Nikolai L. Chepelev

Comparison of toxicogenomics and traditional approaches to inform mode of action and points of departure in human health risk assessment of benzo[a]pyrene in drinking water

Abstract Toxicogenomics is proposed to be a useful tool in human health risk assessment. However, a systematic comparison of traditional risk assessment approaches with those applying toxicogenomics has never been done. We conducted a case study to evaluate the utility of toxicogenomics in the risk assessment of benzo[a]pyrene (BaP), a well-studied carcinogen, for drinking water exposures. Our study was intended to compare methodologies, not to evaluate drinking water safety. We compared traditional (RA1), genomics-informed (RA2) and genomics-only (RA3) approaches. RA2 and RA3 applied toxicogenomics data from human cell cultures and mice exposed to BaP to determine if these data could provide insight into BaPs mode of action (MOA) and derive tissue-specific points of departure (POD). Our global gene expression analysis supported that BaP is genotoxic in mice and allowed the development of a detailed MOA. Toxicogenomics analysis in human lymphoblastoid TK6 cells demonstrated a high degree of consistency in perturbed pathways with animal tissues. Quantitatively, the PODs for traditional and transcriptional approaches were similar (liver 1.2 vs. 1.0 mg/kg-bw/day; lungs 0.8 vs. 3.7 mg/kg-bw/day; forestomach 0.5 vs. 7.4 mg/kg-bw/day). RA3, which applied toxicogenomics in the absence of apical toxicology data, demonstrates that this approach provides useful information in data-poor situations. Overall, our study supports the use of toxicogenomics as a relatively fast and cost-effective tool for hazard identification, preliminary evaluation of potential carcinogens, and carcinogenic potency, in addition to identifying current limitations and practical questions for future work.

Integrating toxicogenomics into human health risk assessment: Lessons learned from the benzo[a]pyrene case study

Abstract The use of short-term toxicogenomic tests to predict cancer (or other health effects) offers considerable advantages relative to traditional toxicity testing methods. The advantages include increased throughput, increased mechanistic data, and significantly reduced costs. However, precisely how toxicogenomics data can be used to support human health risk assessment (RA) is unclear. In a companion paper (Moffat et al. 2014), we present a case study evaluating the utility of toxicogenomics in the RA of benzo[a]pyrene (BaP), a known human carcinogen. The case study is meant as a proof-of-principle exercise using a well-established mode of action (MOA) that impacts multiple tissues, which should provide a best case example. We found that toxicogenomics provided rich mechanistic data applicable to hazard identification, dose–response analysis, and quantitative RA of BaP. Based on this work, here we share some useful lessons for both research and RA, and outline our perspective on how toxicogenomics can benefit RA in the short- and long-term. Specifically, we focus on (1) obtaining biologically relevant data that are readily suitable for establishing an MOA for toxicants, (2) examining the human relevance of an MOA from animal testing, and (3) proposing appropriate quantitative values for RA. We describe our envisioned strategy on how toxicogenomics can become a tool in RA, especially when anchored to other short-term toxicity tests (apical endpoints) to increase confidence in the proposed MOA, and emphasize the need for additional studies on other MOAs to define the best practices in the application of toxicogenomics in RA.

Neurotoxicity may be an overlooked consequence of benzo[a]pyrene exposure that is relevant to human health risk assessment.

Benzo[a]pyrene (BaP) is a well-studied environmental compound that requires metabolic activation to have a carcinogenic effect. The neurotoxicity of BaP has received considerably less attention than its carcinogenicity. Environmental exposure to BaP correlates with impaired learning and memory in adults, and poor neurodevelopment in children. We carried out a comprehensive literature review to examine the neurotoxicity of BaP. The data were used to identify potential point of departure (POD) values for cancer and neurotoxicity endpoints using benchmark dose (BMD) modelling to compare the utility of both endpoints in the risk assessment of BaP. The POD for neurotoxicity in rodents, based on a standard behavioural test (Morris water maze), was 0.025 mg BaP/kg-bw-day compared to 0.54 mg BaP/kg-bw-day for rodent forestomach carcinogenicity, suggesting that neurotoxic endpoints are more sensitive than cancer endpoints for health risks associated with BaP exposure. Using the limited number of published studies on this topic, we propose a preliminary mode of action (MOA) to explain BaP-induced neurotoxicity in rodents. The MOA includes: (1) BaP binding to the aryl hydrocarbon receptor (AHR); (2) AHR-dependent modulation of the transcription of N-methyl-d-aspartate glutamate receptor (NMDAR) subunits; (3) NMDAR-mediated loss of neuronal activity and decreased long-term potentiation; and (4) compromised learning and memory. More data are needed to explore the proposed neurotoxic MOA. In addition, we consider alternative MOAs, including the hypothesis that BaP-mediated DNA damage may lead to either carcinogenicity or neurotoxicity, depending on the tissue. Our proposed MOA is intended to serve as a basis for hypothesis testing in future studies. We emphasise that further studies are needed to validate the proposed MOA, to evaluate its human relevance, and to explore other potential mechanisms of BaP neurotoxicity.

Impact of Genomics Platform and Statistical Filtering on Transcriptional Benchmark Doses (BMD) and Multiple Approaches for Selection of Chemical Point of Departure (PoD)

Many regulatory agencies are exploring ways to integrate toxicogenomic data into their chemical risk assessments. The major challenge lies in determining how to distill the complex data produced by high-content, multi-dose gene expression studies into quantitative information. It has been proposed that benchmark dose (BMD) values derived from toxicogenomics data be used as point of departure (PoD) values in chemical risk assessments. However, there is limited information regarding which genomics platforms are most suitable and how to select appropriate PoD values. In this study, we compared BMD values modeled from RNA sequencing-, microarray-, and qPCR-derived gene expression data from a single study, and explored multiple approaches for selecting a single PoD from these data. The strategies evaluated include several that do not require prior mechanistic knowledge of the compound for selection of the PoD, thus providing approaches for assessing data-poor chemicals. We used RNA extracted from the livers of female mice exposed to non-carcinogenic (0, 2 mg/kg/day, mkd) and carcinogenic (4, 8 mkd) doses of furan for 21 days. We show that transcriptional BMD values were consistent across technologies and highly predictive of the two-year cancer bioassay-based PoD. We also demonstrate that filtering data based on statistically significant changes in gene expression prior to BMD modeling creates more conservative BMD values. Taken together, this case study on mice exposed to furan demonstrates that high-content toxicogenomics studies produce robust data for BMD modelling that are minimally affected by inter-technology variability and highly predictive of cancer-based PoD doses.

Transcriptional profiling of dibenzo[def, p]chrysene-induced spleen atrophy provides mechanistic insights into its immunotoxicity in mutamouse

Dibenzo[def,p]chrysene (DBC) is the most carcinogenic polycyclic aromatic hydrocarbon (PAH) examined to date. We investigated the immunotoxicity of DBC, manifested as spleen atrophy, following acute exposure of adult MutaMouse males by oral gavage. Mice were exposed to 0, 2.0, 6.2, or 20.0 mg DBC /kg-bw per day, for 3 days. Genotoxic endpoints (DBC-DNA adducts and lacZ mutant frequency in spleen and bone marrow, and red blood cell micronucleus frequency) and global gene expression changes were measured. All of the genotoxicity measures increased in a dose-dependent manner in spleen and bone marrow. Gene expression analysis showed that DBC activates p53 signaling pathways related to cellular growth and proliferation, which was evident even at the low dose. Strikingly, the expression profiles of DBC exposed mouse spleens were highly inversely correlated with the expression profiles of the only published toxicogenomics dataset of enlarged mouse spleen. This analysis suggested a central role for Bnip3l, a pro-apoptotic protein involved in negative regulation of erythroid maturation. RT-PCR confirmed expression changes in several genes related to apoptosis, iron metabolism, and aryl hydrocarbon receptor signaling that are regulated in the opposite direction during spleen atrophy versus benzo[a]pyrene-mediated splenomegaly. In addition, benchmark dose modeling of toxicogenomics data yielded toxicity estimates that are very close to traditional toxicity endpoints. This work illustrates the power of toxicogenomics to reveal rich mechanistic information for immunotoxic compounds and its ability to provide information that is quantitatively similar to that derived from standard toxicity methods in health risk assessment.

Transcriptional profiling of the mouse hippocampus supports an NMDAR-mediated neurotoxic mode of action for benzo[a]pyrene

Benzo[a]pyrene (BaP) is a genotoxic carcinogen and a neurotoxicant. The neurotoxicity of BaP is proposed to arise from either genotoxicity leading to neuronal cell death, or perturbed expression of N‐methyl‐d‐aspartate receptor (NMDAR) subunits. To explore these hypotheses, we profiled hippocampal gene expression of adult male Muta™Mouse administered 0, 1, 35, or 70 mg BaP/kg bw per day by oral gavage for 3 days. Transcriptional profiles were examined by RNA‐sequencing (RNA‐seq), DNA microarrays, and real‐time quantitative reverse transcription polymerase chain reaction (RT‐PCR). BaP‐DNA adducts in the cerebellum were quantified by 32P‐post‐labeling to measure genotoxicity. RNA‐seq revealed altered expression of 0, 260, and 219 genes (P‐value < 0.05, fold‐change ≥ ± 1.5) following exposure to the low, medium, and high doses, respectively; 54 genes were confirmed by microarrays. Microarray and RT‐PCR analysis showed increased expression of NMDAR subunits Grina and Grin2a. In contrast, no effects on DNA‐damage response genes were observed despite comparable BaP‐DNA adduct levels in the cerebellum and in the lungs and livers of mice at similar BaP doses in previous studies. The results suggest that DNA‐damage response does not play a major role in BaP‐induced adult neurotoxicity. Meta‐analysis revealed that BaP‐induced transcriptional profiles are highly correlated with those from the hippocampus of transgenic mice exhibiting similar neurotoxicity outcomes to BaP‐exposed mice and rats (i.e., defects in learning and memory). Overall, we suggest that BaP‐induced neurotoxicity is more likely to be a consequence of NMDAR perturbation than genotoxicity, and identify other important genes potentially mediating this adverse outcome. Environ. Mol. Mutagen. 57:350–363, 2016.

Impact of Acrylamide on Calcium Signaling and Cytoskeletal Filaments in Testes From F344 Rat.

Acrylamide (AA) at high exposure levels is neurotoxic, induces testicular toxicity, and increases dominant lethal mutations in rats. RNA-sequencing in testes was used to identify differentially expressed genes (DEG), explore AA-induced pathway perturbations that could contribute to AA-induced testicular toxicity and then used to derive a benchmark dose (BMD). Male F344/DuCrl rats were administered 0.0, 0.5, 1.5, 3.0, 6.0, or 12.0 mg AA/kg bw/d in drinking water for 5, 15, or 31 days. The experimental design used exposure levels that spanned and exceeded the exposure levels used in the rat dominant lethal, 2-generation reproductive toxicology, and cancer bioassays. The time of sample collection was based on previous studies that developed gene expression–based BMD. At 12.0 mg/kg, there were 38, 33, and 65 DEG (P value <.005; fold change >1.5) in the testes after 5, 15, or 31 days of exposure, respectively. At 31 days, there was a dose-dependent increase in the number of DEG, and at 12.0 mg/kg/d the top three functional clusters affected by AA exposure were actin filament organization, response to calcium ion, and regulation of cell proliferation. The BMD lower 95% confidence limit using DEG ranged from 1.8 to 6.8 mg/kg compared to a no-observed-adverse-effect-level of 2.0 mg/kg/d for male reproductive toxicity. These results are consistent with the known effects of AA on calcium signaling and cytoskeletal actin filaments leading to neurotoxicity and suggest that AA can cause rat dominant lethal mutations by these same mechanisms leading to impaired chromosome segregation during cell division.

Application of benchmark dose modeling to protein expression data in the development and analysis of mode of action/adverse outcome pathways for testicular toxicity.

Reliable quantification of gene and protein expression has potential to contribute significantly to the characterization of hypothesized modes of action (MOA) or adverse outcome pathways for critical effects of toxicants. Quantitative analysis of gene expression by benchmark dose (BMD) modeling has been facilitated by the development of effective software tools. In contrast, protein expression is still generally quantified by a less robust effect level (no or lowest [adverse] effect levels) approach, which minimizes its potential utility in the consideration of dose–response and temporal concordance for key events in hypothesized MOAs. BMD modeling is applied here to toxicological data on testicular toxicity to investigate its potential utility in analyzing protein expression relevant to the proposed MOA to inform human health risk assessment. The results illustrate how the BMD analysis of protein expression in animal tissues in response to toxicant exposure: (1) complements other toxicity data, and (2) contributes to consideration of the empirical concordance of dose–response relationships, as part of the weight of evidence for hypothesized MOAs to facilitate consideration and application in regulatory risk assessment. Lack of BMD analysis in proteomics has likely limited its use for these purposes. This paper illustrates the added value of BMD modeling to support and strengthen hypothetical MOAs as a basis to facilitate the translation and uptake of the results of proteomic research into risk assessment. Copyright