Moving Ahead With Mixtures: Environmental Chemical Exposures as Risk Factors for Human Diseases and Disorders

Transcription

1 Moving Ahead With Mixtures: Environmental Chemical Exposures as Risk Factors for Human Diseases and Disorders Deborah A. Cory Slechta Department of Environmental Medicine University of Rochester School of Medicine and Den>stry Rochester, NY 14642

2 Outline Biological ConCnuity and Disease Complexity Disconnect between Experimental and Epidemiological Studies of Chemicals and Disease Complexity PesCcides and Parkinson s disease Phthalates and Fungicides and male reproduccve system disorders Approaching Mixtures: Using Disease and Disorder Endpoints to Define Relevant Mixtures

3 Biological ConCnuity and Disease Complexity Nature is parsimonious: molecular targets are generally well conserved across species. Thus targets of molecules in plants, insects, etc. will oqen generalize to humans, hence the concerns over the role of environmental exposures in human diseases and disorders. It is increasingly apparent that most human diseases and disorders are complex and mulc factorial in ecology rather than the product of a single causacve factor. The importance of non genecc contribucons, including environmental risk factors in complex disease is underscored by escmates that single gene mutacons account for less than 5% of incidence, at least in the case of cancers and cardiovascular related diseases (Willet, Science, 2002; 296:695). Similar consideracons apply to diseases of the nervous system.

4 Parkinson s Disease as a Complex Disorder Factors Enhancing Vulnerability Age GeneCc background (e.g., α synuclein, DJ1, parkin) Boxing Farming Drinking Well Water PesCcide Exposure Gender Diet ProtecCve Factors Gender Smoking Caffeine Exercise Diet

5 InteracCons of Risk Factors Over Time Contribute to an Individual s Health Status Each individual has a unique set of risk factors No individual is exposed to a single chemical; thus there may also be chemical interaccons

6 In Contrast, Most Toxicology Studies Examine Toxicants as Risk Factors in IsolaCon Chemical Exposure Experimental studies: evaluate one chemical in isolacon, typically in a healthy young organism Epidemiological studies generally focus on main effects of an exposure, stacsccally controlling for any potencal modifiers, e.g., gender, other chemical exposures

7 MulCple Chemical Exposures: At Least Two Overlapping Consequences Risk Factor 3 Risk Factor 4 Vesicular long term store Releasable DA Risk Factor 1 Risk Factor 2 Mul>ple Hits and Impairment of Homeostasis Exposure to an individual chemical may be insufficient to induce overt effects, whereas mulcple risks, by provoking changes concurrently at mulcple different target sites of the dopamine system, would impair the operacon of homeostacc mechanisms, leading to dopamine dysfunccon and neuronal cell death. Mul>ple Hits Converging on a Common Adverse Outcome Single insults act by different mechanisms that converge upon a common outcome resulcng in cumulacve toxicity; e.g., male reproduccve toxicity

8 Models Based on Single Risk Factors EffecCvely examine the contribucon of a chemical as a sole ecology for an adverse outcome. This assumes that the disease phenotype results from a single mechanism, which increasingly appears to be inconsistent with many diseases and disorders. This assumes that the impact of a chemical exposure is so severe that it alone produces the phenotype. Under such condicons, much higher exposure levels are likely to be required than would be the case when exposure occurs in conjunccon with other known risk factors. Consequently: We re picking the lowhanging fruit, i.e., the chemical contribucons with the largest effects

9 Importance of Mixtures Does studying chemical exposures in the context of other percnent risk factors, including other chemical exposures: Demonstrate effects of chemical exposures at lower levels or in a synergiscc or potencated capacity? outcomes with significant implicacons for risk assessment and public health proteccon Provide more realiscc models of the disease/disorder phenotype? an outcome with implicacons for neuroproteccve and therapeucc strategies

10 Parkinson s Disease: A Disorder of the Nigrostriatal Dopamine System Loss of dopamine (TH+) cells in substanca nigra Loss of dopamine terminals and associated markers ReducCon in levels of dopamine and metabolites and dopamine turnover Symptoms and signs occur with loss of approximately 80% of dopamine neurons CharacterisCc pathology: Lewy bodies, intracytoplasmic inclusions containing alpha synuclein, ubiquicn and parkin proteins

11 PesCcide Exposure Link to PD AssociaCons of pesccide exposure with PD in numerous epidemiological studies and case reports. Similarity of the herbicide paraquat to MPP +, the accve metabolite of MPTP, a component of illegally manufactured heroin that produced severe progressive PD in young drug addicts. MPP+ + N CH3 Paraquat

12 Risk Factor InteracCons: PesCcides and Parkinson s Disease Pes;cide A Pes;cides A+B Pes;cides A+B+ Gene;c Background Pes;cides A+B + Aging Pes;cides A + B+ Cumula;ve Toxicity Pes;cides A + B + Development + Gender Paraquat and the Parkinson s Disease phenotype

13 Many PesCcides Impact Brain Dopamine Systems Dopaminergic Effects of the Ethylenebisdithiocarbamate Fungicide Maneb Decreases locomotor accvity PotenCates MPTP effects on locomotor accvity and catalepsy Enhances the uptake of MPTP into brain To date, two incidences of Parkinsonism in humans have been related to occupaconal maneb exposures Will combined paraquat and maneb enhance the Parkinson s Disease phenotype? Maneb

14 PesCcides Also Show Overlapping Geographical Use Maneb Paraquat Mancozeb Triadimefon Low Use Based on Pounds per sq. mile High

15 Support for an Environmental Basis of Parkinson s Disease Geographic variacon in age adjusted mortality stacsccs Rate per 100, Based on 1988 data from Lanska et al.

16 Young Adult Animal Model: Combined Paraquat (PQ) and Maneb (MB) and the Parkinson s Disease Phenotype Schedule of i.p. injeccons Week 1 Week 2 Week 3 Week 4 Week 5 Week 6 6 wk male C57Bl6 mice Dose Groups: Saline 10 mg/kg paraquat 30 mg/kg maneb 10 mg/kg paraquat + 30 mg/kg maneb

17 Combined PQ+MB SelecCvely Reduced Nigral TH Dopamine Neurons from control from MB alone STRIATUM N. ACCUMBENS CTRL PQ5 PQ10 MB15 MB30 PQ5+ MB15 PQ5+ MB30 PQ10+ MB15 PQ10+ MB30 Treatment Subsequently confirmed using unbiased stereology

18 OxidaCve Stress and the PQ+MB Induced Parkinson s Disease Phenotype [LOOH] nmol/ug protein Striatum Midbrain Frontal Cortex 0 Saline MB PQ PQ + MB 0 Saline MB PQ PQ + MB 0 Saline MB PQ PQ + MB Treatment Treatment Treatment Levels of lipid peroxidacon in various brain regions in the young adult model with exposures to saline, PQ alone, MB alone or combined PQ+MB

19 MB Increases PQ AccumulaCon and Delays its Clearance from Brain μgmol mg/wet weight Midbrain/PQ Midbrain/PQ+MB Cortex/PQ Cortex/PQ+MB Striatum/PQ Striatum/PQ+MB Cerebellum/PQ 0.3 Cerebellum/PQ+MB Time (Hour) Serum/PQ Serum/PQ+MB Lung/PQ Lung/PQ+MB) Kidney/PQ 20 Kidney/PQ+MB Heart/PQ 15 Heart/PQ+MB Time (Hour) What other food, chemical, drug etc might cause similar toxicokinecc interaccons?

20 Aging and PQ+MB: Enhanced DA Cell Loss and Permanent and Progressive Effects WK TH + Neurons ^ ~ 5 MOS + ^ ~ ~ + ~ ` SALINE PQ MB PQ + MB TREATMENT 18 MOS + ^ ~ + ^ ~ ` 2 WEEKS 3 MONTHS Measured Two Weeks and Three Months Post Dosing

21 Early Development and PD Risk? Developmental Insult Adult Insult Dopamine Function Symptomatic PD Years of Age The ecological risk factors for the disease may occur at a far different Cme than that at which the phenotype is expressed.

22 Experimental Design: Postnatal PesCcide Exposure Can developmental insults lead to a PD phenotype? Can they alter vulnerability to risk factors later in life? PN WEEKS 6 MONTHS 6 1/2 7 1/2 MONTHS 8 MONTHS PN 1 Exposure: Saline, 0.3 mg/kg PQ, 1 mg/kg MB, or PQ + MB Locomotor Ac;vity Locomotor Ac;vity Re Challenge: Saline, 10 mg/kg PQ. 30 mg/kg MB, or PQ + MB + Locomotor Ac;vity Locomotor Ac;vity 2 weeks aher last treatment; saline Neurochemical/ Stereological Determina;ons; Gene expression

23 CumulaCve and Silent Toxicity, Gender ProtecCon Development Only MALES Development +Adult Adult Only FEMALES TH+ NEURONS ^ ^ ~ ~ ~ ^ ~ ~ 0 SAL MB PQ PQ+MB SAL MB PQ PQ+MB SAL MB PQ PQ+MB Treatment Group Decreases in DA neurons following developmental exposures are significantly enhanced When followed by an adult exposure to pesccides as compared to adult only exposures. No changes in numbers of TH neurons

24 Progressive Decline in DA Cell Loss With Age in Males Following Postnatal Exposure MALES FEMALES SNpc TH+ NEURONS ~ ~ ~ ~ AGE (months) AGE (months) SAL MB PQ PQ+MB Stereological counts of substanca nigral DA neurons across the lifecme following PND 5 19 exposure to saline, PQ alone, MB alone or combined PQ+MB

25 SequenCal AdministraCon of Paraquat & Maneb Can sequencal environmental insults produce cumulacve neurotoxicity? GD PN 25 6 WEEKS 7 8 WEEKS 9 WEEKS GD1 Exposure: Saline OR 1 mg/kg MB i.p. PUPS WEANED LOCOMOTOR ACTIVITY Re Challenge: Saline, 5 mg/kg PQ, OR 30 mg/kg MB (every day for 8 days) Locomotor ac;vity 7 days post treatment Neurochemical and stereological analyses

26 SequenCal MB Followed by PQ Later in Life Kills DA Neurons 9 8 1'56 7!"$%&'(!"&)" &!!!! %!!! $!!!!!! "!!!,<0)+":($,<0)!"($3,<0)&"'"=0") +03%)"():"/:;'" /$0'1(/023$'! &'$(")"+,-!"$% &'$(")"!. &'$(")"+,- >$2"$% &'$(")"!. Barlow et al., 2004

27 Parkinson s Disease and ResidenCal Exposure to Paraquat and Maneb from Agricultural ApplicaCons in the Central Valley of California PD risk increased for DAT A clade diplotype OR=1.66) and for 3 VNTR (OR=1.8). High exposure to PQ + MB increased PD risk 3 fold in carriers of one suscepcbility allele (OR=2.99) High exposure to PQ+MB increased PD risk >4 fold in carriers of 2+ alleles (OR=4.53). Same effects were found for occupaconal pesccide exposures Ritz et al., Am. J. Epidemiology, vol. 169:

28 Phthalates are Anti-Androgenic Via their ability to decrease testosterone, some specific phthalates produce a host of adverse Effects on the male Reproductive system, including hypospadias, cryptorchodism, nipple retention, reduced anogenital distance and reductions in fertility But androgen insufficiency can be achieved by several mechanisms, and, regardless of how it occurs, leads to common adverse male reproductive system outcomes

29 Consequently, Other AnC Androgens Should be Included in CumulaCve Risk Assessments e.g., vinclozolin, procydimide, linuron, prochloraz, azole fungicides, polybrominated diphenyl ethers, dioxin, some PCBs

30 New Studies Support These Conclusions CumulaCve Effects of In Utero AdministraCon of Mixtures of AnCandrogens on Male Rat ReproducCve Development We also conducted a mixture study combining seven ancandrogens together. These chemicals elicit ancandrogenic effects at two different sites in the androgen signaling pathway (i.e., AR antagonist or inhibicon of androgen synthesis). In this study, the complex mixture behaved in a dose addicve manner. Our results indicate that compounds that act by disparate mechanisms of toxicity display cumulacve dose addicve effects when present in combinacon. Percent of Top Dose of the Mixture Rider et al., 2009

31 Mixture Effects are Not Considered in Risk Assessment PotenCated effects (no effect of treatment A or B alone, but significant effects when A and B are combined) will never be captured by current risk assessment methodology Do 10 fold safety factors adequately encompass the risks? CumulaCve and silent neurotoxicity are not captured in current neurotoxicology risk assessment How do we modify the risk paradigm to accommodate interaccons? How do we define which interaccons (mixtures) to assess?

32 With So Many Chemicals, Where Do We Begin? In the view of the NRC Commiyee report Phthalates and CumulaCve Risk; The Task Ahead, cumulacve risks strategies should not be specific to phthalates: it is plausible and warranted to extend cumulacve risk assessment to include chemicals associated with common adverse outcomes, as exemplified in this report by inclusion of other ancandrogenic chemicals with phthalates.

33 Moving Ahead with Mixtures Concurrent Risks Pb MeHg Mat Infec SGA PCBs FAS Impaired CogniCve Development To cite another example, EPA could evaluate combined exposures to lead, methylmercury and polychlorinated biphenyls because all contribute to the cumulacve risk of cognicve deficits associated with IQ reduccons in children, although the deficits are produced by different mechanisms of accon. Such a strategy defines the relevant mixtures that should be studied based upon a defined health endpoint, an approach consistent with public health protection and with the mission of the U.S. Environmental Protection Agency