Folate Intake Alters Mutation Frequency and Profiles in a Tissue- and Dose-Specific Manner in MutaMouse Male Mice.

BACKGROUND: While cancer is common, its incidence varies widely by tissue. These differences are attributable to variable risk factors, such as environmental exposure, genetic inheritance, and lifetime number of stem cell divisions in a tissue. Folate deficiency is generally associated with increased risk for colorectal cancer (CRC) and acute lymphocytic leukemia (ALL). Conversely, high folic acid (FA) intake has also been associated with higher CRC risk. OBJECTIVE: Our objective was to compare the effect of folate intake on mutant frequency (MF) and types of mutations in the colon and bone marrow of mice. METHODS: Five-week-old MutaMouse male mice were fed a deficient (0 mg FA/kg), control (2 mg FA/kg), or supplemented (8 mg FA/kg) diet for 20 wk. Tissue MF was assessed using the lacZ mutant assay and comparisons made by 2-factor ANOVA. LacZ mutant plaques were sequenced using next-generation sequencing, and diet-specific mutation profiles within each tissue were compared by Fisher's exact test. RESULTS: In the colon, the MF was 1.5-fold and 1.3-fold higher in mice fed the supplemented diet compared with mice fed the control (P = 0.001) and deficient (P = 0.008) diets, respectively. This contrasted with the bone marrow MF in the same mice where the MF was 1.7-fold and 1.6-fold higher in mice fed the deficient diet compared with mice fed the control (P = 0.02) and supplemented (P = 0.03) diets, respectively. Mutation profiles and signatures (mutation context) were tissue-specific. CONCLUSIONS: Our data indicate that dietary folate intake affects mutagenesis in a tissue- and dose-specific manner in mice. Mutation profiles were generally tissue- but not dose-specific, suggesting that altered cellular folate status appears to interact with endogenous mutagenic mechanisms in each tissue to create a permissive context in which specific mutation types accumulate. These data illuminate potential mechanisms underpinning differences in observed associations between folate intake/status and cancer.

Transcriptomic pathway and benchmark dose analysis of Bisphenol A, Bisphenol S, Bisphenol F, and 3,3',5,5'-Tetrabromobisphenol A in H9 human embryonic stem cells.

Bisphenol A (BPA) is a chemical used in the manufacturing of plastics to which human exposure is ubiquitous. Numerous studies have linked BPA exposure to many adverse health outcomes prompting the replacement of BPA with various analogues including bisphenol-F (BPF) and bisphenol S (BPS). Other bisphenols are used in various consumer applications, such as 3,3',5,5'-Tetrabromobisphenol A (TBBPA), which is used as a flame retardant. Few studies to date have examined the effects of BPA and its analogues in stem cells to explore potential developmental impacts. Here we used transcriptomics to investigate similarities and differences of BPA and three of its analogues in the estrogen receptor negative, human embryonic stem cell line H9 (WA09). H9 cells were exposed to increasing concentrations of the bisphenols and analyzed using RNA-sequencing. Our data indicate that BPA, BPF, and BPS have similar potencies in inducing transcriptional changes and perturb many of the same pathways. TBBPA, the least structurally similar bisphenol of the group, exhibited much lower potency. All bisphenols robustly impacted gene expression in these cells, albeit at concentrations well above those observed in estrogen-positive cells. Overall, we provide a foundational data set against which to explore the transcriptional similarities of other bisphenols in embryonic stem cells, which may be used to assess the suitability of chemical grouping for read-across and for preliminary potency evaluation.

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.

Transcriptional profiling of male CD-1 mouse lungs and Harderian glands supports the involvement of calcium signaling in acrylamide-induced tumors.

Acrylamide (AA) exposure causes increased incidence of forestomach, lung, and Harderian gland tumors in male mice. One hypothesized mode of action (MOA) for AA-carcinogenicity includes genotoxicity/mutagenicity as a key event, possibly resulting from AA metabolism to the direct genotoxic metabolite glycidamide. Alternatively, altered calcium signaling (CS) has been proposed as a central key event in the MOA. To examine the plausibility of these proposed MOAs, RNA-sequencing was performed on tumor target tissues: Harderian glands (the most sensitive tumor target tissue in the rodent 2-year cancer bioassay) and lungs of AA-exposed male CD-1 mice. Animals were exposed to 0.0, 1.5, 3.0, 6.0, 12.0, or 24.0 mg AA/kg bw-day in drinking water for 5, 15, or 31 days. We observed a pronounced effect on genes involved in CS and cytoskeletal processes in both tissues, but no evidence supporting a genotoxic MOA. Benchmark dose modeling suggests transcriptional points of departure (PODs) of 0.54 and 2.21 mg/kg bw-day for the Harderian glands and lungs, respectively. These are concordant with PODs of 0.17 and 1.27 mg/kg bw-day derived from the cancer bioassay data for these tissues in male mice, respectively. Overall, this study supports the involvement of CS in AA-induced mouse carcinogenicity, which is consistent with a recently proposed CS-based MOA in rat thyroid, and with other published reports of aberrant CS in malignant tumors in rodents and humans.

Transcriptional profiling of male F344 rats suggests the involvement of calcium signaling in the mode of action of acrylamide-induced thyroid cancer.

Acrylamide (AA) exposure in 2-year cancer bioassays leads to thyroid, but not liver, adenomas and adenocarcinomas in rats. Hypothesized modes of action (MOAs) include genotoxicity/mutagenicity, or thyroid hormone dysregulation. To examine the plausibility of these two or any alternative MOAs, RNA-sequencing was performed on the thyroids and livers of AA-exposed rats, in parallel with measurement of genotoxicity (blood micronucleus and Pig-a mutant frequency) and serum thyroid hormone levels, following the exposure of male Fischer 344/DuCrl rats to 0.0, 0.5, 1.5, 3.0, 6.0, or 12.0 mg AA/kg bw-day in drinking water for 5, 15, or 31 days. Differentially expressed genes in both tissues provided marginal support for hormonal and genotoxic MOAs, which was consistent with negative/equivocal genotoxicity assay and marginal changes in thyroid hormone levels. Instead, there was a pronounced effect on calcium signaling/cytoskeletal genes in the thyroid. Benchmark dose modeling of RNA-sequencing data for the calcium signaling pathway suggests a point of departure (POD) of 0.68 mg/kg bw-day, which is consistent with a POD of 0.82 mg/kg bw-day derived from the thyroid 2-year cancer bioassay data. Overall, this study suggests a novel MOA for AA-induced thyroid carcinogenicity in male rats centered around perturbation of calcium signaling.

Genetic Toxicity of Complex Mixtures of Polycyclic Aromatic Hydrocarbons: Evaluating Dose-Additivity in a Transgenic Mouse Model.

This study evaluates the risk assessment approach currently employed for polycyclic aromatic hydrocarbon (PAH)-contaminated media, wherein carcinogenic hazards are evaluated using a dose-addition model that employs potency equivalency factors (PEFs) for targeted carcinogenic PAHs. Here, MutaMouse mice were subchronically exposed to PAH mixtures (p.o.), and mutagenic potency (MP) values were determined for five tissues. Predicted dose-additive mixture MPs were generated by summing the products of the concentrations and MPs of the individual targeted PAHs; values were compared to the experimental MPs of the mixtures to evaluate dose-additivity. Additionally, the PEF-determined BaP-equivalent concentrations were compared to those determined using a bioassay-derived method (BDM) (i.e., an additivity-independent approach). In bone marrow, mixture mutagenicity was less than dose-additive and the PEF-method provided higher estimates of BaP-equivalents than the BDM. Conversely, mixture mutagenicity in site-of-contact tissues (e.g., small intestine) was generally more than dose-additive and the PEF-method provided lower estimates of BaP-equivalents than the BDM. Overall, this study demonstrates that dose-additive predictions of mixture mutagenic potency based on the concentrations and potencies of a small number of targeted PAHs results in values that are surprisingly close to those determined experimentally, providing support for the dose-additive assumption employed for human health risk assessment of PAH mixtures.

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 (32) P-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. © 2016 Her Majesty the Queen in Right of Canada. Environmental and Molecular Mutagenesis © 2016 Environmental Mutagen Society.

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.

Adipogenic Mesenchymal Stromal Cells from Bone Marrow and Their Hematopoietic Supportive Role: Towards Understanding the Permissive Marrow Microenvironment in Acute Myeloid Leukemia.

PURPOSE: The role of bone marrow-derived mesenchymal stem/stromal cells (MSCs) in creating a permissive microenvironment that supports the emergence and progression of acute myeloid leukemia (AML) is not well established. We investigated the extent to which adipogenic differentiation in normal MSCs alters hematopoietic supportive capacity and we undertook an in-depth comparative study of human bone marrow MSCs derived from newly diagnosed AML patients and healthy donors, including an assessment of adipogenic differentiation capacity. FINDINGS: MSCs from healthy controls with partial induction of adipogenic differentiation, in comparison to MSCs undergoing partial osteogenic differentiation, expressed increased levels of hematopoietic factors and induced greater proliferation, decreased quiescence and reduced in vitro hematopoietic colony forming capacity of CD34(+) hematopoietic stem and progenitor cells (HSPCs). Moreover, we observed that AML-derived MSCs had markedly increased adipogenic potential and delayed osteogenic differentiation, while maintaining normal morphology and viability. AML-derived MSCs, however, possessed reduced proliferative capacity and decreased frequency of subendothelial quiescent MSCs compared to controls. CONCLUSION: Our results support the notion of a bone marrow microenvironment characterized by increased propensity toward adipogenesis in AML, which may negatively impact normal hematopoiesis. Larger confirmatory studies are needed to understand the impact of various clinical factors. Novel leukemia treatments aimed at normalizing bone marrow niches may enhance the competitive advantage of normal hematopoietic progenitors over leukemia cells.

HPLC Measurement of the DNA Oxidation Biomarker, 8-oxo-7,8-dihydro-2'-deoxyguanosine, in Cultured Cells and Animal Tissues.

Oxidative stress is associated with many physiological and pathological processes, as well as xenobiotic metabolism, leading to the oxidation of biomacromolecules, including DNA. Therefore, efficient detection of DNA oxidation is important for a variety of research disciplines, including medicine and toxicology. A common biomarker of oxidatively damaged DNA is 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dGuo; often erroneously referred to as 8-hydroxy-2'-deoxyguanosine (8-OH-dGuo or 8-oxo-dG)). Several protocols for 8-oxo-dGuo measurement by high pressure liquid chromatography with electrochemical detection (HPLC-ED) have been described. However, these were mainly applied to purified DNA treated with pro-oxidants. In addition, due to methodological differences between laboratories, mainly due to differences in analytical equipment, the adoption of published methods for detection of 8-oxo-dGuo by HPLC-ED requires careful optimization by each laboratory. A comprehensive protocol, describing such an optimization process, is lacking. Here, a detailed protocol is described for the detection of 8-oxo-dGuo by HPLC-ED, in DNA from cultured cells or animal tissues. It illustrates how DNA sample preparation can be easily and rapidly optimized to minimize undesirable DNA oxidation that can occur during sample preparation. This protocol shows how to detect 8-oxo-dGuo in cultured human alveolar adenocarcinoma cells (i.e., A549 cells) treated with the oxidizing agent KBrO3, and from the spleen of mice exposed to the polycyclic aromatic hydrocarbon dibenzo(def,p)chrysene (DBC, formerly known as dibenzo(a,l)pyrene, DalP). Overall, this work illustrates how an HPLC-ED methodology can be readily optimized for the detection of 8-oxo-dGuo in biological samples.

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.

Mutagenic and carcinogenic hazards of settled house dust. I: Polycyclic aromatic hydrocarbon content and excess lifetime cancer risk from preschool exposure.

Settled house dust (SHD) may be a significant source of children's indoor exposure to hazardous substances including polycyclic aromatic hydrocarbons (PAHs). In this study, organic extracts of sieved vacuum cleaner dust from 51 homes were examined for the presence of 13 PAHs via GC/MS. PAHs were found in all samples with levels of total PAHs ranging between 1.5 and 325 microg g(-1). The PAH concentrations in the SHD were correlated with information contained in corresponding household questionnaires. Analyses showed levels of PAHs to be negatively associated with noncombustion activities such as vacuum cleaning frequency. A risk assessment was conducted to evaluate the excess lifetime cancer risks posed to preschool aged children who ingested PAHs in SHD. The assessment revealed that exposure to PAHs at levels found in 90% of the homes (< 40 microg g(-1)) would result in excess cancer risks that are considered acceptable (i.e., 1-100 x 10(-6)). However, exposure to higher levels of PAHs found in five homes yielded risks that could be higher than 1 x 10(-4).

Mutagenic and carcinogenic hazards of settled house dust. II: Salmonella mutagenicity.

Settled house dust (SHD) is a complex mixture that contains numerous chemical contaminants. Very little is known about the hazards of SHD as compared to other complex matrices such as air and soil. In this study, the mutagenic hazards associated with the extracts of sieved dust from 52 homes were examined using the Salmonella Mutagenicity Test. All of the SHD samples displayed mutagenic activity and the mean mutagenic potencies ranged from 2300to 23 600 revertants per gram. Testing with various Salmonella strains revealed a predominance of frameshift mutagens in the dust samples. Analyses showed that polycyclic aromatic hydrocarbons (PAHs) were likely responsible for a quarter of the mutagenic activity of the SHD samples. In an effort to identify factors that influenced dust mutagenicity, the relationships between SHD mutagenicity and household activities were investigated. Mutagenicity was positively correlated with parameters such as the time since last vacuuming (r2 = 0.11, p < 0.05) and the number of people living in the home (r2 = 0.11-0.43, p < 0.05). However, the causative factors responsible for these relationships remain unclear.