Development and validation of the TGx-HDACi transcriptomic biomarker to detect histone deacetylase inhibitors in human TK6 cells.

Transcriptomic biomarkers can be used to inform molecular initiating and key events involved in a toxicant's mode of action. To address the limited approaches available for identifying epigenotoxicants, we developed and assessed a transcriptomic biomarker of histone deacetylase inhibition (HDACi). First, we assembled a set of ten prototypical HDACi and ten non-HDACi reference compounds. Concentration-response experiments were performed for each chemical to collect TK6 human lymphoblastoid cell samples after 4 h of exposure and to assess cell viability following a 20-h recovery period in fresh media. One concentration was selected for each chemical for whole transcriptome profiling and transcriptomic signature derivation, based on cell viability at the 24-h time point and on maximal induction of HDACi-response genes (RGL1, NEU1, GPR183) or cellular stress-response genes (ATF3, CDKN1A, GADD45A) analyzed by TaqMan qPCR assays after 4 h of exposure. Whole transcriptomes were profiled after 4 h exposures by Templated Oligo-Sequencing (TempO-Seq). By applying the nearest shrunken centroid (NSC) method to the whole transcriptome profiles of the reference compounds, we derived an 81-gene toxicogenomic (TGx) signature, referred to as TGx-HDACi, that classified all 20 reference compounds correctly using NSC classification and the Running Fisher test. An additional 4 HDACi and 7 non-HDACi were profiled and analyzed using TGx-HDACi to further assess classification performance; the biomarker accurately classified all 11 compounds, including 3 non-HDACi epigenotoxicants, suggesting a promising specificity toward HDACi. The availability of TGx-HDACi increases the diversity of tools that can facilitate mode of action analysis of toxicants using gene expression profiling.

Dose-response mutation and spectrum analyses reveal similar responses at two microsatellite loci in benzo(a)pyrene-exposed mouse spermatogonia.

Identifying chemical exposures that can cause germline mutations is important as these mutations can be inherited, impacting both individual and population health. However, germline mutations are extremely rare and difficult to detect. Chemically induced germline mutations can be detected through analysis of highly unstable tandem repeat DNA. We recently developed a single-molecule PCR (SM-PCR) approach to quantify mutations at a mouse microsatellite locus (Mm2.2.1) in sperm for such purposes. In this study, we refine this approach through the combined analysis of mouse microsatellites Mm2.2.1 and Mm19.2.3. Mice were exposed to 0, 25, 50 or 100 mg/kg/day benzo(a)pyrene (BaP) by oral gavage for 28 days and sperm sampled 42 days after the end of exposure to measure effects on dividing spermatogonia. DNA was diluted to a single genome per PCR well for amplification of microsatellites in singleplex and multiplex reactions, and alleles were sized to identify mutations using capillary electrophoresis. Analysis of ~300-500 molecules per animal at both microsatellite loci, when tested individually, showed a ~2-fold increase in mutations relative to the controls at both the 50 and 100 mg/kg/day BaP doses. Multiplex SM-PCR revealed similar increases in mutation frequencies in both microsatellites. Comparison with results from a previous lacZ mutation assay conducted on the same mice revealed that although microsatellite mutations are a sensitive endpoint for detecting changes in mutation frequencies at lower doses, they appear to be saturable and thus have a reduced dynamic range. These results confirm that BaP is a male germ cell mutagen that broadly impacts tandem repeat DNA. Likewise, addition of a second hypervariable microsatellite increases the sensitivity of this assay.

Sidestream tobacco smoke is a male germ cell mutagen.

Active cigarette smoking increases oxidative damage, DNA adducts, DNA strand breaks, chromosomal aberrations, and heritable mutations in sperm. However, little is known regarding the effects of second-hand smoke on the male germ line. We show here that short-term exposure to mainstream tobacco smoke or sidestream tobacco smoke (STS), the main component of second-hand smoke, induces mutations at an expanded simple tandem repeat locus (Ms6-hm) in mouse sperm. We further show that the response to STS is not linear and that, for both mainstream tobacco smoke and STS, doses that induced significant increases in expanded simple tandem repeat mutations in sperm did not increase the frequencies of micronucleated reticulocytes and erythrocytes in the bone marrow and blood of exposed mice. These data show that passive exposure to cigarette smoke can cause tandem repeat mutations in sperm under conditions that may not induce genetic damage in somatic cells. Although the relationship between noncoding tandem repeat instability and mutations in functional regions of the genome is unclear, our data suggest that paternal exposure to second-hand smoke may have reproductive consequences that go beyond the passive smoker.

House dust-derived mixtures of organophosphate esters alter the phenotype, function, transcriptome, and lipidome of KGN human ovarian granulosa cells.

Organophosphate esters (OPEs), used as flame retardants and plasticizers, are present ubiquitously in the environment. Previous studies suggest that exposure to OPEs is detrimental to female fertility in humans. However, no experimental information is available on the effects of OPE mixtures on ovarian granulosa cells, which play essential roles in female reproduction. We used high-content imaging to investigate the effects of environmentally relevant OPE mixtures on KGN human granulosa cell phenotypes. Perturbations to steroidogenesis were assessed using ELISA and qRT-PCR. A high-throughput transcriptomic approach, TempO-Seq, was used to identify transcriptional changes in a targeted panel of genes. Effects on lipid homeostasis were explored using a cholesterol assay and global lipidomic profiling. OPE mixtures altered multiple phenotypic features of KGN cells, with triaryl OPEs in the mixture showing higher potencies than other mixture components. The mixtures increased basal production of steroid hormones; this was mediated by significant changes in the expression of critical transcripts involved in steroidogenesis. Further, the total-OPE mixture disrupted cholesterol homeostasis and the composition of intracellular lipid droplets. Exposure to complex mixtures of OPEs, similar to those found in house dust, may adversely affect female reproductive health by altering a multitude of phenotypic and functional endpoints in granulosa cells. This study provides novel insights into the mechanisms of actions underlying the toxicity induced by OPEs and highlights the need to examine the effects of human relevant chemical mixtures.

Transcriptome analysis in mouse skin after exposure to ultraviolet radiation from a canopy sunbed.

Exposure to ultraviolet radiation (UV-R), from both natural and artificial tanning, heightens the risk of skin cancer by inducing molecular changes in cells and tissues. Despite established transcriptional alterations at a molecular level due to UV-R exposure, uncertainties persist regarding UV radiation characterization and subsequent genomic changes. Our study aimed to mechanistically explore dose- and time-dependent gene expression changes, that may drive short-term (e.g., sunburn) and long-term actinic (e.g., skin cancer) consequences. Using C57BL/6N mouse skin, we analyzed transcriptomic expression following exposure to five erythemally weighted UV-R doses (0, 5, 10, 20, and 40 mJ/cm2 ) emitted by a UV-R tanning device. At 96 h post-exposure, 5 mJ/cm2 induced 116 statistically significant differentially expressed genes (DEGs) associated with structural changes from UV-R damage. The highest number of significant gene expression changes occurred at 6 and 48 h post-exposure in the 20 and 40 mJ/cm2 dose groups. Notably, at 40 mJ/cm2 , 13 DEGs related to skin barrier homeostasis were consistently perturbed across all timepoints. UV-R exposure activated pathways involving oxidative stress, P53 signaling, inflammation, biotransformation, skin barrier maintenance, and innate immunity. This in vivo study's transcriptional data offers mechanistic insights into both short-term and potential non-threshold-dependent long-term health effects of UV-R tanning.

Hepatic genotoxicity and toxicogenomic responses in Muta™Mouse males treated with dibenz[a,h]anthracene.

Dibenz[a,h]anthracene (DB[a,h]A) is a polycyclic aromatic hydrocarbon that is a by-product of combustion and a potent carcinogen. Few studies have investigated the effects of DB[a,h]A on mRNA and microRNA expression to dissect the mechanisms involved in carcinogenesis. In this study, mature male mice (Muta(™)Mouse) were exposed to 6.25, 12.5 and 25mg/kg/day DB[a,h]A by oral gavage for 28 consecutive days. Results were compared with mice similarly exposed to benzo[a]pyrene (B[a]P) in our previous work. Liver DNA adduct levels and lacZ mutant frequency increased dose dependently for both chemicals. Aryl hydrocarbon receptor (AhR) potency was greater for DB[a,h]A than B[a]P using the chemical-activated luciferase expression assay. Microarray analysis revealed 19 up-regulated and 22 down-regulated genes (false discovery rate-adjusted P ≤ 0.05; fold change ≥ 1.5) following treatment with 6.25 mg/kg/day DB[a,h]A. Thirteen transcripts were up-regulated and 32 down-regulated in the 12.5mg/kg/day group. The 25mg/kg/day dose had major effects on mRNA expression with 135 up-regulated and 104 down-regulated genes. Overall, perturbations were greater for DB[a,h]A than for B[a]P; in vitro chemical-activated luciferase expression supports that this may be driven by the AhR. Many of the DB[a,h]A-affected genes are implicated in cancer and are essential in vital biological functions including circadian rhythm, glucose metabolism, lipid metabolism, immune response, cell cycle and apoptosis. Although a number of functional groups were similarly affected by B[a]P and DB[a,h]A, in general the responses generated by each chemical were quite distinct. Commonalities included a DNA damage response leading to induction of cell cycle arrest and apoptosis in both Tp53-dependent and Tp53-independent manners. MicroRNA expression was identical for both chemicals, with only miR-34a showing a dose-dependent increase in treated mice.

Dataset on DNA methylation and gene expression changes induced by 5-aza-2'-deoxycytidine in Syrian golden hamster fetal cell cultures.

The Syrian hamster (SH) is an animal model used in virology, toxicology, and carcinogenesis, where a better understanding of epigenetic mechanisms is required. Finding genetic loci regulated by DNA methylation may assist in the development of DNA methylation-based in vitro assays for the identification of carcinogens. This dataset informs on the regulation of gene expression by DNA methylation. Primary cultures of SH male fetal cells (sex determined by differences in kdm5 loci on the X and Y chromosome) were exposed for 7 days to the carcinogen benzo[a]pyrene (20 µM) from which a morphologically transformed colony was collected and reseeded. The colony bypassed senescence and sustained growth. After 210 days of culture, the cells were collected and divided in 16 aliquots to create 4 experimental groups to test the effects of the DNA methylation inhibitor 5-aza-2'-deoxycytidine (5adC). The experiment was initiated 24 h after cell seeding in 10 cm plates. The groups are naïve cells (N), cells exposed for 48 h to either 0.05% DMSO as vehicle (V), or to 5adC at 1 µM and 5 µM. DNA and RNA libraries were sequenced on an Illumina NextSeq 500. Gene expression was analysed by RNAseq and differentially methylated DNA regions (DMRs: clusters of 200 base pairs (bp), read depth >20, q< 0.05, methylation difference >|25%|) were identified by reduce representation bisulfite sequencing (RRBS). Global genome DNA methylation was similar between the N (mean±SD, 47.3%±0.02) and V groups (47.3%±0.01). Although 5adC reduced methylation, the reduction was larger in the 1 µM (39.2%±0.002) than in the 5 µM group (44.3%±0.01). 5adC induced a total of 612 and 190 DMRs by 1 µM and 5 µM, among which 79 and 23 were in the promoter regions (±3,000 bp from the transcription start site), respectively. 5adC induced a total of 1,170 and 1,797 differentially expressed genes (DEGs) by 1 µM and 5 µM, respectively. The 5 µM treatment induced statistically significant toxicity (% cell viability: group N 97%±8, V 98.8%±1.3, 1 µM 97.3%±0.5, 5 µM 93.8%±1.5), which perhaps reduced cell division and daughter cell numbers with inherited changes in methylation, but increased number of DEGs due to both toxicity and methylation changes. As usually observed in the literature, a small portion of DEGs (4% and 4% at 1 µM and 5 µM, respectively) are associated with DMRs in their promoters. These promoter DMRs by themselves are sufficient among other epigenetic marks to induce DEGs. The dataset provides the genomic coordinates of the DMRs and an opportunity to further examine their roles in distal putative promoters or enhancers (yet to be described in the SH) in contributing to gene expression changes, senescence bypass and sustained proliferation as essential carcinogenic events (see companion paper [1]). Finally, this experiment confirms the possibility in future experiments to use 5adC as a positive control for effects on DNA methylation in cells derived from SH.

Toxicological Mechanisms and Potencies of Organophosphate Esters in KGN Human Ovarian Granulosa Cells as Revealed by High-throughput Transcriptomics.

Despite the growing number of studies reporting potential risks associated with exposure to organophosphate esters (OPEs), their molecular mechanisms of action remain poorly defined. We used the high-throughput TempO-Seq™ platform to investigate the effects of frequently detected OPEs on the expression of ∼3000 environmentally responsive genes in KGN human ovarian granulosa cells. Cells were exposed for 48 h to one of five OPEs (0.1 to 50 µM): tris(methylphenyl) phosphate (TMPP), isopropylated triphenyl phosphate (IPPP), tert-butylphenyl diphenyl phosphate (BPDP), triphenyl phosphate (TPHP), or tris(2-butoxyethyl) phosphate (TBOEP). The sequencing data indicate that four OPEs induced transcriptional changes, whereas TBOEP had no effect within the concentration range tested. Multiple pathway databases were used to predict alterations in biological processes based on differentially expressed genes. At lower concentrations, inhibition of the cholesterol biosynthetic pathway was the predominant effect of OPEs; this was likely a consequence of intracellular cholesterol accumulation. At higher concentrations, BPDP and TPHP had distinct effects, primarily affecting pathways involved in cell cycle progression and other stress responses. Benchmark concentration (BMC) modelling revealed that BPDP had the lowest transcriptomic point of departure. However, in vitro to in vivo extrapolation modeling indicated that TMPP was bioactive at lower concentrations than the other OPEs. We conclude that these new approach methodologies provide information on the mechanism(s) underlying the effects of data-poor compounds and assist in the derivation of protective points of departure for use in chemical read-across and decision-making.

In vitro transcriptomic analyses reveal pathway perturbations, estrogenic activities, and potencies of data-poor BPA alternative chemicals.

Since initial regulatory action in 2010 in Canada, bisphenol A (BPA) has been progressively replaced by structurally related alternative chemicals. Unfortunately, many of these chemicals are data-poor, limiting toxicological risk assessment. We used high-throughput transcriptomics to evaluate potential hazards and compare potencies of BPA and 15 BPA alternative chemicals in cultured breast cancer cells. MCF-7 cells were exposed to BPA and 15 alternative chemicals (0.0005-100 µM) for 48 h. TempO-Seq (BioSpyder Inc) was used to examine global transcriptomic changes and estrogen receptor alpha (ERα)-associated transcriptional changes. Benchmark concentration (BMC) analysis was conducted to identify 2 global transcriptomic points of departure: (1) the lowest pathway median gene BMC and (2) the 25th lowest rank-ordered gene BMC. ERα activation was evaluated using a published transcriptomic biomarker and an ERα-specific transcriptomic point of departure was derived. Genes fitting BMC models were subjected to upstream regulator and canonical pathway analysis in Ingenuity Pathway Analysis. Biomarker analysis identified BPA and 8 alternative chemicals as ERα active. Global and ERα transcriptomic points of departure produced highly similar potency rankings with bisphenol AF as the most potent chemical tested, followed by BPA and bisphenol C. Further, BPA and transcriptionally active alternative chemicals enriched similar gene sets associated with increased cell division and cancer-related processes. These data provide support for future read-across applications of transcriptomic profiling for risk assessment of data-poor chemicals and suggest that several BPA alternative chemicals may cause hazards at similar concentrations to BPA.

Per- and polyfluoroalkyl substances (PFAS) in mixtures show additive effects on transcriptomic points of departure in human liver spheroids.

Per- and polyfluoroalkyl substances (PFAS) are a wide range of chemicals that are used in a variety of consumer and industrial products leading to direct human exposure. Many PFAS are chemically nonreactive and persistent in the environment, resulting in additional exposure from water, soil, and dietary intake. While some PFAS have documented negative health effects, data on simultaneous exposures to multiple PFAS (PFAS mixtures) are inadequate for making informed decisions for risk assessment. The current study leverages data from previous work in our group using Templated Oligo-Sequencing (TempO-Seq) for high-throughput transcriptomic analysis of PFAS-exposed primary human liver cell spheroids; herein, we determine the transcriptomic potency of PFAS in mixtures. Gene expression data from single PFAS and mixture exposures of liver cell spheroids were subject to benchmark concentration (BMC) analysis. We used the 25th lowest gene BMC as the point of departure to compare the potencies of single PFAS to PFAS mixtures of varying complexity and composition. Specifically, the empirical potency of 8 PFAS mixtures were compared to predicted mixture potencies calculated using the principal of concentration addition (ie, dose addition) in which mixture component potencies are summed by proportion to predict mixture potency. In this study, for most mixtures, empirical mixture potencies were comparable to potencies calculated through concentration addition. This work supports that the effects of PFAS mixtures on gene expression largely follow the concentration addition predicted response and suggests that effects of these individual PFAS in mixtures are not strongly synergistic or antagonistic.

Integrated Genotoxicity Testing of three anti-infective drugs using the TGx-DDI transcriptomic biomarker and high-throughput CometChip® assay in TK6 cells.

Genotoxicity testing relies on the detection of gene mutations and chromosome damage and has been used in the genetic safety assessment of drugs and chemicals for decades. However, the results of standard genotoxicity tests are often difficult to interpret due to lack of mode of action information. The TGx-DDI transcriptomic biomarker provides mechanistic information on the DNA damage-inducing (DDI) capability of chemicals to aid in the interpretation of positive in vitro genotoxicity data. The CometChip® assay was developed to assess DNA strand breaks in a higher-throughput format. We paired the TGx-DDI biomarker with the CometChip® assay in TK6 cells to evaluate three model agents: nitrofurantoin (NIT), metronidazole (MTZ), and novobiocin (NOV). TGx-DDI was analyzed by two independent labs and technologies (nCounter® and TempO-Seq®). Although these anti-infective drugs are, or have been, used in human and/or veterinary medicine, the standard genotoxicity testing battery showed significant genetic safety findings. Specifically, NIT is a mutagen and causes chromosome damage, and MTZ and NOV cause chromosome damage in conventional in vitro tests. Herein, the TGx-DDI biomarker classified NIT and MTZ as non-DDI at all concentrations tested, suggesting that NIT's mutagenic activity is bacterial specific and that the observed chromosome damage by MTZ might be a consequence of in vitro test conditions. In contrast, NOV was classified as DDI at the second highest concentration tested, which is in line with the fact that NOV is a bacterial DNA-gyrase inhibitor that also affects topoisomerase II at high concentrations. The lack of DNA damage for NIT and MTZ was confirmed by the CometChip® results, which were negative for all three drugs except at overtly cytotoxic concentrations. This case study demonstrates the utility of combining the TGx-DDI biomarker and CometChip® to resolve conflicting genotoxicity data and provides further validation to support the reproducibility of the biomarker.

Germ-line mutations, DNA damage, and global hypermethylation in mice exposed to particulate air pollution in an urban/industrial location.

Particulate air pollution is widespread, yet we have little understanding of the long-term health implications associated with exposure. We investigated DNA damage, mutation, and methylation in gametes of male mice exposed to particulate air pollution in an industrial/urban environment. C57BL/CBA mice were exposed in situ to ambient air near two integrated steel mills and a major highway, alongside control mice breathing high-efficiency air particulate (HEPA) filtered ambient air. PCR analysis of an expanded simple tandem repeat (ESTR) locus revealed a 1.6-fold increase in sperm mutation frequency in mice exposed to ambient air for 10 wks, followed by a 6-wk break, compared with HEPA-filtered air, indicating that mutations were induced in spermatogonial stem cells. DNA collected after 3 or 10 wks of exposure did not exhibit increased mutation frequency. Bulky DNA adducts were below the detection threshold in testes samples, suggesting that DNA reactive chemicals do not reach the germ line and cause ESTR mutation. In contrast, DNA strand breaks were elevated at 3 and 10 wks, possibly resulting from oxidative stress arising from exposure to particles and associated airborne pollutants. Sperm DNA was hypermethylated in mice breathing ambient relative to HEPA-filtered air and this change persisted following removal from the environmental exposure. Increased germ-line DNA mutation frequencies may cause population-level changes in genetic composition and disease. Changes in methylation can have widespread repercussions for chromatin structure, gene expression and genome stability. Potential health effects warrant extensive further investigation.

Potency Ranking of Per- and Polyfluoroalkyl Substances Using High-Throughput Transcriptomic Analysis of Human Liver Spheroids.

Per- and polyfluoroalkyl substances (PFAS) are some of the most prominent organic contaminants in human blood. Although the toxicological implications of human exposure to perfluorooctane sulfonate (PFOS) and perfluorooctanoate (PFOA) are well established, data on lesser-understood PFAS are limited. New approach methodologies (NAMs) that apply bioinformatic tools to high-throughput data are being increasingly considered to inform risk assessment for data-poor chemicals. The aim of this study was to compare the potencies (ie, benchmark concentrations: BMCs) of PFAS in primary human liver microtissues (3D spheroids) using high-throughput transcriptional profiling. Gene expression changes were measured using TempO-seq, a templated, multiplexed RNA-sequencing platform. Spheroids were exposed for 1 or 10 days to increasing concentrations of 23 PFAS in 3 subgroups: carboxylates (PFCAs), sulfonates (PFSAs), and fluorotelomers and sulfonamides. PFCAs and PFSAs exhibited trends toward increased transcriptional potency with carbon chain-length. Specifically, longer-chain compounds (7-10 carbons) were more likely to induce changes in gene expression and have lower transcriptional BMCs. The combined high-throughput transcriptomic and bioinformatic analyses support the capability of NAMs to efficiently assess the effects of PFAS in liver microtissues. The data enable potency ranking of PFAS for human liver cell spheroid cytotoxicity and transcriptional changes, and assessment of in vitro transcriptomic points of departure. These data improve our understanding of the possible health effects of PFAS and will be used to inform read-across for human health risk assessment.

High-Throughput Transcriptomic Analysis of Human Primary Hepatocyte Spheroids Exposed to Per- and Polyfluoroalkyl Substances as a Platform for Relative Potency Characterization.

Per- and poly-fluoroalkyl substances (PFAS) are widely found in the environment because of their extensive use and persistence. Although several PFAS are well studied, most lack toxicity data to inform human health hazard and risk assessment. This study focused on 4 model PFAS: perfluorooctanoic acid (PFOA; 8 carbon), perfluorobutane sulfonate (PFBS; 4 carbon), perfluorooctane sulfonate (PFOS; 8 carbon), and perfluorodecane sulfonate (PFDS; 10 carbon). Human primary liver cell spheroids (pooled from 10 donors) were exposed to 10 concentrations of each PFAS and analyzed at 4 time points. The approach aimed to: (1) identify gene expression changes mediated by the PFAS, (2) identify similarities in biological responses, (3) compare PFAS potency through benchmark concentration analysis, and (4) derive bioactivity exposure ratios (ratio of the concentration at which biological responses occur, relative to daily human exposure). All PFAS induced transcriptional changes in cholesterol biosynthesis and lipid metabolism pathways, and predicted PPARα activation. PFOS exhibited the most transcriptional activity and had a highly similar gene expression profile to PFDS. PFBS induced the least transcriptional changes and the highest benchmark concentration (ie, was the least potent). The data indicate that these PFAS may have common molecular targets and toxicities, but that PFOS and PFDS are the most similar. The transcriptomic bioactivity exposure ratios derived here for PFOA and PFOS were comparable to those derived using rodent apical endpoints in risk assessments. These data provide a baseline level of toxicity for comparison with other known PFAS using this testing strategy.

Cross-platform analysis of global microRNA expression technologies.

BACKGROUND: Although analysis of microRNAs (miRNAs) by DNA microarrays is gaining in popularity, these new technologies have not been adequately validated. We examined within and between platform reproducibility of four miRNA array technologies alongside TaqMan PCR arrays. RESULTS: Two distinct pools of reference materials were selected in order to maximize differences in miRNA content. Filtering for miRNA that yielded signal above background revealed 54 miRNA probes (matched by sequence) across all platforms. Using this probeset as well as all probes that were present on an individual platform, within-platform analyses revealed Spearman correlations of >0.9 for most platforms. Comparing between platforms, rank analysis of the log ratios of the two reference pools also revealed high correlation (range 0.663-0.949). Spearman rank correlation and concordance correlation coefficients for miRNA arrays against TaqMan qRT-PCR arrays were similar for all of the technologies. Platform performances were similar to those of previous cross-platform exercises on mRNA and miRNA microarray technologies. CONCLUSIONS: These data indicate that miRNA microarray platforms generated highly reproducible data and can be recommended for the study of changes in miRNA expression.

Mainstream tobacco smoke causes paternal germ-line DNA mutation.

Despite the presence of known mutagens and carcinogens in cigarette smoke, there is currently no evidence to show that smoking, or exposure to cigarette smoke, can result in heritable genetic mutation. We show that male mice exposed to mainstream tobacco smoke (MTS) exhibit a significant increase in germ-line mutation frequency in spermatogonial stem cells. We exposed mature male mice to MTS for 6 or 12 weeks and investigated mutations arising in exposed spermatogonial stem cells at the expanded simple tandem repeat locus Ms6-hm. A generalized score test showed a significant treatment effect (P = 0.0214). Ms6-hm mutation frequency was 1.4 and 1.7 times higher in mice exposed to MTS for 6 and 12 weeks, respectively, compared with sham controls. The data suggest that mutations accumulate in the spermatogonial stem cells with extended exposures. Mutation spectra were identical between exposed and sham individuals, supporting the hypothesis that tandem repeat mutations arise through indirect mechanisms of mutation. Mutations in sperm that are passed on to offspring cause permanent, irreversible changes in genetic composition and can persist in future generations. Our research suggests that the consequences of smoking extend beyond the smoker to their nonsmoking descendents.

The use of in vitro transcriptional data to identify thresholds of effects in a human lens epithelial cell-line exposed to ionizing radiation.

PURPOSE: The International Commission on Radiological Protection (ICRP) recently recommended reducing the occupational equivalent dose limit for the lens of the eye. Based primarily on a review of epidemiological data, the absorbed dose threshold is now considered to be 0.5 Gy independent of dose-rate and severity of opacification, reduced from the previous threshold of 2 Gy. However, direct mechanistic evidence to support an understanding of the underlying molecular mechanisms of damage is still lacking. To this end, we explored the effects of a broad dose-range of ionizing radiation exposure on gene expression changes in a human lens epithelial (HLE) cell-line in order to better understand the shape of the dose-response relationship and identify transcriptional thresholds of effects. METHODS: HLE cells were exposed to doses of 0, 0.01, 0.05, 0.25, 0.5, 2, and 5 Gy of X-ray radiation at two dose rates (1.62 cGy/min and 38.2 cGy/min). Cell culture lysates were collected 20 h post-exposure and analyzed using whole-genome RNA-sequencing. Pathways and dose-thresholds of biological effects were identified using benchmark dose (BMD) modeling. RESULTS: Transcriptional responses were minimal at doses less than 2 Gy. At higher doses, there were a significant number of differentially expressed genes (DEGs) (p≤.05, fold change≥|1.5|) at both dose rates, with 1308 DEGs for the low dose rate (LDR) and 840 DEGs for the high dose rate (HDR) exposure. Dose-response modeling showed that a number of genes exhibited non-linear bi-phasic responses, which was verified by digital droplet PCR. BMD analysis showed the majority of the pathways responded at BMD median values in the dose range of 1.5-2.5 Gy, with the lowest BMD median value being 0.6 Gy for the HDR exposure. The minimum pathway BMD median value for LDR exposure, however, was 2.5 Gy. Although the LDR and HDR exposures shared pathways involved in extracellular matrix reorganization and collagen production with BMD median value of 2.9 Gy, HDR exposures were more effective in activating pathways associated with DNA damage response, apoptosis, and cell cycling relative to LDR exposure. CONCLUSIONS: Overall, the results suggest that radiation induces complex non-linear transcriptional dose-response relationships that are dose-rate dependent. Pathways shared between the two dose rates may be important contributors to radiation-induced cataractogenesis. BMD analysis suggests that the majority of pathways are activated above 0.6 Gy, which supports current ICRP identified dose thresholds for deterministic effects to the lens of the eye of 0.5 Gy.

Hepatic transcriptional dose-response analysis of male and female Fischer rats exposed to hexabromocyclododecane.

Hexabromocyclododecane (HBCD) is a brominated flame retardant found in the environment and human tissues. The toxicological effects of HBCD exposure are not clearly understood. We employed whole-genome RNA-sequencing on liver samples from male and female Fischer rats exposed to 0, 250, 1250, and 5000 mg technical mixture of HBCD/kg diet for 28 days to gain further insight into HBCD toxicity. HBCD altered 428 and 250 gene transcripts in males and females, respectively, which were involved in metabolism of xenobiotics, oxidative stress, immune response, metabolism of glucose and lipids, circadian regulation, cell cycle, fibrotic activity, and hormonal balance. Signature analysis supported that HBCD operates through the constitutive androstane and pregnane X receptors. The median transcriptomic benchmark dose (BMD) for the lowest statistically significant pathway was within 1.5-fold of the BMD for increased liver weight, while the BMD for the lowest pathway with at least three modeled genes (minimum 5% of pathway) was similar to the lowest apical endpoint BMD. The results show how transcriptional analyses can inform mechanisms underlying chemical toxicity and the doses at which potentially adverse effects occur. This experiment is part of a larger study exploring the use of toxicogenomics and high-throughput screening for human health risk assessment.

Paternal exposure to benzo(a)pyrene induces genome-wide mutations in mouse offspring.

Understanding the effects of environmental exposures on germline mutation rates has been a decades-long pursuit in genetics. We used next-generation sequencing and comparative genomic hybridization arrays to investigate genome-wide mutations in the offspring of male mice exposed to benzo(a)pyrene (BaP), a common environmental pollutant. We demonstrate that offspring developing from sperm exposed during the mitotic or post-mitotic phases of spermatogenesis have significantly more de novo single nucleotide variants (1.8-fold; P < 0.01) than controls. Both phases of spermatogenesis are susceptible to the induction of heritable mutations, although mutations arising from post-fertilization events are more common after post-mitotic exposure. In addition, the mutation spectra in sperm and offspring of BaP-exposed males are consistent. Finally, we report a significant increase in transmitted copy number duplications (P = 0.001) in BaP-exposed sires. Our study demonstrates that germ cell mutagen exposures induce genome-wide mutations in the offspring that may be associated with adverse health outcomes.

Tandem repeat mutation, global DNA methylation, and regulation of DNA methyltransferases in cultured mouse embryonic fibroblast cells chronically exposed to chemicals with different modes of action.

Mutations at expanded simple tandem repeat (ESTR) DNA sequences provide a useful tool for screening germline mutation. However, the mechanisms resulting in induced mutations are unknown and provide an impediment to the utility of the method. Induced ESTR mutations arise through a nontargeted mechanism resulting in destabilization of the repeat locus. We hypothesized that alterations in DNA methylation, or in DNA methyltransferase expression, may be associated with this indirect mechanism of mutation. DNA mutation frequency was measured in C3H/10T1/2 mouse embryonic fibroblast cells following chronic exposure to six chemicals exhibiting different modes of genotoxic action: N-nitroso-N-ethylurea (ENU); benzo(a)pyrene (BaP); etoposide (ETOP); okadaic acid (OA); cisplatin (CisPt); and 5-azacytidine (5azadC). Induced mutation ranged from 2-fold (ENU, BaP, ETOP), to 1.3-1.4 fold (OA, 5azadC), to nonresponsive (CisPt). Global DNA methylation, measured using the cytosine extension assay, revealed hypomethylation following exposure to ENU and 5azadC, hypermethylation following BaP and OA exposure, and no change following treatment with ETOP or CisPt. DNA methyltransferase transcription (Dnmt1, Dnmt3a, Dnmt3b) was significantly affected by all treatments except ETOP, with the vast majority of changes being downregulation. There was no direct correlation between ESTR mutation, global methylation, or DNA methyltransferase transcription. However, 4/5 ESTR mutagens caused changes in global methylation, while the noninducer (CisPt) did not cause changes in methylation. We hypothesize that chemicals that modify chromatin conformation through changes in methylation may compromise the ability of mismatch repair enzymes (or other enzymes) to access and repair secondary structures that may form across ESTR loci resulting in mutation.