lacZ Mutant Research Articles

New vectors and optimal conditions for allelic exchange in hypervirulent Klebsiella pneumoniae

The emergence of antibiotic-resistant Klebsiella pneumoniae is a significant global health threat that has led to increased morbidity and mortality. This resistance also hinders basic research, as many strains are no longer susceptible to antibiotics commonly used in microbial genetics. Addressing this requires the development of new genetic tools with alternative selective markers. In this report, we introduce new allelic exchange vectors for use in drug-resistant strains. These vectors feature a conditional R6K origin of replication, an origin of transfer, SacB counter-selection, and alternative selectable markers. We validated the vectors by generating unmarked deletions in the K. pneumoniae KPPR1S bla (β-lactamase) and lacZ (β-galactosidase) genes. During this process, we defined optimized conditions for SacB-mediated allelic exchange in KPPR1S, significantly enhancing the efficiency of mutant generation. Furthermore, we demonstrated that lacZ is dispensable for virulence and that the lacZ mutant can serve as a surrogate for wild-type strains in competition assays using the Galleria mellonella infection model. Our findings provide new tools for the efficient genetic manipulation of K. pneumoniae and other drug-resistant bacteria.

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.

The functional mutational landscape of the lacZ gene

The lacZ gene of Escherichia coli encodes β-galactosidase (β-gal), a lactose metabolism enzyme of the lactose operon. Previous chemical modification or site-directed mutagenesis experiments have identified 21 amino acids that are essential for β-gal catalytic activity. We have assembled over 10,000 lacZ mutations from published studies that were collected using a positive selection assay to identify mutations in lacZ that disrupted β-gal function. We analyzed 6,465 independent lacZ mutations that resulted in 2,732 missense mutations that impaired β-gal function. Those mutations affected 492 of the 1,023 lacZ codons, including most of the 21 previously known residues critical for catalytic activity. Most missense mutations occurred near the catalytic site and in regions important for subunit tetramerization. Overall, our work provides a comprehensive and detailed map of the amino acid residues affecting the structure and catalytic activity of the β-gal enzyme.

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

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. 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. Five-week-old MutaMouse male mice were fed a deficient (0mg FA/kg), control (2mg FA/kg), or supplemented (8mg 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. 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. 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.

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.

Chemically induced mutations in a MutaMouse reporter gene inform mechanisms underlying human cancer mutational signatures

Transgenic rodent (TGR) models use bacterial reporter genes to quantify in vivo mutagenesis. Pairing TGR assays with next-generation sequencing (NGS) enables comprehensive mutation pattern analysis to inform mutational mechanisms. We used this approach to identify 2751 independent lacZ mutations in the bone marrow of MutaMouse animals exposed to four chemical mutagens: benzo[a]pyrene, N-ethyl-N-nitrosourea, procarbazine, and triethylenemelamine. We also collected published data for 706 lacZ mutations from eight additional environmental mutagens. We report that lacZ gene sequencing generates chemical-specific mutation signatures observed in human cancers with established environmental causes. For example, the mutation signature of benzo[a]pyrene, a carcinogen present in tobacco smoke, matched the signature associated with tobacco-induced lung cancers. Our results suggest that the analysis of chemically induced mutations in the lacZ gene shortly after exposure provides an effective approach to characterize human-relevant mechanisms of carcinogenesis and propose novel environmental causes of mutation signatures observed in human cancers.

Proteasomal degradation within endocytic organelles mediates antigen cross-presentation.

During MHC-I-restricted antigen processing, peptides generated by cytosolic proteasomes are translocated by the transporter associated with antigen processing (TAP) into the endoplasmic reticulum, where they bind to newly synthesized MHC-I molecules. Dendritic cells and other cell types can also generate MHC-I complexes with peptides derived from internalized proteins, a process called cross-presentation. Here, we show that active proteasomes within cross-presenting cell phagosomes can generate these peptides. Active proteasomes are detectable within endocytic compartments in mouse bone marrow-derived dendritic cells. In TAP-deficient mouse dendritic cells, cross-presentation is enhanced by the introduction of human β2 -microglobulin, which increases surface expression of MHC-I and suggests a role for recycling MHC-I molecules. In addition, surface MHC-I can be reduced by proteasome inhibition and stabilized by MHC-I-restricted peptides. This is consistent with constitutive proteasome-dependent but TAP-independent peptide loading in the endocytic pathway. Rab-GTPase mutants that restrain phagosome maturation increase proteasome recruitment and enhance TAP-independent cross-presentation. Thus, phagosomal/endosomal binding of peptides locally generated by proteasomes allows cross-presentation to generate MHC-I-peptide complexes identical to those produced by conventional antigen processing.

Regulation of death receptor signaling by the autophagy protein TP53INP2.

TP53INP2 positively regulates autophagy by binding to Atg8 proteins. Here, we uncover a novel role of TP53INP2 in death-receptor signaling. TP53INP2 sensitizes cells to apoptosis induced by death receptor ligands. In keeping with this, TP53INP2 deficiency in cultured cells or mouse livers protects against death receptor-induced apoptosis. TP53INP2 binds caspase-8 and the ubiquitin ligase TRAF6, thereby promoting the ubiquitination and activation of caspase-8 by TRAF6. We have defined a TRAF6-interacting motif (TIM) and a ubiquitin-interacting motif in TP53INP2, enabling it to function as a scaffold bridging already ubiquitinated caspase-8 to TRAF6 for further polyubiquitination of caspase-8. Mutations of key TIM residues in TP53INP2 abrogate its interaction with TRAF6 and caspase-8, and subsequently reduce levels of death receptor-induced apoptosis. A screen of cancer cell lines showed that those with higher protein levels of TP53INP2 are more prone to TRAIL-induced apoptosis, making TP53INP2 a potential predictive marker of cancer cell responsiveness to TRAIL treatment. These findings uncover a novel mechanism for the regulation of caspase-8 ubiquitination and reveal TP53INP2 as an important regulator of the death receptor pathway.

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.

Integration of sperm DNA damage assessment into OECD test guidelines for genotoxicity testing using the MutaMouse model

The Organisation for Economic Co-operation and Development (OECD) endorses test guidelines (TG) for identifying chemicals that are genotoxic, such as the transgenic rodent gene mutation assay (TG 488). Current OECD TG do not include assays for sperm DNA damage resulting in a critical testing gap. We evaluated the performance of the Sperm Chromatin Structure Assay (SCSA) and the Terminal Deoxynucleotidyl Transferase-Mediated Deoxyuridine Triphosphate Nick end Labeling (TUNEL) assay to detect sperm DNA damage within the recommended TG 488 protocol. MutaMouse males received 0, 0.5, 1, or 2 mg/kg/day triethylenemelamine (TEM), a multifunctional alkylating agent, for 28 days orally and tissues were collected two (blood) and three (sperm and bone marrow) days later. TEM significantly increased the frequency of lacZ mutants in bone marrow, and of micronuclei (MN) in both reticulocytes (%MN-RET) and normochromatic erythrocytes (%MN-NCE) in a dose-dependent manner (P < 0.05). The percentage of DNA fragmentation index (%DFI) and %TUNEL positive cells demonstrated dose-related increases in sperm (P < 0.05), and the two assay results were strongly correlated (R = 0.9298). Within the same animal, a good correlation was observed between %MN-NCE and %DFI (R = 0.7189). Finally, benchmark dose modelling (BMD) showed comparable BMD10 values among the somatic and germ cell assays. Our results suggest that sperm DNA damage assays can be easily integrated into standard OECD designs investigating genotoxicity in somatic tissues to provide key information on whether a chemical is genotoxic in germ cells and impact its risk assessment.

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.

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.

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.

Oral exposure to commercially available coal tar-based pavement sealcoat induces murine genetic damage and mutations.

Coal tar (CT) is a thick black liquid produced as a by‐product of coal carbonization to produce coke or manufactured gas. It is comprised a complex mixture of polycyclic aromatic compounds, including a wide range of polycyclic aromatic hydrocarbons (PAHs), many of which are genotoxic and carcinogenic. CT is used in some pavement sealants (also known as sealcoat), which are applied to pavement in order to seal and beautify the surface. Human exposure is known to occur not only during application, but also as a result of the weathering process, as elevated levels of PAHs have been found in settled house dust in residences adjacent to CT‐sealed surfaces. In this study we examined the genotoxicity of an extract of a commercially available CT‐based sealcoat in the transgenic Muta™Mouse model. Mice were orally exposed to 3 doses of sealcoat extract daily for 28 days. We evaluated genotoxicity by examining: (1) stable DNA adducts and (2) lacZ mutations in bone marrow, liver, lung, small intestine, and glandular stomach, as well as (3) micronucleated red blood cells. Significant increases were seen for each endpoint and in all tissues. The potency of the response differed across tissues, with the highest frequency of adducts occurring in liver and lung, and the highest frequency of mutations occurring in small intestine. The results of this study are the first demonstration of mammalian genotoxicity following exposure to CT‐containing pavement sealcoat. This work provides in vivo evidence to support the contention that there may be adverse health effects in mammals, and potentially in humans, from exposure to coal tar. Environ. Mol. Mutagen. 57:535–545, 2016. © 2016 Her Majesty the Queen in Right of Canada

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.

A Guided Inquiry‐Based Animal Cell Culture Laboratory Course for Undergraduates

The revised version of this course, Tissue Culture (CHE470), includes a series of animal cell culture‐based experiments and lectures on Biochemistry &amp; Molecular Biology (BMB) aspects of cell culture, including cellular transformation and metabolism, growth kinetics, transfection, inducible gene expression and biochemical assays. After receiving lectures on BMB aspects of cell culture and training in fundamental animal cell culture techniques, student undertake a unified series of experiments consists of (1) animal cell transfection by electroporation, (2) clonal selection, isolation and expansion, (3) induction of gene expression with tetracycline, (4) genomic DNA (gDNA) isolation from clonal cell populations and PCR screening of clonal gDNAs, (5) RNA isolation and Real Time‐PCR, (6) protein isolation for western blotting, and (7) beta‐galactosidase enzyme activity assays. In the revised version of this course, first executed in Fall 2015, students were provided with two plasmid DNA samples for transfection into CHO T‐RExTM cells. Each student received a known sample of an unaltered tetracyclin‐inducible beta‐galactosidase expression vector (pcDNA4/TO/mycHIS‐LacZ) and one of four pcDNA4/TO/mycHIS‐lacZ vectors altered by site‐directed mutagenesis. Students were not informed about the nature of the mutation in the mutant vector provided to them. Mutations in the four altered vectors include single nucleotide mutations (SNP) that inactivate the LacZ ATG start codon (ATG‐&gt;TTG), or one of two missense mutations in the beta‐galactosidase active site (E461K and Y503C), or a nonsense mutation that produces a truncated beta‐galactosidase protein (Y503X). After separately transfecting their two vectors into CHO T‐RExTM cells, student used antibiotic selection and isolated clonal cell populations that they molecular characterized. Students utilized their experimental data acquired from TaqMan RT‐PCR, PCR screening of clones and direct DNA sequencing, western blotting and beta‐galactosidase activity assays to determine the exact nature of the mutation in their pcDNA4/TO/mycHIS‐LacZ mutant vector provided. This guided experimental approach provides opportunities for inquiry‐based learning through students’ acquiring and analyses of experimental observations and data. Course grades are based upon a written BMB peer‐review journal style manuscript report of their experimental data and conclusions from their semester‐long series of experiments, three in‐class exams, an oral presentation of their data and findings, and some animal cell culture laboratory exercises. Student outcomes were measured by anonymous feedback through an online survey system using a numeric rating scale (5 = strongly agree; 1 = strongly disagree). Combined Fall 2013 and 2014 survey data from CHE470 student cohorts (n = 7) reported they “know more about this subject” (mean = 5.00, dev. = 0.000), that their “skills in this area have improved” (mean = 5.00, dev. = 0.000), their “appreciation of this subject increased” (mean = 5.00, dev. = 0.000), and that “learning objectives of the course were met” (mean = 4.86, dev. = 0.378). Survey data are consistent with students finding this course informative and beneficial.Support or Funding InformationThis project was supported through generous financial support from the University of Tampa Department of Chemistry, Biochemistry &amp; Physics.

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.