et al., 2010). The divalent ions have similar
ionic radii (Ca2+ 0.97 Å, Cd2+ 0.99 Å) resulting in similar charge-to-radius ratios. Both ions are
therefore able to exert comparable strong electrostatic forces on biological molecules (Jacobson
and Turner, 1980). Cd2+ interacts with the functions of many Ca2+-dependent enzymes such as
2
endonucleases and regulatory proteins such as protein kinase C, the mitogen activated protein
kinases (MAPKs), and phospholipase C (Beyersmann and Hechtenberg, 1997). Based on
theoretical considerations, cellular homeostasis of eukaryotes is maintained as long as cellular
calcium concentrations are about 106-fold higher than Cd concentrations (Thévenod and Lee,
2013a).
The standard enthalpy of hydration (ΔHohydration)
is related to the residence time of water
molecules near an ion (Edsall and McKenzie, 1978). The concept may be helpful in
understanding the process of permeation of ions in ion channels. The higher the ΔHohydration, the
more difficulty an ion faces when traveling through channel pores as it is unable to easily replace
water molecules around itself with dipolar groups of the channel pore. Cd2+ has a relatively high
ΔHohydration, which may contribute to its behavior as a ‘blocker’ of channel proteins as opposed to
a ‘permeator’ like Ca2+ (Thévenod, 2010). However, thanks to the Cd ion’s ability to form
complexes with organic molecules due to its softness, much of its entry into cells is mediated by
the transport of those organic molecules (Thévenod, 2010). Zn2+ transporters for which direct
evidence has been provided in the uptake of Cd2+ include ZIP8 (SLC39A8) and ZIP14
(SLC39A14) (He et al., 2009).
1.1.2 Occurrence and Sources of Exposure
Thanks to the technological advances in the production, use and disposal of Cd and Cd-
containing products, Cd emissions in the air have been constantly decreasing since the 1960s.
This may suggest that the health threat of Cd toxicity has diminished (Moulis and Thévenod,
2010). The worldwide consumption of the metal, however, has increased steadily from 18400
tonnes in 2003 to 20400 tonnes in 2007. Recovery or safe removal of Cd from habitats is
unrealistic and as a result there is an accumulation of the total bioavailable toxic metal. Over the
last 15 years, The Comprehensive Environmental Response, Compensation, and Liability Act
(CER CLA) has permanently listed Cd as number 7 (out of 275) in the priority list of hazardous
materials (US Department of Health and Human Services, 2012).
The earliest observations of Cd exposure were from clinical medicine, first described in
1858. Reports at the time described acute gastrointestinal symptoms as well as delayed
respiratory symptoms among people who were using cadmium carbonate powder as a polishing
agent, and therefore were inhaling and ingesting the toxic metal (Nordberg, 2009). Later,
3
observations in Cd-exposed factory workers were reported to include damage to the lungs, with
acute gastrointestinal effects with vomiting and diarrhea in persons consuming Cd-contaminated
food and drink. Cadmium occurs naturally in zinc ore, so it is not surprising that cases of ‘lead
poisoning’ in workers at zinc smelters in the late 19th century which included proteinuria and
emphysema can be retroactively attributed to Cd (Nordberg, 2009).
Today, the main source of environmental Cd exposure in non-smokers is diet.
Atmospheric deposition of airborne Cd, mining, and the application of Cd-containing fertilizers
on farmland may lead to soil contamination, and uptake by crops including rice, tobacco and
mushrooms (Järup and Åkesson, 2009). High concentrations of Cd are present in molluscs and
crustaceans (1-2 mg/kg). Based on estimations of Cd intake, more than 80% of food-ingested
cadmium comes from cereals, vegetables and potatoes, and the average Cd intake from food in
the United States varies generally between 8 and 25 μg per day (Olsson et al., 2002).
Tobacco smoking is another important source of Cd exposure, with a single cigarette
estimated to contain 1-2 μg of Cd. Roughly 10% of the Cd content of the cigarette is inhaled,
with approximately 50% of absorption into circulation occurring in the lung (Järup and Åkesson,
2009). There is some evidence of environmental or ‘second-hand’ tobacco smoke being a source
of exposure to Cd in children, but this source of exposure does not seem to be an important one
in adults (McElroy et al., 2007).
An emerging source of cadmium exposure is from electronic-waste (e-waste) dumping
grounds in Western Africa and China, among other developing countries (Schmidt, 2006).
Primitive e-waste recycling plants set up in these locations lead the adult and child workers who
handle, assemble and dismantle electronics to be at great risk of Cd exposure. Children living in
Guiyu, China, for instance had significantly higher blood lead and blood Cd levels than those in
comparably sized and urbanized cities. The blood Cd levels of children in Guiyu is 1.58 μg/L,
compared to 0.97 μg/L in Chendian, a city with much lower environmental contamination. The
increased blood levels in Guiyu are linked to e-waste. (Zheng et al., 2008)
4
1.2 Cadmium Toxicity
1.2.1 Routes of Exposure
The extent of Cd absorption into the body depends on the route of exposure.
Approximately 3-10% of Cd is absorbed in the gastrointestinal system when ingested. Unlike the
relatively poor absorption from food, however, approximately 50% of inhaled Cd is absorbed
(Sahmoun et al., 2005). Dermal exposure is not considered a major route of Cd toxicity (US
Department of Health and Human Services, 2012).
The primary route of exposure in the general population is through the diet. Information
on the health effects of ingestion of Cd have been derived mainly from studies of humans living
in Cd-polluted areas. Exposure is estimated in these populations from blood or urinary cadmium
levels. Cadmium is readily found as a free-ionic form in water, but exists in complexes with
proteins including MT in food (US Department of Health and Human Services, 2012). As
mentioned in a previous section, typical dietary intake in the United States is approximately 30
μg daily. Worldwide, however, there are areas with very high Cd in the soil and crop uptake can
lead to significant dietary exposure to individuals living nearby. In the Jinzu and Kakehashi river
basins in Japan, Cd-contaminated rice is a major ingested source of the toxic metal. A lifetime of
eating the Cd-contaminated rice can lead to a serious kidney and bone disease named “Itai-Itai”,
further described in section 2.3 (Shimizu et al., 2006).
Inhalation is a major route of occupational exposure to Cd. The effects of inhaled
exposure to Cd in humans was derived from studies of workers exposed to Cd fumes in
industries including smelting, battery manufacturing, soldering and pigment production. The
primary form of Cd exposure in occupational settings is cadmium oxide (CdO) (US Department
of Health and Human Services, 2012). In general, the different species of cadmium have similar
toxicological effects by inhalation, although some differences do exist for the less soluble
cadmium pigments including cadmium sulfide (CdS) and cadmium selenide (CdSe) (Buckley
and Bassett, 1987). In the non-occupationally exposed general population, smokers have Cd
blood and body burdens double those of non-smokers (Waalkes, 2003).
5
1.2.2 Cadmium Transport in the Body
Cadmium, as a non-essential cation, is not absorbed by any specific mechanism and
therefore crosses membranes by a variety of different means. When inhaled as cadmium oxide
(CdO) or cadmium dichloride (CdCl2), Cd is transported along primary olfactory neurons to the
olfactory bulb, where it accumulates (Sunderman, 2001). Another site of accumulation after
inhalation is the lungs. Large particles (greater than 10 μm in diameter) tend to be deposited in
the upper airway and exhaled, while small particles (approx. 0.1 μm) tend to penetrate the alveoli
(US Department of Health and Human Services, 2012). The specific mechanism by which Cd
reaches the circulation from the alveolar epithelium has not yet been elucidated. It is possible
that Cd binds to chelators such as glutathione or cysteine, or likely uses transporters dedicated to
other biomolecules or ions (Martelli et al., 2006).
After ingestion, the acidic environment of the digestive tract favours Cd transport by a
broad specificity proton-metal cotransporter named divalent metal transporter-1 (DMT-1) at the
apical membrane of enterocytes (Martelli et al., 2006). Repressing DMT-1 by mutation in a
human enterocyte model resulted in diminished Cd entry into cells, whereas overexpression
strongly increased Cd uptake (Kayaaltı et al., 2015). Zinc transporters ZIP8 and ZIP14 may be
involved in cellular entry as well (He et al., 2009). Due to the Cd2+ ion similarity to Ca2+, Cd
transport into cells is also mediated through calcium channels (Leslie et al., 2006). Cadmium
absorption can be increased by dietary deficiencies in calcium or iron, or in diets low in protein
(Erik et al., 2013).
Once inside the cell, Cd encounters proteins with exceptionally high affinity for it:
metallothioneins (MT). MT are considered the major zinc-binding proteins in higher eukaryotes,
and MT-encoded genes are strongly induced by zinc and cadmium (Martelli et al., 2006).
Metallothionein has a much higher affinity for Cd2+ than for Zn2+. Cadmium is then either
retained in the cytoplasm bound to MT in the enterocyte, or transferred to the basolateral
membrane to exit from the cell into circulation (Vesey, 2010). The specific exit mechanism is
not yet known, although it is thought to be through the transporter FPN1 (Kim et al., 2007). In
the circulation, Cd is transported primarily bound to albumin or metallothionein. Storage in the
liver is mainly as Cd-MT, and a small proportion of Cd is released over time as hepatocytes die
off either through normal senescent processes or Cd injury. Due to Cd’s very high affinity for
6
thiol groups of metallothionein and other intracellular molecule including gluthathione, Cd tends
to accumulate in the kidney and liver with a half-life of up to 30 years (Templeton and Liu,
2010). The binding of Cd to MT is a detoxification mechanism that is saturable at high
concentrations, after which renal toxicity becomes apparent (Yang and Shu, 2015). The basic
flow scheme of Cd through the body is diagrammed in Fig. 1, following uptake by liver cells.
Cadmium is very poorly excreted from the body, mainly through urine and feces. In
humans the amount excreted daily in urine represents between 0.005 and 0.015% of the total
body burden (Thévenod and Lee, 2013b).
Figure 1. Cadmium transport in the body. Following uptake into liver cells, small amounts of
Cd are slowly released into the bloodstream either as exchangeable Cd, which then become
bound to albumin, or as Cd-metallothionein. Filtration in the kidneys lead to Cd uptake into renal
tubular cells, where internal lysosomal degradation of Cd complexes result in free Cd2+ to cause
cellular damage. GSH = glutathione, reduced; MT = metallothionein; aa: amino acids; Alb =
albumin (Nordberg, 2009).
7
1.2.3 Organs Affected by Cadmium Toxicity
The liver is the major organ in the in vivo handling of Cd. Liver accumulation of Cd
varies between 1.5 – 8 µg/g dry weight depending on age, sex and environmental exposure. This
accretion is 10 – 20 times lower than the kidney, which is especially surprising as the liver is the
first organ targeted after ingestion of Cd (Thévenod and Lee, 2013b). After chronic exposure, Cd
was found to be hepatotoxic in rodents but not humans. Mild morphological or functional
alterations may be due to induction of MT or glutathione (GSH).
Cadmium concentrations in the lungs vary between 0.4 – 3.0 µg/g dry weight and depend
on age, sex, environmental exposure, and smoking status (Vuori et al., 1979). Long-term
occupational exposure to Cd fumes frequently leads to chronic obstructive lung disease. Chronic
Cd inhalation appears to be associated with a rise in oxidative stress, associated with increased
macrophages, neutrophils and oxidant markers in bronchoalveolar lavage fluid. Proinflammatory
cytokines are elevated in 24h Cd exposed cultured human airway epithelial cells, which include
interleukin-1 beta (IL-1β) and tumor necrosis factor alpha (TNF-α) (Cormet-Boyaka et al.,
2012). Lung cancer also seems to be associated with chronic Cd exposure. A longitudinal
population study in an area close to three zinc smelters found that the risk of lung cancer was
3.58 times higher in a high exposure area compared to a low exposure area (Thévenod and Lee,
2013b). This study has been corroborated with more recent studies that conclude that
occupational exposure to Cd is an important risk factor for lung cancer (Zeka et al., 2006).
In the 1950s, a bone disease was first described in individuals living in the Jinzu river
basin of Japan. People living in the area began developing bone fractures, severe bone pain and
malformations of the long bones. These symptoms were concordant with osteomalacia,
osteoporosis and renal dysfunction, and the collection of symptoms became known as Itai-Itai
disease. It was later discovered that industrial waste containing relatively high Cd discharge from
an upstream zinc mine was to blame (Nogawa and Kido, 1993). At the concentrations
encountered that resulted in Itai-Itai disease, Cd disrupts renal calcium and phosphate transport,
along with decreasing epithelial calcium channels. As a result, excess calcium excretion in the
urine is observed. Even at much lower concentrations, environmental Cd exposure is linked to a
loss of bone density, height loss and increased bone fractures (Kazantzis, 2004). Gene expression
microarray experiments have found that bone demineralization occurs after Cd exposure via a
8
fos-independent, but src- and p38 MAPK-dependent activation of osteoclasts, which may result
in the breakdown of bone matrix (Bhattacharyya, 2009).
The kidney is the major target organ after Cd exposure, and has been classified as the
critical organ for Cd toxicity. Cd-induced nephrotoxicity will be described in detail in the
following section.
1.2.4 Cadmium Nephrotoxicity
Long term exposure to Cd results in accumulation in the proximal tubule of the kidney,
and as such the kidney is the primary critical target organ of chronic Cd toxicity. The term
‘critical organ’ is defined as the particular organ which first attains the critical concentration of a
metal under specified circumstances of exposure for a given population. The critical
concentration in the critical organ is the concentration when adverse reversible or irreversible
changes occur in the most sensitive cells of that organ (Nordberg, 2009). Tubular dysfunction
has been identified as the first sign of adverse effect, with cell death being the primary cause
(Thomas et al., 2014).
In long term exposure to Cd, the level of toxic metal is initially highest in the liver then
gradually increases in the kidney. The major source of renal Cd in chronic exposure is the liver.
This is evidenced in experiments showing that transplanted livers from Cd-exposed rats into
recipient control rats decrease in Cd concentration, with a resulting increase in renal Cd in
recipient rats (Chan et al., 1993).
Circulating Cd is filtered by the glomerulus because of the small molecular mass of the
various species of the metal. It is mainly internalized by the S1 segment of the proximal tubule,
whose cells possess apical transporters that allow for transport of exchangeable or bound Cd
(Thévenod and Lee, 2013b). Even though the Cd-MT complex is nontoxic, after trafficking into
lysosomes and subsequent degradation, exchangeable Cd is released into the cytosol via
lysosomal DMT1-mediated transport. The prevailing thought is that renal damage is prevented
until a stage is reached when the kidney can no longer produce enough MT to protect against Cd.
MTs play a critical role in Cd accumulation in the kidneys, with MT-I/II knockout mice
containing no more than 10% of the renal Cd as wild type controls (Liu et al., 1998). However,
experiments with MT-I/II knockout mice have shown that they are exceedingly more susceptible
9
to Cd-induced nephrotoxicity compared to wild-types. In wild-type mice, renal damage became
evident at concentrations much higher than those that could be quenched with MT (Liu et al.,
2000).
Cd nephropathy is seen in about 10% of the population at renal concentrations of
approximately 200 µg/g, and in 50% of the population at 300 µg/g (Thévenod and Lee, 2013b).
Interestingly, more than 7% of the general population may have significant Cd-induced kidney
alterations due to chronic Cd exposure with levels as low as 50 µg/g. As the critical
concentration for nephropathy is approached in chronic Cd toxicity, the Cd chelating capacities
of cells and adaptive antioxidant defense systems are overwhelmed, resulting in lipid
peroxidation and oxidative damage leading to cell death (Liu et al., 2009).
Cadmium not only damages the proximal tubule, but also is involved in glomerular
damage and distal tubule damage as well. The renal toxicity induced by Cd is reflected in
reabsorptive dysfunction, associated with polyuria, phosphaturia, aminoaciduria, glucosuria and
low molecular weight proteinuria. Reactive oxygen species (ROS) damage the Na/K ATP-ase,
and tubular apoptosis seems to be responsible for the Toni-Debré-Fanconi like syndrome that is
observed (Gonick, 2008). The predominant proteins found in urine after chronic damage include
β2-microglobulin, N-acetyl-β-d-glucosaminidase (NAG), and MT, as well as retinol-binding
protein, lysozyme, ribonuclease, α1-microglobulin, and immunoglobulin light chains. The
excretion of the above proteins as well as Cd in the urine have been used as biomarkers of Cd
exposure (Erik et al., 2013).
The presence of larger proteins such as albumin or transferrin in the urine after
occupational exposure suggest glomerular damage (Prozialeck and Edwards, 2010). Glomerular
damage is also observed with a decreased glomerular filtration rate as demonstrated in studies of
occupationally exposure workers (Järup et al., 1995). Overall, the pathogenesis of glomerular
lesion in Cd nephropathy is not well understood, and that is where the current study aims to fill
some gaps. A more comprehensive introduction to the renal mesangial cell (RMC) of the
glomerulus is given in section 1.5.
10
1.2.5 Environmental Exposure
The first major epidemiological study that investigated the renal effects of Cd on the
general population was the Cadmibel study conducted in Belgium from 1985 – 1989 (Lauwerys
et al., 1990). Belgium is an important producer of Cd (approximately one-fourth of European
production), and certain areas of the country are polluted by the toxic metal due to past emissions
from industry. 1699 subjects aged 20-80 years of age were studied in four regions with varying
degrees of Cd pollution. The World Health Organization (WHO) recommends that Cd
concentration should not exceed 5 – 10 μg Cd/g urinary creatinine. The Cadmibel study,
however, was the first to link urinary Cd with proteinuria in a cross-sectional population study
that showed that there was a 10% chance of developing tubular dysfunction at urinary Cd levels
of 2–3 μg Cd/g urinary creatinine (Thévenod and Lee, 2013b).
The majority of epidemiological studies on the health effects of environmental pollutants
up to the point of the Cadmibel study attempted to assess relationships between external
indicators of pollution (emission sources or environmental monitoring reports) and clinical signs
of health effects. That approach, as detailed by the authors of the Cadmibel study, has limitations
in that environmental pollutants can only be identified as etiological agents of pathology when
their role is overwhelming in comparison to confounding factors, and when the latency period
between exposure and disease in short (Lauwerys et al., 1990). Fortuitously, Cd is a toxic agent
for which there are biological indicators to estimate current and lifetime exposure (Cd in the
blood and kidney, respectively) and also methods to study early nephrotoxic effects. Several
renal biomarkers including retinol binding protein, urinary calcium, β2-microglobulin and N-
acetyl-β-glucosaminidase were positively correlated with urinary Cd, indicating proximal tubule
dysfunction (Buchet et al., 1990). The authors concluded that when urinary excretion of Cd is
below 2 μg/24 h, the risk of occurrence of renal effects remains low. Interestingly, Cd body
burden and diabetes was found to have a synergistic effect on two renal variables (N-acetyl-β-
glucosaminidase and β2-microglobulin) (Buchet et al., 1990).
In addition to Belgium, studies on Cd exposure in Sweden have been undertaken (Järup
et al., 1998). The diet is the main source of Cd exposure in the Swedish general population, with
average daily intake at approximately 15 µg/day. It has been shown that Cd concentrations in
agricultural soil and wheat have increased continuously during the last century in Sweden. From
11
studies conducted in Sweden, data indicate that adverse health effects from Cd exposure may
develop in about 1% of the adult general population at an average daily intake of 30 μg over a
life-span (Järup et al., 1998).
Studies on inhabitants of the Jinzu River basin and other Cd-polluted areas of Japan
demonstrated increased mortality and progression to Itai-Itai disease as described in an earlier
section. Several reports have described an association between increased urinary Cd and
mortality in these Cd-polluted areas of Japan, and further studies have been conducted to
investigate a similar association with lower concentrations of Cd in environmental exposure
(Suwazono et al., 2014). The collection of studies in Japan clarify that urinary Cd is significantly
associated with increased mortality, in both Cd-polluted and Cd-non-polluted areas of the
country.
Environmental studies of Cd exposure and subsequent health outcomes have become
more frequent in the past 15 years as we continue to learn more about the risks of chronic Cd
toxicity. Epidemiologic studies regarding the bioavailability of Cd in food and exposure-related
effects in non-occupationally exposed populations have linked low-level Cd exposure and
adverse effects that are not just restricted to kidney and bone. These data include almost every
organ in which Cd accumulates, including eye tissues (Satarug et al., 2010). Exposure levels
associated with kidney and bone effects from recent environmental studies are summarized in
Table 1.
12
Table 1. Exposure levels associated with kidney and bone defects in recent environmental
studies of Cd exposure.
Study Population, age, reference Exposure/outcomes
Sweden, n = 820, 53-64 years of age,
Åkesson et al. 2005, 2006
Blood and urinary Cd at 0.38 μg/L and 0.67 μg/g
creatinine were associated with tubular impairment.
Urinary Cd at 0.8 μg/g creatinine was associated
with glomerular impairment. Increased body
burden of Cd was associated with lowered bone
mineral density, decreased serum parathyroid
hormone and bone metabolism.
Thailand, n = 200, 16-60 years of age,
Satarug et al. 2005
A 3-fold increase in body burden of Cd associated
with 11% increase in probability of having high
blood pressure, 32% increase in probability of
having renal injury and 61% increase in the
probability of tubular impairment.
Thailand, n = 224, 30-87 years of age,
Teeyakasem et al. 2007
Odds of tubular impairment found to be 10.6 times
higher when comparisons were made between
urinary Cd levels of 1-5 μg/g creatinine to >5 μg/g
creatinine.
United States, n = 4258, ≥ 50 years of age.
Gallagher et al., 2008
A 1.43-fold increase in osteoporosis risk comparing
urinary Cd of 1 μg/g creatinine to <0.5 μg/g
creatinine.
Belgium, n = 294, mean age 49.2 years,
Schutte et al. 2008
A 2-fold increase in body burden of Cd associated
with increased bone resorption, urinary calcium
loss, decreased proximal forearm bone density, and
low serum parathyroid hormone.
China, n = 294, 3-year observation,
Wu et al. 2008
Progressive tubular and glomerular impairment was
observed among those with urinary Cd > 10 μg/g
creatinine.
13
United Kingdom, n = 160, 18-86 years of age,
Thomas et al. 2009
Risk for early renal effects (defined as urinary N-
acetyl-β-glucosaminidase > 2 IU/g creatinine) was
increased by 2.6-fold and 3.6-fold comparing
urinary Cd 0.3 versus <0.5 versus ≥0.5 μg/g
creatinine.
United States, n = 14778, >20 years of age,
Navas-Acien et al. 2009
Risk for albuminuria was 2.34 and risk for lowered
glomerular filtration rate was 1.98, comparing
those in the highest versus lowest quartiles of blood
Cd and blood lead.
1.3 Molecular Changes with Cd Exposure
1.3.1 Reactive Oxygen Species
1.3.1.1 Source of Reactive Oxygen Species in Normally Functioning Cells
One avenue by which Cd displays its toxicity is through the accumulation or formation of
reactive oxygen species (ROS). At the cellular level, many of the damaging and protective
processes induced by Cd require modulation of cellular redox status. The major ROS in cells
include superoxide (O2●-), hydroxyl (HO●), hydrogen peroxide (H2O2), and hydroperoxide
species (ROOH, ROO●) (2008). ROS are potentially dangerous to cells and can be extremely
reactive. They can react with proteins, lipids and nucleic acids, leading to alterations in
macromolecule structure and function. Consequently, ROS can cause lipid peroxidation and
membrane leakiness, enzyme inactivity, DNA breakage and mutation (Halliwell and Gutteridge,
1990). Before investigating the role Cd plays in changes of cellular ROS content, the standard
sources of ROS in mammalian cells will be described in the following section.
Mitochondria are thought to be the largest contributors to intracellular oxidant production
(Holmström and Finkel, 2014). Throughout the process of oxygen-dependent ATP production,
divalent oxygen can undergo a single-electron reduction to generate the superoxide anion.
Specific sites of superoxide production are generally believed to be complex I and III of the
electron transport chain (Brand, 2010). In general, an increase in the proton-motive force, or the
14
combination of electrical and chemical pH gradients across the inner mitochondrial membrane, is
associated with increased ROS production (Mailloux and Harper, 2012).
Another important source of ROS is the family of NADPH oxidases. Initially detected
during neutrophil phagocytosis, it is now known that these enzymes are broadly distributed
among many tissues (Covarrubias et al., 2008). The NADPH oxidase family protein complexes
(NOX1-5, dual oxidase 1 (DUOX1) and DUOX2) function to purposely produce ROS for a
range of host defence and signalling functions by transferring electrons from cytosolic NADPH
to divalent oxygen to create superoxide (Aguirre and Lambeth, 2010).
A wide range of enzymes including xanthine oxidase, nitric acid synthase,
cyclooxygenases, cytochrome p450 enzymes and lipoxygenases can all produce ROS
(Holmström and Finkel, 2014). The prevailing view is that ROS produced by these enzymes are
byproducts of the reactions they catalyze. This idea is slowly changing as redox-signalling
pathways are discovered that require ROS generated by the above enzymes (Kil et al., 2012).
1.3.1.2 Source of ROS during Cd exposure
Redox-active metals such as iron, copper and chromium undergo redox cycling which
leads to disproportionate consumption of O2 and other cellular reducing equivalents. This in turn
results in the formation of ROS, ultimately causing oxidative stress. Cadmium, unlike the above
metals, exists entirely as the Cd2+ ion under biological conditions, and does not redox-cycle due
to the ion having a complete highly stable 4d electron shell. Cadmium is unable to generate free
radicals by itself, but reports have indicated that superoxide, hydroxyl, nitric oxide radical and
lipid peroxides could be generated indirectly (Rani et al., 2014). The ways in which Cd can
generate ROS are multifactorial, and are summarized in Fig. 2.
One route for Cd to increase cellular ROS levels is to alter glutathione (GSH) levels.
Glutathione is the major intracellular antioxidant (Liu et al., 2009). When cells are oxidatively
challenged, GSH synthesis increases in order to buffer changes in cellular redox status.
Alterations in GSH levels have been observed in Cd toxicity, with most suggesting an increase in
GSH levels after exposure (Ercal et al., 2001). Co-treatment in cells with N-acetylcysteine
(NAC), which replenishes cellular GSH, protects against chronic hepatoxicity and
15
nephrotoxicity. By binding to the thiol group of GSH, Cd2+ prevents its action from buffering
changes in cellular ROS (Liu et al., 2009).
Lipid peroxidation has also been observed in Cd toxicity. Reasons for peroxidation have not
been entirely elucidated, but disturbances in GSH or MT levels may allow for free radicals to
react with double bonds in membrane lipids to result in lipid peroxidation (Ercal et al., 2001). As
a consequence of lipid peroxidation, mitochondrial respiration is heightened, thereby enhancing
the potential to produce more superoxide species. In a series of studies in rat adrenal glands, it
was indicated that with an increase in Cd exposure time and concentration, production of lipid
peroxides was also elevated (Yiin et al., 2001).
The mitochondrion is a target of Cd toxicity. It has been proposed that Cd binds to
protein thiols in the mitochondrial membrane, affects mitochondrial permeability, inhibits the
electron transport chain and therefore generates ROS (Liu et al., 2009). By inhibiting complex III
of the electron transport chain, Cd indirectly results in accumulation of the unstable
semiubiquinones, which are prone to transfer single electrons to divalent oxygen, forming
superoxide.
Cadmium can also replace the iron and copper in cytoplasmic membrane proteins, which
would increase in the intracellular concentrations of those metal ions. These ions are then free to
participate in Fenton reactions. Fenton reactions are iron- or copper- catalyzed reactions that
generate hydroxyl radicals (Rani et al., 2014).
16
Figure 2. Cadmium induction of oxidative stress. Cadmium is a redox inactive metal, so its
toxic effects in the cell indirectly lead to accumulation of ROS in acute exposure. Cadmium
inhibits the anti-oxidant system in cells by reducing the activity of superoxide dismutase (SOD),
catalase, and glutathione (GSH). Cadmium has been proposed to interact with mitochondrial
membrane proteins, inadvertently increasing superoxide production. By replacing other metals in
proteins including metallothionein (MT), those free metal ions then can participate in Fenton
reactions to increase ROS (Henkler et al., 2010).
1.3.2 Cd2+ Impact on Ca2+ Signalling
Calcium ions are important intracellular messengers, transducing information from the
extracellular environment into the cell by rapidly changing cytosolic concentration ([Ca2+]cyt)
from a resting level of approximately 0.1 μM to at least a 10-fold increase upon stimulation
(Thévenod and Lee, 2013a). Ca2+ exerts much of its influence by binding to proteins and
17
reversibly altering their conformation, and this process is highly controlled with complex
machinery that ushers Ca2+ to and from different cellular compartments. With the Cd2+ being
structurally similar to Ca2+, and not subject to the same strict control, Cd2+ can interfere with
normal Ca2+ functions.
Acute exposure to Cd2+ has been reported to induce a rapid and sustained increase in
[Ca2+]cyt. Modern aequorin-derived Ca2+ probes have been used to detect Ca2+ without
interference from Cd2+, and experiments with these probes have discovered that Cd2+ treatment
leads to a reduction in endoplasmic reticulum (ER) retention of Ca2+ (Biagioli et al., 2005). This
may be due to Cd2+ inhibition of the sarcoplasmic/endoplasmic reticulum Ca2+-ATPase
(SERCA), which normally transfers Ca2+ from the cytosol into the endoplasmic reticulum
(Thévenod and Lee, 2013a). Prolonged exposure to Cd2+ results in a depletion of ER calcium
pools, and therefore diminishes the response to agonist evoked Ca2+ signalling. Cd2+ exposure
also inhibits calcium-sensitive phospholipase-C activity, which can also contribute to an overall
reduction in Ca2+ signaling with prolonged Cd2+ treatment (Vignes et al., 1996).
Calmodulin is the main mediator of Ca2+ signaling. The ability for Cd2+ to bind to
calmodulin has been demonstrated to occur at C-terminal sites III and IV, which is also where
Ca2+ binds with high affinity (Chmielowska-Bąk et al., 2013). Cd2+ has been shown to
biphasically activate the Ca2+/calmodulin dependent protein kinase II (CaMK-II) in renal
mesangial cells in a concentration-dependent manner (Liu and Templeton, 2007). In addition,
Cd2+ inhibition of membrane calmodulin-dependent Ca2+-ATPases is well documented
(Thévenod and Lee, 2013a).
Cd2+ potently blocks voltage-dependent calcium channels (VDCCs) with an IC50 of 0.3
µM. VDCCs are often expressed in cells that are excitable by extracellular Ca2+ signaling, and
this activity of Cd2+ suggests that it can block this communication (Atchison, 1988). Cd2+ can
also interfere with other Ca2+ channels, including hormone- and neurotransmitter-triggered store-
operated Ca2+ channels (SOCs). SOCs are responsible for non-voltage gated Ca2+ intercellular
communication (Baker et al., 2003). External Cd2+ may also operate as an external ligand of Ca2+
sensing G-protein coupled receptor (Misra et al., 2002).
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1.3.3 Endoplasmic Reticulum Stress
An accumulation of misfolded proteins, depletion of Ca2+ stores, or a buildup of
oxidative species in the ER lumen leads to endoplasmic reticulum (ER) stress. The signalling
proteins protein kinase RNA (PKR)-like ER kinase (PERK), inositol-requiring protein-1 (IRE1)
and activating transcription factor-6 (ATF6) sense ER stress, and collectively activate a
downstream pathway known as the unfolded protein response (UPR). Chronic ER stress can
lead to apoptotic cell death by induction of CHOP, phosphorylation of the Jun-kinase (JNK) or
activation of caspase-12 (Tabas and Ron, 2011).
Cadmium upregulates ER stress response genes, and the pattern of activation appears to
be distinct from other divalent toxic metals (Permenter et al., 2011). Both CHOP and PERK
show heightened activity with Cd2+ exposure as well. GRP78, a protein that indicates an
accumulation of unfolded proteins, is also increased with cadmium toxicity. Cd2+-induced ER
stress can cause phosphorylation of JNK, caspase activation, and crosstalk with mitochondria to
induce apoptosis (Thévenod and Lee, 2013b). The superoxide ion has been suggested to be
responsible for inducing ER stress, and Cd2+ as described in a previous section is capable of
indirectly increasing the cellular concentration of superoxide.
In general, the UPR activated by Cd2+ can be viewed as a survival response in response to
toxicity. However, accumulation of Cd2+ in chronic toxicity and upregulation of CHOP and
caspase-12 gradually lead more cells towards an apoptotic fate even when counteracting
mechanisms are intact (Lee et al., 2012).
1.3.4 Cancer, Proliferation and Survival in Response to Cd2+
The complexity of Cd toxicity becomes more apparent when discussing proliferative and
survival responses to the metal. Cadmium exemplifies the pleiotropic nature of a toxic agent that
has no biological function, and as such it has a multifaceted response in cells that includes not
only cell death effects, but the promotion of cancer, stimulation of cell proliferation and
inhibition of apoptosis.
Cadmium is recognized as a group 1 carcinogen by the International Agency for the
Research on Cancer (IARC), meaning there is enough evidence to conclude that it is
carcinogenic to humans (IARC, 1993). This designation exists due to repeated findings where
19
occupationally exposed humans develop lung cancer after inhalational exposure to Cd, as well as
numerous studies in rodents that suggest the same when they are chronically exposed to airborne
Cd (Waalkes, 2003). Some metals can directly bind to DNA as a prelude to mutation and
therefore cancer. Cadmium, however, only weakly binds to DNA and therefore this initiation of
mutation is unlikely. As discussed in an earlier section, Cd does not participate directly in redox
reactions, but may manifest carcinogenic effects by indirectly leading to the accumulation of
ROS (Bishak et al., 2015). Alternatively, Cd can inhibit DNA repair mechanisms and the
resulting genotoxicity could be a key event in carcinogenesis. Cadmium has been shown to
impair almost all major DNA repair pathways, with evidence of interference of nucleotide
excision repair, base excision repair, and mismatch repair. Frequently these effects were
observed at non-cytotoxic concentrations of the metal (Bishak et al., 2015). The tumor
suppressor protein p53 plays a role in the initiation of apoptosis, in the activation of DNA repair
proteins, and in the inhibition of angiogenesis. Its inactivation is documented as a key factor in
the development of tumours (Zilfou and Lowe, 2009). In MCF7 cells, acute exposure to Cd
disrupts native p53 conformation possibly by replacing zinc within the protein structure
(Waalkes, 2003). It is clear that mutagenesis is not the only mechanism by which carcinogens
can produce cancers. Epigenetic mechanisms may apply in the initiation of tumours as well, and
Cd-transformed prostate epithelial cells were discovered to contain agglomerative DNA
hypermethylation (Luevano and Damodaran, 2014).
With regards to carcinogenesis, Cd can activate proto-oncogenes associated with cell
proliferation, including c-myc, c-jun, or c-fos both in vitro and in vivo (Waalkes, 2003). For
instance, our lab has shown that Cd activates c-fos in mesangial cells (Wang and Templeton,
1998). Being immediate early-response genes (IEGs), the products of c-fos and c-jun constitute
the AP-1 transcription factor, which activates several genes involved in cell growth and division
(Thévenod and Lee, 2013b). Developmental signaling pathways, such as those activated by
secreted Hedgehog and Wnt proteins are also affected by Cd toxicity (Hanson et al., 2010). The
main MAPKs that exist in mammalian cells are ERK, JNK, and p38. In a non-small cell lung
carcinoma cell line, CL3, Cd at low cytotoxic doses (15 – 80 µM) transiently activated JNK and
at the same time reduced ERK activity. At high cytotoxic concentrations (130 – 160 µM), there
was persistent activation of JNK and p38 (Chuang and Yang, 2001). In 9L rat brain tumor cells,
high concentrations of Cd (100 µM) activated p38, and lower concentrations (60 µM) were
20
accompanied with the activation of ERK. All three of the MAPK pathways are activated by Cd
and display a distinct pattern of activity in mesangial cells (Ding and Templeton, 2000). As
mentioned in the previous paragraph, the inactivation of p53 leads to indirect mitogenic activity
by Cd. Furthermore, Cd-induced malignant transformation leads to cells that acquire a resistance
to apoptosis (Qu et al., 2007).
Cadmium’s ability to transform leads to apoptosis-resistant malignant cells. Generally,
sub-micromolar concentrations lead to proliferation and delayed apoptosis. In prostate cancer
cells that have been transformed by Cd (RWPE-1) for instance, pro-apoptotic genes such as
BAX, and caspase-8, -6, -4 and -3 show reduced expression (Luevano and Damodaran, 2014).
This corresponds with the over-expression of the anti-apoptotic protein Bcl-2 (Waalkes, 2003).
Caspase-3 was also shown to be inhibited by Cd exposure (Gunawardana et al., 2006). Previous
work with our group has shown that Cd can inhibit DNA laddering and both intrinsic and
extrinsic apoptotic pathways in rat mesangial cells (RMC), at 10 µM exposure for 8 hours
(Gunawardana et al., 2006). With the transformation to malignancy, constitutive activity of p62
and Nrf2 result in a decrease of ROS generation and general apoptotic resistance (Son et al.,
2014). Suppression of apoptosis facilitates aberrant cell accumulation, which favors pre-
neoplastic or neoplastic cells which may lead to tumour formation.
Overall, Cd acts as a double-edged sword, influencing pathways of cell survival and cell
death. The outcome of exposure to Cd at the cellular level depends not only on the duration and
level of exposure of the metal, but also on intrinsic tissue specificity and metabolic state. What is
a high cytotoxic concentration in one cell line may be of low effect in another. The following
section will discuss mechanisms of cell death that are activated in Cd toxicity.
1.3.5 Cell Death Mechanisms
1.3.5.1 Apoptosis
Programmed cell death is a tightly regulated physiological process that is integral to
physiological homeostasis, removing damaged or unwanted cells in the body. Apoptosis has
been designated as ‘Type I’ cell death, classically thought to be executed by activated caspase
proteins. Caspases are specific enzymes that participate in signaling cascades that culminate in
the rapid removal of organelles and other cellular structures (Mukhopadhyay et al., 2014).
21
Alternatives to caspase-dependent apoptosis include apoptotic cell death mediated by apoptosis-
inducing factor (AIF), endonuclease G (endo G) release from the mitochondria, or cathepsin
release as result of lysosomal membrane permeabilization (Templeton and Liu, 2010). After
uptake by cells, Cd2+ elicits a general cellular stress response that can lead to apoptosis.
Of the MAPKs, the stress activated kinases JNK and p38 can facilitate apoptosis when
the cell is met with environmental stresses. Both JNK and p38 are activated at higher-toxic
concentrations of Cd in HEK293 cells (at 50 µM), with reducd activation at low levels of the
toxic metal (0.5 µM) (Hao et al., 2009). Our lab has observed biphasic responses to JNK in
RMC, with an initial burst followed by a second sustained activation over a few hours (Ding and
Templeton, 2000). JNK activity after hours of Cd exposure may be due to accumulation of ROS,
and also has been considered responsible for Cd-mediated cell death.
The mitochondrial-mediated intrinsic pathway of apoptosis can be initiated by multiple
stimuli, including ROS and increases in cytosolic calcium. The release of pro-apoptotic factor
cytochrome c, followed by caspase-3 and caspase-9 are a prelude to apoptosis. In kidney
proximal tubule cells, both caspase-3 and -9 are activated with Cd exposure, suggesting that
mitochondrial dysfunction by Cd2+ and consequent caspase-dependent apoptosis are related to
the metal’s renal toxicity (Fujiwara et al., 2012). As demonstrated by light scattering, Cd2+ can
induce the swelling of mitochondria and subsequent release of cytochrome c. There is evidence
supporting the view that aquaporin-8 present in the inner mitochondrial membrane is activated
by Cd2+, which is then responsible for the influx of water into the organelle (Thévenod and Lee,
2013b). Cadmium also inhibits the function of electron transport chain complexes, resulting in
mitochondrial uncoupling and dysfunction (Choong et al., 2014). A cell’s fate with regard to
apoptosis seems to be determined by the balance of pro-apoptotic (e.g., Bax, Bak) and anti-
apoptotic (e.g., Bcl-2, Bcl-xL) factors. The anti-apoptotic proteins are localized to the outer
mitochondrial membrane, and work to prevent cytochrome c release from the mitochondria. In
Cd2+ exposed (40 µM treatment) lung epithelial fibroblast (WI-38) cells, anti-apoptotic Bcl-2 is
decreased, and Bax translocates to the mitochondria which then releases cytochrome-c (Oh et al.,
2004). Another interesting observation with Cd exposure is the over-accumulation of p53 in the
cytoplasm. Studies in NRK-52E normal rat kidney epithelial cells demonstrated that Cd
markedly increased p53 phosphorylation as well as intracellular accumulation caused by the
inhibition of p53 degradation (Fujiwara et al., 2012). Cadmium was found to strongly suppress
22
the Ube2d gene family, part of the ubiquitin-proteasome system that degrades p53. The buildup
of p53 would then lead to apoptosis through the release of apoptogenic factors from the
mitochondria (Schuler and Green, 2001). Evidence in vivo was acquired when Cd was found to
inhibit a number of Ube2d genes in the kidneys of mice that were chronically exposed for 12
months, which caused mild renal toxicity (Fujiwara et al., 2012). Cadmium can additionally
induce apoptosis through AIF translocation from the mitochondria to the cytosol in MRC-5
human fibroblasts (Shih et al., 2004).
Along with the activation of the classical intrinsic pathway of apoptosis involving the
mitochondria, Cd can activate the caspase-independent pathways of apoptotic cell death.
Cadmium entry into cells can upregulate the expression of inositol 1,4,5-triphosphate receptor 1
(IP3R1), allowing the release of Ca2+ of the endoplasmic reticulum. An acute increase in Ca2+ in
the cytosol activates the apoptotic protein Calpain, which mobilizes pro-apoptotic factors to
induce DNA fragmentation (Rani et al., 2014). In rat kidney proximal tubule cells, calpain was
active at 6 hours of 10 µM Cd exposure, and apoptosis could be blocked by calpain inhibitors. At
24 hours of 10 µM exposure, calpain activity was reduced and caspase-9 and -3 were active, and
apoptosis could then be repressed by caspase inhibitors (Lee et al., 2006). Phospholipase C
(PLC) produces inositol-1,4,5-triphosphate (IP3) from phosphatidylinositol 4,5-bisphosphate to
induce the release of calcium from the ER. There is evidence that Cd-induced apoptosis can
occur via this pathway, as a PLC-specific inhibitor can abolish Cd-induced calpain and caspase-3
activation (Fujiwara et al., 2012). Moreover, Cd2+ can also activate a G-protein coupled receptor
(GPCR) in renal distal epithelial (A6) cells, which is followed by PLC activation and subsequent
calcium accumulation in the cytoplasm (Nazima et al., 2015). Sphingolipids known as ceramides
have also been shown to be intimately involved in apoptotic signaling. Ceramides can induce
cytochrome c release via ceramide channel proteins. They can also directly activate calpains.
Cd2+ increases ceramide formation, which activates calpain activity be increasing cytosolic Ca2+
(Lee et al., 2007). Lastly, it has been reported that Cd can activate the extrinsic apoptotic
pathway mediated through caspase-8 in U937 human lymphoma cells. It has been speculated that
Cd2+ can accomplish this by induction of Fas expression; Fas is a protein involved in transducing
external apoptotic signals into the cell (Li et al., 2000). In summary, multiple apoptosis signaling
pathways can be affected by Cd2+ to induce apoptotic cell death, shown in Fig. 3.
23
Figure 3. Pathways of Cd-induced apoptosis. Cadmium induces a number of apoptotic
pathways which can be summarized as mitochondrial dependent and mitochondrial-independent.
Cadmium can directly lead to the dysfunction of mitochondria via the electron transport chain
(ETC) dysfunction or by causing swelling of the organelle. Cadmium can activate caspase-8,
possibly by increasing expression of Fas on the cell surface. Cadmium may also decrease Bcl-2
expression, leading to the accumulation of Bax on the inner mitochondrial membrane, or
suppress Ube2d expression, leading to over-accumulation of p53 and consequent signals to
release apoptogenic factors. The apoptogenic factors include AIF and cytochrome c, which lead
to caspase-dependent apoptosis. In the mitochondrial-intendent pathway, Cd can also lead to ER
stress by PLC activation through GPCRs on the cell surface, or ceramide production. This leads
to Ca2+ release from the ER through IP3R, which can activate calpain apoptotic machinery
1.3.5.2 Necrosis/Necroptosis
Necrosis was once thought to be an uncontrolled form of cell death resulting in passive
cell swelling and rupture, although recent evidence has indicated that necrosis can indeed be
finely controlled and programmed (Golstein and Kroemer, 2007). Necrosis is a mode of cell
death that results from a loss of integrity of the cell membrane, the breakdown of organelles, and
24
the release of proteolytic enzymes that result in the destruction of surrounding cells and initiation
of an inflammatory response (Templeton and Liu, 2010).
In general, low concentrations of Cd cause apoptosis, while concentrations greater than
50 µM both in vitro and in vivo cause necrosis (Lee and Thévenod, 2008). At the upper end of
apoptotic concentrations of Cd, a wide variety of cell types have shown both apoptosis and
necrosis simultaneously. The inhibition of apoptotic cell signaling can initiate the re-wiring of
cellular response from apoptosis to necrosis. Depletion of ATP would favour necrosis, which has
less energy demand than apoptosis (Nicotera et al., 1998). There is some evidence that the forms
of accumulated ROS may be the switch between Cd-induced apoptosis and Cd-induced necrosis.
In U-937 human promonocytic cells treated with Cd, co-treatment with H2O2 induced necrosis,
but the same was not observed with other ROS. This may be due to H2O2 reduction of oxidized
gluthathione, leading to an accumulation of GSH (Sancho et al., 2006).
Using pharmacological inhibitors, there is evidence that Ca2+, calpains, mitochondrial
membrane potential, ROS accumulation and NF-κB all have a role in Chinese hamster ovary
(CHO) cell Cd-induced necrosis (Yang et al., 2007). Interestingly, the CHO cells investigated
displayed necrotic characteristics at 4 µM of treatment for 24 hours, calling to attention again the
variability in cellular response to Cd. Later studies with CHO cells determined that necrostatin-1
was active in blocking Cd2+-induced necrosis when cells were co-treated (Yang et al., 2007). In
cultured RMC exposed to Cd in the range of 0.1-20 µM, studies in our lab have found that the
loss of viable cells occurs almost exclusively by non-necrotic mechanisms (Xiao et al., 2009).
1.4 Autophagy
1.4.1 Definition and Purpose of Autophagy
The main point of focus for this dissertation is the process that will be defined in the
following section. Autophagy is the process of self-eating, literally being translated as such from
the Latin words for ‘self’ and ‘eating’. By degrading intracellular components that may be
damaged including proteins and organelles, the cell aims to increase its chances of survival.
Autophagy is a process that occurs in all cell types across all eukaryotes. In Saccharomyces
cerevisiae, with which most autophagy research has been conducted, the process has been
studied as a cellular response for survival during nutrient-limited conditions (Suzuki and
25
Ohsumi, 2007). There are three types of autophagy – macroautophagy, microautophagy, and
chaperone-mediated autophagy, which will be discussed in a later section.
Autophagy requires unique organelles known as autophagosomes, which are double-
membrane-bound vesicles that grow to surround portions of the cytoplasm destined for
degradation. As autophagosomes engulf a bulk portion of the cytoplasm, autophagy is generally
thought to be a nonselective degradation system. This is in stark contrast to the ubiquitin-
proteasome system, which specifically recognizes ubiquitinated proteins for proteasomal
degradation and requires thousands of genes to orchestrate (Mizushima, 2007). Studies over the
past two decades have demonstrated that autophagy has a variety of physiological roles including
starvation adaptation, intracellular protein and organelle clearance, embryonic development, anti-
aging, elimination of invading microorganisms, cell death, tumor suppression and antigen
presentation (Mizushima, 2007). Because autophagy requires a multi-step process to run to
completion, it may be advantageous to base its functions on its elementary processes. Each step
can have a variety of physiological roles, and most of the related functions have not had their
mechanisms elucidated. Of particular importance in this thesis is emerging evidence that
autophagy can protect cells against metal-induced apoptosis, with special attention given to Cd.
Over the past several years, the idea that cells can commit suicide by mechanisms other
than apoptosis or necrosis has been gaining popularity, and the true purpose of autophagy has
been questioned. The idea, however, of autophagic cell death seems to be in direct contradiction
to the evidence determining its importance in Saccharomyces cerevisiae, Caenorhabditis
elegans, Dictyostelium, and plants as a survival mechanism under periods of famine (Edinger
and Thompson, 2004). Interestingly, autophagy can be activated in mammalian cells under
periods of nutrient deprivation, although a number of mammalian cells lines have been observed
to rapidly undergo apoptosis rather than autophagy under starvation. In certain disease states,
including Alzheimer and Parkinson Diseases, cells in an advanced state of autophagy are
frequently observed (Edinger and Thompson, 2004). This may have led to the idea that
autophagic ‘cell suicide’ is of importance in disease states. What complicates the matter is that
cells are often observed to be undergoing apoptosis or necrosis concurrently with autophagy.
Cells can display autophagic vacuolization before or during their death, again confusing the
matter. Another point of complication may be the methods used to study cell death processes,
with frequently used pharmacological inhibitors having multiple targets that can affect two or
26
even all three cell death processes. Evidence for autophagic cell death and survival will be
discussed in a later section. With the possibility of autophagic cell death in mind, however, this
thesis will attempt to explore the question of Cd-induced cell death or survival by means of
autophagy in cultured RMC.
1.4.2 General Process and Molecular Machinery
Autophagy is an intracellular self-degradative pathway by which eukaryotic cells recycle
their own cellular material including proteins and organelles. The process helps maintain cellular
homeostasis and can prevent the organism from damage and disease (Wang et al., 2015a).
Genetic screens in Saccharomyces cerevisiae, Hansenula polymorpha and Pichia pastoris have
led to the identification of autophagy-related genes, or ATG genes. The process progresses in a
very similar fashion in mammals, with a growing number of mammalian autophagy genes being
discovered as yeast counterparts. The analysis of yeast ATG mutants have provided a framework
to divide the autophagic process into distinct steps, including induction, cargo selection and
packaging, vesicle expansion and completion, retrieval, and vesicle docking and fusion. The
simplified process is shown in Fig. 4. To date, 30 ATG genes have been identified in yeast. (Xie
and Klionsky, 2007).
27
Figure 4. Schematic diagram of the stages of autophagy. Autophagy begins with the
formation of an isolation membrane after induction due to cellular stress or starvation.
Autophagy core machinery leads to the expansion of the phagophore around cytoplasmic cargo,
which can be selective or bulk. Further vesicle elongation leads to the completion of the double-
membraned autophagosome structure. When the outer membrane of the autophagosome fuses
with the lysosome, the structure is then known as an autolysosome. Finally, the sequestered
material is degraded inside the autolysosome and recycled (Meléndez and Levine, 2013).
The molecular machinery of autophagy will be described below as it occurs in
mammalian cells. The induction of autophagy often requires the repression of the mammalian
target of rapamycin (mTOR), which normally inhibits autophagy by inactivating the unc-51-like
kinase 1 (ULK1) and ATG13. However, when mTOR is inhibited, there is a change in the
phosphorylation of ULK1, ATG13, and RB1-inducible coiled-coil 1 (RB1CC1, also known as
FIP200). mTOR activity is suppressed upon withdrawal of growth factors, such as insulin or
insulin-like growth factors. AMPK regulates autophagy by inhibiting mTOR with the
phosphorylation of TSC2 and Raptor, and it can directly phosphorylate ULK1 for activation (Das
et al., 2012). When ULK1 becomes active, the protein complex initiates autophagy (Füllgrabe et
al., 2014).
To begin vesicle nucleation which will grow into the autophagosome, there needs to be
activation of the class III phosphatidylinositol 3-kinase (PtdIns3K) complex, which contains
28
vacuolar protein sorting 34 (VPS34). VPS34 activation requires a complex that includes VPS15,
beclin-1, BECN1, ATG14, and Bax-interacting factor 1 (Bif1). Interestingly, Bcl-2, a protein
involved in apoptosis induction, inhibits autophagy by repressing the activity of beclin-1 and
other autophagy related proteins. Displacing Bcl-2 with Bcl-2 homology 3 (BH3)-proteins
activates the VPS34 complex (Füllgrabe et al., 2014).
Two ubiquitin-like conjugation systems exist as part of the vesicle elongation process.
One system involves ATG12 conjugation of ATG5, with the help of ATG7 and ATG10. ATG7
and ATG10 are E1- and E2- like enzymes, respectively. ATG12-ATG5 interacts with ATG16-
like 1 (ATG16L1), which may initiate the second conjugation reaction, which is of
phosphatidylethanolamine (PE) to microtubular-associated protein 1 light chain 3 (LC3-I) by the
action of protease ATG4. LC3-I is converted to LC3-II and attached to the growing phagophore
membrane. Sequestosome 1 (SQSTM1, also known as p62), interacts with ubiquitinylated
proteins on organelles or invading pathogens to shuttle them to the autophagosome. Some of the
proteins involved in the formation of the autophagosome do not stay associated with the
structure, and therefore are available for re-use almost immediately (Xie and Klionsky, 2007).
Phagophore expansion is currently poorly defined, but ATG9-ATG2-WIPI1 (WD repeat
domain, phosphoinositide interacting 1) (or WIP2) complexes have a role in providing lipid
bilayer for the growing membrane. ATG9 cycles between the donor membranes and the growing
autophagosome (Füllgrabe et al., 2014). The completed autophagosome then fuses with the
lysosome, regulated by a set of autophagic SNARE proteins (STX17, SNAP29 and VAMP8) and
ATG14 (Liu et al., 2015). The fusion process also requires Rab7 on the lysosomal surface.
Following fusion, lysosomal hydrolases including cathepsins and lipases degrade the
autophagosomal contents, including LC3-II on the intra-autophagosomal surface (Tanida, 2011).
A schematic of the molecular progression of autophagy is shown in Fig. 5.
29
Figure 5. The molecular execution of autophagy. Initiation of autophagy (1) involves the
activation of ULK1, often due to the inhibition of mTOR. ATG13, ATG101 and RB1CC1
stimulate autophagic membrane nucleation (2). The nucleation of the membrane involves
activation of PtdIns3K, containing VPS34. VPS34 activation depends on a number of factors
including beclin-1 and ATG14. The nucleated vesicle grows under two ubiquitin-like
conjugation systems, one that conjugates ATG12 to ATG5, and the other that conjugates PE to
LC3-I. Lipid conjugation allows LC3-II to become part of the growing autophagosome, and the
protein is notable for continuing the expansion of the structure (3). Phagophore expansion is
poorly defined, but involves ATG9-ATG2-WIPI1 OR WIPI2 complexes to allow donor
membranes to provide the growing phagophore with lipids. ATG9 is involved in retrieving lipids
from membrane sources (4). (5) Refers to the completion of the structure, which then docks and
fuses with the lysosome (6) for degradation of the contents of the autolysosome (7). Further
details on this process are in the text (Füllgrabe et al., 2014).
To date, the only protein consistently identified on the inner autophagosomal membrane
is LC3. As discussed above, one of the two conjugation systems that is required for
autophagosome elongation converts the processed form of LC3, LC3-I, to LC3-II. Initially, the
30
nascent pro-LC3 form is cleaved by protease ATG4 which exposes a carboxy-terminal glycine;
this form, called LC3-I, is then acted upon by ATG7 and the E2-like enzyme ATG3 to conjugate
it to the highly lipophilic PE. At the autophagosome, LC3-II has been shown to play a role both
in cargo selection (with p62), and to promote membrane tethering and fusion (Barth et al., 2010).
LC3-II protein immunoblotting is a technique used widely for the detection of autophagic
activity, and the expression levels serve as an indicator of autophagic activity (Zhang et al.,
2015).
1.4.3 Three Types of Autophagy
To date, three major types of autophagy have been defined: macroautophagy (most often
simply referred to as autophagy), microautophagy, and chaperone-mediated autophagy.
Macroautophagy is the degradation system defined in the previous section, and the progression
of which has been most studied. It accompanies the dynamic process of autophagosome
formation (Tanida, 2011). Microautophagy involves the engulfment of targets of degradation by
the lysosome itself. Both macroautophagy and microautophagy are mostly considered bulk
nonselective degradation of protein, lipids and organelles. In contrast however, chaperone-
mediated autophagy involves selective degradation of proteins with a specific amino acid
sequence: KFERQ. This is dependent on the molecular chaperone Hsc70. Chaperone mediated
autophagy also is not involved in the degradation of lipids or organelles. Targets of degradation
are instead brought to the lysosome to interact with lysosome-associated protein-2A (LAMP2A)
(Tanida, 2011) .
The lack of selectivity of macroautophagy has been called into question, however, when
selected substrates have been seen to be targeted to the phagophore-assembly site (PAS). For
instance, selective autophagy plays an active part in innate immune defense against pathogens.
Streptococcus pyogenes is targeted by selective autophagy after its entry into the cytosol. The
machinery responsible for targeting has not yet been elucidated, but clues can be provided by a
different bacterium, Shigella flexneri, whose mutants without the surface protein IscB become
trapped in autophagosomes. In human cells, p62 selectively targets polyubiquitinated products to
be collected at the PAS and degraded in autophagy (Xie and Klionsky, 2007). The bulk
degradative macroautophagic pathway is mainly focused on survival during starvation conditions
(Hale et al., 2013).
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1.4.4 Autophagy as a Cell Death Mechanism
Autophagy is currently seen as a cell survival mechanism in times of stress as opposed to
a form of cell death. The term ‘autophagic cell death’ has been widely used however to describe
cell death that is accompanied by extensive cytoplasmic vacuolization. There is evidence that
autophagy is a negative regulator of cell survival, some of which will be presented in this
section.
Experimental systems in Dictyostelium discoideum show evidence for autophagic cell
death. The Dictyostelium amoeba lacks apoptotic machinery that could be used in non-apoptotic
cell death. Upon starvation, the unicellular organism aggregates with other cells to form a
fruiting body that serves to protect the most cells possible. It undergoes autophagic cell death, as
mutations in ATG genes prevents the death of ‘stalk’ cells that would feed the rest. However,
due to the lack of apoptotic machinery, this system is limited in its ability to model multicellular
organisms (Das et al., 2012).
Certain examples of cell death have autophagic features, and in an effort to discover a
link between ATG proteins and cell death, Yu et al. treated mouse L929 fibroblastic cells with
zVAD to induce death. The authors found markers of autophagy and necrosis after zVAD
treatment, probably as a result of caspase 8-mediated cleavage of the RIP kinase. However, when
the investigators used RNAi to diminish levels of beclin-1 and ATG7, cell death was
significantly reduced. This suggests that in this particular case, cell death under zVAD
conditions requires beclin-1 and ATG7 (Yu et al., 2004). There are however other interpretations
due to zVAD’s promiscuity in inhibiting not only caspases, but also affecting the lysosomal
degradative pathway (Edinger and Thompson, 2004).
Another instance where autophagy may be responsible for cell death, is during receptor-
induced tissue remodeling. In the breast cell line MCF-10, Mills et al. (2004) found that tumor-
necrosis factor-related apoptosis-inducing ligand (TRAIL) mediated the induction of autophagic
processes associated with lumen formation in vitro. Autophagy was seen to occur both in the
presence and absence of apoptosis, and cell death in the absence of apoptosis was thought to be
from autophagy in the modeling of lumen structure (Mills et al., 2004).
32
In C. elegans, 131 somatic cells and a large number of germline cells undergo
programmed cell death in the process of development. Upon γ-ray treatment, researchers found
that fewer cells underwent the cell death program in mutants that could not undergo autophagy.
A point of note about this study, however, is that the investigators suggest that autophagy
participates in caspase-mediated cell death programs in C. elegans, which would normally be
referred to as caspase-dependent apoptotic death (Wang et al., 2013a).
Autophagy has been observed in dying cells during mammalian development, including
in the regression of the corpus luteum, the involution of the prostate gland, and the regression of
Mullerian duct structures during male genital development. However, the caveats with these
studies is that cell death seems to occur alongside autophagy, rather than due to it. Currently, the
question of whether mammalian cell death can occur by autophagy cannot be answered
definiteively, with numerous examples of cell death occurring with autophagy. The term
autophagic cell death can be considered a misnomer, with overwhelming evidence suggesting
that autophagy’s main role is to protect cells from nutrient stress, aggregates of misfolded
proteins, organelle damage, and microbes (Kroemer and Levine, 2008).
1.4.5 Autophagy as a Homeostatic or Cell Survival Mechanism
Autophagy is known to play multifunctional roles to maintain cellular homeostasis.
Although originally thought to be wholly nonspecific, it is now believed that the process can be
precise in its selection of proteins and organelles bound for degradation (Ryter et al., 2014).
Homeostatic and survival roles of autophagy will be discussed in this section.
Autophagy is known to prolong cell survival under periods of starvation. By replenishing
stores of precursor molecules during nutrient deficient states, cells can focus their attention on
essential processes for survival. Mice deficient in ATG5, which is essential for autophagosome
formation, appear almost normal at birth but die within one day of delivery. When neonates are
starved in that one day, the survival time for ATG5 deficient animals is significantly lower than
their wild type littermates (12h versus 21h). The investigators reached the conclusion that the
production of amino acids by autophagic self-degradation of proteins allowed the maintenance of
energy homeostasis (Kuma et al., 2004). In starved HeLa cells, chemical inhibition of autophagy
promoted apoptosis and caspase-3 activity (Ryter et al., 2014).
33
Autophagy also plays a role in the removal of damaged or dysfunctional mitochondria.
This more selective process is referred to as mitophagy. Mitophagy plays a role in the removal of
mitochondria during erythrocyte maturation (Ryter et al., 2014). A loss of mitochondrial
membrane potential, or an increase in ROS accumulation may be a signal to induce mitophagy,
which involves the Pink1 protein coating the damaged mitochondria for removal via autophagy.
A series of steps leads to the ubiquitination of the organelle, which then allows recognition by
the autophagic cargo protein p62 (Youle and Narendra, 2011). In addition to mitophagy, other
organelle-specific degradative processes have been identified and may contribute to cellular
survival during stress. These include autophagic degradation of peroxisomes, ribosomes, and
endoplasmic reticulum fragments.
Another role of p62 is in the recognition and removal of ubiquitinated protein aggregates.
This process is termed aggrephagy, whereby protein aggregates which are marked for removal
are transported to the autophagosome. The sequestration and subsequent removal by aggrephagy
is coordinated by the selective autophagy adapter NBR1, along with p62 which contains an LC3
interacting motif (Kirkin et al., 2009).
Autophagy can also degrade invading pathogens, which has led to the process being
recognized as part of the innate immune system. In this ‘xenophagy’, bacteria, viral particles and
other parasites can be degraded. The cellular mechanisms of xenophagy are less well studied
than conventional macroautophagy, although many required molecular components are the same.
Intriguingly, xenophagic vacuoles may be considerably bigger than classical autophagosomes,
which reflects the plasticity of the autophagosomal process to accommodate larger cargo
(Mizushima et al., 2008).
In maintaining cellular homeostasis, autophagy protects cells from disease. Growing
evidence has revealed that alterations in the process exist in many human diseases, including
multiple neurodegenerative diseases and cancer. This basal autophagy is especially important in
the liver and in other tissues where the cells (such as neurons or myocytes) do not divide after
maturation (Mizushima et al., 2008). Polyglutamate-containing proteins, for example, that cause
various neurodegenerative diseases such as Huntington disease, are degraded by autophagy
(Martinez-Vicente and Cuervo, 2007).
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1.4.6 Agents that Modulate Autophagy
In the investigation of autophagy, a number of pharmacological manipulators have been
discovered. Different modulating compounds target various steps of the autophagic process, but
ultimately either induce or inhibit autophagy. Many of these chemical manipulators are being
tested in disease states where autophagy is deregulated. In cancers, for instance, autophagy in the
early stages of the disease is cytoprotective through the suppression of chronic inflammation or
tumorigenesis. In the later stages of cancer, autophagy becomes a pro-survival mechanism for
nutrient deprived tumour cells. In these cases, it would be ideal to discover or develop agents to
modulate autophagy in order to treat disease (Duffy et al., 2015). Some of the most common
agents to activate or inhibit autophagy are listed in Table 2 and Table 3, respectively.
Autophagy can be inhibited pharmacologically by targeting multiple stages in the
autophagic process. By targeting the class III PI3K, VPS34, which is involved in the nucleation
of the autophagosomal membrane, the initiation of autophagy can be inhibited. 3-Methyladenine
(3-MA), wortmannin, and LY294002 [2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one
hydrochloride] all suppress the activity of VPS34. Inhibitors that act on the late stage of
autophagy include chloroquine (CQ), bafilomycin A1, leupeptin, and pepstatin. CQ increases
lysosomal pH and prevents the digestive activity of lysosomal hydrolases. This prevents both the
degradation of lysosomal constituents, and the fusion of the lysosome with the autophagosome.
Bafilomycin A1 selectively inhibits the vacuolar proton ATPase responsible for acidifying the
lysosome, leading to a similar result as CQ (Cheng et al., 2013a). Another inhibitor of lysosomal
acidification is ammonium chloride (Mizushima et al., 2010). Leupeptin and pepstatin both
inhibit lysosomal proteolysis, thereby preventing the final step in autophagy. Monensin is an
inhibitor of protein transport, and it inhibits autophagy by preventing the fusion of
autophagosomes and lysosomes (Kondo et al., 2005).
Activating autophagy often requires the suppression of proteins that negatively regulate
autophagy. mTOR is the most well-known repressor of autophagy under nutritionally stable
conditions. Rapamycin and its analogues temsirolimus, everolimus, and deforolimus are strong
inducers of autophagy by inhibiting the activity of mTOR (Cheng et al., 2013a). Other inhibitors
of mTOR signaling include rottlerin, perhexiline, niclosamide and amiodarone, all of which
induce autophagy. mTOR is regulated by the class I PI3K-Akt pathway in regards to cellular
35
nutritional status. There exists a negative feedback loop between mTOR and PI3K-Akt, and dual
inhibitors of PI3K and mTOR are potent inducers of autophagy (O’Reilly et al., 2006). These
dual inhibitors include NVP-BEZ235 and PI-103. Inositol triphosphate (IP3) and its receptor
(IP3R) suppress autophagy and are involved in apoptosis. Lithium and xestospongin B both
inhibit the IP3R, thereby activating autophagy (Cheng et al., 2013a). Proteasome inhibitors like
bortezomib have been shown to increase LC3-II presentation and autophagy. Accumulation of
acetylated histones and proteins promotes autophagosomal formation, and therefore inhibitors of
the histone de-acetylase complex (HDAC) activate autophagy. The mechanism remains unclear,
but vorinostat, and panobinostat inhibit HDAC and induce autophagosomal formation (Duffy et
al., 2015).There are also some agents that pharmacologically induce autophagy by activating the
positive regulators of autophagy. By activating AMPK, metformin can induce autophagy.
Thapsigargin (Tg) and tunicamycin both can induce autophagy by activating the ER stress
response (Cheng et al., 2013b).
Table 2. Compounds known to activate autophagy.
Compound name Type of agent Mechanism of action
Rapamycin (and analogues
Temsiroliums, Evorolimus,
Deforolimus)
Inhibitor of mTORC1 Promotes the initiation of autophagy
Perhexiline Inhibitor of mTORC1 Promotes the initiation of autophagy
Niclosamide Inhibitor of mTORC1 Promotes the initiation of autophagy
Rottlerine Inhibitor of mTORC1 Promotes the initiation of autophagy
Amiodarone Inhibitor of mTORC1 Promotes the initiation of autophagy
PI-103 Dual inhibitor of mTORC1/PI3K Potent promoter of the initiation of
autophagy
NCP-BEZ235 Dual inhibitor of mTORC1/PI3K Potent promoter of the initiation of
autophagy
Lithium Inhibitor of IP3 Lowers inositol and IP3 levels
36
Xestospongin B IP3 receptor antagonist Disrupts interaction between IP3R and
beclin-1, allowing beclin-1 to act in
autophagy
Bortezomib Inhibitor of proteasome Activates autophagy to deal with
clearance of proteins
Vorinostat Inhibitor of HDAC Inhibits mTOR by unknown means
Panobinostat Inhibitor of HDAC Inhibits mTOR by unknown means
Metformin Activator of AMPK Inhibits mTOR by activating AMPK
Tunicamycin Activator of UPR Activates autophagy to deal with
misfolded proteins
Thapsigargin Activator of UPR Activates autophagy to deal with
misfolded proteins
Table 3. Compounds known to inhibit autophagy.
Compound name Type of agent Mechanism of action
3-Methyladenine Inhibitor of the class III PI3K Inhibits the formation of the
autophagosome
Wortmannin Inhibitor of the class III PI3K Inhibits the formation of the
autophagosome
LY294002 Inhibitor of the class III PI3K Inhibits the formation of the
autophagosome
Bafilomycin A1 Inhibitor of lysosomal acidification Increases pH in lysosome, reduces
digestive capacity, and inhibits
autolysosome formation
Chloroquine Inhibitor of lysosomal acidification Increases pH in lysosome, reduces
digestive capacity, and inhibits
autolysosome formation
37
Ammonium chloride Inhibitor of lysosomal acidification Increases pH in lysosome, reduces
digestive capacity, and inhibits
autolysosome formation
Leupeptin Inhibitor of lysosomal proteolysis Prevents the degradation of the
lysosome
Pepstatin A Inhibitor of lysosomal proteolysis Prevents the degradation of the
lysosome
Monensin Inhibitor of protein transport Prevents fusion of autophagosome
and lysosome
1.4.7 Metals and Induction of Autophagy
The ubiquitous nature and distribution of metal contaminants in industrialized societies
has led to the growing interest in mechanisms of metal-induced autophagy. As discussed with
regards to Cd in an earlier section, the use and overuse of metals and metal compounds in the
progress of human civilization has increased their bioaccessibility. Environmental toxicant and
xenobiotic-induced cell death has been extensively studied, but less attention has been paid to
protective mechanisms in metal-induced cell death and tissue injury (Ding, 2012). Autophagy is
one of those mechanisms, and its role in response to toxic metals will be discussed in this
section.
Arsenic is a widely distributed metalloid, occurring in soil, rock, water and air. Inorganic
arsenic compounds are present in groundwater, and organic arsenic compounds are primarily
found in fish (Chatterjee et al., 2014a). Arsenic disrupts energy transduction reactions, ATP
production and capillary integrity, and leads to endothelial damage. Arsenic potently induces
oxidative stress damage. This can lead to DNA damage and apoptosis, but autophagy is another
activated process. As2O3 has been shown to induce autophagy (and possibly autophagic cell
death) in malignant glioma cells, marked by Bcl-2/adenovirus E1B 19kDa interacting protein (a
mitochondrial cell death protein) (BNIP3) and LC3 (Kanzawa et al., 2003). As2O3 also promotes
downregulation of Bax by accumulation of beclin-1 in leukemic cell lines, and extensive
vacuolization was observed (Qian et al., 2007). Autophagosome formation by activation of
38
ERK1/2 (but not Akt/mTOR or JNK) in leukemia cells has been seen in human uroepithelial SV-
HUC-1 cells, along with heightened beclin-1 and LC3 expression after NaAsO2 exposure (Huang
et al., 2009). The major source of autophagic activation following arsenic insult seems to be the
burst of ROS from damaged mitochondria, which then need to be removed via autophagy (Zhang
et al., 2012).
High concentrations of chromium in the cell can lead to DNA damage, especially by its
transformation from Cr(VI) to Cr(III), where hydroxyl and other radicals are byproducts.
Hexavalent chromium (Cr(VI)) is able to induce autophagy in hematopoietic stem cells exposed
to subtoxic and toxic concentrations. At toxic concentrations (10 µM), most of the cells
underwent death processes, but analysis of the surviving cells showed prominent signs of
autophagic vacuolization (Di Gioacchino et al., 2008).
Iron is an essential nutrient vital to energy generation in cells. It cycles in cells between
Fe(II) and Fe(III), and participates as a cofactor in a number of oxidation/reduction reactions.
Most iron is contained within biomolecules, where it is not accessible to hydrogen peroxide.
Excess iron, however, that cannot be sequestered by ferritin, has the capacity to induce
homolytic cleavage of hydrogen peroxide, releasing the hydroxyl radical (Chatterjee et al.,
2014a). Autophagic cell death may be a mechanism of brain injury in iron overload disorders, as
cell death was seen to co-occur with autophagy in adult rats exposed to iron over four months
(Chen et al., 2013). Not much evidence has been found for iron induced autophagy, but critical
for iron homeostasis in the cell is the autophagic turnover of ferritin which releases available iron
for cellular processes (Kurz et al., 2011).
Mercury is a ubiquitous environmental group 12 toxic metal that causes a wide range of
adverse health effects in humans. The three forms of mercury, elemental, inorganic and organic,
all have their own profile of toxicity (Chatterjee et al., 2014a). A number of reports demonstrate
that even low concentrations of mercury cause the induction of cell death in different cell types,
and nonlethal dose mercury is known to induce oxidative stress bursts (Patnaik et al., 2010). In
rat hepatocytes, 5 µM mercury exposure can drive autophagy, following an ATG5-ATG12
covalent conjugation pathway (Chatterjee et al., 2014b).
39
1.4.8 Cadmium-induced Autophagy
Of greatest interest in this dissertation is the investigation of Cd-induced autophagy.
Cadmium-induced autophagy has been shown to be mediated by ROS formation, glycogen
synthase kinase-3 β (GSK-3 β), AMPK, P38 and ERK activation (Wang et al., 2009). These all
were shown to occur in various cell lines following Cd exposure by the presence of
autophagosomes, LC3-II, and increase in ATG gene expression. What has not been fully
elucidated, however, is the consequence of Cd-induced autophagy.
In MES-13 cells, both autophagy and apoptosis were observed through the elevation of
Ca2+-mitochondrial caspase and Ca2+-ERK-LC3 signaling pathways (Wang et al., 2008). At
concentrations lower than 10 µM of Cd2+, vascular endothelial cells showed signs of autophagy
and depressed levels of integrin β4, caveolin-1 and PC-PLC (Dong et al., 2009). There also
exists an interesting link between ER stress and downstream Cd2+-induced autophagy. Some
evidence points towards autophagy being involved in Cd2+ adaptation by counterbalancing ER
stress in RWI38 lung epithelial fibroblast cells (Lim et al., 2010). Other experiments on ER
stress and Cd were conducted using salubrinal, a selective inhibitor of complexes that
dephosphorylate eukaryotic translation initiation factor 2 subunit α (eIF2α). Salubrinal therefore
activates the UPR which protects HK-2 cells from Cd2+-induced apoptosis, but had no effect on
autophagy. Very few studies have been undertaken regarding Cd2+ exposure, ER stress and
autophagy, and the multiple levels of complexity are now becoming more apparent.
In some cases, autophagy by Cd2+ treatment seems to suppress apoptosis. In rat kidney
NRK-52E cells, rapamycin induction of autophagy seemed to prevent Cd-induced cell death at
10 µM of exposure (Kato et al., 2013). The data were supported in two reports in rat neuronal
PC12 and human neuroblastoma SH-SY5Y cells, where apoptosis was reduced in Cd-exposed
cells. Additionally, downregulation of mTOR by RNA interference rescued cells from Cd-
induced apoptosis (Chen et al., 2008). However, recent repetitions of the experiment in proximal
tubule cells could not confirm that rapamycin-induced-autophagy protected against cell death by
Cd (Thévenod and Lee, 2015). Instead, 3-methyladenine, a blocker of autophagy, was protective
against cell death in NRK-52E cells suggesting that death was a consequence of autophagy. The
discrepancy in the data requires clarification, especially because all exposure times and
40
concentrations were equivalent in repeated experiments. As such, the relationship between
autophagy and cell death remains unclear.
1.5 The Mesangial Cell Model
1.5.1 Physiology of Mesangial Cells
The major functions of the kidney are to maintain blood volume, blood pressure, blood
osmolarity and blood pH in addition to filtration and excretion of waste. Each human kidney
contains over a million functioning units called nephrons, with each nephron being composed of
a structure known as the glomerulus, and a tubule. The glomerulus filters large proteins and cells
from the blood, producing an ultrafiltrate. The ultrafiltrate passes along the tubule, which at
various segments either removes substances into the kidney parenchyma or allows them to
remain in the ultrafiltrate for excretion (Preuss, 1993).
Cadmium is a potent nephrotoxic substance known to cause damage to the proximal
tubular epithelium in vivo. The renal glomerulus, however, is sensitive to Cd in vitro with the
mesangial cell being the major target for Cd2+ toxicity in isolated glomeruli (Chin and
Templeton, 1992). Mesangial cells are specialized smooth muscle cells, with their contractility
dependent on molecules such as angiotensin II and endothelin-1. They support the glomerular
capillaries and act as an extension of the filtration system. Mesangial cells are directly exposed to
the plasma and are not separated from the vascular lumen by a basement membrane. They are
therefore the kidney cells that are exposed first to noxious substances from the blood, and in
response they acquire a proliferative, myoblast phenotype (Johnson, 1994). The mesangial cell
maintains the structural integrity of the glomerulus and can regulate the glomerular filtration rate
(GFR), which is a good indicator of kidney function and health. The anatomy of the mesangial
cell and surrounding glomerulus is diagrammed below (Fig. 6).
41
Figure 6. Anatomy of the glomerulus. The mesangial cells, which are in dark red, are the focus
of this thesis and are labeled above. The mesangial cell is unprotected by the glomerular
basement membrane (in red) and is therefore in direct contact with any noxious substances in the
blood plasma (Seret et al., 2012).
1.5.2 Relevance to Cd Nephrotoxicity
Animal experiments and occupational studies place the glomerulus as an important target
in Cd toxicity. In rats, repeated intraperitoneal injection of Cd results in mixed or tubular
proteinuria, but prolonged oral administration results mainly in glomerular proteinuria. The
former was found to be reversible, but the glomerular proteinuria was irreversible. Additionally,
in both humans and rats chronically exposed to Cd, circulating anti-glomerular basement
membrane antibodies were found (Lauwerys et al., 1984) . Other studies also found similar
glomerular involvement in chronically exposed Cd pigment workers (Roels et al., 1993). Chronic
42
exposure to inhaled Cd fumes has also been linked to mesangial glomerulonephritis, with no
renal tubule or pulmonary involvement (Nogué et al., 2004). The Cadmibel study, described in
an earlier section, found evidence of glomerular damage in humans living in industrially polluted
regions of Belgium (Buchet et al., 1990). Although the reasons remain unknown, it is interesting
to note that individuals with diabetes are more susceptible to adverse glomerular effects of
environmental Cd exposure (Buchet et al., 1990).
In toxicity studies in our lab, therefore, the effects of Cd on cultured mesangial cells is an
attempt to model the biochemical and anatomical changes seen in the renal glomerulus. Studies
in our lab have utilized Cd concentrations ranging from 0.1 µM to 40 µM. We have observed
striking effects of Cd on actin assembly, glutathione synthesis, MAPK activation, oncogene
activation and cell death and survival pathways, among others. Concentrations of Cd utilized in
our studies may seem high, but in comparison to concentrations measured in human studies of
Cd body burden, they are surprisingly very low. The cadmium concentation in the blood of a
non-smoking white collar worker is 0.0007 µM. However, a different perspective is the
concentration of Cd in the renal cortex of a typical 50 year old in Liège, Belgium measured in
the Cadmibel study, which is 310 µM (Lauwerys et al., 1990). We have therefore studied effects
in cultured RMC at concentrations potentially below those observed in chronically exposed
humans. From results in our lab, cultured mesangial cells experience an intracellular
concentration of 1.6 pM when they were exposed to 10 µM Cd for 10 hours (Wang et al., 1996).
1.6 Hypotheses and Objectives
Mechanisms of cellular Cd toxicity are incompletely understood. Given how little is
known about Cd-induced autophagy, we embarked on the investigation of autophagy and its
effect on mesangial cell survival or cell death. This dissertation will describe the studies
undertaken to elucidate mechanisms of Cd-induced autophagy in primary glomerular cells. Our
lab has studied multiple outcomes of Cd toxicity in RMC (Liu and Templeton, 2007, 2008; Xiao
et al., 2009), but we have yet to investigate Cd-induced autophagy. We hypothesize that:
1) Cadmium-induced autophagy acts to protect Cd-exposed mesangial cells from cell death.
2) Cadmium induces autophagy through a JNK-dependent mechanism in mesangial cells.
43
The objectives of this thesis were to i) Establish the time and concentration of Cd at
which RMC respond with autophagy, ii) Undertake studies to investigate the causal factors of
Cd-induced autophagy, iii) Determine if Cd-induced autophagy is protective or harmful to cells,
and iv) Develop baselines techniques for measuring autophagy in Cd-exposed RMC for future
experiments.
44
2 Materials and Methods
2.1 Materials
Fetal bovine serum (FBS), RPMI-1640 culture medium, and DMEM culture medium
were purchased from Wisent Biocenter (Quebec, Canada). Cadmium chloride (CdCl2), thiazolyl
blue tetrazolium bromide (MTT), protease inhibitors (aprotinin, leupeptin, pepstatin and
phenylmethylsulfonyl fluoride (PMSF)), thapsigargin (Tg), camptothecin (Cpt), butylated
hydroxyanisole (BHA), and the utilized inducers and suppressors of autophagy, rapamycin and
CQ, respectively, were purchased from Sigma Aldrich (St. Louis, MO). Kinase inhibitors with
blocked kinases in parentheses, LY294002 (PI3K) (#440202), KN93 (CAMKII) (#422708),
AG1478 (EGFRK) (#658552), PD98059 (ERK) (#513000), SB203580 (p38) (#559389), and
SP600125 (JNK) (#420119) were all purchased from Calbiochem (Billerica, MA).
Mouse monoclonal anti β-actin (#A1978) antibody was purchased from Sigma-Aldrich.
Secondary horseradish peroxidase (HRP)-conjugated anti-rabbit and anti-mouse antibodies
(#7074) and (#7076), respectively, were obtained from Cell Signaling Technology (Danvers,
MA). Polyclonal rabbit anti-caspase-3 antibody (#9662) and the monoclonal antibody to the p17
fragment of caspase-3 (#9664) were also products of Cell Signaling Technology. For detection of
autophagy, the anti-LC3 antibody that recognizes both LC3-I and –II was purchased from Cell
Signaling Technologies. The anti-p62 antibody (#E2413) was obtained from Santa Cruz
Biotechnology (Dallas, TX). Cyto-ID autophagy detection kit (#51031) was purchased from
Enzo Biosciences (Brockville, ON).
2.2 Cell Culture
Rat mesangial cells cultures were used from frozen stocks maintained in our lab. They
had been prepared from glomeruli of 100 g male Wistar rats (Charles River; Saint Constant,
Quebec) with the following procedure (Wang and Templeton, 1996). The decapsulated renal
cortex was minced and sieved through graded stainless steel sieves (180, 125, 106 and 90 µm)
and washed with 0.9% saline. Cells were collected from the 106 and 90 µm sieves and grown in
20% FBS RPMI medium with penicillin G (100 IU/ml), streptomycin (100 g/ml). Mesangial
cells were subcultured by trypsinization until cells reach passage 3. Cells were characterized by
their morphology and positive staining for desmin and smooth muscle actin. They were used
45
between passages 5 and 15 when they begin to contract in response to angiotensin II and
endothelin and exhibit growth suppression in the presence of heparin (1 µg/ml).
ATG16-/- mouse embryonic fibroblast immortalized (MEF) cell lines and matched wild
type immortalized MEF cell lines were a kind gift from Dr. Stephen Girardin (Dooley et al.,
2014). MEF were grown in 10% FBS DMEM.
2.3 Cell Treatments
Cells were cultured in RPMI-1640 (for RMC) or DMEM (for MEF and MEF ATG16-/-)
with 10% FBS, incubated in 5% CO2 at 37ºC. Cells were passaged by trypsinization at 70 – 80%
confluence (approximately 5x105 cells per 10 cm culture dish or 2x105 cells per 6 cm dish).
Experiments on RMC were conducted on cells between passages 8 and 13. After RMC were
attached overnight, they were rendered quiescent by growing in 0.2% FBS serum for 48 h prior
to transfer to serum-free (SF) medium followed by addition of CdCl2 in SF medium for times
indicated in figure legends. Certain treatments were also conducted in full-serum (10% FBS)
medium with CdCl2 treatment. MEF cells were serum-starved for 24 h in order to more easily
observe robust cellular response prior to administering CdCl2 treatment in SF DMEM. For
studies with treatments in addition to CdCl2 (Rapamycin, CQ, or kinase inhibitors), cells were
pre-treated with the specified reagents for 1 h before Cd treatments.
Cells were treated with the following agents at the concentrations indicated here:
Rapamycin 50 nM, CQ 50 µM, LY294002 20 µM, KN93 10 µM, AG1478 0.5 µM, PD98059 10
µM, SB203580 10 µM, SP600125 10 µM, Tg 50 nM and 150 nM, BHA 33 µM, 100 µM, and
150 µM, Cpt 100 nM.
2.4 1% NP-40 whole-cell lysate for detection of p62, β-actin and caspase-3 proteins
To obtain whole cell lysates, adherent cells in monolayer cultures were washed twice
with ice-cold phosphate-buffered saline (PBS) and scraped with 1% Nonidet P-40 (NP-40) lysis
buffer (20 mM Tris-HCl, pH 8, 150 mM NaCl, 1.5 mM MgCl2, 2mM EGTA, 1mM NaF, 1mM
Na3VO4, 1 mM PMSF, and 1 µg/ml each of aprotinin, leupeptin, and pepstatin). Cell lysates
were vortexed 3 times for 5 seconds each, then incubated at 0ºC for 30 minutes. Whole cell
lysates were then centrifuged at 8,000 g for 10 minutes at 4ºC to pellet insoluble material. The
supernatant was taken as the whole cell lysate.
46
2.5 Triton X-100 whole cell lysate for detection of LC3-II proteins
In order to obtain whole cell lysates for LC3-II detection by Western blot, adherent cells
in monolayer were washed 3 times with ice-cold PBS and scraped with X-100 lysis buffer (0.5%
Triton X-100, 10 mM HEPES pH 7.9, 50 µM NaCl, 100 mM EDTA, 0.5 M sucrose, 1mM NaF,
1mM Na3VO4, 1 mM PMSF, and 1 µg/ml each of aprotinin, leupeptin, and pepstatin). Cell
lysates were sonicated 3 times for 5 seconds each, then incubated at 0ºC for 30 minutes. Whole
cell lysates were then centrifuged at 10,000 g for 10 minutes at 4ºC to pellet insoluble material.
The supernatant was taken as the whole cell lysate.
2.6 Western Blotting
For the detection of proteins by Western blot, supernatant concentrations of whole cell
lysates were measured by the Bradford assay using Bio-Rad Protein Assay reagent. For
separation by gel electrophoresis, 6x SDS-loading buffer (0.375 M Tris pH 6.8, 12% SDS, 60%
Glycerol, 0.6 M Dithiothreitol (DTT), 0.06% bromophenol blue) was mixed with lysates
containing 40 µg of protein and incubated for 5 minutes at 95ºC to avoid aggregation of proteins
and accelerate denaturation of proteins. Samples were resolved on a 12% SDS-PAGE gel.
Proteins were transferred to nitrocellulose membranes. Blots were blocked with 5% BSA in
TBS-T (10 mM Tris-HCl pH 7.5, 15 mM NaCl, 0.05% Tween 20 ) for 1 hour prior to incubation
with primary antibody overnight at 4ºC.
Antibody dilutions are as follows: anti-LC3 (1:1000); anti-p62 (1:1000); anti-caspase-3
(1:1000), anti-β-actin (1:10,000). After incubation, membranes were washed with tris-buffered
saline-tween (TBS-T) and incubated with HRP-conjugated secondary antibody for 1 h. Blots
were visualized using enhanced chemiluminescence. Blots to be re-probed were stripped in
stripping buffer (2% SDS, 62.5 mM Tris pH 6.7, 100 mM β-mercaptoethanol) for 15 minutes at
room temperature. Film was scanned and band intensities analyzed using ImageJ software (NIH,
Bethesda, MD).
2.7 Immunofluorescence
RMC, MEFATG-/-, and MEF cells were seeded on 12 mm cover-slips, grown overnight
and starved for 48 h (for RMC) and 24 h (for MEF) with 0.2% FBS RPMI-1640 or DMEM to
prepare cells for treatment. After treatment, cells were washed twice with 1 x Assay buffer (from
Cyto-ID detection kit). 100 µl of microscopy dual detection reagent (including both green
47
fluorescent autophagy lysosomotropic dye and Hoescht nuclear stain) was used to cover each
sample of monolayer cells, and cells were incubated in the dark at 37ºC for 30 minutes. Cells
were then washed with 1 x PBS and fixed with 4% formaldehyde for 20 minutes at room
temperature. Cells were then washed 3 times with 1 x PBS and mounted on slides with
Vectashield Mounting Medium. Slides were viewed on a Nikon Eclipse 80i microscope and
images were taken with QIimaging Qicam Fast 1394 camera using QCapture Pro 6.0 software.
2.8 Viability Assay
Cells growing in 96-well plates (seeded at 1 x 103 cells per well) were exposed to
1 mg/ml MTT in 100 μl 1 x PBS for 1 h at 37 °C. The medium was removed, and 100 μl of
DMSO was added with shaking for 30 min at room temperature. Sample absorbance was read at
570 nm with background subtraction at 650 nm.
2.9 Statistical Analyses
Values combined from three or more experiments are expressed as mean ± s.d. Pairwise
comparisons are performed with Student's unpaired t-test. When multiple comparisons are made,
analysis was by one-way or two-way ANOVA followed by Dunnett's post hoc test against the
specified control treatment. Analyses were performed with Prism Software (GraphPad, San
Diego, CA).
48
3 Results
3.1 Cadmium induces autophagy in RMC
3.1.1 Cadmium-induced autophagy is time and concentration dependent
Rat mesangial cells were starved for two days in 0.2% fetal bovine serum (FBS) RPMI
medium, and then treated with a range of Cd concentrations from 0.5 µM to 20 µM CdCl2. Cells
were harvested at 1, 3, 6 and 24 hours. The cells were either treated in serum-replete conditions,
in the five right-most lanes or in SF conditions in the five left-most lanes (Fig. 7). 1% NP-40
whole cell lysates were collected and separated on a gel to be probed by an anti-LC3 antibody.
The conversion of microtubule-associated protein 1A/1B-light chain 3 (LC3-I) to LC3-II is
characteristic of autophagy, and the presence of the converted form can be tracked by Western
blot. A direct comparison of band intensities at different time points is not performed as each of
the time points are independent experiments.
None of the cells in serum-replete conditions display LC3-II accumulation significantly
above control levels. In SF conditions, however, Cd induces autophagy from 3 hours of
treatment in comparison to the SF, Cd-free control. Autophagy as marked by the presence of
LC3-II is maximum in this experiment at 24 hours.
49
Figure 7. Time- and concentration-dependent autophagy in Cd-exposed RMC. RMC were
treated with CdCl2 (0 – 20 µM) over a period of 24 hours. The cells were starved in 0.2% FBS
RPMI for 48 hours before treatment. Cell lysates were collected at 1, 3, 6 and 24 hours. Lysates
were separated by SDS PAGE and probed using an anti-LC3 antibody. Presence or absence of
serum (10% FBS) is denoted by a + or – across the lanes of the gel. The upper band visible at all
time points on the membrane is LC3-I, which may be cleaved and lipidated to become LC3-II
(lower band). The density of the LC3-II band indicates the extent of autophagy. Next to each
Western blot image is the quantitated result of densitometric analysis of the LC3-II band. Bars
are mean ± s.d (n= 3 - 4) (One-way ANOVA followed by post-hoc Dunnet test, *p < 0.05, **p <
0.01 vs. SF control).
50
The results above show that Cd-induced autophagy as indicated by LC3-II expression
peaks at 10 µM Cd treatment at 24 hours. Subsequent experiments were performed with cells
incubated at that time and concentration to define autophagy further.
3.1.2 At 24 hours of Cd exposure, autophagy is not the only cell fate
In order to visualize autophagic vacuolization, RMC were stained with green fluorescent
dye using the Cyto-ID system (Fig. 8). The Cyto-ID fluorescent dye partitions into cells and
accumulates in vacuoles associated with the autophagy pathway. In Fig. 8A, cells were grown in
10% FBS conditions and stained, and exhibit autophagic vacuolization at numerous points
around the cell body. While autophagic vacuoles are observed in all regions of the cell, the dye
mainly aggregates in vacuoles in the perinuclear region. In experiments visualized in panels 8B-
8F, cells were rendered quiescent by growing in 0.2% FBS for 48 hours, then treated
accordingly. In Fig. 8B, where cells were grown in FBS-free medium, more intense localization
of the dye is visible. This is expected, as nutrient starvation induces autophagy in many types of
cultured cells (Mizushima, 2007).
Following rapamycin treatment (Fig. 8C), a single cell seems to show large autophagic
vacuolization, with surrounding cells displaying a much reduced extent of autophagy. Cells that
were treated with 10 µM Cd for 24 hours are displayed in panels 8D, 8E and 8F, by scanning . In
panel 8D, widespread autophagy is visible with large vacuoles being targeted by the Cyto-ID
dye. This is comparable to the extent of autophagy measured by immunoblot in section 3.1.1.
However, in panels 8E and 8F, the cells appear very different from those in full-serum
conditions, either with cellular boundaries misshapen and in disarray, or with shrunken nuclei
and no visible boundaries. Autophagy is visible in Cd-treated cells, but due to the pleiotropic
effects of Cd at 24 hours of treatment, cells undergoing death are also obvious. Interestingly, the
cells undergoing autophagy all seem to be have round, unperturbed nuclei and visible cellular
boundaries.
51
Figure 8. Effects of Cd on autophagic vacuolization as visualized by fluorescent dye. All
panels show RMC at 600x magnification, with the Cyto-ID dye (green) accumulating in vacuoles
associated with autophagy. Nuclei are stained with Hoescht nuclear fluorescent dye (blue). Other
than in panel A, all treatments are 24 h in length in serum free RPMI media, after 48 h of 0.2%
FBS conditions. A) Cells grown in 10% FBS. B) Cells grown in SF conditions. C) Cells treated
with 50 nM rapamycin. D, E and F) Cells treated with 10 µM CdCl2.
A D
B E
C F
52
3.2 Cadmium-induced autophagy is decreased by JNK inhibition
Rat mesangial cells were starved for 48 hours in 0.2% FBS RPMI medium, and treated
with various kinase inhibitors for one hour, followed by Cd treatment for 24 hours. An
experiment was conducted to investigate the pathway leading to autophagy induced by Cd
treatment (Fig. 9). A diminution of autophagy after suppression of a particular kinase would
suggest the importance of a pathway dependent on that kinase. The kinases being targeted by
specific inhibitors are PI3K, CAMK II, EGFR K, ERK, p38 and JNK. The lane labeled quiescent
in Fig. 9 describes the treatment wherein cells were left in 0.2% FBS medium for the duration of
24 hours. The final lane is of a sample from cells exposed to 50 µM of CQ for 24 hours.
Chloroquine is a lysosomotropic agent that prevents the acidification of lysosomal
compartments, serving as a positive control for LC3-II which no longer is degraded efficiently.
53
Figure 9. Extent of autophagy when Cd is co-treated with various kinase inhibitors. Rat
mesangial cells were starved for 48 hours in 0.2% FBS RPMI media, and then treated for an hour
with the following inhibitor treatments, with the targets of inhibition given in parentheses:
LY294002 (PI3 kinase), 20 µM. KN93 (CAMK-II), 10 µM. AG1478 (EGFR), 0.5 µM. PD98059
(ERK), 10 µM. SB203580 (p38), 10 µM. SP600125 (Jun-N-terminal kinase), 10 µM. After the
hour-long treatment with inhibitor, cells were then exposed to Cd for 24 hours. The lysates were
separated by SDS-PAGE and the gel probed with an anti-LC3 antibody. Beneath the Western
blot is a densitometric analysis of the lanes that visually showed the greatest disparity in LC3-II
cleavage from the serum-free (SF) + Cd treatments. Bars are mean ± s.d (n=5) (One-way
ANOVA followed by post-hoc Dunnet test, *p < 0.05 vs. SF + Cd10 control).
54
In the above experiment, the Jun N-terminal kinase (JNK) inhibitor SP600125, when
added to Cd-treated cells, resulted in reduced autophagy as measured by LC3-II accumulation.
Addition of KN93 (an inhibitor of CAMK-II) with Cd produced an apparent increase in LC3-II
cleavage, but after repeated experiments it was found that this result was variable and not
significant. Regardless, the results indicate a role for JNK in Cd-induced autophagy.
3.3 Cadmium-induced autophagy is suppressed with Tg treatment
After starvation, RMC were treated with two combinations of Tg and CdCl2.
Thapsigargin is a specific and non-competitive inhibitor of the sarcoplasmic/endoplasmic
reticulum Ca2+ ATPase (SERCA). Ca2+ is therefore inhibited from entering the organelle, leading
to its transient elevation in the cytosol. Prolonged Tg treatment, however, leads to a loss in
cellular Ca2+ (Kaneko and Tsukamoto, 1994), and also initiates the ER stress response known as
the UPR. Because heightened autophagy has been observed as a consequence of the UPR (Ding
et al., 2007), we determined whether modulation of Ca2+ in coordination with Cd treatments
affects Cd-induced autophagy (Fig. 10).
As before, the results show significant accumulation of LC3-II after 10 µM Cd of 24 hour
treatment compared to the SF control. If the UPR increased the extent of autophagy, then Tg
treatment should result in a more obvious LC3-II band by Western blot. The results indicate,
however, that neither the lower (50 nM) nor higher (150 nM) concentration of Tg alone or with
Cd enhances autophagy beyond SF conditions. Instead, Cd-induced autophagy is suppressed by
24 hour Tg treatment.
55
Figure 10. Determining the extent of autophagy with co-treatment of Cd and Tg. Rat
mesangial cells were starved for 48 hours in 0.2% FBS RPMI and then treated either with Tg
alone, or with Tg and Cd in serum-free (SF) . In co-treatments, Tg is added to the for 1 hour
before Cd. The final lane is treatment with 50 nM rapamycin, a suppressor of the mammalian
target of rapamycin (mTOR), inducing autophagy and acting as a positive control. Cells were
treated for 24 hours. The lysates were probed with an anti-LC3 antibody. The bar graph below
the Western blot figure is a densitometric analysis of the LC3-II bands. Bars are mean ± s.d
(n=3) (One-way ANOVA followed by post-hoc Dunnet test, *p < 0.05 vs. SF control).
56
3.4 Cadmium-induced autophagy progresses independently of ROS accumulation
Following starvation, RMC were treated with either 10 µM or 20 µM Cd, in combination
with 33 µM to 100 µM of the ROS scavenger butylated hydroxyanisole. It is not yet clear to
what extent ROS participates in various aspects of Cd-mediated cell death, and the following
experiment (Fig. 11) was devised to investigate the role of ROS in Cd-induced autophagy. BHA
may be expected to reduce the extent of autophagy by depleting cellular ROS. On the contrary,
in isolation 100 µM BHA actually induces autophagy to a greater degree than 10 µM Cd. Also of
interest is that co-treatment with Cd and 100 µM BHA results in the greatest increase in
autophagy.
57
Figure 11. Extent of autophagy when cells are treated with a ROS scavenger. Rat mesangial
cells were starved for 48 hours in 0.2% FBS RPMI medium, and then treated with either 33 µM
or 100 µM BHA in addition to 10 µM or 20 µM Cd for 24 hours in SF conditions. The final lane
displays treatment with 100 µM BHA alone. After treatment, lysates were collected and
separated on a gel to be probed by an anti-LC3 antibody. The bar graph below is the
densitometric analysis of the LC3-II band from the Western blot antibody probing. Bars are
mean ± s.d (n=3) (One-way ANOVA followed by post-hoc Dunnet test, * p < 0.05, ** p < 0.01
vs. 10 µM Cd, 0 µM BHA control)
58
Autophagy was heightened significantly when both Cd and BHA are co-treated,
suggesting that its progression is unrelated to ROS in Cd-treated cells. ROS accumulation may
be suppressing autophagy, although further experiments would be needed to arrive at that
conclusion.
3.5 Selective autophagy protein p62 expression declines with increasing Cd
concentration
Selective autophagy is one process whereby autophagy receptors such as p62 or NBR1
bind to substrates bound for destruction at the growing autophagosome (Kirkin et al., 2009). The
following experiment was performed to check the extent of p62 expression under different Cd
concentrations for 24 hours in RMC (Fig. 12).
After 24 hours of treatment, there is a decline in p62 expression as Cd concentrations
increase. Rapamycin exposure resulted in a decrease in p62 expression, showing that induction
of autophagy by rapamycin leads to the degradation of endogenous p62. Camptothecin, which
induces apoptosis by inhibiting DNA repair enzyme topoisomerase I, displays the greatest
decrease in p62 expression.
59
Figure 12. Monitoring p62-assisted selective autophagy in Cd-treated mesangial cells. Rat
mesangial cells were starved for 48 hours in 0.2% FBS RPMI medium, and then treated with Cd
in a range from 0.5 µM to 20 µM in serum-free (SF) conditions. The final two lanes are from
samples treated with 50 nM rapamycin (Rapa) and 25 µM Cpt, specific inducers of autophagy
and apoptosis, respectively. Lysates were probed with an anti-p62 antibody. Below the Western
blot image is a densitometric analysis of the p62 band from three independent experiments. Bars
are mean ± s.d (n=3) (One-way ANOVA followed by post-hoc Dunnet test, * p < 0.05, ** p <
0.01 vs. SF control).
60
3.6 Cells that cannot undergo autophagy are less viable
3.6.1 Rat mesangial cell viability after 24h of Cd exposure
In the following experiments, cells were seeded equivalently, starved for 48 hours, and
then treated with a range of Cd concentrations up to 10 µM. RMC were treated for 24 hours (Fig.
13). Cell viability, measured by the MTT assay, declines as Cd concentrations increase, as
expected. At a concentration of 10 µM Cd, 52% of cells are viable as determined by metabolic
activity.
Figure 13. Rat mesangial cell viability measured by MTT assay after 24h of Cd treatment.
Rat mesangial cells were seeded in a 96 well plate at 300 cells per well. After 48 hours of 0.2%
FBS RPMI starvation, the cells were either switched to serum-replete medium (10% FBS),
maintained in low-serum conditions (0.2% FBS), or treated with 2 - 10 µM CdCl2 in SF
medium. Bars are mean ± s.d (n=3) (One-way ANOVA followed by post-hoc Dunnet test, * p <
0.05, vs. SF control).
61
3.6.2 Viability of autophagy-deficient mouse embryonic fibroblasts exposed to Cd
Mouse embryonic fibroblast (MEF) cells that have the ATG16 gene knocked-out
homozygously were acquired from Dr. Stephen Girardin. The ATG16 gene product is required
for the progression of autophagy, involved with autophagic vesicle elongation and LC3-II
turnover (Xie and Klionsky, 2007). These MEFATG16-/- cells were compared to wild type
MEFs, and their viability was examined with the MTT assay (Fig. 14). All treatments were 4
hours in length, and in all cases the autophagy deficient cells were less viable.
62
Figure 14. MEFWT and MEFATG-/- viability measured by MTT. MEF cells were seeded in
a 96 well plate at 300 cells per well. After 24 hours of 0.2% FBS DMEM low serum treatment,
cells were either switched to serum-replete medium, maintained in serum-free (SF) medium,
starved in amino acid-free KRB buffer (starve), treated with 50 nM rapamycin to induce
autophagy, 50 µM CQ to interrupt autophagy, or 10 µM Cd, all for 4 hour treatments. All
treatments are in SF conditions except for serum-replete and starve treatments. In all treatments,
the autophagy-deficient cells were less viable. (Two-way ANOVA, n=4, * p<0.05 pairwise
comparisons within each treatment).
63
The MTT assay conducted with MEF cells shows that cells that can undergo autophagy
are more protected from Cd2+ than those cells that cannot under autophagy. Interestingly, being
forced to undergo a heightened level of autophagy as in the rapamycin treatment is not
conducive to greater survival. Wild type MEF cells treated with rapamycin remain equally as
viable as matched cells in SF conditions.
3.7 MEF cells that cannot undergo autophagy experience greater apoptosis
A remaining question about MEF cells that cannot undergo autophagy is whether or not
they endure amplified apoptosis. The answer to this question would shed more light on the
interplay between autophagy and apoptosis, and whether one process can overtake cell fate when
the other is suppressed. MEF cells were treated with 50 nM rapamycin, µM of CQ and 10 µM
Cd, as well as being maintained in serum-replete, serum-depleted, and amino acid-free media
(Fig. 15). All treatments are 4 hours in length, and cell lysates were resolved by SDS PAGE and
probed for LC3 and the caspase-3-cleaved product.
64
Figure 15. Monitoring apoptosis and autophagy in MEFWT and MEFATG-/- cells. After 24
hours of 0.2% FBS DMEM, cells were either switched to serum-replete medium, maintained in
SF medium , starved in amino acid-free KRB buffer (starve), treated with 50 nM rapamycin
(rapa) to induce autophagy, 50 µM CQ (chloro) to interrupt autophagy, or 10 µM Cd. All
treatments are in SF conditions except for serum-replete and serum-starved treatments. After the
treatments, lysates were collected and separated on a gel to be probed by an anti-LC3 antibody
and anti-caspase-3 cleaved antibody. Cyto-ID staining for autophagic vacuoles is displayed
below the Western blots.
65
As expected, LC3-II is significantly reduced in MEFATG16-/- cells compared to MEFWT
cells. ATG16 is a protein that is involved with pre-autophagosomal membrane construction, and
LC3-II resides in the autophagosomal membrane. What is interesting, however, is that in wild
type MEF cells, there is no visible caspase-3 cleavage product, unlike the result in the ATG16-
deficient cells. Specifically, MEFATG16-/- cells display caspase-3 cleavage after SF, rapamyicn,
CQ and Cd exposures. It seems that an inability to undergo autophagy in some cases results in
apoptosis, while the same treatments do not lead to apoptosis in MEFWT cells. This lends
credence to the idea that cells that cannot undergo autophagy engage apoptotic signals and die,
possibly due to autophagy being cytoprotective.
66
4 Discussion
Cadmium is an important metal with numerous industrial uses. Its increased availability
due to human activities has exposed the pleiotropic nature of its toxicity (Templeton and Liu,
2010). Cadmium can cause tissue injury by creating oxidative stress, disrupting the cytoskeleton,
forcing changes in cell epigenetic regulation of DNA, inhibiting or upregulating transport
pathways, and affecting cell death and survival pathways (Bernhoft, 2013). Cell death and
survival pathways are of greatest concern to this investigation. This study explores Cd-induced
autophagy and the role the process plays in protecting cells from the toxic effects of the metal.
Numerous toxic effects are observed when cells are exposed to Cd, and these effects lead to renal
injury. Understanding the basis of renal injury by Cd exposure will require the investigation of
each of Cd’s cellular effects. Determining the role of Cd-induced autophagy and its other
intersecting pathways will provide insight into the mechanisms of toxicity, and the importance of
the process in renal injury.
The results from this dissertation show that Cd induces autophagy in RMC in a time- and
dose-dependent manner, with the extent of the process still increasing at the longest time studied
at 10 µM exposure. At 10 µM Cd exposure, due to the multiple toxic effects, RMC display not
only autophagy but also caspase-dependent and caspase-independent apoptosis (Xiao et al.,
2009). Cd-induced autophagy is suppressed with Tg treatment, and progresses independently of
ROS accumulation. Results also indicate that Cd-induced autophagy is protective, with enhanced
Cd-induced apoptosis evident in MEF cells that cannot undergo autophagy.
4.1 Cadmium and autophagy
Autophagy is an evolutionarily conserved process implicated in the degradation and
recycling of cytoplasmic proteins, macromolecules, and organelles. The vast majority of
evidence points to autophagy being critically important in cell fate, removing damaged
organelles and harmful substances to maintain cellular homeostasis (Zou et al., 2015). In most
studies where Cd-induced autophagy is being investigated in mammalian cells, researchers tend
to look at particular markers including LC3, and the ATGs including Beclin-1, Atg-5, Atg-7, and
Atg-12 in order to measure the extent of the process (Zou et al., 2015). Another common method
67
to measure Cd-induced autophagy is flow cytometry to quantify the level of a vital dye that
accumulates in lysosomal compartments (Wang et al., 2008).
Our lab is unique in being one the few to perform experiments on primary RMC, giving
us a unique insight on the extent of autophagy in cells that in vivo are surrounded by glomerular
capillaries. RMC in vivo exist without a protective basement membrane, being therefore
especially sensitive to functional compromise by circulating toxic substances like Cd (Chin and
Templeton, 1993). In the above results, Cd was seen to induce autophagy significantly over
control SF conditions at 24 hours of exposure from 2 – 20 µM (Fig. 7). Numerous studies exist
to show that Cd does in fact induce autophagy in many model systems. In cultured, SV40-
transformed mouse mesangial (MES-13) cells, evidence has shown that Cd induces both
autophagy and apoptosis as measured by flow cytometric measurement of acridine orange and
annexin V/PI. MES-13 cells were treated for 28 hours with various levels of Cd, displaying a
maximum autophagic response at 12 µM (Wang et al., 2008). It is important to note however that
acridine orange is not specific to autophagic vacuoles, staining all acidic compartments which
may add uncertainty to the measurements. In PC-12 rat medulla neuronal cells, LC3-II
accumulation was measured by Western blot after 2.5 – 20 µM Cd treatment for 4 hours of
exposure. It was also observed that from 8 hours of exposure onwards, LC3-II presence
diminished and annexin V/PI double staining increased, suggesting autophagy was being
overtaken by apoptosis (Wang et al., 2013b). In LKB1 skin epidermal cells, autophagy was
visualized a number of ways including LC3-II Western blot, GFP-LC3 fluorescence, and
transmission electron microscopy. The researchers found that similar to our results, autophagy
was observed after 12 hours of 10 µM Cd treatment, further augmented at 24 hours of exposure
(Son et al., 2011). Additionally, human cord blood hematopoietic stem cells treated with Cd for
48 hours were observed to contain autophagosomes at 10 µM exposure (Di Gioacchino et al.,
2008).
Results from this dissertation are in line with those discussed above. For the most part,
increasing Cd exposure and treatment time results in increasing autophagy. However, most of the
above studies show autophagy still increasing at the greatest time point and highest Cd
concentration used in the experiments. In our work, autophagy is higher at 10 µM, and decreases
by 20 µM at 24 hours of exposure. Apoptosis can account for this, which can take over when the
cellular burden of Cd is too high for autophagic protection. Our lab has found cells undergoing
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apoptosis in mouse mesangial cultures with 24 h of Cd treatment in full-serum conditions (Liu
and Templeton, 2008). In fact, the study in MES-13 cells discussed in the previous paragraph
showed that apoptosis peaked at a later time point than autophagy, after the extent of autophagy
was receding (Wang et al., 2008). Our results visually display the different pathways at 10 µM
exposure (Fig. 8), with possible apoptosis occurring alongside autophagy. In displaying this
immunofluorescence data, we provide evidence for the pluralistic way that Cd can display its
toxicity. Within the same experimental treatment, cells are observed that are undergoing very
different processes.
Multiple cell fates occurring simultaneously have been observed in rat liver (BRL 3A)
cells. At 6 hours of exposure to 2.5 to 10 µM Cd, cells showed typical apoptotic nuclear
morphological changes. Autophagy was activated in a dose-dependent manner from 2.5 to 10
µM as well in 6 hour treatments (Zou et al., 2015). Our lab in the past has observed suppression
of apoptosis by Cd in RMC. These studies involved treating cells with either Cpt or TNF-α,
initiators of intrinsic and extrinsic apoptotic pathways, respectively, followed by 10 µM Cd for 8
hours. Cd reduced DNA laddering, chromatin condensation, and caspase-3 activation in response
to both the extrinsic and intrinsic stimuli (Gunawardana et al., 2006). Evaluating those results in
the light of the current observations, it may be possible that Cd-induced autophagy is the process
that is subduing apoptotic signaling. While our results augment past knowledge, they would be
much stronger with multiple corroborating pieces of evidence for autophagy. We have data with
LC3-II and p62 currently, and plan to continue this work with flow cytometry. Beclin-1 protein
accumulation has also been marked by Western blot, but preliminary results indicate that it is not
an effective marker for autophagy in RMC. This is intriguing because beclin-1 is normally
thought to be a strong marker. Beclin-1 is involved in the nucleation of the autophagosomal
membrane, while LC3-II is a marker for whole autophagy. It is possible that Cd2+ enhances
autophagy at later stage of autophagy and therefore beclin-1 is not as involved, although further
investigation will be required to conclude this.
4.2 JNK activation and autophagy
There are three classes of the mitogen activated protein kinase (MAPK) family. These
include the extracellular signal-activated protein kinase (ERK), Jun N-terminal kinase (JNK) and
p38. Originally, it was thought that ERKs are important for cell survival, whereas JNK and p38
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are stress-responsive and involved in apoptosis or inflammatory signaling (Thévenod, 2009).
However, the regulation of apoptosis and autophagy by the MAPKs are much more complicated
than once thought. All three families of MAPKs, along with some other kinase families, were
tested for their involvement in Cd-induced autophagy (Fig. 9). Our results indicate that JNK has
a significant role to play in Cd-induced autophagy. What is intriguing is that treatment of RMC
with the specific JNK inhibitor SP600125 suppresses Cd-induced autophagy to a level very close
to background homeostatic autophagy taking place in full serum conditions. Inhibiting the other
MAPKs, CAMKII, or EGFRK did not result in a significant reduction or enhancement of Cd-
induced autophagy.
The experiment in Fig. 9 aims to investigate the kinases involved with Cd-induced
autophagy. However, the experiment does not aim to identify which kinases are activated by Cd.
In various models and assays, Cd has been confirmed to activate the MAPKs. In renal proximal
tubule cells (LLC-PK1), JNK is phosphorylated after an hour of Cd treatment at 10 µM. It was
observed that JNK phosphorylation was abolished when cells were pre-incubated with BAPTA-
AM, which would chelate intracellular Ca2+, but not when pre-treated with TPEN, which has a
low affinity for Ca2+. Therefore it was concluded that Cd-induced JNK activation is dependent
on Ca2+ (Matsuoka and Igisu, 1998). Our lab showed activation of JNK, ERK and c-fos by Cd2+
in RMC at 8 hours of 10 µM exposure (Ding and Templeton, 2000). Additionally, we have
shown in RMC that 10 – 40 µM Cd for 6 hours in serum free medium increases the
phosphorylation of all three MAPK, with only p38 and CAMK-II activation leading to cell death
(Liu and Templeton, 2008). With the most intriguing result from Fig. 9 being JNK involvement
in Cd-induced autophagy, it is curious that there is support from multiple studies that JNK
signaling is involved in Cd-induced apoptosis. Strong evidence for this has been shown in jnk1-
and jnk2-null fibroblasts, which cannot undergo the mitochondrial apoptotic pathway following
exposure to 10 µM Cd for 15 – 25 hours (Papadakis et al., 2006). The absence of apoptotic death
by Cd correlates with a defect in the activation of Bax, which would occur through JNK
activation.
The JNK pathway has been linked to autophagy, especially in regards to acute oxidative
or xenobiotic insults (Zhou et al., 2015), but this connection has not been made in Cd toxicology
as of yet. In regards to Cd toxicity, one could argue that oxidative stress could be the instigator of
the pathway, but results from Fig. 11 suggest that Cd-induced autophagy can progress without
70
ROS. Proponents of the notion of autophagic cell death suggest that hyperactive autophagy in the
absence of apoptosis leads to JNK activation, and then cell death (Shimizu et al., 2014).
Nevertheless, our results indicate the importance of JNK in the progression of Cd-induced
autophagy in RMC. It would be prudent to identify the time points of JNK activation in relation
to the start of autophagy. One problem with relying on measurement by LC3-II for studying
autophagy is that the whole autophagic process is measured, because LC3-II accumulation
occurs near the final stages of the process. By measuring multiple autophagic markers at
different stages of the process while continuing to pharmacologically inhibit JNK activity, the
importance of the kinase on specific phases of the process could be determined.
4.3 Ca2+ and Cd-induced autophagy
Calcium ions transduce information by rapidly changing their cytosolic concentration
from approximately 100 nM to at least a 10-fold increase. Cells invest a lot of their energy to
maintain a low cytosolic concentration of Ca2+ (Thévenod, 2009). Exposure to Cd leads to
cytosolic elevations in Ca2+. Our lab has observed elevations over 250 nM after 8 hours of 10
µM Cd exposure in RMC (Wang et al., 1996). Transient elevations in intracellular Ca2+ are
observed with Cd exposure, but prolonged exposure (48 hours in NIH 3T3 cells) leads to the
depletion of the ER Ca2+ pool, and later the depletion of cellular Ca2+ (Biagioli et al., 2008). The
rise in intracellular Ca2+ is implicated in the mediation of Cd toxicity. In regards to autophagy, it
is established that intracellular Ca2+ is one of the regulators of autophagy. However, this control
of autophagy by intracellular Ca2+ is the subject of two major opposing views. One is that
intracellular Ca2+ signals, mainly IP3Rs activation, suppress autophagy, while the other is that
elevated cytosolic Ca2+ promotes autophagy (Decuypere et al., 2011). The depletion of
intracellular stores of Ca2+ with Tg specifically has been reported to have both a stimulatory
(Høyer-Hansen et al., 2007) and an inhibitory (Gordon et al., 1993) effect on autophagy.
Evidence for Ca2+ acting as an inhibitor of autophagy mainly focuses on the IP3R, a
ubiquitously expressed intracellular Ca2+-release channel, located mainly on the ER. IP3R
isoforms mediate Ca2+ release from the ER in response to stimulation by hormones, growth
factors or antibodies (Berridge, 2009). Autophagic stimulation by Li+ in HeLa cells has been
shown to take place by inhibiting inositol monophosphatases, reducing IP3 levels and decreasing
the activity of IP3R. Chemical inhibition of IP3Rs with xestospongin also induced autophagy in
71
HeLa cells (Sarkar et al., 2005). IP3R knock outs of all three isoforms in chicken DT40 B
lymphocytes showed higher levels of autophagy than wild type counterparts (Khan and Joseph,
2010).
In contrast, reports considering Ca2+ as an activator of autophagy mainly make use of
Ca2+-mobilizing agents like Tg, ionomycin, ATP (Høyer-Hansen et al., 2007), resveratrol
(Vingtdeux et al., 2010), and Cd2+ (Wang et al., 2008). Treatment of cells with these compounds
resulted in increased autophagy in a number of cell systems (Decuypere et al., 2011). There is
evidence that removal of Ca2+ by chelation results in a block to autophagy at an early stage prior
to LC3-II accumulation at the autophagosome. The localized release of Ca2+ from the ER in the
vicinity of autophagosomes and lysosomes may be needed for vesicular fusion (Ganley et al.,
2011). Addition of BAPTA-AM, a potent intracellular chelator of Ca2+, prevented the induction
of autophagy, indicating the importance of cytosolic Ca2+. Controversy surrounds the use of
BAPTA-AM, however, with evidence indicating inhibitory, stimulatory, or non-significant
effects on autophagy depending on the cell type (Decuypere et al., 2011). It is interesting to note
that both sides of the argument often utilized an entirely different set of pharmacological
inhibitors and cell types, further obfuscating Ca2+ involvement in autophagy.
Preliminary experiments to determine the effects on depleted Ca2+ on Cd-induced
autophagy were conducted with the help of Tg (Fig. 10). Our results indicate a reduction in
autophagic activity with any treatment of Tg. Cd-induced autophagy at 10 µM treatment for 24
hours was our standard of autophagic activity, and the extent of the process was suppressed to
basal full-serum levels with Tg. We provide evidence that Cd-induced autophagy is decreased
with Tg treatment, presumably due to the absence of cytosolic Ca2+, which has been measured
previously (Wang et al., 1996). While current conflicting ideas of Ca2+-induced autophagy may
lead to confusion, the models need not necessarily represent conflicting ideas. Different Ca2+
signaling modes may depend on the state of the cells, and may have opposite effects depending if
the cells are normal or stressed. ER stress, which may result from the depletion of ER Ca2+
stores, has been implicated in both autophagy and apoptosis. In order to determine whether ER
stress plays a role in Cd-induced autophagy, the UPR needs to be measured during Cd treatment.
One method is to perform RT-PCR on the splice variant of xbp-1 mRNA, which is cleaved by
IRE-1 when the cell is experiencing ER stress. The alternate splice variant of xbp-1 when
translated acts as a transcription factor to initiate expression of various chaperones and the ER
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protein degradation pathway in response to the UPR (Biagioli et al., 2008). Thus, Cd-induced
autophagy may be reduced by Tg treatment due to the cell undergoing heightened apoptotic
activity.
4.4 Selective autophagy with Cd exposure
Macroautophagy has been considered an unselective process for bulk degradation of
long-lived proteins and organelles, often during nutrient deprivation. In contrast to the bulk
degradation process, especially in times of cellular stress, a number of recent reports have
observed selective autophagic degradation of substrates by recognition by cargo receptors such
as p62 (Johansen and Lamark, 2011). The p62 protein is an ubiquitin-binding adapter protein that
serves multiple cellular functions for autophagy, apoptosis, ROS signaling and cancer (Johansen
and Lamark, 2011; Son et al., 2014). In the process of autophagy, a substrate must first be
recognized by cell systems to be sorted into the autophagy degradation pathway. Next, the
substrate must be ubiquitinated. Lastly, p62 is recruited to the ubiquitinated substrates. The role
of p62 and other adapter proteins like NBR1 and HDAC6 is to sequester autophagy substrates
into larger aggregates before being degraded within the autophagosome (Bjørkøy et al., 2005).
It has been demonstrated, both in culture and in vivo, that the mammalian p62 and the
Drosophila homologue Ref2P is required for the formation of ubiquitinated protein aggregates to
be degraded via autophagy (Nezis et al., 2008). It is also interesting to note that the presence of
p62 in nuclear or cytoplasmic aggregates is a common feature found in human disease including
Alzheimer disease, Parkinson disease and various chronic liver diseases (Strnad et al., 2008).
This suggests, at least in part, a dysfunction of the autophagic machinery to properly degrade p62
in these disease states.
Our results in Fig. 12 indicate a decline in p62 protein as Cd concentrations increase,
suggestive of heightened selective autophagy. This is in agreement with previous studies that
have shown that p62 expression correlates with increased autophagy (Johansen and Lamark,
2011). Rapamycin exposure resulted in a decrease in p62 expression, an observation that has
been made before (Bjørkøy et al., 2005), showing that induction of autophagy by rapamycin
leads to the degradation of endogenous p62. What is still to be determined is the nature of the
protein aggregates during Cd toxicity. With major toxic effects of Cd being targeted to the
mitochondria, it would be interesting to study p62-mediated autophagic degradation of
73
mitochondria. Cadmium significantly induces mitochondrial membrane potential collapse and
has been shown to induce mitophagy in whole mouse kidneys through a ROS-mediated,
PINK1/Parkin system (Wei et al., 2014). While p62 has been observed to localize to the
mitochondria in periods of mitochondrial stress, the expression and localization of p62 was not
investigated in that particular study.
As mentioned earlier, p62 is involved in cellular signaling processes that are unrelated to
autophagy. The degradation of p62 by autophagy may be an important mechanism to regulate the
levels of this signaling molecule. Aggregate bodies of p62 have been suggested to act as
signaling hubs to interact with the E3 ligase TRAF6 and caspase-8, affecting both pro-survival
and pro-apoptotic signaling pathways (Johansen and Lamark, 2011). According to the results in
Fig. 12, one could suggest that p62 is anti-apoptotic due to its diminution when RMC are treated
with Cpt.
4.5 ROS and Cd-induced autophagy
Cadmium induces significant oxidative damage in cells. The toxic metal does not redox
cycle, but can increase cellular levels of ROS by a variety of mechanisms including the depletion
of antioxidant defenses, displacement of Fenton-active metals from other sites, and inhibition of
mitochondrial electron transport (Templeton and Liu, 2010). Little is known about the role of
ROS in Cd-induced autophagy, however (Wang et al., 2015b). Both ROS and reactive nitrogen
species (RNS) irreversibly oxidize DNA and other cellular biomolecules, representing the
primary source of damage in biological systems. Autophagy, as has been discussed, contributes
to the clearing of cells of dysfunctional proteins and organelles, including irreversibly oxidized
biomolecules, and may be involved in the antioxidant defense of the cell (Filomeni et al., 2015).
ROS have been reported as early inducers of autophagy upon nutrient deprivation. While
it is unclear which species most drives the process, the prevailing hypothesis is that ROS are
crucial for autophagic execution as treatment with antioxidants partially or completely reverses
the process (Filomeni et al., 2015). In line with that assumption is that ROS (or RNS) may act as
alarm molecules to initiate autophagy. ROS is one of the stressors that activates the stress-
activated protein kinase, JNK. Studies with ciclopirox olamine, a fungicide, have found that
autophagy can be induced in human rhabdomyosarcoma RD and Rh30 cells through a ROS-
activated JNK pathway (Zhou et al., 2014). In regards to Cd toxicity, autophagy was found to
74
protect against Cd-induced reduction in mitochondrial membrane potential as a result of ROS
generation in primary rat cerebral cortical neurons (Wang et al., 2015b).
In order to examine the role of accumulated ROS in the induction of autophagy, with the
connection to JNK activation, RMC were treated with Cd and BHA. BHA is an antioxidant that
scavenges ROS by donating a labile hydrogen to oxygen radicals (Festjens et al., 2005).
Curiously, it was found that increasing the concentration of BHA in 24 hour RMC treatment led
to a resultant increase in autophagy (Fig. 11). With most previous studies indicating the
importance of ROS in autophagic activity, with defective autophagy leading to ROS
accumulation, this result came as a surprise. We have measured ROS in Cd treatments with and
without BHA, and found BHA to be an effective scavenger of ROS (Liu et al., 2014). It is
surprising that the depletion of ROS by 100 µM BHA in treatment with Cd increased the
accumulation of LC3-II to more than double that of simple Cd treatment. However, the
instigation of autophagy by BHA is not limited to Cd and BHA co-treatment, as isolated BHA
treatment also induced autophagy significantly over SF control. This result is most interesting
because it does not integrate with the multitude of studies that show evidence for ROS-mediated
autophagy (Filomeni et al., 2015). To thoroughly examine ROS accumulation and Cd-induced
autophagy, it would be interesting to induce autophagy with Cd in the presence of ROS
stimulators such as buthionine sulfoximine. To explain the results from Fig. 11, one could claim
that by depleting the cell of ROS, ROS dependent apoptosis is mitigated, potentially increasing
Cd-induced autophagy.
4.6 Autophagy and apoptosis
Autophagy controls the turnover of organelles and proteins within cells, with apoptosis
controlling the turnover of cells within organisms. Many stress pathways sequentially elicit
autophagy and then apoptosis within the same cell (Mariño et al., 2014). Autophagic initiation
before apoptosis has been observed in MES-13 cells (Wang et al., 2013b). Mesangial cell studies
in our lab have shown that Cd can kill mesangial cells by multiple pathways, including caspase-
dependent, caspase-independent, and apoptosis-like cell death after 6 hours of exposure (Liu and
Templeton, 2008). It is interesting, then, that maximal autophagy was observed in RMC at 24
hours of exposure, rather than at a point preceding apoptosis, visualized in Fig 7. Without
measuring both apoptosis and autophagy within the same experiment, however, it is difficult to
75
compare the time periods of each process. This can be accomplished with acridine orange
staining in conjunction with Annexin V/PI staining by flow cytometry.
Generally, it is observed that autophagy blocks the induction of apoptosis, and apoptotic
caspase activation suppresses the autophagic process. However, what complicates the
communication between the two processes is that they share numerous regulators (Thorburn,
2008). In preliminary studies to explore the role of autophagic cell death in Cd toxicity,
MEFATG16-/- cells were treated with Cd and compared to wild type cells (Fig. 14). It was found
that in all cases, autophagy deficiency was a detriment to cell survival. This indicates that
autophagy, or at least ATG16 specifically, has a role in protecting cells under stress.
Investigating caspase-dependent apoptosis in MEFWT and MEFATG-/- cells resulted in caspase-
3 cleavage in cells that were unable to undergo autophagy, compared to WT cells that displayed
no caspase-3 cleavage. This preliminary work agrees with observations made before, many of
which were made in cell systems that were nutrient starved followed by investigation of
apoptosis and autophagy (Mariño et al., 2014) . One of the principal mechanisms by which
autophagy can reduce the tendency of cells to undergo apoptosis is through mitophagy of
dysfunctional mitochondria (Youle and Narendra, 2011). Therefore, studying the extent of
mitophagy in our MEFATG16-/- system could lead to answers both about the extent of
mitophagy in Cd-exposed cells, as well as whether heightened mitophagy specifically can abate
apoptosis. Thus, our results suggest that Cd-induced autophagy and Cd-induced caspase-
dependent apoptosis exist in a tug-of-war, where the enhancement of one process suppresses the
other.
76
5 Summary and Significance
This study examines Cd-induced autophagy with a focus on investigating the mechanism
behind the process and whether or not autophagy is protecting cells against cell death. We have
shown that at mid-levels of Cd exposure (10 µM), there is extensive autophagy taking place in
RMC. The process is suppressed with Tg treatment, suggesting that autophagy requires Ca2+
signaling in order to activate. Additionally, Cd-induced autophagy is heightened when cells are
co-treated with BHA, indicating that depleting ROS results in increased autophagy. This study
also highlights that autophagy is a protective mechanism, with improved viability in cells that
can undergo autophagy versus those that cannot. Cells that cannot undergo autophagy are
observed to undergo caspase-dependent apoptosis in its stead, suggesting that autophagy again is
a protective mechanism against apoptosis.
Chronic Cd toxicity has emerged as a previously underestimated health hazard for
essentially all human populations. So far, the role of autophagy in Cd induced nephrotoxicity has
remained unsettled due to contradictory results. In reference to our own studies in this
dissertation, we find preliminary results to suggest that autophagy is a protective mechanism that
protects cells against the harmful apoptotic effects of Cd. While many studies have shown the
pro-apoptotic actions of Cd, work in our lab and others have found that Cd also elicits an anti-
apoptotic response, which could be related to its autophagic response. Studies on RMC have
shown that prolonged exposure to 10 µM Cd results in autophagy. While this may suggest the
use of autophagic markers as a biomarker for subtoxic Cd exposure, this may not be practical.
Sampling biomarkers should be easy and efficient, without expensive, invasive or damaging
procedures. More aggressive methods may be required when cells are undergoing cytoprotective
autophagy in the glomerulus or proximal tubule without leaving any remnants of their fate in the
blood or urine. However, a detailed understanding of the mechanisms of activation and
regulation of Cd-induced autophagy and its role in cell fate can lead to the development of novel
strategies including the modulation of protective autophagy for prevention and therapy of acute
and chronic Cd toxicity.
77
Bibliography
Aguirre, J., and Lambeth, J.D. (2010). Nox enzymes from fungus to fly to fish and what they tell us
about Nox function in mammals. Free Radic. Biol. Med. 49, 1342–1353.
Åkesson, A., Bjellerup, P., Lundh, T., Lidfeldt, J., Nerbrand, C., Samsioe, G., Skerfving, S., and
Vahter, M. (2006). Cadmium-induced effects on bone in a population-based study of women.
Environ. Health Perspect. 114, 830–834.
Atchison, W.D. (1988). Effects of neurotoxicants on synaptic transmission: lessons learned from
electrophysiological studies. Neurotoxicol. Teratol. 10, 393–416.
Baker, T.K., VanVooren, H.B., Smith, W.C., and Carfagna, M.A. (2003). Involvement of calcium
channels in the sexual dimorphism of cadmium-induced hepatotoxicity. Toxicol. Lett. 137, 185–192.
Barth, S., Glick, D., and Macleod, K.F. (2010). Autophagy: assays and artifacts. J. Pathol. 221, 117–
124.
Bernhoft, R.A. (2013). Cadmium toxicity and treatment. Sci. World J. 2013, 394652.
Berridge, M.J. (2009). Inositol trisphosphate and calcium signalling mechanisms. Biochim. Biophys.
Acta 1793, 933–940.
Beyersmann, D., and Hechtenberg, S. (1997). Cadmium, gene regulation, and cellular signalling in
mammalian cells. Toxicol. Appl. Pharmacol. 144, 247–261.
Bhattacharyya, M.H. (2009). Cadmium osteotoxicity in experimental animals: mechanisms and
relationship to human exposures. Toxicol. Appl. Pharmacol. 238, 258–265.
Biagioli, M., Pinton, P., Scudiero, R., Ragghianti, M., Bucci, S., and Rizzuto, R. (2005). Aequorin
chimeras as valuable tool in the measurement of Ca2+ concentration during cadmium injury.
Toxicology 208, 389–398.
Biagioli, M., Pifferi, S., Ragghianti, M., Bucci, S., Rizzuto, R., and Pinton, P. (2008). Endoplasmic
reticulum stress and alteration in calcium homeostasis are involved in cadmium-induced apoptosis.
Cell Calcium 43, 184–195.
Bishak, Y.K., Payahoo, L., Osatdrahimi, A., and Nourazarian, A. (2015). Mechanisms of cadmium
carcinogenicity in the gastrointestinal tract. Asian Pac. J. Cancer Prev. 16, 9–21.
Bjørkøy, G., Lamark, T., Brech, A., Outzen, H., Perander, M., Overvatn, A., Stenmark, H., and
Johansen, T. (2005). p62/SQSTM1 forms protein aggregates degraded by autophagy and has a
protective effect on huntingtin-induced cell death. J. Cell Biol. 171, 603–614.
Brand, M.D. (2010). The sites and topology of mitochondrial superoxide production. Exp. Gerontol.
45, 466–472.
78
Buchet, J.P., Lauwerys, R., Roels, H., Bernard, A., Bruaux, P., Claeys, F., Ducoffre, G., de Plaen, P.,
Staessen, J., and Amery, A. (1990). Renal effects of cadmium body burden of the general population.
Lancet 336, 699–702.
Buckley, B.J., and Bassett, D.J. (1987). Pulmonary cadmium oxide toxicity in the rat. J. Toxicol.
Environ. Health 21, 233–250.
Chan, H.M., Zhu, L.F., Zhong, R., Grant, D., Goyer, R.A., and Cherian, M.G. (1993). Nephrotoxicity
in rats following liver transplantation from cadmium-exposed rats. Toxicol. Appl. Pharmacol. 123,
89–96.
Chatterjee, S., Sarkar, S., and Bhattacharya, S. (2014a). Toxic metals and autophagy. Chem. Res.
Toxicol. 27, 1887–1900.
Chatterjee, S., Ray, A., Mukherjee, S., Agarwal, S., Kundu, R., and Bhattacharya, S. (2014b). Low
concentration of mercury induces autophagic cell death in rat hepatocytes. Toxicol. Ind. Health 30,
611–620.
Chen, J.-H., Shahnavas, S., Singh, N., Ong, W.-Y., and Walczyk, T. (2013). Stable iron isotope
tracing reveals significant brain iron uptake in adult rats. Metallomics 5, 167–173.
Chen, L., Liu, L., Luo, Y., and Huang, S. (2008). MAPK and mTOR pathways are involved in
cadmium-induced neuronal apoptosis. J. Neurochem. 105, 251–261.
Cheng, Y., Ren, X., Hait, W.N., and Yang, J.-M. (2013a). Therapeutic targeting of autophagy in
disease: biology and pharmacology. Pharmacol. Rev. 65, 1162–1197.
Cheng, Y., Ren, X., Zhang, Y., Shan, Y., Huber-Keener, K.J., Zhang, L., Kimball, S.R., Harvey, H.,
Jefferson, L.S., and Yang, J.-M. (2013b). Integrated regulation of autophagy and apoptosis by
EEF2K controls cellular fate and modulates the efficacy of curcumin and velcade against tumor cells.
Autophagy 9, 208–219.
Chin, T.A., and Templeton, D.M. (1992). Effects of CdCl2 and Cd-metallothionein on cultured
mesangial cells. Toxicol. Appl. Pharmacol. 116, 133–141.
Chin, T.A., and Templeton, D.M. (1993). Protective elevations of glutathione and metallothionein in
cadmium-exposed mesangial cells. Toxicology 77, 145–156.
Chmielowska-Bąk, J., Izbiańska, K., and Deckert, J. (2013). The toxic doppelganger: on the ionic
and molecular mimicry of cadmium. Acta Biochim. Pol. 60, 369–374.
Choong, G., Liu, Y., and Templeton, D.M. (2014). Interplay of calcium and cadmium in mediating
cadmium toxicity. Chem. Biol. Interact. 211, 54–65.
Chuang, S.M., and Yang, J.L. (2001). Comparison of roles of three mitogen-activated protein kinases
induced by chromium(VI) and cadmium in non-small-cell lung carcinoma cells. Mol. Cell. Biochem.
222, 85–95.
Cormet-Boyaka, E., Jolivette, K., Bonnegarde-Bernard, A., Rennolds, J., Hassan, F., Mehta, P.,
Tridandapani, S., Webster-Marketon, J., and Boyaka, P.N. (2012). An NF-κB-independent and
79
Erk1/2-dependent mechanism controls CXCL8/IL-8 responses of airway epithelial cells to cadmium.
Toxicol. Sci. 125, 418–429.
Covarrubias, L., Hernández-García, D., Schnabel, D., Salas-Vidal, E., and Castro-Obregón, S.
(2008). Function of reactive oxygen species during animal development: passive or active? Dev.
Biol. 320, 1–11.
Das, G., Shravage, B.V., and Baehrecke, E.H. (2012). Regulation and function of autophagy during
cell survival and cell death. Cold Spring Harb. Perspect. Biol. 4, a008813.
Decuypere, J.-P., Bultynck, G., and Parys, J.B. (2011). A dual role for Ca2+ in autophagy regulation.
Cell Calcium 50, 242–250.
Di Gioacchino, M., Petrarca, C., Perrone, A., Martino, S., Esposito, D.L., Lotti, L.V., and Mariani-
Costantini, R. (2008). Autophagy in hematopoietic stem/progenitor cells exposed to heavy metals:
biological implications and toxicological relevance. Autophagy 4, 537–539.
Ding, W.-X. (2012). Autophagy in toxicology: defense against xenobiotics. Drug Metab. Toxicol. 3,
e108.
Ding, W., and Templeton, D.M. (2000). Activation of parallel mitogen-activated protein kinase
cascades and induction of c-fos by cadmium. Toxicol. Appl. Pharmacol. 162, 93–99.
Ding, W.-X., Ni, H.-M., Gao, W., Hou, Y.-F., Melan, M.A., Chen, X., Stolz, D.B., Shao, Z.-M., and
Yin, X.-M. (2007). Differential effects of endoplasmic reticulum stress-induced autophagy on cell
survival. J. Biol. Chem. 282, 4702–4710.
Dong, Z., Wang, L., Xu, J., Li, Y., Zhang, Y., Zhang, S., and Miao, J. (2009). Promotion of
autophagy and inhibition of apoptosis by low concentrations of cadmium in vascular endothelial
cells. Toxicol. In Vitro 23, 105–110.
Dooley, H.C., Razi, M., Polson, H.E.J., Girardin, S.E., Wilson, M.I., and Tooze, S.A. (2014). WIPI2
links LC3 conjugation with PI3P, autophagosome formation, and pathogen clearance by recruiting
Atg12–5-16L1. Mol. Cell 55, 238–252.
Duffy, A., Le, J., Sausville, E., and Emadi, A. (2015). Autophagy modulation: a target for cancer
treatment development. Cancer Chemother. Pharmacol. 75, 439–447.
Edinger, A.L., and Thompson, C.B. (2004). Death by design: apoptosis, necrosis and autophagy.
Curr. Opin. Cell Biol. 16, 663–669.
Edsall, J.T., and McKenzie, H.A. (1978). Water and proteins. I. The significance and structure of
water; its interaction with electrolytes and non-electrolytes. Adv. Biophys. 10, 137–207.
Ercal, N., Gurer-Orhan, H., and Aykin-Burns, N. (2001). Toxic metals and oxidative stress part I:
mechanisms involved in metal-induced oxidative damage. Curr. Top. Med. Chem. 1, 529–539.
Erik J., T., Windy A., B., Jonathan H., F., and Michael P., W. (2013). Toxic effects of metals. In
Casarett & Doull's Toxicology: The Basic Science of Poisons 8th edition, C. Klaassen, ed. (New
York, US: McGraw-Hill Education)
80
Festjens, N., Kalai, M., Smet, J., Meeus, A., Van Coster, R., Saelens, X., and Vandenabeele, P.
(2005). Butylated hydroxyanisole is more than a reactive oxygen species scavenger. Cell Death
Differ. 13, 166–169.
Filomeni, G., De Zio, D., and Cecconi, F. (2015). Oxidative stress and autophagy: the clash between
damage and metabolic needs. Cell Death Differ. 22, 377–388.
Fujiwara, Y., Lee, J.-Y., Tokumoto, M., and Satoh, M. (2012). Cadmium renal toxicity via apoptotic
pathways. Biol. Pharm. Bull. 35, 1892–1897.
Füllgrabe, J., Klionsky, D.J., and Joseph, B. (2014). The return of the nucleus: transcriptional and
epigenetic control of autophagy. Nat. Rev. Mol. Cell Biol. 15, 65–74.
Gallagher, C.M., Kovach, J.S., and Meliker, J.R. (2008). Urinary cadmium and osteoporosis in U.S.
Women ≥50 years of age: NHANES 1988-1994 and 1999-2004. Environ. Health Perspect. 116,
1338–1343.
Ganley, I.G., Wong, P.-M., Gammoh, N., and Jiang, X. (2011). Distinct autophagosomal-lysosomal
fusion mechanism revealed by thapsigargin-induced autophagy arrest. Mol. Cell 42, 731–743.
Golstein, P., and Kroemer, G. (2007). Cell death by necrosis: towards a molecular definition. Trends
Biochem. Sci. 32, 37–43.
Gonick, H.C. (2008). Nephrotoxicity of cadmium & lead. Indian J. Med. Res. 128, 335–352.
Gordon, P.B., Holen, I., Fosse, M., Røtnes, J.S., and Seglen, P.O. (1993). Dependence of hepatocytic
autophagy on intracellularly sequestered calcium. J. Biol. Chem. 268, 26107–26112.
Gunawardana, C.G., Martinez, R.E., Xiao, W., and Templeton, D.M. (2006). Cadmium inhibits both
intrinsic and extrinsic apoptotic pathways in renal mesangial cells. Am. J. Physiol. Renal Physiol.
290, F1074–F1082.
Hale, A.N., Ledbetter, D.J., Gawriluk, T.R., and Rucker, E.B. (2013). Autophagy: regulation and role
in development. Autophagy 9, 951–972.
Halliwell, B., and Gutteridge, J.M. (1990). Role of free radicals and catalytic metal ions in human
disease: an overview. Meth. Enzymol. 186, 1–85.
Hanson, M.L., Brundage, K.M., Schafer, R., Tou, J.C., and Barnett, J.B. (2010). Prenatal cadmium
exposure dysregulates sonic hedgehog and Wnt/beta-catenin signaling in the thymus resulting in
altered thymocyte development. Toxicol. Appl. Pharmacol. 242, 136–145.
Hao, C., Hao, W., Wei, X., Xing, L., Jiang, J., and Shang, L. (2009). The role of MAPK in the
biphasic dose-response phenomenon induced by cadmium and mercury in HEK293 cells. Toxicol. In
Vitro 23, 660–666.
He, L., Wang, B., Hay, E.B., and Nebert, D.W. (2009). Discovery of ZIP transporters that participate
in cadmium damage to testis and kidney. Toxicol. Appl. Pharmacol. 238, 250–257.
Henkler, F., Brinkmann, J., and Luch, A. (2010). The role of oxidative stress in carcinogenesis
induced by metals and xenobiotics. Cancers (Basel) 2, 376–396.
81
Holmström, K.M., and Finkel, T. (2014). Cellular mechanisms and physiological consequences of
redox-dependent signalling. Nat. Rev. Mol. Cell Biol. 15, 411–421.
Høyer-Hansen, M., Bastholm, L., Szyniarowski, P., Campanella, M., Szabadkai, G., Farkas, T.,
Bianchi, K., Fehrenbacher, N., Elling, F., Rizzuto, R., Mathiasen, I.S., Jäättelä, M. (2007). Control of
macroautophagy by calcium, calmodulin-dependent kinase kinase-β, and Bcl-2. Mol. Cell 25, 193–
205.
Huang, Y.-C., Hung, W.-C., Chen, W.-T., Yu, H.-S., and Chai, C.-Y. (2009). Sodium arsenite-
induced DAPK promoter hypermethylation and autophagy via ERK1/2 phosphorylation in human
uroepithelial cells. Chem. Biol. Interact. 181, 254–262.
IARC (1993). Meeting of the IARC working group on beryllium, cadmium, mercury and exposures
in the glass manufacturing industry. Scand. J. Work Environ. Health 19, 360–363.
Jacobson, K.B., and Turner, J.E. (1980). The interaction of cadmium and certain other metal ions
with proteins and nucleic acids. Toxicology 16, 1–37.
Järup, L., and Åkesson, A. (2009). Current status of cadmium as an environmental health problem.
Toxicol. Appl. Pharmacol. 238, 201–208.
Järup, L., Persson, B., and Elinder, C.G. (1995). Decreased glomerular filtration rate in solderers
exposed to cadmium. Occup. Environ. Med. 52, 818–822.
Järup, L., Berglund, M., Elinder, C.G., Nordberg, G., and Vahter, M. (1998). Health effects of
cadmium exposure--a review of the literature and a risk estimate. Scand. J. Work Environ. Health 24
Suppl 1, 1–51.
Johansen, T., and Lamark, T. (2011). Selective autophagy mediated by autophagic adapter proteins.
Autophagy 7, 279–296.
Johnson, R.J. (1994). The glomerular response to injury: progression or resolution? Kidney Int. 45,
1769–1782.
Joseph, P. (2009). Mechanisms of cadmium carcinogenesis. Toxicol. Appl. Pharmacol. 238, 272–
279.
Kaneko, Y., and Tsukamoto, A. (1994). Thapsigargin-induced persistent intracellular calcium pool
depletion and apoptosis in human hepatoma cells. Cancer Lett. 79, 147–155.
Kanzawa, T., Kondo, Y., Ito, H., Kondo, S., and Germano, I. (2003). Induction of autophagic cell
death in malignant glioma cells by arsenic trioxide. Cancer Res. 63, 2103–2108.
Kato, H., Katoh, R., and Kitamura, M. (2013). Dual regulation of cadmium-induced apoptosis by
mTORC1 through selective induction of IRE1 branches in unfolded protein response. PLoS One 8,
e64344.
Kayaaltı, Z., Akyüzlü, D.K., and Söylemezoğlu, T. (2015). Evaluation of the effect of divalent metal
transporter 1 gene polymorphism on blood iron, lead and cadmium levels. Environ. Res. 137, 8–13.
Kazantzis, G. (2004). Cadmium, osteoporosis and calcium metabolism. Biometals 17, 493–498.
82
Khan, M.T., and Joseph, S.K. (2010). Role of inositol trisphosphate receptors in autophagy in DT40
cells. J. Biol. Chem. 285, 16912–16920.
Kil, I.S., Lee, S.K., Ryu, K.W., Woo, H.A., Hu, M.-C., Bae, S.H., and Rhee, S.G. (2012). Feedback
control of adrenal steroidogenesis via H2O2-dependent, reversible inactivation of peroxiredoxin III in
mitochondria. Mol. Cell 46, 584–594.
Kim, D.-W., Kim, K.-Y., Choi, B.-S., Youn, P., Ryu, D.-Y., Klaassen, C.D., and Park, J.-D. (2007).
Regulation of metal transporters by dietary iron, and the relationship between body iron levels and
cadmium uptake. Arch. Toxicol. 81, 327–334.
Kirkin, V., Lamark, T., Sou, Y.-S., Bjørkøy, G., Nunn, J.L., Bruun, J.-A., Shvets, E., McEwan, D.G.,
Clausen, T.H., Wild, P., Bilusic, I., Theurillat, J.P., Øvervatn, A., Ishii, T., Elazar, Z., Komatsu, M.,
Dikic, I., Johansen, T. (2009). A role for NBR1 in autophagosomal degradation of ubiquitinated
substrates. Mol. Cell 33, 505–516.
Kondo, Y., Kanzawa, T., Sawaya, R., and Kondo, S. (2005). The role of autophagy in cancer
development and response to therapy. Nat. Rev. Cancer 5, 726–734.
Kroemer, G., and Levine, B. (2008). Autophagic cell death: the story of a misnomer. Nat. Rev. Mol.
Cell Biol. 9, 1004–1010.
Kuma, A., Hatano, M., Matsui, M., Yamamoto, A., Nakaya, H., Yoshimori, T., Ohsumi, Y.,
Tokuhisa, T., and Mizushima, N. (2004). The role of autophagy during the early neonatal starvation
period. Nature 432, 1032–1036.
Kurz, T., Gustafsson, B., and Brunk, U.T. (2011). Cell sensitivity to oxidative stress is influenced by
ferritin autophagy. Free Radic. Biol. Med. 50, 1647–1658.
Lauwerys, R., Amery, A., Bernard, A., Bruaux, P., Buchet, J.P., Claeys, F., De Plaen, P., Ducoffre,
G., Fagard, R., and Lijnen, P. (1990). Health effects of environmental exposure to cadmium:
objectives, design and organization of the Cadmibel Study: a cross-sectional morbidity study carried
out in Belgium from 1985 to 1989. Environ. Health Perspect. 87, 283–289.
Lauwerys, R.R., Bernard, A., Roels, H.A., Buchet, J.P., and Viau, C. (1984). Characterization of
cadmium proteinuria in man and rat. Environ. Health Perspect. 54, 147–152.
Lee, W.-K., and Thévenod, F. (2008). Novel roles for ceramides, calpains and caspases in kidney
proximal tubule cell apoptosis: lessons from in vitro cadmium toxicity studies. Biochem. Pharmacol.
76, 1323–1332.
Lee, W.-K., Abouhamed, M., and Thévenod, F. (2006). Caspase-dependent and -independent
pathways for cadmium-induced apoptosis in cultured kidney proximal tubule cells. Am. J. Physiol.
Renal Physiol. 291, F823–F832.
Lee, W.-K., Torchalski, B., and Thévenod, F. (2007). Cadmium-induced ceramide formation triggers
calpain-dependent apoptosis in cultured kidney proximal tubule cells. Am. J. Physiol., Cell Physiol.
293, C839–C847.
83
Lee, W.-K., Chakraborty, P.K., Roussa, E., Wolff, N.A., and Thévenod, F. (2012). ERK1/2-
dependent bestrophin-3 expression prevents ER-stress-induced cell death in renal epithelial cells by
reducing CHOP. Biochim. Biophys. Acta 1823, 1864–1876.
Leslie, E.M., Liu, J., Klaassen, C.D., and Waalkes, M.P. (2006). Acquired cadmium resistance in
metallothionein-I/II(-/-) knockout cells: role of the T-type calcium channel Cacnalpha1G in cadmium
uptake. Mol. Pharmacol. 69, 629–639.
Li, M., Kondo, T., Zhao, Q.L., Li, F.J., Tanabe, K., Arai, Y., Zhou, Z.C., and Kasuya, M. (2000).
Apoptosis induced by cadmium in human lymphoma U937 cells through Ca2+-calpain and caspase-
mitochondria- dependent pathways. J. Biol. Chem. 275, 39702–39709.
Lim, S.-C., Hahm, K.-S., Lee, S.-H., and Oh, S.-H. (2010). Autophagy involvement in cadmium
resistance through induction of multidrug resistance-associated protein and counterbalance of
endoplasmic reticulum stress WI38 lung epithelial fibroblast cells. Toxicology 276, 18–26.
Liu, Y., and Templeton, D.M. (2007). Cadmium activates CaMK-II and initiates CaMK-II-dependent
apoptosis in mesangial cells. FEBS Lett. 581, 1481–1486.
Liu, Y., and Templeton, D.M. (2008). Initiation of caspase-independent death in mouse mesangial
cells by Cd2+: involvement of p38 kinase and CaMK-II. J. Cell. Physiol. 217, 307–318.
Liu, J., Liu, Y., Habeebu, S.S., and Klaassen, C.D. (1998). Susceptibility of MT-null mice to chronic
CdCl2-induced nephrotoxicity indicates that renal injury is not mediated by the CdMT complex.
Toxicol. Sci. 46, 197–203.
Liu, J., Qu, W., and Kadiiska, M.B. (2009). Role of oxidative stress in cadmium toxicity and
carcinogenesis. Toxicol. Appl. Pharmacol. 238, 209–214.
Liu, R., Zhi, X., and Zhong, Q. (2015). ATG14 controls SNARE-mediated autophagosome fusion
with a lysosome. Autophagy 11, 847–849.
Liu, Y., Liu, J., Habeebu, S.M., Waalkes, M.P., and Klaassen, C.D. (2000). Metallothionein-I/II null
mice are sensitive to chronic oral cadmium-induced nephrotoxicity. Toxicol. Sci. 57, 167–176.
Liu, Y., Xiao, W., and Templeton, D.M. (2014). Cadmium-induced aggregation of iron regulatory
protein-1. Toxicology 324, 108–115.
Luevano, J., and Damodaran, C. (2014). A review of molecular events of cadmium-induced
carcinogenesis. J. Environ. Pathol. Toxicol. Oncol. 33, 183–194.
Mailloux, R.J., and Harper, M.-E. (2012). Mitochondrial proticity and ROS signaling: lessons from
the uncoupling proteins. Trends Endocrinol. Metab. 23, 451–458.
Mariño, G., Niso-Santano, M., Baehrecke, E.H., and Kroemer, G. (2014). Self-consumption: the
interplay of autophagy and apoptosis. Nat. Rev. Mol. Cell Biol. 15, 81–94.
Martelli, A., Rousselet, E., Dycke, C., Bouron, A., and Moulis, J.-M. (2006). Cadmium toxicity in
animal cells by interference with essential metals. Biochimie 88, 1807–1814.
84
Martinez-Vicente, M., and Cuervo, A.M. (2007). Autophagy and neurodegeneration: when the
cleaning crew goes on strike. Lancet Neurol. 6, 352–361.
Matsuoka, M., and Igisu, H. (1998). Activation of c-Jun NH2-terminal kinase (JNK/SAPK) in LLC-
PK1 cells by cadmium. Biochem. Biophys. Res. Commun. 251, 527–532.
McElroy, J.A., Shafer, M.M., Trentham-Dietz, A., Hampton, J.M., and Newcomb, P.A. (2007).
Urinary cadmium levels and tobacco smoke exposure in women age 20-69 years in the United States.
J. Toxicol. Environ. Health A 70, 1779–1782.
Meléndez, A., and Levine, B. (2015). Autophagy in C. elegans. In Wormbook. The Online Review
of C. elegans Biology, J. Kramer, and D. Moerman, ed. (The C. elegans Research Community)
Mills, K.R., Reginato, M., Debnath, J., Queenan, B., and Brugge, J.S. (2004). Tumor necrosis factor-
related apoptosis-inducing ligand (TRAIL) is required for induction of autophagy during lumen
formation in vitro. Proc. Natl. Acad. Sci. U.S.A. 101, 3438–3443.
Misra, U.K., Gawdi, G., Akabani, G., and Pizzo, S.V. (2002). Cadmium-induced DNA synthesis and
cell proliferation in macrophages: the role of intracellular calcium and signal transduction
mechanisms. Cell. Signal. 14, 327–340.
Mizushima, N. (2007). Autophagy: process and function. Genes Dev. 21, 2861–2873.
Mizushima, N., Levine, B., Cuervo, A.M., and Klionsky, D.J. (2008). Autophagy fights disease
through cellular self-digestion. Nature 451, 1069–1075.
Mizushima, N., Yoshimorim, T., and Levine, B. (2010). Methods in mammalian autophagy research.
Cell 140, 313–326.
Moulis, J.-M., and Thévenod, F. (2010). New perspectives in cadmium toxicity: an introduction.
Biometals 23, 763–768.
Mukhopadhyay, S., Panda, P.K., Sinha, N., Das, D.N., and Bhutia, S.K. (2014). Autophagy and
apoptosis: where do they meet? Apoptosis 19, 555–566.
Navas-Acien, A., Tellez-Plaza, M., Guallar, E., Muntner, P., Silbergeld, E., Jaar, B., and Weaver, V.
(2009). Blood cadmium and lead and chronic kidney disease in US adults: a joint analysis. Am. J.
Epidemiol. 170, 1156–1164.
Nazima, B., Manoharan, V., and Miltonprabu, S. (2015). Grape seed proanthocyanidins ameliorates
cadmium-induced renal injury and oxidative stress in experimental rats through the up-regulation of
nuclear related factor 2 and antioxidant responsive elements. Biochem. Cell Biol. 93, 210–226.
Nezis, I.P., Simonsen, A., Sagona, A.P., Finley, K., Gaumer, S., Contamine, D., Rusten, T.E.,
Stenmark, H., and Brech, A. (2008). Ref(2)P, the Drosophila melanogaster homologue of
mammalian p62, is required for the formation of protein aggregates in adult brain. J. Cell Biol. 180,
1065–1071.
Nicotera, P., Leist, M., and Ferrando-May, E. (1998). Intracellular ATP, a switch in the decision
between apoptosis and necrosis. Toxicol. Lett. 102-103, 139–142.
85
Nogawa, K., and Kido, T. (1993). Biological monitoring of cadmium exposure in itai-itai disease
epidemiology. Int. Arch. Occup. Environ. Health 65, S43–S46.
Nogué, S., Sanz-Gallén, P., Torras, A., and Boluda, F. (2004). Chronic overexposure to cadmium
fumes associated with IgA mesangial glomerulonephritis. Occup. Med. (Lond) 54, 265–267.
Nordberg, G.F. (2009). Historical perspectives on cadmium toxicology. Toxicol. Appl. Pharmacol.
238, 192–200.
Oh, S.-H., Lee, B.-H., and Lim, S.-C. (2004). Cadmium induces apoptotic cell death in WI 38 cells
via caspase-dependent Bid cleavage and calpain-mediated mitochondrial Bax cleavage by Bcl-2-
independent pathway. Biochem. Pharmacol. 68, 1845–1855.
Olsson, I.-M., Bensryd, I., Lundh, T., Ottosson, H., Skerfving, S., and Oskarsson, A. (2002).
Cadmium in blood and urine--impact of sex, age, dietary intake, iron status, and former smoking--
association of renal effects. Environ. Health Perspect. 110, 1185–1190.
O’Reilly, K.E., Rojo, F., She, Q.-B., Solit, D., Mills, G.B., Smith, D., Lane, H., Hofmann, F.,
Hicklin, D.J., Ludwig, D.L., Baselga, J., Rosen, N. (2006). mTOR inhibition induces upstream
receptor tyrosine kinase signaling and activates Akt. Cancer Res. 66, 1500–1508.
Papadakis, E.S., Finegan, K.G., Wang, X., Robinson, A.C., Guo, C., Kayahara, M., and Tournier, C.
(2006). The regulation of Bax by c-Jun N-terminal protein kinase (JNK) is a prerequisite to the
mitochondrial-induced apoptotic pathway. FEBS Lett. 580, 1320–1326.
Patnaik, B.B., Roy, A., Agarwal, S., and Bhattacharya, S. (2010). Induction of oxidative stress by
non-lethal dose of mercury in rat liver: possible relationships between apoptosis and necrosis. J.
Environ. Biol. 31, 413–416.
Permenter, M.G., Lewis, J.A., and Jackson, D.A. (2011). Exposure to nickel, chromium, or cadmium
causes distinct changes in the gene expression patterns of a rat liver derived cell line. PLoS ONE 6,
e27730.
Preuss, H.G. (1993). Basics of renal anatomy and physiology. Clin. Lab. Med. 13, 1–11.
Prozialeck, W.C., and Edwards, J.R. (2010). Early biomarkers of cadmium exposure and
nephrotoxicity. Biometals 23, 793–809.
Qian, W., Liu, J., Jin, J., Ni, W., and Xu, W. (2007). Arsenic trioxide induces not only apoptosis but
also autophagic cell death in leukemia cell lines via up-regulation of Beclin-1. Leuk. Res. 31, 329–
339.
Qu, W., Ke, H., Pi, J., Broderick, D., French, J.E., Webber, M.M., and Waalkes, M.P. (2007).
Acquisition of apoptotic resistance in cadmium-transformed human prostate epithelial cells: Bcl-2
overexpression blocks the activation of JNK signal transduction pathway. Environ. Health Perspect.
115, 1094–1100.
Rani, A., Kumar, A., Lal, A., and Pant, M. (2014). Cellular mechanisms of cadmium-induced
toxicity: a review. Int. J. Environ. Health Res. 24, 378–399.
86
Roels, H., Bernard, A.M., Cárdenas, A., Buchet, J.P., Lauwerys, R.R., Hotter, G., Ramis, I., Mutti,
A., Franchini, I., and Bundschuh, I. (1993). Markers of early renal changes induced by industrial
pollutants. III. Application to workers exposed to cadmium. Br. J. Ind. Med. 50, 37–48.
Ryter, S.W., Mizumura, K., and Choi, A.M.K. (2014). The impact of autophagy on cell death
modalities. Int. J. of Cell Biol. 2014, e502676.
Sahmoun, A.E., Case, L.D., Jackson, S.A., and Schwartz, G.G. (2005). Cadmium and prostate
cancer: a critical epidemiologic analysis. Cancer Invest. 23, 256–263.
Sancho, P., Fernández, C., Yuste, V.J., Amrán, D., Ramos, A.M., de Blas, E., Susin, S.A., and Aller,
P. (2006). Regulation of apoptosis/necrosis execution in cadmium-treated human promonocytic cells
under different forms of oxidative stress. Apoptosis 11, 673–686.
Sarkar, S., Floto, R.A., Berger, Z., Imarisio, S., Cordenier, A., Pasco, M., Cook, L.J., and
Rubinsztein, D.C. (2005). Lithium induces autophagy by inhibiting inositol monophosphatase. J. Cell
Biol. 170, 1101–1111.
Satarug, S., Nishijo, M., Ujjin, P., Vanavanitkun, Y., and Moore, M.R. (2005). Cadmium-induced
nephropathy in the development of high blood pressure. Toxicol. Lett. 157, 57–68.
Satarug, S., Garrett, S.H., Sens, M.A., and Sens, D.A. (2010). Cadmium, environmental exposure,
and health outcomes. Environ. Health Perspect. 118, 182–190.
Schmidt, C.W. (2006). Unfair trade: e-waste in Africa. Environ. Health Perspect. 114, a232–a235.
Schuler, M., and Green, D.R. (2001). Mechanisms of p53-dependent apoptosis. Biochem. Soc. Trans.
29, 684–688.
Schutte, R., Nawrot, T.S., Richart, T., Thijs, L., Vanderschueren, D., Kuznetsova, T., Van Hecke, E.,
Roels, H.A., and Staessen, J.A. (2008). Bone resorption and environmental exposure to cadmium in
women: a population study. Environ. Health Perspect. 116, 777–783.
Seret, G., Le Meur, Y., Renaudineau, Y., and Youinou, P. (2012). Mesangial cell-specific antibodies
are central to the pathogenesis of lupus nephritis. Clin. Dev. Immunol. 2012, e579670.
Shih, C.-M., Ko, W.-C., Wu, J.-S., Wei, Y.-H., Wang, L.-F., Chang, E.-E., Lo, T.-Y., Cheng, H.-H.,
and Chen, C.-T. (2004). Mediating of caspase-independent apoptosis by cadmium through the
mitochondria-ROS pathway in MRC-5 fibroblasts. J. Cell. Biochem. 91, 384–397.
Shimizu, A., Kobayashi, E., Suwazono, Y., Uetani, M., Oishi, M., Inaba, T., Kido, T., and Nogawa,
K. (2006). Estimation of benchmark doses for urinary cadmium based on beta2-microglobulin
excretion in cadmium-polluted regions of the Kakehashi River basin, Japan. Int. J. Environ. Health
Res. 16, 329–337.
Shimizu, S., Yoshida, T., Tsujioka, M., and Arakawa, S. (2014). Autophagic cell death and cancer.
Int. J. Mol. Sci. 15, 3145–3153.
Son, Y.-O., Lee, J.-C., Hitron, J.A., Pan, J., Zhang, Z., and Shi, X. (2010). Cadmium induces
intracellular Ca2+- and H2O2-dependent apoptosis through JNK- and p53-mediated pathways in skin
epidermal cell line. Toxicol. Sci. 113, 127–137.
87
Son, Y.-O., Wang, X., Hitron, J.A., Zhang, Z., Cheng, S., Budhraja, A., Ding, S., Lee, J.-C., and Shi,
X. (2011). Cadmium induces autophagy through ROS-dependent activation of the LKB1-AMPK
signaling in skin epidermal cells. Toxicol. Appl. Pharmacol. 255, 287–296.
Son, Y.-O., Pratheeshkumar, P., Roy, R.V., Hitron, J.A., Wang, L., Zhang, Z., and Shi, X. (2014).
Nrf2/p62 signaling in apoptosis resistance and its role in cadmium-induced carcinogenesis. J. Biol.
Chem. 289, 28660–28675.
Strnad, P., Zatloukal, K., Stumptner, C., Kulaksiz, H., and Denk, H. (2008). Mallory-Denk-bodies:
lessons from keratin-containing hepatic inclusion bodies. Biochim. Biophys. Acta 1782, 764–774.
Sunderman, F.W. (2001). Nasal toxicity, carcinogenicity, and olfactory uptake of metals. Ann. Clin.
Lab. Sci. 31, 3–24.
Suwazono, Y., Nogawa, K., Morikawa, Y., Nishijo, M., Kobayashi, E., Kido, T., Nakagawa, H., and
Nogawa, K. (2014). Impact of urinary cadmium on mortality in the Japanese general population in
cadmium non-polluted areas. Int. J. Hyg. Environ. Health 217, 807–812.
Suzuki, K., and Ohsumi, Y. (2007). Molecular machinery of autophagosome formation in yeast,
Saccharomyces cerevisiae. FEBS Lett. 581, 2156–2161.
Tabas, I., and Ron, D. (2011). Integrating the mechanisms of apoptosis induced by endoplasmic
reticulum stress. Nat. Cell Biol. 13, 184–190.
Tanida, I. (2011). Autophagosome formation and molecular mechanism of autophagy. Antioxid.
Redox Signal. 14, 2201–2214.
Teeyakasem, W., Nishijo, M., Honda, R., Satarug, S., Swaddiwudhipong, W., and Ruangyuttikarn,
W. (2007). Monitoring of cadmium toxicity in a Thai population with high-level environmental
exposure. Toxicol. Lett. 169, 185–195.
Templeton, D.M., and Liu, Y. (2010). Multiple roles of cadmium in cell death and survival. Chem.
Biol. Interact. 188, 267–275.
Thévenod, F. (2009). Cadmium and cellular signaling cascades: To be or not to be? Toxicol. Appl.
Pharmacol. 238, 221–239.
Thévenod, F. (2010). Catch me if you can! Novel aspects of cadmium transport in mammalian cells.
Biometals 23, 857–875.
Thévenod, F., and Lee, W.-K. (2013a). Cadmium and cellular signaling cascades: interactions
between cell death and survival pathways. Arch. Toxicol. 87, 1743–1786.
Thévenod, F., and Lee, W.-K. (2013b). Toxicology of cadmium and its damage to mammalian
organs. In Cadmium: From Toxicity to Essentiality, A. Sigel, H. Sigel, and R.K. Sigel, eds.
(Dordrecht, NL: Springer Netherlands), pp. 415–490.
Thévenod, F., and Lee, W.-K. (2015). Live and let die: roles of autophagy in cadmium
nephrotoxicity. Toxics 3, 130–151.
88
Thomas, L.D.K., Hodgson, S., Nieuwenhuijsen, M., and Jarup, L. (2009). Early kidney damage in a
population exposed to cadmium and other heavy metals. Environ. Health Perspect. 117, 181–184.
Thomas, L.D.K., Elinder, C.-G., Wolk, A., and Åkesson, A. (2014). Dietary cadmium exposure and
chronic kidney disease: a population-based prospective cohort study of men and women. Int. J. Hyg.
Environ. Health 217, 720–725.
Thorburn, A. (2008). Apoptosis and Autophagy: regulatory connections between two supposedly
different processes. Apoptosis 13, 1–9.
US Department of Health and Human Services, ASTDR (2012). Toxicological Profile for Cadmium.
Vesey, D.A. (2010). Transport pathways for cadmium in the intestine and kidney proximal tubule:
focus on the interaction with essential metals. Toxicol. Lett. 198, 13–19.
Vignes, M., Blanc, E., Davos, F., Guiramand, J., and Récasens, M. (1996). Cadmium rapidly and
irreversibly blocks presynaptic phospholipase C-linked metabotropic glutamate receptors.
Neurochem. Int. 29, 371–381.
Vingtdeux, V., Giliberto, L., Zhao, H., Chandakkar, P., Wu, Q., Simon, J.E., Janle, E.M., Lobo, J.,
Ferruzzi, M.G., Davies, P., Marambaud, P. (2010). AMP-activated protein kinase signaling activation
by resveratrol modulates amyloid-beta peptide metabolism. J. Biol. Chem. 285, 9100–9113.
Vuori, E., Huunan-Seppälä, A., Kilpiö, J.O., and Salmela, S.S. (1979). Biologically active metals in
human tissues. II. The effect of age on the concentration of cadmium in aorta, heart, kidney, liver,
lung, pancreas and skeletal muscle. Scand. J. Work Environ. Health 5, 16–22.
Waalkes, M.P. (2003). Cadmium carcinogenesis. Mutat. Res. 533, 107–120.
Wang, A., and Templeton, D.M. (1996). Inhibition of mitogenesis and c-fos induction in mesangial
cells by heparin and heparan sulfates. Kidney Int. 49, 437–448.
Wang, Z., and Templeton, D.M. (1998). Induction of c-fos proto-oncogene in mesangial cells by
cadmium. J. Biol. Chem. 273, 73–79.
Wang, H., Lu, Q., Cheng, S., Wang, X., and Zhang, H. (2013a). Autophagy activity contributes to
programmed cell death in Caenorhabditis elegans. Autophagy 9, 1975–1982.
Wang, Q., Zhu, J., Zhang, K., Jiang, C., Wang, Y., Yuan, Y., Bian, J., Liu, X., Gu, J., and Liu, Z.
(2013b). Induction of cytoprotective autophagy in PC-12 cells by cadmium. Biochem. Biophys. Res.
Commun. 438, 186–192.
Wang, Q.-W., Wang, Y., Wang, T., Zhang, K.-B., Yuan, Y., Bian, J.-C., Liu, X.-Z., Gu, J.-H., Zhu,
J.-Q., and Liu, Z.-P. (2015a). Cadmium-induced autophagy is mediated by oxidative signaling in PC-
12 cells and is associated with cytoprotection. Mol. Med. Rep. 12, 4448–4454.
Wang, S.H., Shih, Y.L., Ko, W.C., Wei, Y.H., and Shih, C.M. (2008). Cadmium-induced autophagy
and apoptosis are mediated by a calcium signaling pathway. Cell. Mol. Life Sci. 65, 3640–3652.
Wang, S.H., Shih, Y.-L., Kuo, T.-C., Ko, W.-C., and Shih, C.-M. (2009). Cadmium toxicity toward
autophagy through ROS-activated GSK-3beta in mesangial cells. Toxicol. Sci. 108, 124–131.
89
Wang, T., Wang, Q., Song, R., Zhang, Y., Zhang, K., Yuan, Y., Bian, J., Liu, X., Gu, J., and Liu, Z.
(2015b). Autophagy plays a cytoprotective role during cadmium-induced oxidative damage in
primary neuronal cultures. Biol. Trace Elem. Res. 168, 481–489.
Wang, Z., Chin, T.A., and Templeton, D.M. (1996). Calcium-independent effects of cadmium on
actin assembly in mesangial and vascular smooth muscle cells. Cell Motil. Cytoskeleton 33, 208–
222.
Wei, X., Qi, Y., Zhang, X., Qiu, Q., Gu, X., Tao, C., Huang, D., and Zhang, Y. (2014). Cadmium
induces mitophagy through ROS-mediated PINK1/Parkin pathway. Toxicol. Mech. Methods 24,
504–511.
Wu, X., Liang, Y., Jin, T., Ye, T., Kong, Q., Wang, Z., Lei, L., Bergdahl, I.A., and Nordberg, G.F.
(2008). Renal effects evolution in a Chinese population after reduction of cadmium exposure in rice.
Environ. Res. 108, 233–238.
Xiao, W., Liu, Y., and Templeton, D.M. (2009). Pleiotropic effects of cadmium in mesangial cells.
Toxicol. Appl. Pharmacol. 238, 315–326.
Xie, Z., and Klionsky, D.J. (2007). Autophagosome formation: core machinery and adaptations. Nat.
Cell Biol. 9, 1102–1109.
Yang, H., and Shu, Y. (2015). Cadmium transporters in the kidney and cadmium-induced
nephrotoxicity. Int. J. Mol. Sci. 16, 1484–1494.
Yang, P.-M., Chen, H.-C., Tsai, J.-S., and Lin, L.-Y. (2007). Cadmium induces Ca2+-dependent
necrotic cell death through calpain-triggered mitochondrial depolarization and reactive oxygen
species-mediated inhibition of nuclear factor-kappaB activity. Chem. Res. Toxicol. 20, 406–415.
Yiin, S.J., Sheu, J.Y., and Lin, T.H. (2001). Lipid peroxidation in rat adrenal glands after
administration cadmium and role of essential metals. J. Toxicol. Environ. Health 62, 47–56.
Youle, R.J., and Narendra, D.P. (2011). Mechanisms of mitophagy. Nat. Rev. Mol. Cell Biol. 12, 9–
14.
Yu, L., Alva, A., Su, H., Dutt, P., Freundt, E., Welsh, S., Baehrecke, E.H., and Lenardo, M.J. (2004).
Regulation of an ATG7-beclin 1 program of autophagic cell death by caspase-8. Science 304, 1500–
1502.
Zeka, A., Mannetje, A., Zaridze, D., Szeszenia-Dabrowska, N., Rudnai, P., Lissowska, J., Fabiánová,
E., Mates, D., Bencko, V., Navratilova, M., Cassidy, A., Janout, V., Travier, N., Fevotte, J., Fletcher,
T., Brennan, P., Boffetta, P. (2006). Lung cancer and occupation in nonsmokers: a multicenter case-
control study in Europe. Epidemiology 17, 615–623.
Zhang, N., Li, L., Wang, J., Cao, M., Liu, G., Xie, G., Yang, Z., and Li, Y. (2015). Study of
autophagy-related protein light chain 3 (LC3)-II expression levels in thyroid diseases. Biomed.
Pharmacother. 69, 306–310.
Zhang, T., Qi, Y., Liao, M., Xu, M., Bower, K.A., Frank, J.A., Shen, H.-M., Luo, J., Shi, X., and
Chen, G. (2012). Autophagy is a cell self-protective mechanism against arsenic-induced cell
transformation. Toxicol. Sci. 130, 298–308.
90
Zheng, L., Wu, K., Li, Y., Qi, Z., Han, D., Zhang, B., Gu, C., Chen, G., Liu, J., Chen, S., Xu, X.,
Huo, X. (2008). Blood lead and cadmium levels and relevant factors among children from an e-waste
recycling town in China. Environ. Res. 108, 15–20.
Zhou, H., Shen, T., Shang, C., Luo, Y., Liu, L., Yan, J., Li, Y., and Huang, S. (2014). Ciclopirox
induces autophagy through reactive oxygen species-mediated activation of JNK signaling pathway.
Oncotarget 5, 10140–10150.
Zhou, Y.-Y., Li, Y., Jiang, W.-Q., and Zhou, L.-F. (2015). MAPK/JNK signalling: a potential
autophagy regulation pathway. Biosci. Rep. 35, e00199.
Zilfou, J.T., and Lowe, S.W. (2009). Tumor suppressive functions of p53. Cold Spring Harb.
Perspect. Biol. 1, a001883.
Zou, H., Zhuo, L., Han, T., Hu, D., Yang, X., Wang, Y., Yuan, Y., Gu, J., Bian, J., Liu, X., Liu, Z.
(2015). Autophagy and gap junctional intercellular communication inhibition are involved in
cadmium-induced apoptosis in rat liver cells. Biochem. Biophys. Res. Commun. 459, 713–719.