Douglas C. Wolf

Mode of action and human relevance analysis for nuclear receptor-mediated liver toxicity: A case study with phenobarbital as a model constitutive androstane receptor (CAR) activator

Abstract The constitutive androstane receptor (CAR) and pregnane X receptor (PXR) are important nuclear receptors involved in the regulation of cellular responses from exposure to many xenobiotics and various physiological processes. Phenobarbital (PB) is a non-genotoxic indirect CAR activator, which induces cytochrome P450 (CYP) and other xenobiotic metabolizing enzymes and is known to produce liver foci/tumors in mice and rats. From literature data, a mode of action (MOA) for PB-induced rodent liver tumor formation was developed. A MOA for PXR activators was not established owing to a lack of suitable data. The key events in the PB-induced liver tumor MOA comprise activation of CAR followed by altered gene expression specific to CAR activation, increased cell proliferation, formation of altered hepatic foci and ultimately the development of liver tumors. Associative events in the MOA include altered epigenetic changes, induction of hepatic CYP2B enzymes, liver hypertrophy and decreased apoptosis; with inhibition of gap junctional intercellular communication being an associative event or modulating factor. The MOA was evaluated using the modified Bradford Hill criteria for causality and other possible MOAs were excluded. While PB produces liver tumors in rodents, important species differences were identified including a lack of cell proliferation in cultured human hepatocytes. The MOA for PB-induced rodent liver tumor formation was considered to be qualitatively not plausible for humans. This conclusion is supported by data from a number of epidemiological studies conducted in human populations chronically exposed to PB in which there is no clear evidence for increased liver tumor risk.

A 21st century roadmap for human health risk assessment

Abstract The Health and Environmental Sciences Institute (HESI)-coordinated Risk Assessment in the 21st Century (RISK21) project was initiated to develop a scientific, transparent, and efficient approach to the evolving world of human health risk assessment, and involved over 120 participants from 12 countries, 15 government institutions, 20 universities, 2 non-governmental organizations, and 12 corporations. This paper provides a brief overview of the tiered RISK21 framework called the roadmap and risk visualization matrix, and articulates the core principles derived by RISK21 participants that guided its development. Subsequent papers describe the roadmap and matrix in greater detail. RISK21 principles include focusing on problem formulation, utilizing existing information, starting with exposure assessment (rather than toxicity), and using a tiered process for data development. Bringing estimates of exposure and toxicity together on a two-dimensional matrix provides a clear rendition of human safety and risk. The value of the roadmap is its capacity to chronicle the stepwise acquisition of scientific information and display it in a clear and concise fashion. Furthermore, the tiered approach and transparent display of information will contribute to greater efficiencies by calling for data only as needed (enough precision to make a decision), thus conserving animals and other resources.

Formaldehyde-induced gene expression in F344 rat nasal respiratory epithelium

Formaldehyde (FA), an occupational and environmental toxicant used extensively in the manufacturing of many household and personal use products, is known to induce squamous cell carcinomas in the nasal turbinates of rats and mice and squamous metaplasia in monkey noses. Tissue responses to FA include a dose dependent epithelial degeneration, respiratory cell hypertrophy, and squamous metaplasia. The primary target for FA-induced toxicity in both rodents and monkeys is the respiratory nasal epithelium. FA increases nasal epithelial cell proliferation and DNA-protein crosslinks (DPX) that are associated with subsequent nasal cancer development. To address the acute effects of FA exposure that might contribute to known pathological changes, cDNA gene expression analysis was used. Two groups of male F344 rats received either 40 ul of distilled water or FA (400 mM) instilled into each nostril. Twenty-four hours following treatment, nasal epithelium was recovered from which total RNA was used to generate cDNA probes. Significance analysis of microarrays (SAM) hybridization data using Clontech Rat Atlas 1.2 arrays revealed that 24 of the 1185 genes queried were significantly up-regulated and 22 genes were significantly downregulated. Results for ten of the differentially expressed genes were confirmed by quantitative real time RT PCR. The identified genes with FA-induced change in expression belong to the functional gene categories xenobiotic metabolism, cell cycle, apoptosis, and DNA repair. These data suggest that multiple pathways are dysregulated by FA exposure, including those involved in DNA synthesis/repair and regulation of cell proliferation. Differential gene expression profiles may provide clues that could be used to define mechanisms involved in FA-induced nasal cancer.

A mixture of ammonium perchlorate and sodium chlorate enhances alterations of the pitutary-thyroid axis caused by the individual chemicals in adult male F344 rats

Ammonium perchlorate (AP) and sodium chlorate (SC) have been detected in public drinking water supplies in many parts of the United States. These chemicals cause perturbations in pituitary-thyroid homeostasis in animals by competitively inhibiting iodide uptake, thus hindering the synthesis of thyroglobulin and reducing circulating T4 (thyroxine). Little is known about the short-term exposure effects of mixtures of perchlorate and chlorate. The present study investigated the potential for the response to a mixture of these chemicals on the pituitary-thyroid axis in rats to be greater than that induced by the individual chemicals. Adult male F-344 rats were exposed, via their drinking water, to the nominal concentrations of 0.1, 1.0, 10 mg/L AP or 10, 100, 1000 mg/L SC and their mixtures for 7 days. Serum T4 levels were significantly (p < 0.05) reduced in rats following exposure to the mixtures, but not after exposure to the individual chemicals. Serum T3 (triiodothyronine) was not altered by treatment and TSH (thyroid stimulating hormone) was only increased after the high-dose chlorate treatment. Histological examination of the thyroid gland showed colloid depletion and hypertrophy of follicular epithelial cells in high-dose single chemical and all mixture-treated rats, while hyperplasia was observed only in some of the rats treated with mixtures (AP 10 + SC 100, AP 0.1 + SC 1000, and AP 10 + SC 1000 mg/L). These data suggest that short-term exposure to the mixture of AP and SC enhances the effect of either chemical alone on the pituitary-thyroid axis in rats.

Transcriptomic analysis of F344 rat nasal epithelium suggests that the lack of carcinogenic response to glutaraldehyde is due to its greater toxicity compared to formaldehyde.

Formaldehyde is cytotoxic and carcinogenic to the rat nasal respiratory epithelium inducing tumors after 12 months. Glutaraldehyde is also cytotoxic but is not carcinogenic to nasal epithelium even after 24 months. Both aldehydes induce similar acute and subchronic histopathology that is characterized by inflammation, hyperplasia, and squamous metaplasia. Because early aldehyde-induced lesions are microscopically similar, we investigated whether transcriptional patterns using cDNA technology could explain the different cancer outcomes. Treatments included 1-, 5-, or 28-day exposure by nasal instillation of formaldehyde solution (400 mM) or glutaraldehyde solution (20 mM). Animals were euthanized and the nasal respiratory epithelium removed for gene expression analysis and a subset of rats treated for 28 days was processed for microscopic examination. RNA was isolated and processed for expression assessment using Clontech ® Atlas Toxicology II Arrays. Both aldehydes induced hyperplasia, squamous metaplasia, and inflammatory infiltrates with scattered apoptotic bodies in the epithelium covering luminal surfaces of the nasoturbinate, maxilloturbinate, and nasal septum. A subset of 80 genes that were the most variant between the treated and control included the functional categories of DNA repair and apoptosis. Hierarchical clustering discriminated chemical treatment effects after 5 days of exposure, with 6 clusters of genes distinguishing formaldehyde from glutaraldehyde. These data suggest that although both aldehydes induced similar short-term cellular phenotypes, gene expression could distinguish glutaraldehyde from formaldehyde. The gene expression patterns suggest that glutaraldehyde’s lack of carcinogenicity may be due to its greater toxicity from lack of DNA-repair, greater mitochondrial damage, and increased apoptosis.

Classification schemes for carcinogenicity based on hazard-identification have become outmoded and serve neither science nor society

Classification schemes for carcinogenicity based solely on hazard-identification such as the IARC monograph process and the UN system adopted in the EU have become outmoded. They are based on a concept developed in the 1970s that chemicals could be divided into two classes: carcinogens and non-carcinogens. Categorization in this way places into the same category chemicals and agents with widely differing potencies and modes of action. This is how eating processed meat can fall into the same category as sulfur mustard gas. Approaches based on hazard and risk characterization present an integrated and balanced picture of hazard, dose response and exposure and allow informed risk management decisions to be taken. Because a risk-based decision framework fully considers hazard in the context of dose, potency, and exposure the unintended downsides of a hazard only approach are avoided, e.g., health scares, unnecessary economic costs, loss of beneficial products, adoption of strategies with greater health costs, and the diversion of public funds into unnecessary research. An initiative to agree upon a standardized, internationally acceptable methodology for carcinogen assessment is needed now. The approach should incorporate principles and concepts of existing international consensus-based frameworks including the WHO IPCS mode of action framework.

Analysis of preneoplastic and neoplastic renal lesions in Tsc2 mutant Long-Evans (Eker) rats following exposure to a mixture of drinking water disinfection by-products.

Disinfection of surface water for human consumption results in the generation of a complex mixture of chemicals in potable water. Cancer risk assessment methodology assumes additivity of carcinogenic effects in the regulation of mixtures. A rodent model of hereditary renal cancer was used to investigate the carcinogenic response to a mixture of drinking water disinfection by-products (DBPs). Rats carrying a mutation in the Tsc2 tumor suppressor gene (Eker rats) readily develop renal preneoplastic and neoplastic lesions, and are highly susceptible to the effects of renal carcinogens. Male and female Eker rats were exposed via drinking water to individual or a mixture of DBPs for 4 or 10 months. Potassium bromate, 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX), chloroform, and bromodichloromethane were administered at low concentrations of 0.02, 0.005, 0.4 and 0.07 g/l, respectively, and high concentrations of 0.4, 0.07, 1.8 and 0.7 g/l, respectively. Low and high dose mixture solutions were comprised of all four chemicals at either low concentrations or high concentrations, respectively, Following necropsy, each kidney was examined microscopically for preneoplastic lesions (atypical tubules and hyperplasias) and tumors. While some of the mixture responses observed in male rats did fall within the range expected for an additive response, especially at the high dose, predominantly antagonistic effects on renal lesions were observed in response to the low dose mixture in male rats and the high dose mixture in female rats. These data suggest that current default risk assessments assuming additivity may overstate the cancer risk associated with exposure to mixtures of DBPs at low concentrations.

Transcriptional Modulation of the ERK1/2 MAPK and NF-κB Pathways in Human Urothelial Cells After Trivalent Arsenical Exposure: Implications for Urinary Bladder Cancer.

Chronic exposure to drinking water contaminated with inorganic arsenic (iAs) is associated with an increased risk of urinary bladder (UB) cancers in humans. The exact role of specific iAs metabolite(s) in As-mediated carcinogenesis remains largely unknown. Experimental evidence suggests that trivalent arsenicals, namely arsenite (iAsIII) and two of its metabolites, monomethylarsonous acid (MMAIII) and dimethylarsinous acid (DMAIII), are possible proximate UB carcinogens. Here, we used a transcriptomics approach to examine perturbed molecular pathways in a human urothelial cell line (UROtsa) after short-term exposure to iAsIII, MMAIII and DMAIII. Molecular pathways containing genes that encode proteins implicated in UB cancer development were perturbed by both MMAIII and DMAIII. These pathways included those of the extracellular signal-regulated kinase 1/2 mitogen-activated protein kinase (ERK 1/2 MAPK) and nuclear factor kappa beta (NF-κB). Together, these results may inform the current understanding of effects in the UB induced by acute As exposure and the relationship of these effects with As-mediated carcinogenesis.

Finfish and aquatic invertebrate pathology resources for now and the future.

Utilization of finfish and aquatic invertebrates in biomedical research and as environmental sentinels has grown dramatically in recent decades. Likewise the aquaculture of finfish and invertebrates has expanded rapidly worldwide as populations of some aquatic food species and threatened or endangered aquatic species have plummeted due to overharvesting or habitat degradation. This increasing intensive culture and use of aquatic species has heightened the importance of maintaining a sophisticated understanding of pathology of various organ systems of these diverse species. Yet, except for selected species long cultivated in aquaculture, pathology databases and the workforce of highly trained pathologists lag behind those available for most laboratory animals and domestic mammalian and avian species. Several factors must change to maximize the use, understanding, and protection of important aquatic species: 1) improvements in databases of abnormalities across species; 2) standardization of diagnostic criteria for proliferative and nonproliferative lesions; and 3) more uniform and rigorous training in aquatic morphologic pathology.

A predictive data-driven framework for endocrine prioritization: a triazole fungicide case study

Abstract The US Environmental Protection Agency Endocrine Disruptor Screening Program (EDSP) is a tiered screening approach to determine the potential for a chemical to interact with estrogen, androgen, or thyroid hormone systems and/or perturb steroidogenesis. Use of high-throughput screening (HTS) to predict hazard and exposure is shifting the EDSP approach to (1) prioritization of chemicals for further screening; and (2) targeted use of EDSP Tier 1 assays to inform specific data needs. In this work, toxicology data for three triazole fungicides (triadimefon, propiconazole, and myclobutanil) were evaluated, including HTS results, EDSP Tier 1 screening (and other scientifically relevant information), and EPA guideline mammalian toxicology study data. The endocrine-related bioactivity predictions from HTS and information that satisfied the EDSP Tier 1 requirements were qualitatively concordant. Current limitations in the available HTS battery for thyroid and steroidogenesis pathways were mitigated by inclusion of guideline toxicology studies in this analysis. Similar margins (3–5 orders of magnitude) were observed between HTS-predicted human bioactivity and exposure values and between in vivo mammalian bioactivity and EPA chronic human exposure estimates for these products’ registered uses. Combined HTS hazard and human exposure predictions suggest low priority for higher-tiered endocrine testing of these triazoles. Comparison with the mammalian toxicology database indicated that this HTS-based prioritization would have been protective for any potential in vivo effects that form the basis of current risk assessment for these chemicals. This example demonstrates an effective, human health protective roadmap for EDSP evaluation of pesticide active ingredients via prioritization using HTS and guideline toxicology information.