Environmental exposure to metals and the development of tauopathies, synucleinopathies, and TDP-43 proteinopathies: A systematic evidence map protocol.

BACKGROUND: Neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis are incurable and expected to increase in prevalence in the upcoming decades. Environmental exposure to metals has been suggested as a contributing factor to the development of neurodegenerative disease. This systematic evidence map will identify and characterize the epidemiological and experimental data available on the intersection of eighteen metals of environmental concern (i.e., aluminum, antimony, arsenic, barium, beryllium, cadmium, chromium, cobalt, copper, lead, manganese, mercury, nickel, palladium, radium, silver, vanadium, and zinc) and three neurodegenerative disease clusters (i.e., tauopathies, synucleinopathies, and TDP-43 proteinopathies). We aim to describe the type and amount of evidence available (or lack thereof) for each metal and neurodegenerative disease combination and highlight important knowledge gaps and knowledge clusters for future research. METHODS: We will conduct a thorough search using two databases (MEDLINE and Web of Science Core Collection) and grey literature resources. Pre-defined criteria have been developed to identify studies which evaluate at least one of the selected metals and neurodegenerative disease-relevant outcomes (e.g., neuropathology, cognitive function, motor function, disease mortality). At each phase of review, studies will be evaluated by two reviewers. Studies determined to be relevant will be extracted for population, exposure, and outcome information. We will conduct a narrative review of the included studies, and the extracted data will be available in a database hosted on Tableau Public. CONCLUSION: This protocol documents the decisions made a priori to data collection regarding these objectives.

Genomic comparisons between hepatocarcinogenic and non-hepatocarcinogenic organophosphate insecticides in the mouse liver.

Short-term biomarkers of toxicity have an increasingly important role in the screening and prioritization of new chemicals. In this study, we examined early indicators of liver toxicity for three reference organophosphate (OP) chemicals, which are among the most widely used insecticides in the world. The OP methidathion was previously shown to increase the incidence of liver toxicity, including hepatocellular tumors, in male mice. To provide insights into the adverse outcome pathway (AOP) that underlies these tumors, effects of methidathion in the male mouse liver were examined after 7 and 28 day exposures and compared to those of two other OPs that either do not increase (fenthion) or possibly suppress liver cancer (parathion) in mice. None of the chemicals caused increases in liver weight/body weight or histopathological changes in the liver. Parathion decreased liver cell proliferation after 7 and 28 days while the other chemicals had no effects. There was no evidence for hepatotoxicity in any of the treatment groups. Full-genome microarray analysis of the livers from the 7 and 28 day treatments demonstrated that methidathion and fenthion regulated a large number of overlapping genes, while parathion regulated a unique set of genes. Examination of cytochrome P450 enzyme activities and use of predictive gene expression biomarkers found no consistent evidence for activation of AhR, CAR, PXR, or PPARα. Parathion suppressed the male-specific gene expression pattern through STAT5b, similar to genetic and dietary conditions that decrease liver tumor incidence in mice. Overall, these findings indicate that methidathion causes liver cancer by a mechanism that does not involve common mechanisms of liver cancer induction.

Predictive Analysis Using Chemical-Gene Interaction Networks Consistent with Observed Endocrine Activity and Mutagenicity of U.S. Streams.

In a recent U.S. Geological Survey/U.S. Environmental Protection Agency study assessing more than 700 organic compounds in 38 streams, in vitro assays indicated generally low estrogen, androgen, and glucocorticoid receptor activities, with 13 surface waters with 17ß-estradiol-equivalent (E2Eq) activities greater than a 1-ng/L estimated effects-based trigger value for estrogenic effects in male fish. Among the 36 samples assayed for mutagenicity in the Salmonella bioassay (reported here), 25% had low mutagenic activity and 75% were not mutagenic. Endocrine and mutagenic activities of the water samples were well correlated with each other and with the total number and cumulative concentrations of detected chemical contaminants. To test the predictive utility of knowledge-base-leveraging approaches, site-specific predicted chemical-gene (pCGA) and predicted analogous pathway-linked (pPLA) association networks identified in the Comparative Toxicogenomics Database were compared with observed endocrine/mutagenic bioactivities. We evaluated pCGA/pPLA patterns among sites by cluster analysis and principal component analysis and grouped the pPLA into broad mode-of-action classes. Measured E2eq and mutagenic activities correlated well with predicted pathways. The pPLA analysis also revealed correlations with signaling, metabolic, and regulatory groups, suggesting that other effects pathways may be associated with chemical contaminants in these waters and indicating the need for broader bioassay coverage to assess potential adverse impacts.

From the Cover: Genomic Effects of Androstenedione and Sex-Specific Liver Cancer Susceptibility in Mice.

Current strategies for predicting carcinogenic mode of action for nongenotoxic chemicals are based on identification of early key events in toxicity pathways. The goal of this study was to evaluate short-term key event indicators resulting from exposure to androstenedione (A4), an androgen receptor agonist and known liver carcinogen in mice. Liver cancer is more prevalent in men compared with women, but androgen-related pathways underlying this sex difference have not been clearly identified. Short-term hepatic effects of A4 were compared with reference agonists of the estrogen receptor (ethinyl estradiol, EE) and glucocorticoid receptor (prednisone, PRED). Male B6C3F1 mice were exposed for 7 or 28 days to A4, EE, or PRED. EE increased and PRED suppressed hepatocyte proliferation, while A4 had no detectable effects. In a microarray analysis, EE and PRED altered >3000 and >670 genes, respectively, in a dose-dependent manner, whereas A4 did not significantly alter any genes. Gene expression was subsequently examined in archival liver samples from male and female B6C3F1 mice exposed to A4 for 90 days. A4 altered more genes in females than males and did not alter expression of genes linked to activation of the mitogenic xenobiotic receptors AhR, CAR, and PPARα in either sex. A gene expression biomarker was used to show that in female mice, the high dose of A4 activated the growth hormone-regulated transcription factor STAT5b, which controls sexually dimorphic gene expression in the liver. These findings suggest that A4 induces subtle age-related effects on STAT5b signaling that may contribute to the higher risk of liver cancer in males compared with females.

Dose and Effect Thresholds for Early Key Events in a PPARα-Mediated Mode of Action.

Current strategies for predicting adverse health outcomes of environmental chemicals are centered on early key events in toxicity pathways. However, quantitative relationships between early molecular changes in a given pathway and later health effects are often poorly defined. The goal of this study was to evaluate short-term key event indicators using qualitative and quantitative methods in an established pathway of mouse liver tumorigenesis mediated by peroxisome proliferator-activated receptor alpha (PPARα). Male B6C3F1 mice were exposed for 7 days to di (2-ethylhexyl) phthalate (DEHP), di-n-octyl phthalate (DNOP), and n-butyl benzyl phthalate (BBP), which vary in PPARα activity and liver tumorigenicity. Each phthalate increased expression of select PPARα target genes at 7 days, while only DEHP significantly increased liver cell proliferation labeling index (LI). Transcriptional benchmark dose (BMDT) estimates for dose-related genomic markers stratified phthalates according to hypothetical tumorigenic potencies, unlike BMDs for non-genomic endpoints (relative liver weights or proliferation). The 7-day BMDT values for Acot1 as a surrogate measure for PPARα activation were 29, 370, and 676 mg/kg/day for DEHP, DNOP, and BBP, respectively, distinguishing DEHP (liver tumor BMD of 35 mg/kg/day) from non-tumorigenic DNOP and BBP. Effect thresholds were generated using linear regression of DEHP effects at 7 days and 2-year tumor incidence values to anchor early response molecular indicators and a later phenotypic outcome. Thresholds varied widely by marker, from 2-fold (Pdk4 and proliferation LI) to 30-fold (Acot1) induction to reach hypothetical tumorigenic expression levels. These findings highlight key issues in defining thresholds for biological adversity based on molecular changes.

Identification of developmentally toxic drinking water disinfection byproducts and evaluation of data relevant to mode of action.

Reactions between chemicals used to disinfect drinking water and compounds present in source waters produce chemical mixtures containing hundreds of disinfection byproducts (DBPs). Although the results have been somewhat inconsistent, some epidemiological studies suggest associations may exist between DBP exposures and adverse developmental outcomes. The potencies of individual DBPs in rodent and rabbit developmental bioassays suggest that no individual DBP can account for the relative risk estimates reported in the positive epidemiologic studies, leading to the hypothesis that these outcomes could result from the toxicity of DBP mixtures. As a first step in a mixtures risk assessment for DBP developmental effects, this paper identifies developmentally toxic DBPs and examines data relevant to the mode of action (MOA) for DBP developmental toxicity. We identified 24 developmentally toxic DBPs and four adverse developmental outcomes associated with human DBP exposures: spontaneous abortion, cardiovascular defects, neural tube defects, and low birth weight infancy. A plausible MOA, involving hormonal disruption of pregnancy, is delineated for spontaneous abortion, which some epidemiologic studies associate with total trihalomethane and bromodichloromethane exposures. The DBP data for the other three outcomes were inadequate to define key MOA steps.

Development of an inhalation physiologically based pharmacokinetic (PBPK) model for 2,2, 4-trimethylpentane (TMP) in male Long-Evans rats using gas uptake experiments.

2,2,4-Trimethylpentane (TMP) is a volatile colorless liquid used primarily to increase the octane rating of combustible fuels. TMP is released in the environment through the manufacture, use, and disposal of products associated with the gasoline and petroleum industry. Short-term inhalation exposure to TMP (< 4 h; > 1000 ppm) caused sensory and motor irritations in rats and mice. Like many volatile hydrocarbons, acute exposure to TMP may also be expected to alter neurological functions. To estimate in vivo metabolic kinetics of TMP and to predict its target tissue dosimetry during inhalation exposures, a physiologically based pharmacokinetic (PBPK) model was developed for the chemical in Long-Evans male rats using closed-chamber gas-uptake experiments. Gas-uptake experiments were conducted in which rats (80-90 days old) were exposed to targeted initial TMP concentrations of 50, 100, 500, and 1000 ppm. The model consisted of compartments for the closed uptake chamber, lung, fat, kidney, liver, brain, and rapidly and slowly perfused tissues. Physiological parameters were obtained from literature. Partition coefficients for the model were experimentally determined for air/blood, fat, liver, kidney, muscle, and brain using vial equilibration methods. Common to other hydrocarbons, metabolism of TMP via oxidative reactions is assumed to mainly occur in the liver. The PBPK model simulations of the closed chamber data were used to estimate in vivo metabolic parameters for TMP in male Long-Evans rats.

Research issues underlying the four-lab study: integrated disinfection by-products mixtures research.

Chemical disinfection of drinking water is a major public health triumph of the 20th century, resulting in significant decreases in morbidity and mortality from waterborne diseases. Disinfection by-products (DBP) are chemicals formed by the reaction of oxidizing disinfectants with inorganic and organic materials in the source water. To address potential health concerns that cannot be answered directly by toxicological research on individual DBPs or defined DBP mixtures, scientists residing within the various organizations of the U.S. Environmental Protection Agency's Office of Research and Development (the National Health and Environmental Effects Research Laboratory, the National Risk Management Research Laboratory, the National Exposure Research Laboratory, and the National Center for Environmental Assessment) engaged in joint investigation of environmentally realistic complex mixtures of DBP. Research on complex mixtures of DBP is motivated by three factors: (a) DBP exposure is ubiquitous to all segments of the population; (b) some positive epidemiologic studies are suggestive of potential developmental, reproductive, or carcinogenic health effects in humans exposed to DBP; and (c) significant amounts of the material that makes up the total organic halide portion of the DBP have not been identified. The goal of the Integrated Disinfection Byproducts Mixtures Research Project (the 4Lab Study) is provision of sound, defensible, experimental data on environmentally relevant mixtures of DBP and an improved estimation of the potential health risks associated with exposure to the mixtures of DBP formed during disinfection of drinking water. A phased research plan was developed and implemented. The present series of articles provides the results from the first series of experiments.

Integrated disinfection by-products research: salmonella mutagenicity of water concentrates disinfected by chlorination and ozonation/postchlorination.

Although chemical disinfection of drinking water is a highly protective public health practice, the disinfection process is known to produce toxic contaminants. Epidemiological studies associate chlorinated drinking water with quantitatively increased risks of rectal, kidney, and bladder cancer. One study found a significant exposure-response association between water mutagenicity and relative risk for bladder and kidney cancer. A number of studies found that several types of disinfection processes increase the level of mutagens detected by the Salmonella assay. As part of a comprehensive study to examine chlorinated and ozonated/postchlorinated drinking water for toxicological contaminants, the Salmonella mutagenicity assay was used to screen both volatile and nonvolatile organic components. The assay also compared the use of reverse osmosis and XAD resin procedures for concentrating the nonvolatile components. Companion papers provide the results from other toxicological assays and chemical analysis of the drinking water samples. The volatile components of the ozonated/postchlorinated and chlorinated water samples and a trihalomethane mixture were mutagenic to a Salmonella tester strain transfected with a rat theta-class glutathione S-transferase and predominantly nonmutagenic in the control strain. In this study, the nonvolatile XAD concentrate of the untreated water possessed a low level of mutagenic activity. However, compared to the levels of mutagenicity in the finished water XAD concentrates, the contribution from the settled source water was minimal. The mutagenicity seen in the reverse osmosis concentrates was < 50% of that seen in the XAD concentrates. Overall, mutagenic responses were similar to those observed in other North American studies and provide evidence that the pilot plant produced disinfection by-products similar to that seen in other studies.

Developing an exposure-dose-response model for the acute neurotoxicity of organic solvents: overview and progress on in vitro models and dosimetry.

We are developing an exposure-dose-response (EDR) model for volatile organic compounds (VOCs) to predict acute effects of VOCs on nervous system function from exposure data (concentration and duration of inhalation). This model contains both toxicokinetic and toxicodynamic components. One advantage of the EDR model will be its ability to relate in vitro effects of solvents on cellular ion channels (putative targets) to in vivo effects, using a combination of physiologically-based toxicokinetic (PBTK) modeling (to estimate VOC concentrations in the blood and brain) and in vitro studies to clarify the mode of action of the VOCs. Recent work in vitro has focused on quantifying the inhibitory effects of toluene, trichloroethylene (TCE) and perchloroethylene (PERC) on ion channel currents. All three VOCs inhibit current through voltage-sensitive calcium channels (VSCCs) in pheochromocytoma cells; PERC blocked calcium currents and altered the current-voltage relationship at lower concentrations than did toluene or TCE. Recombinant nicotinic acetylcholine receptors (nAChRs), expressed in Xenopus oocytes, were also inhibited by PERC and toluene in a concentration-dependent manner. PERC inhibited α7 receptors more than α4ß2 receptors in recombinant human and rat nAChRs. However, human and rat α7 receptors were equally sensitive to PERC and TOL. These in vitro studies will be used to identify an appropriate neuronal receptor system to serve as an index of acute effects of VOCs in vivo. The PBTK model incorporates physiological input parameters derived from radiotelemetered heart rate data from rats performing operant tests of cognitive and motor functions. These studies should improve predictions of target organ concentrations of inhaled VOCs in subjects actively performing behavioral tests over a range of physical activity levels.