a new antioxidant prevents toxicity of hiv proteins with methamphetamine
DESCRIPTION
A New Antioxidant Prevents Toxicity of HIV Proteins with Methamphetamine. Nuran Ercal MD PhD Department of Chemistry, Missouri University of Science and Technology, Rolla, MO & Department of Internal Medicine, St. Louis University, St. Louis, MO. Our Hypothesis:. Toxic HIV Proteins. - PowerPoint PPT PresentationTRANSCRIPT
A New Antioxidant Prevents Toxicity of HIV Proteins with Methamphetamine
Nuran Ercal MD PhDDepartment of Chemistry,
Missouri University of Science and Technology, Rolla, MO
&
Department of Internal Medicine, St. Louis University, St. Louis, MO
Our Hypothesis:
Toxic HIV ProteinsAddictive Drugs
(METH)
OXIDATIVESTRESS
BBB
BBB integrity disrupted
NEURONs are exposed to TOXINS!
What is Oxidative Stress?
Increased generation of ROS and/or a decrease in the antioxidant capacity of cells result in “Oxidative Stress”.
Oxidative stress compromises crucial cellular functions.
Sources of Reactive Oxygen Species
1) Non mitochondrial: NADPH OxidasesMicrosomal cytochrome P-450CyclooxygenasesMonoamine oxidases
Peroxisomal oxidation of fatty acids
Phagocytes
2) >90% is mitochondrial electron transport chain contains several redox centers
that may leak electrons to oxygen----- Superoxide radical!
ROS Superoxide (O2
¯• ) – No direct effects on targets Penetrates important sites Subsequently converted to other ROI
Hydrogen Peroxide (H2O2) – Dismutation of superoxide radical
2H + + 2O2¯• H2O2 + O2
Reacts with thiols Bacteriocidal only at higher concentrations Secondary oxidants from H2O2 responsible for
killing
SOD
Hydroxyl Radicals (OH•) – Fenton Reaction
Fe 2+ +H2O2 Fe 3+ + OH¯ + OH•
OH• as a major component of neutrophil bacteriocidal arsenal is controversial
Limited radius of action
Consequences of oxidative stress
Membrane peroxidationDecreased membrane fluidity
Oxidative Damage to Lipids
8-iso-PGF2 F2-Isoprostanes
Oxidative Damage
to DNA
MutationsDeletions O
H
N
NH
NH
N
NH2
Sugar
oxo8dG
Oxidative Damage
to Proteins
Oxidation of sulfhydryl groupsReactions with aldehydesProtein aggregation
Protein carbonylsPROTEIN
Marker
Outline
Introduction (background) HIV Associated Dementia
Methamphetamine
Blood Brain Barrier (BBB)
Proposed Research Goals
Experimental Methods Previous Results: RBE4
Recent Results: HBMVEC
Future Studies: immortal HBMVEC and transgenic animals
Conclusion
What is the problem?
Neurological disorders are serious complications of human immunodeficency virus type 1 (HIV-1).
What are these neurological complications? HIV-associated dementia (HAD) HIV-related encephalitis (HIVE) More commonly minor neurocognitive
problems DEMENTIA is the most challenging
complications of HIV infection.
Avindra Nath et al International Review of Psychiatry, Feb 2008, 20 (1), 25-31.
What is HAD?
A neurological syndrome characterized by cognitive deficits and motor and behavioral dysfunction.
Symptoms include:Sluggish mental capabilities and motor controlMarked apathyLoss of interest in previously enjoyable activitiesTremors and impaired balance, slow eye
movement, abnormal reflexes..The disorder continues to escalate, eventually resulting in severe dementiaSome experience mania or even psychosis
What are the symptoms of HAD?
How often is HAD seen?
One third of adults and half of children with HIV have dementia in the western countries
HAD is the most common case of dementia among people aged 40 or less and a significant independent risk factor for death due to AIDS!
Steiner J et al, Antioxidants &Redox Signaling, 2006; McArthur JC et al, Neurology, 1993; Bouwman FH et al, Neurology, 1998
HAART (Highly Active AntiRetroviral Therapy) therapy has reduced HAD in infected population some but it does not seem to be effective.
In the era of HAART, the course of HIV dementia appears to have changed.
Avindra Nath et al International Review of Psychiatry, Feb 2008, 20 (1), 25-31.
Treatment of AIDS/HIV
Highly active antiretroviral therapy (HAART) is introduced in the mid-90s.
HAART consists of combination of nucleoside reverse transcriptase inhibitors non-nucleoside RT inhibitors and protease inhibitors
There are more than 20 (zidovudine, lamivudine, stavudine, emtriva, crixivan, kaletra and more) approved antiretroviral drugs.
HAART therapy has been shown to prolong survival in AIDS patients.
Current Pharmaceutical Design, 2006, 12,2031-2055
How about: World Wide AIDS Statistics
Over 22 million people have died from AIDS Over 42 million people are living with HIV/AIDS Over 19 million women are living with HIV/AIDS There are over 14,000 new infections every day
(95% are in developing countries)
http://www.until.org/statistics.shtml
World Wide Future Estimates
The UN estimates that, currently, there are 14 million AIDS orphans and that by 2010 there will be 25 million
By the year 2010, five countries (Ethiopia, Nigeria, China, India, and Russia) with 40% of the world’s population will add 50 to 75 million infected people to the worldwide pool of HIV disease if nothing is done to help stop the spread of HIV/AIDS
http://www.until.org/statistics.shtml
United States AIDS Statistics
One million people are currently living with HIV in the U.S., with approximately 40,000 new infections occurring each year
70% of these new infections occur in men and 30% occur in women
75% of the new infections in women are heterosexually transmitted
Half of all new infections occur in people 25 years old or younger
http://www.until.org/statistics.shtml
What is the possible mechanism of HIV dementia?
HIV envelope protein (gp120) and transregulatory protein (Tat) may play a role in the development of HAD by increasing the production of reactive oxygen species (ROS) in blood brain barrier (BBB).
Oxidative stress may be involved in HIV neuropathogenesis.
Oxidative Stress Related Disorders
Parkinson’s Disease Alzheimer’s Disease Stroke Rheumatoid arthritis Atherosclerosis Vascular dysfunction Multiple sclerosis Inflammatory bowel disease H. pylori-associated gastritis Systemic inflammatory
response syndrome
And the list is growing…
Autoimmune thyroid disease Cystic fibrosis Diabetes Aging Macular degeneration HIV/AIDS Cancer Septic shock Heavy metal toxicity Nanoparticle toxicity EtOH abuse Meth ALS
Is there “Oxidative Stress” in HIV Dementia? Analyses of brain tissue and CSF of patients with
HIV-1 dementia shows evidence for oxidative stress correlated with disease pathogenesis and cognitive impairment.
Evidences are: 4-HNE is high Protein carbonyls are high Nitrated tyrosine residues are increased in HIV
dementia brains
Another important concern: Methamphetamine (METH)
It is widely known that many people with HIV-1 use addictive drugs. Among them: Alcohol Methamphetamine Cocaine Nitrite Inhalants Hallucinogens
Nearly 50% of HIV positive women in the US contract the infection via drug use!! Methamphetamine abuse is common
9.4 million people in the US Midwest – 90% of all drug cases Methamphetamine (METH) induces ROS.
Crystal
PowderPill
Missouri – Highest Rate of METH Lab Activity in the Country
Picture source: US Department of Justice (www.usdoj.gov)
More on Methamphetamine
Street names of methamphetamine (METH) Speed Ice Crystal Meth Chalk
Consumption Smoke Snort Inject Oral Ingestion
National Drug Intelligence Center, National Drug Threat Assessment 2008, October 2007; Picture Source: http://cerhr.niehs.nih.gov/chemicals/stimulants/amphetamines/methamphetamine.gif
Surge in catecholamine levels: Dopamine (DA) Serotonin Norepinephrine (NE)
Meth and Oxidative Stress
Oxidative stress has been shown to play an important role in the toxic effects of METH.
METH increases levels of dopamine.
Dopamine can react to form ROS through several different pathways.
Josephine W.S et al., Annals of the New York Academy of Sciences (2000)Zecca, L., et al., Nature Reviews (2004)
Dopamine autoxidation
DA + O2 SQ· + O2· - + H+
DA + O2· - + 2 H+ SQ· + H2O2
Dopamine enzymatic oxidation MAO
DA + O2 + H2O-------------------
3,4,dihydroxyphenylacetic acid + NH3 +
H2O2
What are toxic HIV proteins?
HIV envelope protein (gp120) and transregulatory protein (Tat) may play a role in the development of HAD by increasing the production of reactive oxygen species (ROS) in blood brain barrier (BBB).
What exactly is happening?
The BBB has been implicated in the development of HIV Dementia.
The BBB is a barrier between the blood and the fluid that surrounds the cells of the brain.
The BBB is selectively permeable, allowing some substances to cross and not others. It is generally more permeable to lipophilic substances.
The cells that line the capillaries of the rest of the body usually have small gaps between them, that allow substance exchange. The BBB lacks these small gaps, prohibiting much exchange.
Blood Brain Barrier (BBB)
What is the function of the BBB?
The BBB maintains that delicate balance by regulating the entry and exit of substances.
The BBB also provides protection by preventing toxic chemicals from entering the brain.
The structural integrity of the BBB is compromised in HIV/AIDs demented patients.
What is the cause of this destruction?
Studies have found that HIV-1 envelope protein gp120 and/or Tat may have a role in the structural damage that the BBB experiences.
What’s causing the blood-brain barrier to fail?
Our Hypothesis:
Toxic HIV ProteinsGp120 and tat
Addictive Drugs(METH)
OXIDATIVESTRESS
BBB
BBB integrity disrupted
NEURONs are exposed to TOXINS..
Let’s study HIV-1 and its proteins
gp120 gp120 is a protein on the outer envelope of the HIV-1 virus.
It binds to a receptor on CD4 cells and aides in the injection of viral nucleic acid into the host cell.
The amino acids responsible for this binding interaction are highly conserved amongst strains of the virus.
Gp120 (red) in complex with a CD4 surface protein (yellow)
gp120 is composed of an outer and inner domain made of α helices and β sheets.
The negatively charged central cavity contains the binding site for a positively charged region of a surface protein on CD4 (electrostatic interactions).
More on gp120
Molecular Structure of gp120
gp120 changes confirmation when bound to different proteins
Picture Source: Tongqing Zhou et al. Nature 2007
Transregulatory Protein (Tat)
It is a viral protein released from HIV-1-infected T cells and monocytes/macrophages
Transactivator of Transcription (Tat) protein consists of between 86 and 101 amino acids depending on the subtype
Jeang, K. T. (1996) In: Human Retroviruses and AIDS: A Compilation and Analysis of Nucleic Acid and Amino Acid Sequences. Los Alamos National Laboratory (Ed.) pp. III-3–III-18; Picture Source: Grant R. Campbell et al. J. Biol. Chem. 2004
Tat and gp120-Induced Oxidative Stress
Although the mechanism is not explicitly known, it has been shown that both gp120 and tat increase oxidative stress levels in cells.
Tat Decreases GSH by inhibiting GSH synthetase Increases NO secretion which induces apoptosis Increases calcium uptake resulting in production of ROS
gp120 Decreases GSH by inhibiting GSH synthetase Increases cell permeability Promotes calcium overload Oxyradical production Mitochondrial dysfunction by altering ion channels
Choi J et al, J Biol Chem 2000; Toneatto S et al, AIDS 1999; Visalli, Valeria, Neuroscience 2007
METH
Gp120TAT
Oxidative Stress
Microglialactivation
NOO¯
Dopamine
Glutamate mobilization by NMDARactivation
Autooxidation
Oxidation by MAO
iNOSeNOS
•NO
Ca2+
overload
nNOS
ROS
O2•¯
DNA oxidationProtein oxidationLipid Peroxidation
H2O2
QuinonesO2
•¯, OH•
Cytokines & Chemokines IL-1B,IL-8,TNF-a CCL2, CCL4,CXCL8
ERK1/2
MAPK
RNS
Antioxidants
Oxidative Stress: Possible Pathways
Oxidative Stress in HIV Dementia
Analyses of brain tissue and CSF of patients with HIV-1 dementia shows evidence for oxidative stress correlated with disease pathogenesis and cognitive impairment.
Evidences are: 4-HNE is high Protein carbonyls are high Nitrated tyrosine residues are increased in HIV
dementia brains
HIV Associated Dementia and METH
Overlap of pathways leading to neurodegeneration in HAD
METH use enhances gp120 and Tat mediated neurotoxicity
Oxidative stress common to viral protein and METH mediated toxicity
Therapeutic approaches Block rise in intracellular calcium Antagonize NMDA receptors Target oxidative damage
Disruption of Blood Brain Barrier
Disruption of the BBB is seen more in AIDS/HIV dementia patients then in non-demented AIDS/HIV patients.
The disruption of the BBB may be due to an increase in ROS by viral proteins
Tat and gp120 induce oxidative stress Stimulation of iNOS and production of NO Glutamate mediated excitotoxicity Disruption of calcium homeostasis
METH may potentiate Tat and gp120 induced oxidative stress on BBB.
Antioxidants in Neurodegeneration
Highly active antiretroviral therapy (HAART) does not prevent BBB disruption nor does it decrease ROS production.
Therefore, ANTIOXIDANTS should be included in the treatment to prevent HAD.
Dealing with BBB Most antioxidants (AO) unable to cross BBB Design AO capable of crossing BBB Low MW thiol antioxidants with ability to cross BBB
Thiols as Antioxidants
The most important biological thiol, Glutathione (GSH), protects cells against ROS.
GSH deficiency has been associated with various neurodegenerative diseases.
A significant decrease in GSH levels in patients with AIDS/HIV in various biological samples (blood, liver, brain).
Thiol antioxidants in HAD: NAC (decreased the mortality rate of HIV-infected patients) NAC analogs (N-acetyl-L-cysteinyl)-S-acetylcysteamine (NACA, possible candidate)
Pocernich CB et al., Brain Research Reviews, 2005 .
Structures of NAC and NACA
NAC NACA
NACA related projects
NACA in radiation damage NACA in neurological complications of
HIV Dementia Lead poisoning (possibly via Glu)
NACA in medicinal and abusive drug complications Acetaminophen poisoning METH neurotoxicity
NACA in macular degeneration
30-day percentage survival rates of SD-rats after irradiation with pre-treatment of NAC or NACA and post-treatment of NAC or NACA.
A: XRT only; B: XRT + NAC (pre-treated); C: XRT + NACA (pre-treated);D: Control (no XRT and any treatment); E:NAC only; F: NACA only; G:XRT + NAC (post-treated); H: XRT + NACA (post-treated).
0%
25%
50%
75%
100%%
30-
day
su
rviv
al r
ate
A B C D E F G H
Animal groups
Control NACA only (750 μM)
Glutamate only (10 mM)Glutamate (10 mM) + NACA (750 μM)
PC12 cells were plated at a density 25 x 103 cells/well in a 24 well plate and grown for 24 h in culture medium; then they were treated or not (control) with 10 mM Glu with or without NACA.. Twenty four h later, cells were examined and photographed.
Immortalized HBMVEC
Control METH 1mM METH +NACA NACA0.5mM0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
1.1
***
###
HCMVEC/D3 Cells were pretreated with 1mM NACA for 2 h, followed by24 h incubation with METH 1mM. NACA protected HCMVEC/D3 cells fromcytotoxicity caused by METH. Values were the means +SD of threesamples.Statistically different values of ***p <0.0001 were determined,compared to control. ###p < 0.0001 compared to METH 1mM group.
Ce
ll V
iab
ility
(%
of
co
ntr
ol)
Leading cause of blindness in people over 55
Degeneration of macular region of the eye
Macula – oval yellow spot near the center of the retina of the eye
Near its center is the fovea, a small pit containing the largest concentration of cone cells of the eye. Responsible for central vision
Disease begins when transport of nutrients and waste via retinal pigment epithelial cells begins to slow and down, leading to accumulation of waste products
Age Related Macular degeneration
Changes in Macula in ARMD
Image Source: www.sfn.org (Society for Neuroscience)
Retinal Pigment Epithelium
RPE affected in AMD
Undergoes oxidative stress: Metabolically active cells Large oxygen fluxes across its boundary Exposure to sunlight RPE phagocytose photoreceptor outer segments
containing PUFAS which can undergo lipid peroxidation
Age decreases antioxidants in RPE, increasing effect of oxidative insults
Experimental design Cell model – Adult retinal pigment epithelial cells
(ARPE-19)
Oxidative stress induction – tert butylhydroperoxide (tBHP), a cell permeant oxidant that causes lipid peroxidation and loss of GSH
Antioxidant – Various concentrations of N-acetycysteine amide
Pretreat cells for 24 h with antioxidant, followed by treatment with tBHP (0.4 mM) for 4 h
Experimental design Cell model – Adult retinal pigment epithelial cells
Oxidative stress induction – tert butylhydroperoxide (tBHP), a cell permeant oxidant that causes lipid peroxidation and loss of GSH
Antioxidant – Various concentrations of N-acetycysteine amide
Pretreat cells for 24 h with antioxidant, followed by treatment with tBHP (0.4 mM) for 4 h
Cell Viability
tBHP induced cell death and protection by NACA pretreatment
n=3, *p<0.0001 as compared to control
0
20
40
60
80
100
*
%
Cell
Su
rviv
al
Reactive oxygen species (ROS) production
tBHP induced production of ROS (incubation with tBHP for 45 min)
0
100
200
300
400
*
****
Concentration of tBHP(M)
Re
lati
ve
Flu
ore
sc
en
ce
(%
Co
ntr
ol)
n=3, *p<0.00 as compared to control, **p<0.005 as compared to control
Reactive oxygen species production
Protection by NACA pretreatment
0
50
100
150
200*
#
Rela
tive F
luo
rescen
ce (
% C
on
tro
l)
n=3, *p<0.0001 as compared to control, #p<0.005 as compared to tBHP treated group
GSH levels
0
20
40
60
80
*
#
GS
H (
nm
ole
s/m
g p
rote
in)
n=3, * p<0.005 as compared to control, # p<0.0001 as compared to tBHP treated group.
Restoration of GSH levels by NACA pretreatment
Conclusions NACA is capable of supplying GSH to cells
undergoing tBHP induced oxidative stress
NACA has a protective effect against tBHP induced cell death and reactive oxygen species production
NACA protected cells against
XRT-induced oxidative stress Glutamate-toxicity HIV-proteins (gp120and Tat)-METH-induced
oxidative stress Acetaminophen-toxicity Macular degeneration
NACA protected cells against
XRT-induced oxidative stress Glutamate-toxicity HIV-proteins (gp120and Tat)-METH-induced
oxidative stress Acetaminophen-toxicity Macular degeneration
HPLC Analysis of Thiol Antioxidants
A reversed-phase HPLC method developed to separate and quantify NAC and NACA by fluorescence detection by using N-(1-pyrenyl) maleimide (NPM) as the derivatizing agent.
Biological thiols such as glutathione (GSH), cysteine (CYS), and homocysteine (HCYS) can be determined simultaneously.
NPM-thiol derivative
N OO
HS R
N OO
SHH H
R
Wu W et al., Biomedical Chromatography, 20: 415-422 (2006)
NPM
thiol
Chromatogram of a Plasma Sample from an Animal Sacrificed 30 min After Administration of 500 mg/kg Body Weight NACA.
Minutes0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
mV
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0
5.5
mV
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0
5.5
NA
CN
AC
A Hyd
roly
sis
peaks
GS
H
CY
S
HC
YS
Antioxidants: N-acetylcysteine (NAC)
Replenishes GSH through deacetylation to cysteine
Low oral bioavailability (30%) due to negative charge
Low solubility and tissue distribution May cause a severe, anaphylaxis-like allergic
reaction when given intravenously
http://www.medicinenet.com/acetylcysteine-injection/article.htm
Antioxidants: NACA
Neutral charge: Lipophilic Easily crosses the cell membranes Crosses blood brain barrier, scavenges free-radicals,
chelates copper, and protects red blood cells from oxidative stress.
Current research areas using NACA include: Parkinson’s treatment cerebral ischemia other neurodegenerative diseases
Penugonda S et.al., Brain Research, September 21,
2005
HIV Dementia and Antioxidants
A few antioxidants have been used in clinical trials in HIV dementia.
OPC-14117 (lipophilic compound structurally similar to Vit E), Selegiline (L-Deprenyl, MAO inhibitor), and CPI-1189 (lipophilic antioxidant scavenging superoxide radicals).
Unfortunately, the findings are very disappointing with side effects of cataract formation, elevation of hepatic enzymes and decrease in mean corpuscular volume.
Steiner J et al, Antioxidants & Redox Signaling, 2006
Our Hypothesis:
Toxic HIV ProteinsAddictive Drugs
(METH)
OXIDATIVESTRESS
BBB
BBB integrity disrupted
NEURONs are exposed to TOXINS
AIMS
Aim I: To determine whether oxidative stress induced by gp120 and Tat at the BBB is potentiated by METH.
Aim II: To determine whether the potent antioxidant NACA protects the BBB from gp120, Tat, or METH alone and in combination.
Cell Models
RBE4-rat blood brain barrier cell model (Banks Lab)
HBMEC-primary human brain microvascular endothelial cells (Banks Lab)
Immortilized HBMEC (Banks Lab)
Oxidative Stress Parameters
Reduced glutathione (GSH) and oxidized glutathione (GSSG) assay by HPLC method
ROS measurement by DCF fluorescence-DCFH2DA converted by intracellular ROS into fluorescent DCF
Lipid peroxidation by-product (MDA) by HPLC Caspase-3 for apoptosis Antioxidant enzyme levels
GSH (glutathione) -glutamyl-cyteinyl-glycine
HOOC CH CH2 CH2 CHC N C N CH2 COOH
NH3+ O O
HH CH2
SH
Importance of glutathione GSH is the most abundant non-protein thiol Critical in maintaining the redox environment Cellular GSH is increased in times of stress, and
down-regulated after a challenge has been faced. GSH is crucial antioxidant in the BRAIN because:
20% oxygen is used in the brain (2% of the BW) Result: large quantities of ROS in the brain
In addition, There is high levels of iron in some parts of the brain Brain is rich in pufas Brain has very low amounts of SOD, CAT and GPx
So, defense against ROS by GSH becomes crucial in the brain.
Treatment of RBE4 Cells RBE4 cells divided into groups:
Control METH only (100M) Tat only (40nM) gp120 only (3nM) NACA only (1mM) Tat (40nM)+ METH (100M) gp120 (3nM) + METH (100M) Tat (40nM)+ METH (100M) + NACA (1mM) gp120 (3nM) + METH (100M) + NACA (1mM)
Incubation times – 3 h METH + 12 h for gp120 and Tat groups
Serine-Borate buffer used to prevent artifactual oxidation
Effects of increasing concentrations of METH on GSH levels in RBE4 cells.
Results (RBE4 Cells)
N=4; *Significantly different when compared with control p<0.005, **p<0.0005, ***p<0.00003. (n=3/group).
Effect of METH on GSH Levels
Gp120+Meth+NACA treated cells have similar GSH levels as controls.
N=4; *p<0.05, **p<0.05, ***p<0.05, #p<0.05, ##p<0.003, ###p<0.001.
Effect of NACA on GSH Levels in gp120 & METH Treated Cells
GSH Levels (% control) ***###*
#******
***
0
20
40
60
80
100
120
140
NACA significantly increased the GSH levels in Tat+METH-incubated RBE4 cells.
N=4; *p<0.05 compared with control, **p<0.05, ***p<0.02, #p<0.03, ##p<0.02.
Effect of NACA on GSH Levels in Tat and METH Treated Cells
Measurement of Intracellular ROS by DCF Method
Intracellular ROS production was measured using the dye 2,7-dichlorofulrescin diacetate (DCFH2-DA) method.
This measurement of cell oxidation is based on ROS-mediated conversion of nonfluorescent compound (DCFH2-DA) into a highly fluorescent compound (DCF).
Effects of NACA on the Generation of ROS in gp120 Treated Cells
N=4; *p<0.05, **p<0.05
1 nM gp120 treated cells have significantly higher intracellular ROS as compared to
controls. The increase of ROS was inhibited in the presence of ROS scavenger NACA (1 mM)
N=4; *p<0.05, **p<0.05
Effects of NACA on the Generation of ROS in Tat Treated Cells
10nm Tat treated cells have significantly higher intracellular ROS as compared to controls. The increase of ROS was inhibited in the presence of ROS scavenger
NACA (1mM).
Oxidative Stress Parameter-MDA
Malondialdehyde (MDA) levels: Polyunsaturated fatty acids, exposed to free radicals, can be oxidized to hydroperoxides which decompose (in the presence of metals) to hydrocarbons and aldehydes such as malondialdehyde (MDA).
N=4; *p<0.05, **p<0.05
NACA (1mM) attenuated lipid peroxidation in gp120 treated cells.1nM gp120 induced significant increase in MDA levels
Lipid Peroxidation Levels in gp120 Treated Cells
N=4; ***p<0.05, **p<0.05, #p<0.01
Lipid Peroxidation Levels in Tat Treated Cells
NACA (1mM) attenuated lipid peroxidation in Tat treated cells. 10 nM Tat induced significant increases in MDA levels.
Caspase-3 apoptotic Activity
Caspase-3 activity was measured by a spectrophotometric assay kit. The protease activity was measured by the addition of a specific peptide substrate for caspsase-3. The cleavage of peptide by the caspase releases the chromophore, which can be quantitated spectrophotometrically at a wavelength of 405nm.
N=4; *p<0.05, **p<0.05
Effect of NACA on Caspase 3 Activity in gp120 Treated Cells
NACA (1mM) treated cells have significantly lower caspase-3 activity as compared to the gp120 alone treated cells.
N=4; *p<0.05
Effect of NACA on caspase 3 activity in Tat treated cells
NACA (1mM) treated cells have significantly lower caspase-3 activity as compared to the Tat alone treated cells.
Effect of NACA on Antioxidant Enzyme Levels in RBE4 Cells
Groups Catalase(mU/mg protein)
GPx(mU/mg protein)
GR(mU/mg protein)
Control 6.5 ± 0.7 6.25 ± 0.73 13.94 ± 1.4
gp120 3.38 ± 0.14* 3.62 ± 0.25* 9.82 ± 0.09*
Tat 5.02 ± 0.16 3.96 ± 0.52* 9.73 ± 0.33*
gp120+NACA 8.45 ± 0.68** 8.87 ± 0.47** 11.82 ± 0.23**
Tat+NACA 9.2 ± 0.8# 10.12 ± 0.51# 11.71 ± 1.44#
N=4*p<0.005-0.01, Compared with control group **p<0.005-0.01, Compared with gp120 group#p<0.005-0.01, Compared with Tat group
Further Studies
Determine whether the potent antioxidant NACA protects the BBB from gp120, Tat, or METH (alone and/or in combination) In vitro: Use Human brain microvascular
endothelial cells (HBMVECs) New cell line :immortilized Human brain
microvascular endothelial cells (HBMVECs) In vivo: Use transgenic mice over expressing
gp120 or Tat. Permeability experiments in BBB models both in
HBMVECs and cells isolated from the transgenic mice
Treatment of HBMVEC Cells
HBMVEC cells divided into groups: Control METH only (100M) Tat only (40nM) gp120 only (3nM) NACA only (1mM) Tat (40nM)+ METH (100M) gp120 (3nM) + METH (100M) Tat (40nM)+ METH (100M) + NACA (1mM) gp120 (3nM) + METH (100M) + NACA (1mM)
Incubation times – 3 h METH + 12 h for gp120 and Tat groups
Serine-Borate buffer used to prevent artifactual oxidation
Cytotoxicity Assays
MTS tetrazolium compound
NADH or NADPH (in living cells)
NAD+ or NADP+
Colored MTS Formazan Product
(The absorbance at 490nm is proportional to the number of living cells in culture.)
Toxicological Profile of NAC and NACA in HBMVEC (MTS Assay)
0
20
40
60
80
100
120
140
0.5 5 10
Concentration (mM)
Cel
l via
bili
ty (
% u
ntr
eate
d c
on
tro
l)NACA
NAC
*
* p<0.05 as compared to NACA
NACA treated cells have significantly higher cell viability as compared to NAC treated cells.
N=4
NACA + gp120 treated cells have significantly higher cell viability as compared to gp120 alone treated cells. (Cells were treated with gp120 for 12 hours, followed by NACA
treatment for 12 hours)
Recent Results in HBMVEC Cells
N=4; *P<0.05 Control vs. 5 nM gp120, **P<0.05 5 nM gp120 vs. 5 nM gp120+ 1 mM NACA
Effect of gp120 on cell viability
Protective Effect of NACA on Tat and METH Treated cells
0
10
20
30
40
50
60
70
80
90C
ell v
iab
ilit
y (
% u
ntr
eate
d c
on
tro
l)
*
#
* p<0.005 as compared to METH only group# p < 0.05 as compared to METH + Tat group
Tat (100nM) +METH (4nM)+NACA treated cells have significantly higher cell viability as compared to Tat+METH alone treated group. Cell viability was measured using MTS assay. Cells were exposed to Tat and/or METH for 24 hours.
Immortalized HBMVEC
Control METH 1mM METH +NACA NACA0.5mM0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
1.1
***
###
HCMVEC/D3 Cells were pretreated with 1mM NACA for 2 h, followed by24 h incubation with METH 1mM. NACA protected HCMVEC/D3 cells fromcytotoxicity caused by METH. Values were the means +SD of threesamples.Statistically different values of ***p <0.0001 were determined,compared to control. ###p < 0.0001 compared to METH 1mM group.
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ll V
iab
ility
(%
of
co
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ol)
Therefore
The BBB cells (rat and human) do show oxidative stress when exposed to HIV proteins (gp120 and tat) and Meth.
NACA reverses all the oxidative stress parameters to their control levels.
How does it affect the functionality of BBB? Meaning, what happens to its permeability?
Permeability assay: Transepithelial Electric Resistance (TEER)
TEER was measured using Millicell- electric resistance system. The cells were placed in the upper chamber of 24-well tissue inserts and were cultured for 4 days before use. Change in TEER during experimental conditions was calculated as a percentage of the corresponding baseline values. Unit: ohm/cm2 (Ohm: resistance, cm2: surface area of the monolayer).
N=3
Transepithelial Electrical Resistance
NACA treated cells have higher electric resistance as compared to Tat and gp120 alone treated groups.
Permeability results
TEER decreases with gp120, tat and both NACA again returns it to its control level. Meaning:
BBB permeability increases in the presence of gp120, tat or both.
BBB integrity DISRUPTED. NACA helps keep BBB integrity.
Glutathione Peroxidase (GPx) Increases in brains of gp120 Transgenic mice.
Gp120 transgenic (SJL/(C57BL/6/SV129) and non-transgenic (C57BL/6) mice were used. Genotyping was done by PCR using DNA isolated from tail cuts. Brain samples were removed and GPx activities were measured.
Groups GPx (mU/mg protein)
Non-transgenic 17.22 ± 1.83
gp120-transgenic 20.49 ± 1.64*
*p<0.05 compared to the control group. (N = 4/group)
Review
HIV proteins, gp120 and tat, along with many addictive drugs, specifically methamphetamine, can induce oxidative stress in the blood-brain barrier.
Oxidative stress disturbs the integrity of BBB. New thiol antioxidants may protect the BBB
against oxidative stress and help reduce the risk of neurodegenerative diseases.
Publications on NACA1. Ates B, Abraham LS, Ercal N. In vitro Antioxidant and Free-Radical
Scavenging Properties of N-Acetylcysteine Amide: A Novel Thiol Antioxidant. (Accepted, Free Radical Research).
2. Wu W, Abraham LS, Ogony J, Matthews R, Ercal N. Effects of N-Acetylcysteine Amide, a Novel Thiol Antioxidant on Radiation Induced Cytotoxicity in Chinese Hamster Ovary cells. (Accepted, Life Sciences).
3. Yan M, Shen J, Person MD, Kuang X, Lynn WS, Atlas D, Wong PK. Endoplasmic reticulum stress and unfolded protein response in Atm-deficient thymocytes and thymic lymphoma cells are attributable to oxidative stress. Neoplasia. 2008;10(2):160-7.
4. Lee KS, Kim SR, Park HS, Park SJ, Min KH, Lee KY, Choe YH, Hong SH, Han HJ, Lee YR, Kim JS, Atlas D, Lee YC. A novel thiol compound, N-acetylcysteine amide, attenuates allergic airway disease by regulating activation of NF-kappaB and hypoxia-inducible factor-1alpha. Exp Mol Med. 200;39(6):756-68.
5. Amer J, Atlas D, Fibach E. N-acetylcysteine amide (AD4) attenuates oxidative stress in beta-thalassemia blood cells. Biochim Biophys Acta. 2008;1780(2):249-55.
6. Penugonda S, Mare S, Lutz P, Banks WA, Ercal N. Potentiation of lead-induced cell death in PC12 cells by glutamate: protection by N-acetylcysteine amide (NACA), a novel thiol antioxidant. Toxicol Appl Pharmacol. 2006;216(2):197-205.
7. Price TO., Uras F, Banks WA, Ercal N. A novel antioxidant N-acetylcysteine amide prevents gp120- and Tat-induced oxidative stress in brain endothelial cells. Exp Neurol. 2006;201(1):193-202.
8. Bartov O, Sultana R, Butterfield DA, Atlas D. Low molecular weight thiol amides attenuate MAPK activity and protect primary neurons from Abeta(1-42) toxicity. Brain Res. 2006;1069(1):198-206.
9. Wu W, Goldstein G, Adams C, Matthews RH, Ercal N. Separation and quantification of N-acetyl-l-cysteine and N-acetyl-cysteine-amide by HPLC with fluorescence detection. Biomed Chromatogr. 2006; 20(5):415-22.
10. Sadan O, Bahat-Stromza M, Gilgun-Sherki Y, Atlas D, Melamed E, Offen D. A novel brain-targeted antioxidant (AD4) attenuates haloperidol-induced abnormal movement in rats: implications for tardive dyskinesia. Clin Neuropharmacol. 2005;28(6):285-8.
Publications on NACA
Publications on NACA11. Penugonda S, Mare S, Goldstein G, Banks WA, Ercal N. Effects of
N-acetylcysteine amide (NACA), a novel thiol antioxidant against glutamate-induced cytotoxicity in neuronal cell line PC12. Brain Res. 2005; 1056(2): 132-8.
12. Gilgun-Sherki Y, Barhum Y, Atlas D, Melamed E, Offen D. Analysis of gene expression in MOG-induced experimental autoimmune encephalomyelitis after treatment with a novel brain-penetrating antioxidant. J Mol Neurosci. 2005;27(1):125-35.
13. Grinberg L, Fibach E, Amer J, Atlas D. N-acetylcysteine amide, a novel cell-permeating thiol, restores cellular glutathione and protects human red blood cells from oxidative stress. Free Radic Biol Med. 2005;38(1):136-45.
14. Offen D, Gilgun-Sherki Y, Barhum Y, Benhar M, Grinberg L, Reich R, Melamed E, Atlas D. A low molecular weight copper chelator crosses the blood-brain barrier and attenuates experimental autoimmune encephalomyelitis. J Neurochem. 2004;89(5):1241-51.
Acknowledgements
NIH R15 AIDS Reagent Program
Tulin Price-Otamis William A. Banks Glenn Goldstein Linu Abraham Xinsheng Zhang Karissa Braaten Atrayee Banerjee Shinya Dohgu Burhan Ates Wei Wu
Thank You
Thank You !