A multi-megabase copy number gain causes maternal transmission ratio distortion on mouse chromosome 2.

Significant departures from expected Mendelian inheritance ratios (transmission ratio distortion, TRD) are frequently observed in both experimental crosses and natural populations. TRD on mouse Chromosome (Chr) 2 has been reported in multiple experimental crosses, including the Collaborative Cross (CC). Among the eight CC founder inbred strains, we found that Chr 2 TRD was exclusive to females that were heterozygous for the WSB/EiJ allele within a 9.3 Mb region (Chr 2 76.9 - 86.2 Mb). A copy number gain of a 127 kb-long DNA segment (designated as responder to drive, R2d) emerged as the strongest candidate for the causative allele. We mapped R2d sequences to two loci within the candidate interval. R2d1 is located near the proximal boundary, and contains a single copy of R2d in all strains tested. R2d2 maps to a 900 kb interval, and the number of R2d copies varies from zero in classical strains (including the mouse reference genome) to more than 30 in wild-derived strains. Using real-time PCR assays for the copy number, we identified a mutation (R2d2WSBdel1) that eliminates the majority of the R2d2WSB copies without apparent alterations of the surrounding WSB/EiJ haplotype. In a three-generation pedigree segregating for R2d2WSBdel1, the mutation is transmitted to the progeny and Mendelian segregation is restored in females heterozygous for R2d2WSBdel1, thus providing direct evidence that the copy number gain is causal for maternal TRD. We found that transmission ratios in R2d2WSB heterozygous females vary between Mendelian segregation and complete distortion depending on the genetic background, and that TRD is under genetic control of unlinked distorter loci. Although the R2d2WSB transmission ratio was inversely correlated with average litter size, several independent lines of evidence support the contention that female meiotic drive is the cause of the distortion. We discuss the implications and potential applications of this novel meiotic drive system.

R2d2 Drives Selfish Sweeps in the House Mouse.

A selective sweep is the result of strong positive selection driving newly occurring or standing genetic variants to fixation, and can dramatically alter the pattern and distribution of allelic diversity in a population. Population-level sequencing data have enabled discoveries of selective sweeps associated with genes involved in recent adaptations in many species. In contrast, much debate but little evidence addresses whether "selfish" genes are capable of fixation-thereby leaving signatures identical to classical selective sweeps-despite being neutral or deleterious to organismal fitness. We previously described R2d2, a large copy-number variant that causes nonrandom segregation of mouse Chromosome 2 in females due to meiotic drive. Here we show population-genetic data consistent with a selfish sweep driven by alleles of R2d2 with high copy number (R2d2(HC)) in natural populations. We replicate this finding in multiple closed breeding populations from six outbred backgrounds segregating for R2d2 alleles. We find that R2d2(HC) rapidly increases in frequency, and in most cases becomes fixed in significantly fewer generations than can be explained by genetic drift. R2d2(HC) is also associated with significantly reduced litter sizes in heterozygous mothers, making it a true selfish allele. Our data provide direct evidence of populations actively undergoing selfish sweeps, and demonstrate that meiotic drive can rapidly alter the genomic landscape in favor of mutations with neutral or even negative effects on overall Darwinian fitness. Further study will reveal the incidence of selfish sweeps, and will elucidate the relative contributions of selfish genes, adaptation and genetic drift to evolution.

The use of genetically modified mice in cancer risk assessment: challenges and limitations.

The use of genetically modified (GM) mice to assess carcinogenicity is playing an increasingly important role in the safety evaluation of chemicals. While progress has been made in developing and evaluating mouse models such as the Trp53⁺/⁻, Tg.AC and the rasH2, the suitability of these models as replacements for the conventional rodent cancer bioassay and for assessing human health risks remains uncertain. The objective of this research was to evaluate the use of accelerated cancer bioassays with GM mice for assessing the potential health risks associated with exposure to carcinogenic agents. We compared the published results from the GM bioassays to those obtained in the National Toxicology Program's conventional chronic mouse bioassay for their potential use in risk assessment. Our analysis indicates that the GM models are less efficient in detecting carcinogenic agents but more consistent in identifying non-carcinogenic agents. We identified several issues of concern related to the design of the accelerated bioassays (e.g., sample size, study duration, genetic stability and reproducibility) as well as pathway-dependency of effects, and different carcinogenic mechanisms operable in GM and non-GM mice. The use of the GM models for dose-response assessment is particularly problematic as these models are, at times, much more or less sensitive than the conventional rodent cancer bioassays. Thus, the existing GM mouse models may be useful for hazard identification, but will be of limited use for dose-response assessment. Hence, caution should be exercised when using GM mouse models to assess the carcinogenic risks of chemicals.

Toxicology and carcinogenesis study of senna in C3B6.129F1-Trp53 tm1Brd N12 haploinsufficient mice.

Senna is a pod or leaf of Senna alexandrina P. Mill and is used as a stimulant laxative. In the large intestine, bacterial enzymes reduce sennosides to rhein-9-anthrone, the active form for the laxative effect. To determine the potential toxic effects of senna, a 5-week dose range finding study in the C57BL/6N mouse and a 40-week toxicology and carcinogenesis study in the C3B6.129F1-Trp53 (tm1Brd) N12 haploinsufficient (p53(+/-)) mouse were conducted. In the 5-week study, C57BL/6N mice were exposed to up to 10,000 ppm senna in feed. Increased incidences of epithelial hyperplasia of the cecum and colon were observed in males and females exposed to 5,000 or 10,000 ppm senna. These intestinal lesions were not considered to be of sufficient severity to cause mortality and, thus, in the p53(+/-) mouse 40-week study, the high dose of 10,000 ppm was selected. Significant increases in the incidences of epithelial hyperplasia of the colon and cecum were observed at 10,000 ppm in p53(+/-) males and females, and the incidence of hyperplasia of the colon was significantly increased at 3,000 ppm in females. In conclusion, the large intestine was the major target of senna-induced toxicity in both wild-type and the p53(+/-) mouse model. There was no neoplastic change when senna was administered to p53(+/-) mouse.

Gender differences in chemical carcinogenesis in National Toxicology Program 2-year bioassays.

Differences in cancer incidences between men and women are often explained by either differences in environmental exposures or by influences of sex hormones. However, there are few studies on intrinsic gender differences in susceptibility to chemical carcinogens. We have analyzed the National Toxicology Program (NTP) database for sex differences in rat responses to chemical carcinogens. We found that the odds that male rat bioassays were assigned a higher level of evidence than female rat bioassays was 1.69 (p < .001). Of 278 carcinogenic chemicals in the database, 201 (72%) exhibited statistical gender differences (p ≤ .05) in at least one nonreproductive organ. One hundred thirty of these 201 chemicals induced gender-specific tumors in male rats and 59 in female rats. Sixty-eight chemicals induced tumors in males but no tumors in females. Less than one third (i.e., 19 chemicals) induced tumors in females but not males. Male-specific tumors included pancreatic and skin tumors, and female-specific tumors included lung tumors. For some tumor sites, these differences in gender susceptibility can be associated with literature data on sex hormone receptor expression. In conclusion, gender-specific tumors were common. The male dominance is in line with recent human data, and the male susceptibility to carcinogens should be further studied.

The utility of naphthyl-keratin adducts as biomarkers for jet-fuel exposure.

We investigated the association between biomarkers of dermal exposure, naphthyl-keratin adducts (NKA), and urine naphthalene biomarker levels in 105 workers routinely exposed to jet-fuel. A moderate correlation was observed between NKA and urine naphthalene levels (p = 0.061). The NKA, post-exposure breath naphthalene, and male gender were associated with an increase, while CYP2E1*6 DD and GSTT1-plus (++/+-) genotypes were associated with a decrease in urine naphthalene level (p < 0.0001). The NKA show great promise as biomarkers for dermal exposure to naphthalene. Further studies are warranted to characterize the relationship between NKA, other exposure biomarkers, and/or biomarkers of biological effects due to naphthalene and/or PAH exposure.

Epigenetic Markers Are Associated With Differences in Isocyanate Biomarker Levels in Exposed Spray-Painters.

Isocyanates are respiratory and skin sensitizers that are one of the main causes of occupational asthma globally. Genetic and epigenetic markers are associated with isocyanate-induced asthma and, before asthma develops, we have shown that genetic polymorphisms are associated with variation in plasma and urine biomarker levels in exposed workers. Inter-individual epigenetic variance may also have a significant role in the observed biomarker variability following isocyanate exposure. Therefore, we determined the percent methylation for CpG islands from DNA extracted from mononuclear blood cells of 24 male spray-painters exposed to 1,6-hexamethylene diisocyanate (HDI) monomer and HDI isocyanurate. Spray-painters' personal inhalation and skin exposure to these compounds and the respective biomarker levels of 1,6-diaminohexane (HDA) and trisaminohexyl isocyanurate (TAHI) in their plasma and urine were measured during three repeated industrial hygiene monitoring visits. We controlled for inhalation exposure, skin exposure, age, smoking status, and ethnicity as covariates and performed an epigenome-wide association study (EWAS) using likelihood-ratio statistical modeling. We identified 38 CpG markers associated with differences in isocyanate biomarker levels (Bonferroni < 0.05). Annotations for these markers included 18 genes: ALG1, ANKRD11, C16orf89, CHD7, COL27A, FUZ, FZD9, HMGN1, KRT6A, LEPR, MAPK10, MED25, NOSIP, PKD1, SNX19, UNC13A, UROS, and ZFHX3. We explored the functions of the genes that have been published in the literature and used GeneMANIA to investigate gene ontologies and predicted protein-interaction networks. The protein functions of the predicted networks include keratinocyte migration, cell-cell adhesions, calcium transport, neurotransmitter release, nitric oxide production, and apoptosis regulation. Many of the protein pathway functions overlap with previous findings on genetic markers associated with variability both in isocyanate biomarker levels and asthma susceptibility, which suggests there are overlapping protein pathways that contribute to both isocyanate toxicokinetics and toxicodynamics. These predicted protein networks can inform future research on the mechanism of allergic airway sensitization by isocyanates and aid in the development of mitigation strategies to better protect worker health.

Synergizing Mouse and Human Studies to Understand the Heterogeneity of Obesity.

Obesity is routinely considered as a single disease state, which drives a "one-size-fits-all" approach to treatment. We recently convened the first annual University of North Carolina Interdisciplinary Nutrition Sciences Symposium to discuss the heterogeneity of obesity and the need for translational science to advance understanding of this heterogeneity. The symposium aimed to advance scientific rigor in translational studies from animal to human models with the goal of identifying underlying mechanisms and treatments. In this review, we discuss fundamental gaps in knowledge of the heterogeneity of obesity ranging from cellular to population perspectives. We also advocate approaches to overcoming limitations in the field. Examples include the use of contemporary mouse genetic reference population models such as the Collaborative Cross and Diversity Outbred mice that effectively model human genetic diversity and the use of translational models that integrate -omics and computational approaches from pre-clinical to clinical models of obesity. Finally, we suggest best scientific practices to ensure strong rigor that will allow investigators to delineate the sources of heterogeneity in the population with obesity. Collectively, we propose that it is critical to think of obesity as a heterogeneous disease with complex mechanisms and etiologies, requiring unique prevention and treatment strategies tailored to the individual.

Exposure to naphthalene induces naphthyl-keratin adducts in human epidermis in vitro and in vivo.

We observed naphthyl-keratin adducts and dose-related metabolic enzyme induction at the mRNA level in reconstructed human epidermis in vitro after exposure to naphthalene. Immunofluorescence detection of 2-naphthyl-keratin-1 adducts confirmed the metabolism of naphthalene and adduction of keratin. We also observed naphthyl-keratin adducts in dermal tape-strip samples collected from naphthalene-exposed workers at levels ranging from 0.004 to 6.104 pmol adduct microg(-1) keratin. We have demonstrated the ability of the human skin to metabolize naphthalene and to form naphthyl-keratin adducts both in vitro and in vivo. The results indicate the potential use of keratin adducts as biomarkers of dermal exposure.

Panel discussion: alternative mouse models for carcinogenicity assessment.

This article summarizes key points from Dr. Bernard Leblanc's presentation European Perspectives on Alternative Mouse Carcinogenicity Models and a distillation of questions and answers from a panel discussion following presentations on Alternative Mouse Models for Carcinogenicity Assessment at the Society of Toxicologic Pathology's annual symposium on June 23, 2009, in Washington, DC.

Influence of Genetic Variance on Biomarker Levels After Occupational Exposure to 1,6-Hexamethylene Diisocyanate Monomer and 1,6-Hexamethylene Diisocyanate Isocyanurate.

We evaluated the impact of genetic variance on biomarker levels in a population of workers in the automotive repair and refinishing industry who were exposed to respiratory sensitizers 1,6-hexamethylene diisocyanate (HDI) monomer and one of its trimers, HDI isocyanurate. The exposures and respective urine and plasma biomarkers 1,6-diaminohexane (HDA) and trisaminohexyl isocyanurate (TAHI) were measured in 33 workers; and genome-wide microarrays (Affymetrix 6.0) were used to genotype the workers' single-nucleotide polymorphisms (SNPs). Linear mixed model analyses have indicated that interindividual variations in both inhalation and skin exposures influenced these biomarker levels. Using exposure values as covariates and a false discovery rate < 0.10 to assess statistical significance, we observed that seven SNPs were associated with HDA in plasma, five were associated with HDA in urine, none reached significance for TAHI in plasma, and eight were associated with TAHI levels in urine. The different genotypes for the 20 significant SNPs accounted for 4- to 16-fold changes observed in biomarker levels. Associated gene functions include transcription regulation, calcium ion transport, vascular morphogenesis, and transforming growth factor beta signaling pathway, which may impact toxicokinetics indirectly by altering inflammation levels. Additionally, in an expanded analysis using a minor allele cutoff of 0.05 instead of 0.10, there were biomarker-associated SNPs within three genes that have been associated with isocyanate-induced asthma: ALK, DOCK2, and LHPP. We demonstrate that genetic variance impacts the biomarker levels in workers exposed to HDI monomer and HDI isocyanurate and that genetics can be used to refine exposure predictions in small cohorts when quantitative personal exposure and biomarker measurements are included in the models.

Aflatoxin B1 and/or hepatitis B virus induced tumor spectrum in a genetically engineered hepatitis B virus expression and Trp53 haploinsufficient mouse model system for hepatocarcinogenesis.

The authors investigated the spectrum of tumors and Trp53 mutations in genetically engineered models using the FVB/N mouse that expressed the hepatitis B virus genome and/or carried a Trp53 null and wildtype allele and/or were exposed to aflatoxin B1. Liver tumor incidence was increased when all three risk factors were present. Without aflatoxin B1 exposure, neither Trp53 haploinsufficiency nor HBV expression affected liver tumor development. Liver tumor prevalence increased with aflatoxin B1 exposure (p < .001), as thirteen of fourteen mice with liver tumors were initiated with aflatoxin B1. Liver tumors were more frequent in males (12/190) than females (2/170). Seventy-three mice developed sarcomas. Trp53 haploinsufficiency was associated with increased sarcoma incidence in males and females (p < .001). In Trp53 haploinsufficient mice, the HBV transgene increased the risk of sarcoma in males and females (p < .001). Lymphoma was significantly increased in Trp53 haploinsufficient FVB/N mice. There was no loss of heterozygosity at the wildtype Trp53 locus in twenty-five sarcomas or four hepatocellular tumors examined. No mutations were identified in the mRNA (exons 2-11) of Trp53 in six liver neoplasms or twenty-four sarcomas. In this model system, HBV expression affected only hepatocellular neoplasia in association with both aflatoxin B1 initiation and p53 haploinsufficiency.

DNA methylation in mice is influenced by genetics as well as sex and life experience.

DNA methylation is an essential epigenetic process in mammals, intimately involved in gene regulation. Here we address the extent to which genetics, sex, and pregnancy influence genomic DNA methylation by intercrossing 2 inbred mouse strains, C57BL/6N and C3H/HeN, and analyzing DNA methylation in parents and offspring using whole-genome bisulfite sequencing. Differential methylation across genotype is detected at thousands of loci and is preserved on parental alleles in offspring. In comparison of autosomal DNA methylation patterns across sex, hundreds of differentially methylated regions are detected. Comparison of animals with different histories of pregnancy within our study reveals a CpG methylation pattern that is restricted to female animals that had borne offspring. Collectively, our results demonstrate the stability of CpG methylation across generations, clarify the interplay of epigenetics with genetics and sex, and suggest that CpG methylation may serve as an epigenetic record of life events in somatic tissues at loci whose expression is linked to the relevant biology.

Notch1 is a frequent mutational target in chemically induced lymphoma in mouse.

Activating Notch1 mutations have been reported in human T-lineage acute lymphoblastic leukemia (T-ALL) and lymphomas from genetically modified mice. We report that Notch1 is a prevalent and major mutational target in chemically induced mouse lymphoma. The regions of the gene that are frequently mutated are the heterodimerization domain and the N-terminal ligand-binding region, important for protein stability, and the polypeptide rich in proline, glutamate, serine and threonine (PEST) domains, which is critical for protein degradation. Another gene, CDC4, is also involved in Notch1 degradation and shows frequent mutations. Mutations in the heterodimerization and the ligand-binding regions may cause ligand-independent signaling, whereas mutations preventing protein degradation result in accumulation of intracellular Notch1. We analyzed 103 chemical-induced mouse lymphomas for mutations in the Notch1 gene using single strand conformation analysis (SSCA) and DNA sequencing. Genetic alterations resulting in premature truncation of Notch1 were identified in 28 tumors, whereas 8 revealed alterations in the heterodimerization and 16 harbored deletions in the ligand-binding region. Dideoxycytidine-induced lymphomas displayed the highest frequency of Notch1 mutations (49%), whereas in butadiene- and phenolphthalein-induced tumors showed lower frequencies (26 and 10%, respectively). In total, 26 novel and 3 previously reported mutations were detected. This report shows that Notch1 is a prevalent and major mutational target for 2',3'-dideoxycytidine and butadiene-induced lymphoma.

Erratum: "Diversity Outbred Mice Identify Population-Based Exposure Thresholds and Genetic Factors that Influence Benzene-Induced Genotoxicity".

[This corrects the article DOI: http://dx.doi.org/10.1289/ehp.1408202.].

Acquisition of apoptotic resistance in cadmium-transformed human prostate epithelial cells: Bcl-2 overexpression blocks the activation of JNK signal transduction pathway.

BACKGROUND: We have recently shown that cadmium can induce malignant transformation of the human prostate epithelial cell line (RWPE-1) and that these cadmium-transformed prostate epithelial (CTPE) cells acquire apoptotic resistance concurrently with malignant phenotype. OBJECTIVE: The present study was designed to define the mechanism of acquired apoptotic resistance in CTPE cells. METHODS: Various molecular events associated with apoptosis were assessed in control and CTPE cells that were obtained after 8 weeks of continuous cadmium exposure. RESULTS: Compared with control, CTPE cells showed a generalized resistance to apoptosis induced by cadmium, cisplatin, or etoposide. Signal-regulated mitogen-activated protein kinases, extracellular signal-regulated kinases 1 and 2, c-Jun N-terminal kinases (JNK1 and JNK2), and p38 were phosphorylated in a cadmium concentration-dependent fashion in CTPE and control cells. However, phosphorylated JNK1/2 levels and JNK kinase activity were much lower in CTPE cells. The pro-apoptotic gene Bax showed lower transcript and protein levels, whereas the anti-apoptotic gene Bcl-2 showed higher levels in CTPE cells. The ratio of Bcl-2/Bax, a key determinant in apoptotic commitment, increased more than 4-fold in CTPE cells. In Bcl-2-transfected PT-67 cells, phosphorylated JNK1/2 levels were much lower after apoptogenic stimulus, and apoptosis induced by cadmium or etoposide was reduced compared with control. Mutation of tyrosine to serine at the 21st amino acid of the Bcl-2 protein BH4 domain resulted in a loss both of suppression of JNK1/2 phosphorylation and its anti-apoptotic function. CONCLUSIONS: CTPE cells become resistant to apoptosis during malignant transformation, and disruption of the JNK pathway and Bcl-2 overexpression play important roles in this resistance. Bcl-2 BH4 domain is required for modulating JNK phosphorylation and anti-apoptotic function.

QTL Mapping and Identification of Candidate Genes in DO Mice: A Use Case Model Derived from a Benzene Toxicity Experiment.

Diversity Outbred (DO) mice are a multiparental advanced generation intercross population derived from eight inbred strains which are genetically very diverse. They are maintained as an outbred population using a randomized mating design. Thus DO mice represent an ideal population to map phenotypic traits. Here, we provide a case study in which male DO mice were exposed to benzene and phenotyped for the number of micronucleated reticulocytes. We provide step-by-step R scripts for the analysis of phenotypes, genotypes, mapping of resistance gene loci and identification of candidate genes.

Diversity Outbred Mice Identify Population-Based Exposure Thresholds and Genetic Factors that Influence Benzene-Induced Genotoxicity.

BACKGROUND: Inhalation of benzene at levels below the current exposure limit values leads to hematotoxicity in occupationally exposed workers. OBJECTIVE: We sought to evaluate Diversity Outbred (DO) mice as a tool for exposure threshold assessment and to identify genetic factors that influence benzene-induced genotoxicity. METHODS: We exposed male DO mice to benzene (0, 1, 10, or 100 ppm; 75 mice/exposure group) via inhalation for 28 days (6 hr/day for 5 days/week). The study was repeated using two independent cohorts of 300 animals each. We measured micronuclei frequency in reticulocytes from peripheral blood and bone marrow and applied benchmark concentration modeling to estimate exposure thresholds. We genotyped the mice and performed linkage analysis. RESULTS: We observed a dose-dependent increase in benzene-induced chromosomal damage and estimated a benchmark concentration limit of 0.205 ppm benzene using DO mice. This estimate is an order of magnitude below the value estimated using B6C3F1 mice. We identified a locus on Chr 10 (31.87 Mb) that contained a pair of overexpressed sulfotransferases that were inversely correlated with genotoxicity. CONCLUSIONS: The genetically diverse DO mice provided a reproducible response to benzene exposure. The DO mice display interindividual variation in toxicity response and, as such, may more accurately reflect the range of response that is observed in human populations. Studies using DO mice can localize genetic associations with high precision. The identification of sulfotransferases as candidate genes suggests that DO mice may provide additional insight into benzene-induced genotoxicity.

Importance of investigating epigenetic alterations for industry and regulators: An appraisal of current efforts by the Health and Environmental Sciences Institute.

Recent technological advances have led to rapid progress in the characterization of epigenetic modifications that control gene expression in a generally heritable way, and are likely involved in defining cellular phenotypes, developmental stages and disease status from one generation to the next. On November 18, 2013, the International Life Sciences Institute (ILSI) Health and Environmental Sciences Institute (HESI) held a symposium entitled "Advances in Assessing Adverse Epigenetic Effects of Drugs and Chemicals" in Washington, D.C. The goal of the symposium was to identify gaps in knowledge and highlight promising areas of progress that represent opportunities to utilize epigenomic profiling for risk assessment of drugs and chemicals. Epigenomic profiling has the potential to provide mechanistic information in toxicological safety assessments; this is especially relevant for the evaluation of carcinogenic or teratogenic potential and also for drugs that directly target epigenetic modifiers, like DNA methyltransferases or histone modifying enzymes. Furthermore, it can serve as an endpoint or marker for hazard characterization in chemical safety assessment. The assessment of epigenetic effects may also be approached with new model systems that could directly assess transgenerational effects or potentially sensitive stem cell populations. These would enhance the range of safety assessment tools for evaluating xenobiotics that perturb the epigenome. Here we provide a brief synopsis of the symposium, update findings since that time and then highlight potential directions for future collaborative efforts to incorporate epigenetic profiling into risk assessment.