Chemical Toxicants and Chronic

InflammationKristen DostieApril 8, 2015

Cadmium (Cd)• Toxic heavy metal • Found naturally in environment• Extremely long biological half-life of 18-33 years• Intoxication leads to a variety of adverse effects

• Oxidative stress• Genotoxicity• Nephrotoxicity• Osteotoxicity• Pulmonary toxicity• Hepatoxocity• Reproductive toxicity• Carcenogenicity

• Currently no treatment for Cd-intoxication

What are the main sources of Cd?• Natural sources• Erosion of rocks and soils, forest fires,

volcanic eruptions

• Man-made processes• Usage of phosphate fertilizers, presence in

sewage sludge, industrial uses

• Industrial Uses• Metal plating, producing pigments, NiCd

batteries, stabilizers in plastics

Routes of Cd Exposure

• Inhalation• Occupational exposure- mining, metal

plating, paint production, welding• Non-occupational exposure- cigarette

smoking

• Ingestion• Plant products that take up Cd in the soil

• Skin absorption• Negligible amounts absorbed through

the skin

Known adverse effects of Cd on human health• Toxic to many organs such as kidney, liver, lungs, pancreas, testes, placenta, brain, and

bones

• Cellular mechanisms of Cd toxicity in cells:• Decrease in antioxidant potential• Decrease in thiol status• Activation of signaling pathways• Inhibition of DNA methylation and DNA repair• Cell damage

• Class I human carcinogen

• Can induce inflammatory response

Cd and oxidative stress

Cuypers et al 2010

Transcription factors• NF-κB• AP-1

Proinflammatory cytokines• IL-6• TNF-α• IL-1β• IL-8

Adhesion molecules• ICAM-1• PECAM-1

Other inflammatory mediators• MPO• iNOS• MMPs• COX-2 and PGE2

• CRP

Cd exposure leads to upregulation of many inflammatory mediators such as:

Miley Cyrus poisons the youth of America in more ways than one

Quick Background

• Acute inhalation of Cd as soluble ion or particle• Bronchial inflammation, ↑ MMP-9 in BALF (Bolognin et al 2009)• ↑IL-6, ↑ macrophage inflammatory protein-2 (MIP-2) in primary rat lung epithelial cells (Lag et

al 2010)• ↑TNF-α, ↑IL-1β, ↑MIP-2 in alveolar macrophages (Lag et al 2010)

• Chronic Cd exposure• 90% inhaled dose absorbed in lungs (Kundu et al 2009)• Lung cell proliferation (Kundu et al 2009)• Lung inflammation (Kundu et al 2009)

• Cadmium oxide nanoparticles (CdO NPs)• Nanoparticle: has at least 1 dimension <100nm• Used for medical imaging and directing pharmaceutical agents to target sites

Approach1. Exposed mice to ~1.7µg CdO NP per day

(below OSHA Action level)

2. Euthanized 24h after final exposure or allowed to recover for 6 days prior to euthanasia

3. Measured various markers of inflammation and tissue damage

Objective: to determine the effects of inhaled CdO NPs at an occupationally relevant concentration on pulmonary injury and repair, and on systemic immunity in adult mice.

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Effects of inhaled CdO NP on body and lung weights

Cd exposure causes an increase in relative lung weight that persists for longer than 7 days after cessation of exposure.

Inhaled CdO NP leads to pulmonary inflammation

Filtered air 1d post-cessation of Cd exposure 7d post-cessation of Cd exposure

Fluid/inflammatory cell infiltration

Site of septum wall thickening

Area of increased BALT

BALT= bronchus-associated lymphoid tissue

BALT and septum wall thickening persist 7 days after cessation of CdO NP exposure.

Effects of inhaled CdO NPs on lavagable cell number and viability

Number of cells in BALF increase 7 days after cessation of CdO exposure consistent with leukocyte infiltration into lung tissues associated with inflammation.

Effects of inhaled CdO NP on lavagable protein and LDH activity

Although total protein in BALF decreases 7 days following cessation of Cd treatment, indicators of cell damage persist.

Effects of exposure to NPs on lavagable pro-inflammatory cytokines

Short-term inhalation of CdO NPs continues to alter the release of pro-inflammatory cytokines 7 days after exposure has ceased.

Effects of inhaled CdO NPs on BALF MMP-2 and MMP- activity

ND= none detected?

Metalloproteinase activity persists 7 days after CdO NP exposure has ceased.

Effects of CdO NP inhalation on peripheral white blood cell profiles

Circulating neutrophils decrease 7 days post cessation of exposure compared to 24h timepoint and to control; this could be attributed to neutrophils adhering to/exiting vasculature.

Effects of inhaled CdO NPs on blood leukocyte populations and bacterial uptake by circulating phagocytes

Phagocytic index of circulating blood phagocytes suggests that inhaled CdO may be translocating from lung tissue into the blood

Conclusions of Paper 1

• Short-term inhalation of CdO NPs can:• Lead to persistent pulmonary cell damage• Affect lung histology and systemic immune function

• Some markers of inflammation were reversible• Others persisted for ≥7 days

• LDH• Release of certain proinflammatory cytokines• MMP-2

Short-term CdO NP inhalation can lead to continued lung tissue injury or decreased ability to repair damage

Dietary sources of Cadmium• Little is known about the impact of

chronic ingestion of Cd on small intestine and colon health

• We do not know how passage of Cd influences mucosal homeostasis

• Link between Cd ingestion and IBD?

Wolk et al, 2012

Approach

• Age- and sex-matched BALB/c mice• Exposed mice to various doses of Cd and Pb chloride salts in drinking

water • Cd: 5, 20, and 100 ppm• Pb: 100 and 500 ppm• 4, 8, or 12 weeks of exposure

• Measured: • Cd and Pb accumulation in various tissues• Essential metal content (Zn, Fe, Cu) in various tissues• DNA damage in intestinal tissues

Cd and Pb contents in various regions of small and large intestine

Cd accumulates in the ileum and duodenum, but is cleared 4 weeks after cessation of Cd exposure.

Impact of Cd and Pb on essential metal content in distinct tissues

Cd accumulates in the ileum and duodenum, but is cleared 4 weeks after cessation of Cd exposure.

Genotoxic effects of chronic Cd exposure in the intestine of mice

Cd has genotoxic effects on colonic cells and at low concentrations in the duodenum

Paper 2 Conclusions

• Continuous exposure to low and intermediate levels of Cd and Pb have minimal biological impact on the intestinal microenvironment• Cannot conclude impact of environmental Cd or Pb on susceptibility to

IBD• Cannot conclude potential role of Cd or Pb exposure in the

pathogenesis of IBD

• Chronic Cd ingestion does disturb essential metal homeostasis in the gut• Zn, Fe• These could have potential implications on intestinal immunity

What we still don’t know about Cd and inflammation

From Paper #1:• Mechanisms associated with CdO NP-induced pulmonary injury• Mechanisms leading to the onset and persistence of these effects• Relationship between Cd exposure, induction of MMP-2 and -9 and pulmonary

disease

From Paper #2:• Consequences of Zn and Fe homeostasis on mucosal immune function• Relationship between Cd exposure and pathogenesis of colitis• If metal homeostasis and metallothioneins influences susceptibility to chronic

inflammatory diseases

Specific aim and future directions

Approach• Mouse model

• C57BL/6J (wild type)• C57BL/6J-Mt1tmBriMt2tmBri mice (Mt1 and Mt2 deficient)• C57BL/6J-TgN(Mt1)174Bri mice (overexpress Mt1)

• Chronic exposure of mice to Cd• Inhaled-aerosol • Ingested- spike drinking water

• Measure molecular and clinical markers of inflammation during and after cessation of exposure• Histology• PMN infiltration• Proteinase activity• Adhesion molecule expression• Inflammatory cytokine expression

Specific AimTo determine the role of the metal-binding protein metallothionein in the pathogenesis of chronic

pulmonary inflammation induced by Cd.

Questions? Comments? Suggestions?