lacZ Mutant Frequency Research Articles

Lack of in vivo mutagenicity of carbendazim in the liver and glandular stomach of MutaMice

BackgroundCarbendazim (methyl 2-benzimidazolecarbamate, CASRN: 10605-21-7) exhibits spindle poisoning effects and is widely used as a fungicide. With respect to genotoxicity, carbendazim is deemed to be non-mutagenic in vitro, but it causes indicative DNA damage in vivo and chromosome aberrations in vitro and in vivo. In this study, we examined the mutagenicity of carbendazim in vivo.ResultsMutaMice were treated with carbendazim orally at doses of 0 (corn oil), 250, 500, and 1,000 mg/kg/day once a day for 28 days. A lacZ assay was used to determine the mutant frequency (MF) in the liver and glandular stomach of mice. MutaMice were administered up to the maximum dose recommended by the Organization for Economic Co-operation and Development Test Guidelines for Chemicals No. 488 (OECD TG488). The lacZ MFs in the liver and glandular stomach of carbendazim-treated animals were not significantly different from those in the negative control animals. In contrast, positive control animals exhibited a significant increase in MFs in both the liver and glandular stomach.ConclusionsCarbendazim is non-mutagenic in the liver and glandular stomach of MutaMice following oral treatment.

N-Nitrosodimethylamine investigations in Muta™Mouse define point-of-departure values and demonstrate less-than-additive somatic mutant frequency accumulations.

The N-nitrosamine, N-nitrosodimethylamine (NDMA), is an environmental mutagen and rodent carcinogen. Small levels of NDMA have been identified as an impurity in some commonly used drugs, resulting in several product recalls. In this study, NDMA was evaluated in an OECD TG-488 compliant Muta™Mouse gene mutation assay (28-day oral dosing across seven daily doses of 0.02-4 mg/kg/day) using an integrated design that assessed mutation at the transgenic lacZ locus in various tissues and at the endogenous Pig-a gene-locus, along with micronucleus frequencies in peripheral blood. Liver pathology was determined together with NDMA exposure in blood and liver. The additivity of mutation induction was assessed by including two acute single-dose treatment groups (i.e. 5 and 10 mg/kg dose on Day 1), which represented the same total dose as two of the repeat dose treatment groups. NDMA did not induce statistically significant increases in mean lacZ mutant frequency (MF) in bone marrow, spleen, bladder, or stomach, nor in peripheral blood (Pig-a mutation or micronucleus induction) when tested up to 4 mg/kg/day. There were dose-dependent increases in mean lacZ MF in the liver, lung, and kidney following 28-day repeat dosing or in the liver and kidney after a single dose (10 mg/kg). No observed genotoxic effect levels (NOGEL) were determined for the positive repeat dose-response relationships. Mutagenicity did not exhibit simple additivity in the liver since there was a reduction in MF following NDMA repeat dosing compared with acute dosing for the same total dose. Benchmark dose modelling was used to estimate point of departure doses for NDMA mutagenicity in Muta™Mouse and rank order target organ tissue sensitivity (liver > kidney or lung). The BMD50 value for liver was 0.32 mg/kg/day following repeat dosing (confidence interval 0.21-0.46 mg/kg/day). In addition, liver toxicity was observed at doses of ≥ 1.1 mg/kg/day NDMA and correlated with systemic and target organ exposure. The integration of these results and their implications for risk assessment are discussed.

In vitro mutagenicity of selected environmental carcinogens and their metabolites in MutaMouse FE1 lung epithelial cells.

Chemicals in commerce or under development must be assessed for genotoxicity; assessment is generally conducted using validated assays (e.g. Tk mouse lymphoma assay) as part of a regulatory process. Currently, the MutaMouse FE1 cell mutagenicity assay is undergoing validation for eventual use as a standard in vitro mammalian mutagenicity assay. FE1 cells have been shown to be metabolically competent with respect to some cytochrome P450 (CYP) isozymes; for instance, they can convert the human carcinogen benzo[a]pyrene into its proximate mutagenic metabolite. However, some contradictory results have been noted for other genotoxic carcinogens that require two-step metabolic activation (e.g. 2-acetylaminofluorene and 2-amino-3-methylimidazo[4,5-f]quinoxaline). Here, we examined three known or suspected human carcinogens, namely acrylamide, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and 4-aminobiphenyl (4-ABP), together with their proximate metabolites (i.e. glycidamide, N-OH-PhIP and N-OH-4-ABP), to aid in the validation of the FE1 cell mutagenicity assay. Assessments of the parent compounds were conducted both in the presence and absence of an exogenous metabolic activation mixture S9; assessments of the metabolites were in the absence of S9. The most potent compound was N-OH-PhIP -S9, which elicited a mutant frequency (MF) level 5.3-fold over background at 5 µM. There was a 4.3-fold increase for PhIP +S9 at 5 µM, a 1.7-fold increase for glycidamide -S9 at 3.5 mM and a 1.5-fold increase for acrylamide +S9 at 4 mM. Acrylamide -S9 elicited a marginal 1.4-fold MF increase at 8 mM. Treatment with PhIP -S9, 4-ABP ±S9 and N-OH-4-ABP -S9 failed to elicit significant increases in lacZ MF with any of the treatment conditions tested. Gene expression of key CYP isozymes was quantified by RT-qPCR. Cyp1a1, 1a2 and 1b1 are required to metabolise PhIP and 4-ABP. Results showed that treatment with both compounds induced expression of Cyp1a1 and Cyp1b1 but not Cyp1a2. Cyp2e1, which catalyses the bioactivation of acrylamide to glycidamide, was not induced after acrylamide treatment. Overall, our results confirm that the FE1 cell mutagenicity assay has the potential for use alongside other, more traditional in vitro mutagenicity assays.

Mutagenicity of 2-hydroxyamino-1-methyl-6-phenylimidazo[4,5-b]pyridine (N–OH-PhIP) in human TP53 knock-in (Hupki) mouse embryo fibroblasts

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is a possible human carcinogen formed in cooked fish and meat. PhIP is bioactivated by cytochrome P450 enzymes to form 2-hydroxyamino-1-methyl-6-phenylimidazo[4,5-b]pyridine (N–OH-PhIP), a genotoxic metabolite that reacts with DNA leading to the mutation-prone DNA adduct N-(deoxyguanosin-8-yl)-PhIP (dG-C8-PhIP). Here, we studied N–OH-PhIP-induced whole genome mutagenesis in human TP53 knock-in (Hupki) mouse embryo fibroblasts (HUFs) immortalised and subjected to whole genome sequencing (WGS). In addition, mutagenicity of N–OH-PhIP in TP53 and the lacZ reporter gene were assessed. TP53 mutant frequency in HUF cultures treated with N–OH-PhIP (2.5 μM for 24 h, n = 90) was 10% while no TP53 mutations were found in untreated controls (DMSO for 24 h, n = 6). All N–OH-PhIP-induced TP53 mutations occurred at G:C base pairs with G > T/C > A transversions accounting for 58% of them. TP53 mutations characteristic of those induced by N–OH-PhIP have been found in human tumours including breast and colorectal, which are cancer types that have been associated with PhIP exposure. LacZ mutant frequency increased 25-fold at 5 μM N–OH–PHIP and up to ~350 dG-C8-PhIP adducts/108 nucleosides were detected by ultra-performance liquid chromatography-electrospray ionisation multistage scan mass spectrometry (UPLC-ESI-MS3) at this concentration. In addition, a WGS mutational signature defined by G > T/C > A transversions was present in N–OH-PhIP-treated immortalised clones, which showed similarity to COSMIC SBS4, 18 and 29 signatures found in human tumours.

Integrated In Vivo Genotoxicity Assessment of Procarbazine Hydrochloride Demonstrates Induction of Pig-a and LacZ Mutations, and Micronuclei, in MutaMouse Hematopoietic Cells.

Procarbazine hydrochloride (PCH) is a DNA‐reactive hematopoietic carcinogen with potent and well‐characterized clastogenic activity. However, there is a paucity of in vivo mutagenesis data for PCH, and in vitro assays often fail to detect the genotoxic effects of PCH due to the complexity of its metabolic activation. We comprehensively evaluated the in vivo genotoxicity of PCH on hematopoietic cells of male MutaMouse transgenic rodents using a study design that facilitated assessments of micronuclei and Pig‐a mutation in circulating erythrocytes, and lacZ mutant frequencies in bone marrow. Mice were orally exposed to PCH (0, 6.25, 12.5, and 25 mg/kg/day) for 28 consecutive days. Blood samples collected 2 days after cessation of treatment exhibited significant dose‐related induction of micronuclei in both immature and mature erythrocytes. Bone marrow and blood collected 3 and 70 days after cessation of treatment also showed significantly elevated mutant frequencies in both the lacZ and Pig‐a assays even at the lowest dose tested. PCH‐induced lacZ and Pig‐a (immature and mature erythrocytes) mutant frequencies were highly correlated, with R2 values ≥0.956, with the exception of lacZ vs. Pig‐a mutants in mature erythrocytes at the 70‐day time point (R2 = 0.902). These results show that PCH is genotoxic in vivo and demonstrate that the complex metabolism and resulting genotoxicity of PCH is best evaluated in intact animal models. Our results further support the concept that multiple biomarkers of genotoxicity, especially hematopoietic cell genotoxicity, can be readily combined into one study provided that adequate attention is given to manifestation times. Environ. Mol. Mutagen. 60:505–512, 2019. © 2018 Her Majesty the Queen in Right of Canada

In Utero Exposure to Benzo[a]pyrene Induces Ovarian Mutations at Doses That Deplete Ovarian Follicles in Mice.

Polycyclic aromatic hydrocarbons like benzo[a]pyrene (BaP) are ubiquitous environmental contaminants formed during incomplete combustion of organic materials. Our prior work showed that transplacental exposure to BaP depletes ovarian follicles and increases prevalence of epithelial ovarian tumors later in life. We used the MutaMouse transgenic rodent model to address the hypothesis that ovarian mutations play a role in tumorigenesis caused by prenatal exposure to BaP. Pregnant MutaMouse females were treated with 0, 10, 20, or 40 mg/(kg day) BaP orally on gestational days 7-16, covering critical windows of ovarian development. Female offspring were euthanized at 10 weeks of age; some ovaries with oviducts were processed for follicle counting; other ovaries/oviducts and bone marrow were processed for determination of lacZ mutant frequency (MF). Mutant plaques were pooled within dose groups and sequenced to determine the mutation spectrum. BaP exposure caused highly significant dose-related decreases in ovarian follicles and increases in ovarian/oviductal and bone marrow mutant frequencies at all doses. Absence of follicles, cell packets, and epithelial tubular structures were observed with 20 and 40 mg/(kg day) BaP. Depletion of ovarian germ cells was inversely associated with ovarian MF. BaP induced primarily G > T and G > C transversions and deletions in ovaries/oviducts and bone marrow cells and produced a mutation signature highly consistent with that of tobacco smoking in human cancers. Overall, our results show that prenatal BaP exposure significantly depletes ovarian germ cells, causes histopathological abnormalities, and increases the burden of ovarian/oviductal mutations, which may be involved in pathogenesis of epithelial ovarian tumors. Environ. Mol. Mutagen. 60:410-420, 2019. © 2018 Her Majesty the Queen in Right of Canada.

Folate deficiency increases chromosomal damage and mutations in hematopoietic cells in the transgenic mutamouse model

Folate deficiency causes megaloblastic anemia and neural tube defects, and is also associated with some cancers. In vitro, folate deficiency increases mutation frequency and genome instability, as well as exacerbates the mutagenic potential of known environmental mutagens. Conversely, it remains unclear whether or not elevated folic acid (FA) intakes are beneficial or detrimental to the induction of DNA mutations and by proxy human health. We used the MutaMouse transgenic model to examine the in vivo effects of FA deficient, control, and supplemented diets on somatic DNA mutant frequency (MF) and genome instability in hematopoietic cells. We also examined the interaction between FA intake and exposure to the known mutagen N-ethyl-N-nitrosourea (ENU) on MF. Male mice were fed the experimental diets for 20 weeks from weaning. Half of the mice from each diet group were gavaged with 50 mg/kg body weight ENU after 10 weeks on diet and remained on their respective diet for an additional 10 weeks. Mice fed a FA-deficient diet had a 1.3-fold increase in normochromatic erythrocyte micronucleus (MN) frequency (P = 0.034), and a doubling of bone marrow lacZ MF (P = 0.035), compared to control-fed mice. Mice exposed to ENU showed significantly higher bone marrow lacZ and Pig-a MF, but there was no effect of FA intake on ENU-induced MF. These data indicate that FA deficiency increases mutations and MN formation in highly proliferative somatic cells, but that FA intake does not mitigate ENU-induced mutations. Also, FA intake above adequacy had no beneficial or detrimental effect on mutations or MN formation. Environ. Mol. Mutagen. 59:366-374, 2018. © 2018 Her Majesty the Queen in Right of Canada 2018.

Benchmark dose analyses of multiple genetic toxicity endpoints permit robust, cross-tissue comparisons of MutaMouse responses to orally delivered benzo[a]pyrene

Genetic damage is a key event in tumorigenesis, and chemically induced genotoxic effects are a human health concern. Although genetic toxicity data have historically been interpreted using a qualitative screen-and-bin approach, there is increasing interest in quantitative analysis of genetic toxicity dose–response data. We demonstrate an emerging use of the benchmark dose (BMD)-approach for empirically ranking cross-tissue sensitivity. Using a model environmental carcinogen, we quantitatively examined responses for four genetic damage endpoints over an extended dose range, and conducted cross-tissue sensitivity rankings using BMD100 values and their 90% confidence intervals (CIs). MutaMouse specimens were orally exposed to 11 doses of benzo[a]pyrene. DNA adduct frequency and lacZ mutant frequency (MF) were measured in up to 8 tissues, and Pig-a MF and micronuclei (MN) were assessed in immature (RETs) and mature red blood cells (RBCs). The cross-tissue BMD pattern for lacZ MF is similar to that observed for DNA adducts, and is consistent with an oral route-of-exposure and differences in tissue-specific metabolism and proliferation. The lacZ MF BMDs were significantly correlated with the tissue-matched adduct BMDs, demonstrating a consistent adduct conversion rate across tissues. The BMD CIs, for both the Pig-a and the MN endpoints, overlapped for RETs and RBCs, suggesting comparable utility of both cell populations for protracted exposures. Examination of endpoint-specific response maxima illustrates the difficulty of comparing BMD values for a fixed benchmark response across endpoints. Overall, the BMD-approach permitted robust comparisons of responses across tissues/endpoints, which is valuable to our mechanistic understanding of how benzo[a]pyrene induces genetic damage.

Multi-walled carbon nanotube-induced genotoxic, inflammatory and pro-fibrotic responses in mice: Investigating the mechanisms of pulmonary carcinogenesis

The International Agency for Research on Cancer has classified one type of multi-walled carbon nanotubes (MWCNTs) as possibly carcinogenic to humans. However, the underlying mechanisms of MWCNT- induced carcinogenicity are not known. In this study, the genotoxic, mutagenic, inflammatory, and fibrotic potential of MWCNTs were investigated. Muta™Mouse adult females were exposed to 36±6 or 109±18μg/mouse of Mitsui-7, or 26±2 or 78±5μg/mouse of NM-401, once a week for four consecutive weeks via intratracheal instillations, alongside vehicle-treated controls. Samples were collected 90days following the first exposure for measurement of DNA strand breaks, lacZ mutant frequency, p53 expression, cell proliferation, lung inflammation, histopathology, and changes in global gene expression. Both MWCNT types persisted in lung tissues 90days post-exposure, and induced lung inflammation and fibrosis to similar extents. However, there was no evidence of DNA damage as measured by the comet assay following Mitsui-7 exposure, or increases in lacZ mutant frequency, for either MWCNTs. Increased p53 expression was observed in the fibrotic foci induced by both MWCNTs. Gene expression analysis revealed perturbations of a number of biological processes associated with cancer including cell death, cell proliferation, free radical scavenging, and others in both groups, with the largest response in NM-401-treated mice. The results suggest that if the two MWCNT types were capable of inducing DNA damage, strong adaptive responses mounted against the damage, resulting in efficient and timely elimination of damaged cells through cell death, may have prevented accumulation of DNA damage and mutations at the post-exposure time point investigated in the study. Thus, MWCNT-induced carcinogenesis may involve ongoing low levels of DNA damage in an environment of persisting fibres, chronic inflammation and tissue irritation, and parallel increases or decreases in the expression of genes involved in several pro-carcinogenic pathways.

Quantitative relationships between lacZ mutant frequency and DNA adduct frequency in Muta™Mouse tissues and cultured cells exposed to 3-nitrobenzanthrone.

The frequency of stable DNA adducts in a target tissue can be used to assess biologically effective dose; however, the utility of the metric in a risk assessment context depends on the likelihood that the DNA damage will be manifested as mutation. Previously, we employed the Muta™Mouse system to examine the induction of lacZ mutants and DNA adducts following exposure to the well-studied mutagenic carcinogen 3-nitrobenzanthrone (3-NBA). In this follow-up work, we examined the empirical relationships between total adduct frequency and mutant frequency (MF) in tissues and cultured cells following acute 3-NBA exposure. The results show a significant induction of DNA damage and lacZ mutants in liver, colon and bone marrow, as well as FE1 pulmonary epithelial cells. In contrast, lung and small intestine samples had low, but significantly elevated adduct levels, with no significant increases in lacZ MF. Additional analyses showed a significant relationship between the mutagenic efficiency of total adducts, measured as the slope of the relationships between MF and total adduct frequency, and tissue-specific mitotic index (MI). The lack of mutation response in lung, in contrast to the high in vitro MF in FE-1 lung cells, is likely related to the 100-fold difference in MI. The lack of small intestine mutagenic response may be related to limited metabolic capacity, differences in DNA repair, and /or chemically induced apoptosis that has been observed for other potent mutagens. The results indicate that interpretation of adduct frequency values in a risk assessment context can be improved by considering the MI of the target tissue; however, more generalised interpretation is hampered by tissue-specific variations in metabolic capacity and damage processing. The work provides a proof of principle regarding the use of the Muta™Mouse system to critically examine the health risks associated with tissue-specific adduct loads.

Next generation sequencing of benzo(a)pyrene-induced lacZ mutants identifies a germ cell-specific mutation spectrum.

De novo mutations are implicated in a variety of genetic diseases and arise primarily in the male germline. We investigated whether male germ cells have unique mechanisms for spontaneous or chemically-induced mutation relative to somatic cells using the MutaMouse model. We recovered lacZ transgenes from sperm 42 days after a 28-day exposure to benzo(a)pyrene (BaP, 100 mg/kg/day) to assess mutations arising in dividing spermatogonia. BaP caused a 3.4-fold increase in lacZ mutant frequency over controls which increased to 4.1-fold after clonal correction. We then used next generation sequencing to compare the spontaneous and BaP-induced mutation spectra in sperm and bone marrow. The spontaneous spectrum in sperm had significantly more G:C to A:T transitions and fewer mutations at A:T basepairs than bone marrow. BaP predominantly induced G:C to T:A transversions in both cell types, and both were enriched for mutations at CpG dinucleotides. However, BaP induced significantly more deletions in sperm, but more G:C to A:T transitions and G:C to C:G transversions in bone marrow. Differences in error-prone translesion DNA synthesis polymerases may underlie the observed spectrum differences between sperm and bone marrow. These findings suggest that mutations in sperm can arise via mechanisms that are unique to male germ cells.

Benzo(a)pyrene Is Mutagenic in Mouse Spermatogonial Stem Cells and Dividing Spermatogonia.

Although many environmental agents are established male germ cell mutagens, few are known to induce mutations in spermatogonial stem cells. Stem cell mutations are of great concern because they result in a permanent increase in the number of mutations carried in sperm. We investigated mutation induction during mouse spermatogenesis following exposure to benzo(a)pyrene (BaP). MutaMouse males were given 0, 12.5, 25, 50, or 100 mg/kg bw/day BaP for 28 days by oral gavage. Germ cells were collected from the cauda epididymis and seminiferous tubules 3 days after exposure and from cauda epididymis 42 and 70 days after exposure. This design enabled targeted investigation of effects on post-spermatogonia, dividing spermatogonia, and spermatogonial stem cells, respectively. BaP increased lacZ mutant frequency (MF) in cauda sperm after exposure of dividing spermatogonia (4.2-fold at highest dose, P < .01) and spermatogonial stem cells (2.1-fold at highest dose, P < .01). No significant increases in MF were detected in cauda sperm or seminiferous tubule cells collected 3 days post-exposure. Dose-response modelling suggested that the mutational response in male germ cells to BaP is sub-linear at low doses. Our results demonstrate that oral exposure to BaP causes spermatogonial stem cell mutations, that different phases of spermatogenesis exhibit varying sensitivities to BaP, with dividing spermatogonia representing a window of peak sensitivity, and that sampling spermatogenic cells from the seminiferous tubules at earlier time-points may underestimate germ cell mutagenicity. This information is critical to optimize the use of the international test guideline for transgenic rodent mutation assays for detecting germ cell mutagens.

Empirical analysis of BMD metrics in genetic toxicology part II: in vivo potency comparisons to promote reductions in the use of experimental animals for genetic toxicity assessment.

Genotoxicity tests have traditionally been used only for hazard identification, with qualitative dichotomous groupings being used to identify compounds that have the capacity to induce mutations and/or cytogenetic alterations. However, there is an increasing interest in employing quantitative analysis of in vivo dose-response data to derive point of departure (PoD) metrics that can be used to establish human exposure limits or margins of exposure (MOEs), thereby supporting human health risk assessments and regulatory decisions. This work is an extension of our companion article on in vitro dose-response analyses and outlines how the combined benchmark dose (BMD) approach across included covariates can be used to improve the analyses and interpretation of in vivo genetic toxicity dose-response data. Using the BMD-covariate approach, we show that empirical comparisons of micronucleus frequency dose-response data across multiple studies justifies dataset merging, with subsequent analyses improving the precision of BMD estimates and permitting attendant potency ranking of seven clastogens. Similarly, empirical comparisons of Pig-a mutant phenotype frequency data collected in males and females justified dataset merging across sex. This permitted more effective scrutiny regarding the effect of post-exposure sampling time on the mutagenicity of N-ethyl-N-nitrosourea observed in reticulocytes and erythrocytes in the Pig-a assay. The BMD-covariate approach revealed tissue-specific differences in the induction of lacZ transgene mutations in Muta™Mouse specimens exposed to benzo[a]pyrene (BaP), with the results permitting the formulation of mechanistic hypotheses regarding the observed potency ranking. Lastly, we illustrate how historical dose-response data for assessments that examined numerous doses (i.e. induced lacZ mutant frequency (MF) across 10 doses of BaP) can be used to improve the precision of BMDs derived from datasets with far fewer doses (i.e. lacZ MF for 3 doses of dibenz[a,h]anthracene). Collectively, the presented examples illustrate how innovative use of the BMD approach can permit refinement of the use of in vivo data; improving the efficacy of experimental animal use in genetic toxicology without sacrificing PoD precision.

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.

Comparative transcriptomic analyses to scrutinize the assumption that genotoxic PAHs exert effects via a common mode of action.

In this study, the accuracy of the assumption that genotoxic, carcinogenic polycyclic aromatic hydrocarbons (PAHs) act via similar mechanisms of action as benzo(a)pyrene (BaP), the reference PAH used in the human health risk assessment of PAH-containing complex mixtures, was investigated. Adult male Muta™Mouse were gavaged for 28 days with seven individual, genotoxic PAHs. Global gene expression profiles in forestomach, liver, and lung (target tissues of exposure) were determined at 3 days post-exposure. The results are compared with our previously published results from mice exposed to BaP via the same exposure regimen. Although all PAHs showed enhanced ethoxyresorufin-O-deethylase activity, DNA adduct formation, and lacZ mutant frequency in the lungs, the unsupervised cluster analysis of differentially expressed genes revealed that the transcriptional changes are both PAH- and tissue-specific, with lung showing the most response. Further bioinformatics-/pathway-based analysis revealed that all PAHs induce expression of genes associated with carcinogenic processes, including DNA damage response, immune/inflammatory response, or cell signaling processes; however, the type of pathways and the magnitude of change varied for each PAH and were not the same as those observed for BaP. Benchmark dose modeling showed transcriptomic data closely reflected the known tumor incidence for the individual PAHs in each tissue. Collectively, the results suggest that the underlying mechanisms of PAH-induced toxicity leading to tumorigenesis are tissue-specific and not the same for all PAHs; based on the tissue type considered, use of BaP as a reference chemical may overestimate or underestimate the carcinogenic potential of PAHs.Electronic supplementary materialThe online version of this article (doi:10.1007/s00204-015-1595-5) contains supplementary material, which is available to authorized users.

Sublinear response in lacZ mutant frequency of Muta™ Mouse spermatogonial stem cells after low dose subchronic exposure to N-ethyl-N-nitrosourea.

The transgenic rodent mutation assay was used to compare the dose–response relationship of lacZ mutant frequency (MF) in spermatogonial stem cells exposed acutely or subchronically to N‐ethyl‐N‐nitrosourea (ENU). Muta™Mouse males were exposed orally to 0, 25, 50, or 100 mg/kg ENU for acute exposures and 0, 1, 2, or 5 mg/(kg day) for 28‐day subchronic exposures. LacZ MF was measured in sperm collected 70 days post‐exposure to target spermatogonial stem cells. Dose–response data were fit to linear, quadratic, exponential, or power models. Acute exposure resulted in a dose‐dependent increase in MF that was significant (P < 0.05) at all doses tested and was best described by a quadratic dose–response model that was linear in the low dose range. In contrast, similar total doses fragmented over a 28‐day subchronic exposure only resulted in a significant increase in lacZ MF at the highest dose tested. Therefore, the subchronic no observable genotoxic effect level (NOGEL) was 2 mg/(kg day) (or 56 mg/kg total dose). The subchronic dose–response was best described by the exponential and power models, which were sublinear in the low dose range. Benchmark dose lower confidence limits (BMDLs) for acute and subchronic exposure were 3.0 and 1.0 mg/(kg day) (or 27.4 mg/kg total dose), respectively. These findings are supportive of a saturable DNA repair mechanism as the mutagenic mode of action for ENU in spermatogonia and imply that sufficiently low exposures would not cause appreciable genotoxic effects over background. This may have important implications for the quantitative risk assessment of germ cell mutagens. Environ. Mol. Mutagen. 56:347–355, 2015. © 2015 The Authors. Environmental and Molecular Mutagenesis Published by Wiley Periodicals, Inc.

The progeroid phenotype of Ku80 deficiency is dominant over DNA-PKCS deficiency.

Ku80 and DNA-PKCS are both involved in the repair of double strand DNA breaks via the nonhomologous end joining (NHEJ) pathway. While ku80−/− mice exhibit a severely reduced lifespan and size, this phenotype is less pronounced in dna-pkcs−/− mice. However, these observations are based on independent studies with varying genetic backgrounds. Here, we generated ku80−/−, dna-pkcs−/− and double knock out mice in a C57Bl6/J*FVB F1 hybrid background and compared their lifespan, end of life pathology and mutation frequency in liver and spleen using a lacZ reporter. Our data confirm that inactivation of Ku80 and DNA-PKCS causes reduced lifespan and bodyweights, which is most severe in ku80−/− mice. All mutant mice exhibited a strong increase in lymphoma incidence as well as other aging-related pathology (skin epidermal and adnexal atrophy, trabacular bone reduction, kidney tubular anisokaryosis, and cortical and medullar atrophy) and severe lymphoid depletion. LacZ mutation frequency analysis did not show strong differences in mutation frequencies between knock out and wild type mice. The ku80−/− mice had the most severe phenotype and the Ku80-mutation was dominant over the DNA-PKCS-mutation. Presumably, the more severe degenerative effect of Ku80 inactivation on lifespan compared to DNA-PKCS inactivation is caused by additional functions of Ku80 or activity of free Ku70 since both Ku80 and DNA-PKCS are essential for NHEJ.

Quantitative dose–response analysis of ethyl methanesulfonate genotoxicity in adult gpt‐delta transgenic mice

The assumption that mutagens have linear dose-responses recently has been challenged. In particular, ethyl methanesulfonate (EMS), a DNA-reactive mutagen and carcinogen, exhibited sublinear or thresholded dose-responses for LacZ mutation in transgenic Muta™Mouse and for micronucleus (MN) frequency in CD1 mice (Gocke E and Müller L [2009]: Mutat Res 678:101-107). In order to explore variables in establishing genotoxicity dose-responses, we characterized the genotoxicity of EMS using gene mutation assays anticipated to have lower spontaneous mutant frequencies (MFs) than Muta™Mouse. Male gpt-delta transgenic mice were treated daily for 28 days with 5 to 100 mg/kg EMS, and measurements were made on: (i) gpt MFs in liver, lung, bone marrow, kidney, small intestine, and spleen; and (ii) Pig-a MFs in peripheral blood reticulocytes (RETs) and total red blood cells. MN induction also was measured in peripheral blood RETs. These data were used to calculate Points of Departure (PoDs) for the dose responses, i.e., no-observed-genotoxic-effect-levels (NOGELs), lower confidence limits of threshold effect levels (Td-LCIs), and lower confidence limits of 10% benchmark response rates (BMDL10 s). Similar PoDs were calculated from the published EMS dose-responses for LacZ mutation and CD1 MN induction. Vehicle control gpt and Pig-a MFs were 13-40-fold lower than published vehicle control LacZ MFs. In general, the EMS genotoxicity dose-responses in gpt-delta mice had lower PoDs than those calculated from the Muta™Mouse and CD1 mouse data. Our results indicate that the magnitude and possibly the shape of mutagenicity dose responses differ between in vivo models, with lower PoDs generally detected by gene mutation assays with lower backgrounds.

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.

No evidence for transgenerational genomic instability in the F1 or F2 descendants of Muta™Mouse males exposed to N-ethyl-N-nitrosourea

Exposure of male mice to genotoxic agents can increase mutation frequencies in their unexposed descendants. This phenomenon, known as transgenerational genomic instability (TGI), can persist for several generations. However, little is known about the underlying mechanisms. Chemically-induced TGI has been demonstrated in non-coding unstable tandem repeat DNA regions, but it is unclear whether it extends to other genetic endpoints. We investigated whether exposure of Muta™Mouse males to a single dose of 75mg/kg N-ethyl-N-nitrosourea (ENU) increased the spontaneous frequency of gene mutations or chromosome damage in their offspring. Treated males were mated with untreated females 3 days, 6 weeks or 10 weeks post-exposure to produce the F1 generation. Offspring were thus conceived from germ cells exposed to ENU as mature spermatozoa, dividing spermatogonia, or spermatogonial stem cells, respectively. F2 mice were generated by mating F1 descendants with untreated partners. Mutations in the lacZ transgene were quantified in bone marrow and micronucleus frequencies were evaluated in red blood cells by flow-cytometry for all F0 and their descendants. LacZ mutant frequencies were also determined in sperm for all exposed males and their male descendants. In F0 males, lacZ mutant frequencies were significantly increased in bone marrow at least 10-fold at all three time points investigated. In sperm, lacZ mutant frequency was significantly increased 7–11-fold after exposure of dividing and stem cell spermatogonia, but not in replication-deficient haploid sperm. Micronucleus frequencies assessed two days after ENU treatment were increased 5-fold in F0 males, but returned to control levels after 10 weeks. Despite the strong mutagenic response in F0 males, pre- and post-meiotic ENU exposure did not significantly increase lacZ mutant or micronucleus frequencies in F1 or F2 offspring. These findings suggest that TGI may not extend to all genetic endpoints and that further investigation of this phenomenon and its health relevance will require multiple measures of genomic damage.