1. Chemical Toxicants and Chronic Inflammation Kristen Dostie April 8, 2015

2. 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

3. 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

4. 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

5. Cadmium compounds and their sources (maybe move to the end)

6. 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

7. Cd and oxidative stress Cuypers et al 2010

8. 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 PGE 2 CRP Cd exposure leads to upregulation of many inflammatory mediators such as:

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

11. 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

12. Approach 1.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. CdO

13. 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.

14. Inhaled CdO NP leads to pulmonary inflammation Filtered air1d post-cessation of Cd exposure7d 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.

15. 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.

16. 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.

17. 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.

18. 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.

19. 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.

20. 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

21. 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

23. 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

24. 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

25. 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.

26. 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.

27. 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

28. 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

29. 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

30. Specific aim and future directions Approach Mouse model C57BL/6J (wild type) C57BL/6J-Mt1 tmBri Mt2 tmBri 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 Aim To determine the role of the metal-binding protein metallothionein in the pathogenesis of chronic pulmonary inflammation induced by Cd.

31. Questions? Comments? Suggestions?