Application of the adverse outcome pathway (AOP) approach to inform mode of action (MOA): A case study with inorganic arsenic.

The aim of this study was to establish a process for deriving a chemical-specific mode of action (MOA) from chemical-agnostic adverse outcome pathway (AOPs), using inorganic arsenic (iAs) as a case study. The AOP developed for this case study are related to disruption of cellular signaling by chemicals that strongly bind to vicinal dithiols in cellular proteins, leading to disruption of inflammatory and oxidative stress signaling along with inhibition of the DNA damage responses. The proposed MOA for iAs incorporates this AOP, overlaid on a background of increasing oxidative stress and/or co-exposure to mutagenic chemicals or radiation. The most challenging aspect of developing a MOA from AOP is the incorporation of metabolism and dose-response, neither of which may be considered in the development of an AOP. The cellular responses to relatively low concentrations (below 100 parts per billion) of iAs in drinking water appear to be secondary to binding of trivalent arsenite and its trivalent metabolite, monomethyl arsenous acid to key cellular vicinal dithiols in target tissues, resulting in a co-carcinogenic MOA. The proposed AOP may also be applied to non-cancer endpoints, enabling an integrated approach to conducting a risk assessment for iAs.

Arsenic and ultraviolet radiation exposure: melanoma in a New Mexico non-Hispanic white population.

Cases of cutaneous melanoma and controls were enrolled in a New Mexico population-based study; subjects were administered questionnaires concerning ultraviolet (UV) and inorganic arsenic (iAs) exposure. Historical iAs exposure was estimated. UV exposure estimates were also derived using geospatial methods. Drinking water samples were collected for iAs analysis. Blood samples were collected for DNA repair (Comet) and DNA repair gene polymorphism assays. Arsenic concentrations were determined in urine and toenail samples. UV exposures during the previous 90 days did not vary significantly between cases and controls. Mean (±SD) current home iAs drinking water was not significantly different for cases and controls [3.98 µg/L (±3.67) vs. 3.47 µg/L (±2.40)]. iAs exposure showed no effect on DNA repair or association with melanoma. Results did not corroborate a previously reported association between toenail As and melanoma risk. Arsenic biomarkers in urine and toenail were highly significantly correlated with iAs in drinking water. A UV-DNA repair interaction for UV exposure over the previous 7-90 days was shown; cases had higher DNA damage than controls at low UV values. This novel finding suggests that melanoma cases may be more sensitive to low-level UV exposure than are controls. A UV-APEX1 interaction was shown. Subjects with the homozygous rare APEX1 DNA repair gene allele had a higher risk of early melanoma diagnosis at low UV exposure compared with those with the homozygous wild type or the heterozygote. Notably, a UV-arsenic interaction on inhibition of DNA repair was not observed at iAs drinking water concentrations below 10 ppb (µg/L).

The impact of recent advances in research on arsenic cancer risk assessment.

Scientific debate surrounds the regulatory approach for evaluating carcinogenic risk of arsenic compounds. The arsenic ambient water quality criteria (AWQC), based on the assumption of a linear mode of action for skin cancer risk, results in an allowable limit of 0.018ppb in ambient waters; the drinking water Maximum Contaminant Level (MCL) was determined using a similar linear approach. Integration of results from recent studies investigating arsenic's mode of action provide the basis for a change in the approach for conducting an arsenic cancer risk assessment. Results provide support for a concentration demonstrating a dose-dependent transition in response from those representing adaptive changes to those that may be key events in the development of cancer endpoints. While additional information is needed, integration of current research results provides insight for a new quantitative cancer risk assessment methodology as an alternative toxicologically-based dose response (BBDR) cancer modeling. Integration of the new experimental results, combined with epidemiological evidence, support a dose-dependent transition concentration of approximately 0.1µM arsenic. Some uncertainties remain; additional information from chronic in vitro studies underway is needed. Results to date also provide initial insight into variability in population response at low arsenic exposures.

Time-dependent genomic response in primary human uroepithelial cells exposed to arsenite for up to 60 days.

Evidence from both in vivo and in vitro studies suggests that gene expression changes from long-term exposure to arsenite evolve markedly over time, including reversals in the direction of expression change in key regulatory genes. In this study, human uroepithelial cells from the ureter segments of 4 kidney-donors were continuously treated in culture with arsenite at concentrations of 0.1 or 1 µM for 60 days. Gene expression at 10, 20, 30, 40, and 60 days was determined using Affymetrix human genome microarrays and signal pathway analysis was performed using GeneGo Metacore. Arsenic treated cells continued to proliferate for the full 60-day period, whereas untreated cells ceased proliferating after approximately 30 days. A peak in the number of gene changes in the treated cells compared to untreated controls was observed between 30 and 40 days of exposure, with substantially fewer changes at 10 and 60 days, suggesting remodeling of the cells over time. Consistent with this possibility, the direction of expression change for a number of key genes was reversed between 20 and 30 days, including CFOS and MDM2. While the progression of gene changes was different for each subject, a common pattern was observed in arsenic treated cells over time, with early upregulation of oxidative stress responses (HMOX1, NQ01, TXN, TXNRD1) and down-regulation of immune/inflammatory responses (IKKα). At around 30 days, there was a transition to increased inflammatory and proliferative signaling (AKT, CFOS), evidence of epithelial-to-mesenchymal transition (EMT), and alterations in DNA damage responses (MDM2, ATM). A common element in the changing response of cells to arsenite over time appears to involve up-regulation of MDM2 by inflammatory signaling (through AP-1 and NF-κB), leading to inhibition of P53 function.

Factors that distinguish serious versus less severe strain and sprain injuries: an analysis of electric utility workers.

BACKGROUND: Occupational sprain and strain injuries are one of the most common types of nonfatal occupational injuries and a significant source of lost workdays. This study examines factors associated with severe work-related sprain/strain injuries to the back, shoulder, and knees. METHODS: A synthetic case-control study was performed (controls were selected from the same pool of utility workers as cases). Cases included all electric utility workers who had experienced a severe work-related sprain/strain injury to the back, knee, or shoulder. Primary controls were selected from all workers who had sustained a minor injury. Secondary controls were selected from employees with a minor sprain/strain injury to the back, knee, or shoulder. Multivariate logistic regression models were used to estimate odds ratios and 95% confidence intervals. RESULTS: Workers 41 years and older were more likely to have experienced severe shoulder sprain/strain injuries [Age 41-50: OR = 3.62, 95% CI: 1.71-7.65; age 51 and older: OR = 4.49, 95% CI: 1.89-10.67] and severe back sprain/strain injuries [Age 41-50: OR = 1.70, 95% CI: 1.06-2.33; age 51 and older: OR = 1.5, 95% CI: 0.90-2.52]. Line workers and maintenance workers had an increased risk of serious sprain/strain injuries. Gender and day of week were not significantly associated with sprain/strain injuries. DISCUSSION: Though this study is limited by available data, future studies may benefit from this preliminary examination of occupational and demographic characteristics associated with serious sprain/strain injuries among electric utility workers.

Biomonitoring Equivalents (BE) dossier for cadmium (Cd) (CAS No. 7440-43-9).

Recent efforts by the US Centers for Disease Control and Prevention and other researchers have resulted in a growing database of measured concentrations of chemical substances in blood or urine samples taken from the general population. However, few tools exist to assist in the interpretation of the measured values in a health risk context. Biomonitoring Equivalents (BEs) are defined as the concentration or range of concentrations of a chemical or its metabolite in a biological medium (blood, urine, or other medium) that is consistent with an existing health-based exposure guideline. This document reviews available pharmacokinetic data and models for cadmium and applies these data and models to existing health-based exposure guidance values from the US Environmental Protection Agency, the Agency for Toxic Substances and Disease Registry, Health Canada, and the World Health Organization, to estimate corresponding BE values for cadmium in blood and urine. These values can be used as screening tools for evaluation of biomonitoring data for cadmium in the context of existing risk assessments for cadmium and for prioritization of the potential need for additional risk assessment and risk management efforts for cadmium.

Guidelines for the derivation of Biomonitoring Equivalents: report from the Biomonitoring Equivalents Expert Workshop.

Biomonitoring Equivalents (BEs) are defined as the concentration of a chemical (or metabolite) in a biological medium (blood, urine, human milk, etc.) consistent with defined exposure guidance values or toxicity criteria including reference doses and reference concentrations (RfD and RfCs), minimal risk levels (MRLs), or tolerable daily intakes (TDIs) [Hays, S.M., Becker, R.A., Leung, H.W., Aylward, L.L., Pyatt, D.W., 2007. Biomonitoring equivalents: a screening approach for interpreting biomonitoring results from a public health risk perspective. Regul. Toxicol. Pharmacol. 47(1), 96-109]. The utility of the BE is to provide a screening tool for placing biomonitoring data into a health risk context. A Panel of experts took part in the Biomonitoring Equivalents Expert Workshop to discuss the various technical issues associated with calculating BEs and developed a set of guidelines for use in the derivation of BEs. Issues addressed included the role of the point of departure (POD) in BE derivation, the appropriate application of human and animal kinetic data and models, consideration of default uncertainty factor components in the context of internal dose-based extrapolations, and relevance of mode of action to technical choices in kinetic modeling and identification of screening values. The findings from this Expert Panel Workshop on BE derivation are presented and provide a set of guidelines and considerations for use in BE derivation.

Guidelines for the communication of Biomonitoring Equivalents: report from the Biomonitoring Equivalents Expert Workshop.

Biomonitoring Equivalents (BEs) are screening tools for interpreting biomonitoring data. However, the development of BEs brings to the public a relatively novel concept in the field of health risk assessment and presents new challenges for environmental risk communication. This paper provides guidance on methods for conveying information to the general public, the health care community, regulators and other interested parties regarding how chemical-specific BEs are derived, what they mean in terms of health, and the challenges and questions related to interpretation and communication of biomonitoring data. Key communication issues include: (i) developing a definition of the BE that accurately captures the BE concept in lay terms, (ii) how to compare population biomonitoring data to BEs, (iii) interpreting biomonitoring data that exceed BEs for a specific chemical, (iv) how to best describe the confidence in chemical-specific BEs, and (v) key requirements for effective communication with health care professionals. While the risk communication literature specific to biomonitoring is sparse, many of the concepts developed for traditional risk assessments apply, including transparency and discussions of confidence and uncertainty. Communication of BEs will require outreach, education, and development of communication materials specific to several audiences including the lay public and health care providers.

Estimating uncertainty in observational studies of associations between continuous variables: example of methylmercury and neuropsychological testing in children.

BACKGROUND: We suggest that the need to account for systematic error may explain the apparent lack of agreement among studies of maternal dietary methylmercury exposure and neuropsychological testing outcomes in children, a topic of ongoing debate. METHODS: These sensitivity analyses address the possible role of systematic error on reported associations between low-level prenatal exposure to methylmercury and neuropsychological test results in two well known, but apparently conflicting cohort studies: the Faroe Islands Study (FIS) and the Seychelles Child Development Study (SCDS). We estimated the potential impact of confounding, selection bias, and information bias on reported results in these studies using the Boston Naming Test (BNT) score as the outcome variable. RESULTS: Our findings indicate that, assuming various degrees of bias (in either direction) the corrected regression coefficients largely overlap. Thus, the reported effects in the two studies are not necessarily different from each other. CONCLUSION: Based on our sensitivity analysis results, it is not possible to draw definitive conclusions about the presence or absence of neurodevelopmental effects due to in utero methylmercury exposure at levels reported in the FIS and SCDS.

Thermal burn and electrical injuries among electric utility workers, 1995-2004.

This study describes the occurrence of work-related injuries from thermal-, electrical- and chemical-burns among electric utility workers. We describe injury trends by occupation, body part injured, age, sex, and circumstances surrounding the injury. This analysis includes all thermal, electric, and chemical injuries included in the Electric Power Research Institute (EPRI) Occupational Health and Safety Database (OHSD). There were a total of 872 thermal burn and electric shock injuries representing 3.7% of all injuries, but accounting for nearly 13% of all medical claim costs, second only to the medical costs associated with sprain- and strain-related injuries (38% of all injuries). The majority of burns involved less than 1 day off of work. The head, hands, and other upper extremities were the body parts most frequently injured by burns or electric shocks. For this industry, electric-related burns accounted for the largest percentage of burn injuries, 399 injuries (45.8%), followed by thermal/heat burns, 345 injuries (39.6%), and chemical burns, 51 injuries (5.8%). These injuries also represented a disproportionate number of fatalities; of the 24 deaths recorded in the database, contact with electric current or with temperature extremes was the source of seven of the fatalities. High-risk occupations included welders, line workers, electricians, meter readers, mechanics, maintenance workers, and plant and equipment operators.

Hershey Medical Center Technical Workshop Report: optimizing the design and interpretation of epidemiologic studies for assessing neurodevelopmental effects from in utero chemical exposure.

Neurodevelopmental disabilities affect 3-8% of the 4 million babies born each year in the U.S. alone, with known etiology for less than 25% of those disabilities. Numerous investigations have sought to determine the role of environmental exposures in the etiology of a variety of human neurodevelopmental disorders (e.g., learning disabilities, attention deficit-hyperactivity disorder, intellectual disabilities) that are manifested in childhood, adolescence, and young adulthood. A comprehensive critical examination and discussion of the various methodologies commonly used in investigations is needed. The Hershey Medical Center Technical Workshop: Optimizing the design and interpretation of epidemiologic studies for assessing neurodevelopmental effects from in utero chemical exposure provided such a forum for examining these methodologies. The objective of the Workshop was to develop scientific consensus on the key principles and considerations for optimizing the design and interpretation of epidemiologic studies of in utero exposure to environmental chemicals and subsequent neurodevelopmental effects. (The Panel recognized that the nervous system develops post-natally and that critical periods of exposure can span several developmental life stages.) Discussions from the Workshop Panel generated 17 summary points representing key tenets of work in this field. These points stressed the importance of: a well-defined, biologically plausible hypothesis as the foundation of in utero studies for assessing neurodevelopmental outcomes; understanding of the exposure to the environmental chemical(s) of interest, underlying mechanisms of toxicity, and anticipated outcomes; the use of a prospective, longitudinal cohort design that, when possible, runs for periods of 2-5 years, and possibly even longer, in an effort to assess functions at key developmental epochs; measuring potentially confounding variables at regular, fixed time intervals; including measures of specific cognitive and social-emotional domains along with non-cognitive competence in young children, as well as comprehensive measures of health; consistency of research design protocols across studies (i.e., tests, covariates, and analysis styles) in an effort to improve interstudy comparisons; emphasis on design features that minimize introduction of systematic error at all stages of investigation: participant selection, data collection and analysis, and interpretation of results; these would include (but not be limited to) reducing selection bias, using double-blind designs, and avoiding post hoc formulation of hypotheses; a priori data analysis strategies tied to hypotheses and the overall research design, particularly for methods used to characterize and address confounders in any neurodevelopmental study; actual quantitative measurements of exposure, even if indirect, rather than methods based on subject recall; careful examination of standard test batteries to ensure that the battery is tailored to the age group as well as what is known about the specific neurotoxic effects on the developing nervous system; establishment of a system for neurodevelopmental surveillance for tracking the outcomes from in utero exposure across early developmental time periods to determine whether central nervous system injuries may be lying silent until developmentally challenged; ongoing exploration of computerized measures that are culturally and linguistically sensitive, and span the age range from birth into the adolescent years; routine incorporation of narrative in manuscripts concerning the possibility of spurious (i.e., false positive and false negative) test results in all research reportage (this can be facilitated by detailed, transparent reporting of design, covariates, and analyses so that others can attempt to replicate the study); forthright, disciplined, and intellectually honest treatment of the extent to which results of any study are conclusive--that is, how generalizable the results of the study are in terms of the implications for the individual study participants, the community studied, and human health overall; confinement of reporting to the actual research questions, how they were tested, and what the study found, and avoiding, or at least keeping to a minimum, any opinions or speculation concerning public health implications; education of clinicians and policymakers to critically read scientific reports, and to interpret study findings and conclusions appropriately; and recognition by investigators of their ethical duty to report negative as well as positive findings, and the importance of neither minimizing nor exaggerating these findings.

Kinetics of arsenic methylation by freshly isolated B6C3F1 mouse hepatocytes.

The toxic and carcinogenic effects of arsenic may be mediated by both inorganic and methylated arsenic species. The methylation of arsenic(III) is thought to take place via sequential oxidative methylation and reduction steps to form monomethylarsenic (MMA) and dimethylarsenic (DMA) species, but recent evidence indicates that glutathione complexes of arsenic(III) can be methylated without oxidation. The kinetics of arsenic methylation were determined in freshly isolated hepatocytes from male B6C3F1 mice. Hepatocytes (>90% viability) were isolated by collagenase perfusion and suspended in Williams' Medium E with various concentrations of arsenic(III) (sodium m-arsenite). Aliquots of the lysed cell suspension were analyzed for arsenic species by hydride generation-atomic absorption spectrometry. The formation of MMA(III) from sodium arsenite (1 microM) was linear with respect to time for >90 min. DMA(III) formation did not become significant until 60 min. MMA(V) and DMA(V) were not consistently observed in the incubations. These results suggest that the glutathione complex mechanism of methylation plays an important role in arsenic biotransformation in mouse hepatocytes. Metabolism of arsenic(V) was not observed in mouse hepatocytes, consistent with inhibition of arsenic(V) active cellular uptake by phosphate in the medium. The formation of MMA(III) increased with increasing arsenic(III) concentrations up to approximately 2 microM and declined thereafter. The concentration dependence is consistent with a saturable methylation reaction accompanied by uncompetitive substrate inhibition of the reaction by arsenic(III). Kinetic analysis of the data suggested an apparent K(M) of approximately 3.6 microM arsenic(III), an apparent V(max) of approximately 38.9 microg MMA(III) formed/L/h/million cells, and an apparent K(I) of approximately 1.3 microM arsenic(III). The results of this study can be used in the physiologically based pharmacokinetic model for arsenic disposition in mice to predict the concentration of MMA(III) in liver and other tissues.

Comparison of tissue dosimetry in the mouse following chronic exposure to arsenic compounds.

Several chronic bioassays have been conducted in multiple strains of mice in which various concentrations of arsenate or arsenite were administered in the drinking water without a tumorigenic effect. However, one study (Ng et al., 1999) reported a significant increase in tumor incidence in C57Bl/6J mice exposed to arsenic in their drinking water throughout their lifetime, with no tumors reported in controls. A physiologically based pharmacokinetic model for arsenic in the mouse has previously been developed (Gentry et al., 2004) to investigate potential differences in tissue dosimetry of arsenic species across various strains of mice. Initial results indicated no significant differences in blood, liver, or urine dosimetry in B6C3F1 and C57Bl/6 mice for acute or subchronic exposure. The current work was conducted to compare model-predicted estimates of tissue dosimetry to additional kinetic information from the (C57Bl/6 xCBA)F1 and TgAc mouse. The results from the current modeling indicate that the pharmacokinetic parameters derived based on information in the B6C3F1 mouse adequately describe the measured concentrations in the blood/plasma, liver, and urine of both the (C57Bl/6 x CBA)F1 and TgAc mouse, providing further support that the differences in response observed in the chronic bioassays are not related to strain-specific differences in pharmacokinetics. One significant finding was that no increases in skin or lung concentrations of arsenic species in the (C57Bl/6 x CBA)F1 strain were observed following administration of low concentrations (0.2 or 2 mg/U of arsenate in the drinking water, even though differences in response in the skin were reported. These data suggest that pharmacodynamic changes may be observed following exposure to arsenic compounds without an observable change in tissue dosimetry. These results provided further indirect support for the existence of inducible arsenic efflux in these tissues.

Arsenic relative bioavailability from diet and airborne exposures: Implications for risk assessment.

Major human environmental health concern has been associated with inorganic arsenic (iAs) in drinking water in which dissolved iAs is highly bioavailable. More recently health concerns have been raised regarding the extent of iAs exposure via food and other potential sources. Arsenic relative bioavailability (RBA) in soil is known to be variable; the extent and role of iAs bioavailability in food are not well characterized. iAs in coal fly ash and bottom ash are other potential exposure media for which RBA has not been well characterized. A comprehensive literature search was conducted to support evaluation of the contribution of food and coal fly ash to iAs exposure. Few studies were found that investigated bioavailability associated with As-containing coal ash or airborne As-containing particles; estimated bioavailability in these studies ranged from 11% to 50%. The implications and potential usefulness of iAs bioavailability associated with inhalation exposure to human health risk assessment remain unknown at this time. Main sources of dietary iAs intake in the U.S. include rice and other grains, vegetables, and fruits. Due to low concentrations of iAs, seafood is not a primary contributor to dietary iAs intake. Three general kinds of food studies were identified: studies of As bioaccessibility in composites, As bioavailability and bioaccessibility in specific foods, and As consumption and urinary excretion in human volunteers. One in vivo study was identified that examined As bioavailability in food. A variety of experimental in vitro gastro-intestinal protocols have been used, however, few studies have included As speciation before and after the in vitro extraction. Current data suggest that the bioaccessibility of iAs in rice is quite high, typically 70% or more indicating that iAs in rice is highly bioavailable. Adjusting for RBA may not have a meaningful impact on iAs exposure estimates for rice-based foods.

Evaluation of gene expression changes in human primary lung epithelial cells following 24-hr exposures to inorganic arsenic and its methylated metabolites and to arsenic trioxide.

The concentration response for altered gene expression in primary lung epithelial cells was determined following two treatments with arsenicals: (1) a mixture of trivalent arsenic compounds representative of urinary arsenic concentrations in exposed human populations, and (2) arsenite (As2 O3 ) a common form of inhaled arsenic dust that is frequently used in both in vivo and in vitro experimental exposures. Biochemical assays did not detect any evidence of cytotoxicity at the concentrations used, apart from a concentration-related increase in cellular heme oxygenase that was also indicated by the genomic analysis. Cell signal pathway enrichment analysis indicated similar responses to both treatments, with concentration-related responses in pathways related to cell adhesion, cytoskeleton remodeling, development (morphogenesis), cell cycle control, and to a lesser extent inflammatory responses. These cellular responses to arsenic were consistent with those observed in a previous study with primary uroepithelial cells. Benchmark dose analysis also demonstrated similar potency of the two treatments as well as comparable sensitivity of the two cell types. A number of genes showing similar concentration-dependent expression across individuals in both bladder and lung cells were identified, including heme oxygenase 1, thioredoxin reductase, DNA damage binding protein 2, and thrombomodulin. The data on human primary lung cells from this study, together with the data from human primary uroepithelial cells, support a conclusion that biological responses to arsenic by human cells under study conditions are unlikely to occur at concentrations below 0.1 µM. Environ. Mol. Mutagen. 56:477-490, 2015. © 2015 Wiley Periodicals, Inc.

Occupational injury surveillance among electric utility employees.

Currently, there are only limited injury surveillance data for the electric utility workforce. To address this gap, an Occupational Health Surveillance Database for electric power utilities was established for epidemiologic monitoring and intervention program evaluation. Injury rates varied across utility occupations, such as, managers, line workers, and meter readers, ranging from 0.18 to 9.63 per 100 employee-years based on more than 500,000 employee-years of observation. Compared with male workers, the risk of injury among female workers was lower overall, although their risk was higher in nonoffice occupations than their male counterparts. Across the period 2000 to 2002, three of four companies that experienced decreases in workforce size also experienced noticeable increases in injury rates. Our results suggest that benchmarking and prevention efforts should be directed at specific occupational groups and specific injury types.

Physiologically based pharmacokinetic modeling of arsenic in the mouse.

A remarkable feature of the carcinogenicity of inorganic arsenic is that while human exposures to high concentrations of inorganic arsenic in drinking water are associated with increases in skin, lung, and bladder cancer, inorganic arsenic has not typically caused tumors in standard laboratory animal test protocols. Inorganic arsenic administered for periods of up to 2 yr to various strains of laboratory mice, including the Swiss CD-1, Swiss CR:NIH(S), C57Bl/6p53(+/-), and C57Bl/6p53(+/+), has not resulted in significant increases in tumor incidence. However, Ng et al. (1999) have reported a 40% tumor incidence in C57Bl/6J mice exposed to arsenic in their drinking water throughout their lifetime, with no tumors reported in controls. In order to investigate the potential role of tissue dosimetry in differential susceptibility to arsenic carcinogenicity, a physiologically based pharmacokinetic (PBPK) model for inorganic arsenic in the rat, hamster, monkey, and human (Mann et al., 1996a, 1996b) was extended to describe the kinetics in the mouse. The PBPK model was parameterized in the mouse using published data from acute exposures of B6C3F1 mice to arsenate, arsenite, monomethylarsonic acid (MMA), and dimethylarsinic acid (DMA) and validated using data from acute exposures of C57Black mice. Predictions of the acute model were then compared with data from chronic exposures. There was no evidence of changes in the apparent volume of distribution or in the tissue-plasma concentration ratios between acute and chronic exposure that might support the possibility of inducible arsenite efflux. The PBPK model was also used to project tissue dosimetry in the C57Bl/6J study, in comparison with tissue levels in studies having shorter duration but higher arsenic treatment concentrations. The model evaluation indicates that pharmacokinetic factors do not provide an explanation for the difference in outcomes across the various mouse bioassays. Other possible explanations may relate to strain-specific differences, or to the different durations of dosing in each of the mouse studies, given the evidence that inorganic arsenic is likely to be active in the later stages of the carcinogenic process.

Evaluation of gene expression changes in human primary uroepithelial cells following 24-hr exposures to inorganic arsenic and its methylated metabolites.

Gene expression changes in primary human uroepithelial cells exposed to arsenite and its methylated metabolites were evaluated to identify cell signaling pathway perturbations potentially associated with bladder carcinogenicity. Cells were treated with mixtures of inorganic arsenic and its pentavalent or trivalent metabolites for 24 hr at total arsenic concentrations ranging from 0.06 µM to 18 µM. One series (five samples) was conducted with arsenite and pentavalent metabolites and a second (10 samples) with arsenite and trivalent metabolites. Similar gene expression responses were obtained for pentavalent or trivalent metabolites. A suite of eight gene changes was consistently identified across individuals that reflect effects on key signaling pathways: oxidative stress, protein folding, growth regulation, metallothionine regulation, DNA damage sensing, thioredoxin regulation, and immune response. No statistical significance of trend (NOSTASOT) analysis of these common genes identified lowest observed effect levels (LOELs) from 0.6 to 6.0 µM total arsenic and no observed effect levels (NOELs) from 0.18 to 1.8 µM total arsenic. For the trivalent arsenical mixture, benchmark doses (BMDs) ranged from 0.13 to 0.92 µM total arsenic; benchmark dose lower 95% confidence limits (BMDLs) ranged from 0.09 to 0.58 µM total arsenic. BMDs ranged from 0.53 to 2.7 µM and BMDLs from 0.35 to 1.7 µM for the pentavalent arsenical mixture. Both endpoints varied by a factor of 3 across individuals. Thisstudy is the first to examine gene expression response in primary uroepithelial cells from multiple individuals and to identify no effect levels for arsenical-induced cell signaling perturbations in normal human cells exposed to a biologically plausible concentration range.

Analysis of genomic dose-response information on arsenic to inform key events in a mode of action for carcinogenicity.

A comprehensive literature search was conducted to identify information on gene expression changes following exposures to inorganic arsenic compounds. This information was organized by compound, exposure, dose/concentration, species, tissue, and cell type. A concentration-related hierarchy of responses was observed, beginning with changes in gene/protein expression associated with adaptive responses (e.g., preinflammatory responses, delay of apoptosis). Between 0.1 and 10 microM, additional gene/protein expression changes related to oxidative stress, proteotoxicity, inflammation, and proliferative signaling occur along with those related to DNA repair, cell cycle G2/M checkpoint control, and induction of apoptosis. At higher concentrations (10-100 microM), changes in apoptotic genes dominate. Comparisons of primary cell results with those obtained from immortalized or tumor-derived cell lines were also evaluated to determine the extent to which similar responses are observed across cell lines. Although immortalized cells appear to respond similarly to primary cells, caution must be exercised in using gene expression data from tumor-derived cell lines, where inactivation or overexpression of key genes (e.g., p53, Bcl-2) may lead to altered genomic responses. Data from acute in vivo exposures are of limited value for evaluating the dose-response for gene expression, because of the transient, variable, and uncertain nature of tissue exposure in these studies. The available in vitro gene expression data, together with information on the metabolism and protein binding of arsenic compounds, provide evidence of a mode of action for inorganic arsenic carcinogenicity involving interactions with critical proteins, such as those involved in DNA repair, overlaid against a background of chemical stress, including proteotoxicity and depletion of nonprotein sulfhydryls. The inhibition of DNA repair under conditions of toxicity and proliferative pressure may compromise the ability of cells to maintain the integrity of their DNA.