Chemically induced mutations in a MutaMouse reporter gene inform mechanisms underlying human cancer mutational signatures.
ABSTRACT: 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.
Project description: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.
Project description: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.
Project description:We have previously shown that acute oral exposure to the environmental carcinogen benzo(a)pyrene (BaP) elicits comparable levels of DNA adducts, but distinct transcriptomic changes, in mouse lungs and livers, the two main BaP bioactivating organs. Oral BaP exposure is predominantly associated with lung cancer and not hepatic cancer in some animal models, suggesting that gene expression differences may provide insight into the drivers of tissue-specific carcinogenesis. In the present study, we examine pulmonary DNA adduct formation, lacZ mutant frequency, and mRNA profiles in adult male MutaMouse following subchronic (28 day) oral exposure to BaP (0, 25, 50, and 75 mg/kg/day) and sacrificed 3 days postexposure. The results are compared with those obtained from livers of the same mice (previously published). Although there was a 1.8- to 3.3-fold increase in the levels of DNA adducts in lung compared with liver, the lacZ transgene mutant frequency was similar in both tissues. At the transcriptomic level, a transition from activation of the DNA damage response p53 pathway at the low dose to the induction of genes involved in angiogenesis, evasion of apoptosis and growth signals at the high doses was evident only in the lungs. These results suggest that tissue DNA adducts and mutant frequency are sensitive markers of target tissue exposure and mode of action, whereas early changes in gene expression may provide a better indication of the likelihood of carcinogenic transformation in selected tissues. Moreover, the study provides new information on the underlying mechanisms that contribute to tissue-specific responses to BaP.
Project description:Whole genome sequencing of human tumours has revealed distinct patterns of mutation that hint at the causative origins of cancer. Experimental investigations of the mutations and mutation spectra induced by environmental mutagens have traditionally focused on single genes. With the advent of faster cheaper sequencing platforms, it is now possible to assess mutation spectra in experimental models across the whole genome. As a proof of principle, we have examined the whole genome mutation profiles of mouse embryo fibroblasts immortalised following exposure to benzo[a]pyrene (BaP), ultraviolet light (UV) and aristolochic acid (AA). The results reveal that each mutagen induces a characteristic mutation signature: predominantly G?T mutations for BaP, C?T and CC?TT for UV and A?T for AA. The data are not only consistent with existing knowledge but also provide additional information at higher levels of genomic organisation. The approach holds promise for identifying agents responsible for mutations in human tumours and for shedding light on the aetiology of human cancer.
Project description:Various kind of chemical substances, including man-made chemical products and unintended products, are emitted to ambient air. Some of these substances have been shown to be mutagenic and therefore to act as a carcinogen in humans. National pollutant inventories (e.g., Pollutant Release and Transfer Registration in Japan) have estimated release amounts of man-made chemical products, but a major concern is the release of suspended particulate matter containing potent mutagens, for example, polycyclic aromatic hydrocarbons and related compounds generated by the combustion of fossil fuel, which are not estimated by PRTR system. In situ exposure studies have revealed that DNA adducts in the lung, and possibly mutations in germline cells are induced in rodents by inhalation of ambient air, indicating that evaluating in vivo mutations is important for assessing environmental health risks. Transgenic rodent systems (Muta, Big Blue, and gpt delta) are good tools for analyzing in vivo mutations induced by a mixture of chemical substances present in the environment. Following inhalation of diesel exhaust (used as a model mixture), mutation frequency was increased in the lung of gpt delta mice and base substitutions were induced at specific guanine residues (mutation hotspots) on the target transgenes. Mutation hotspots induced by diesel exhaust were different from those induced by benzo[a]pyrene, a typical mutagen in ambient air, but nearly identical to those induced by 1,6-dinitropyrene contained in diesel exhaust. Comparison between mutation hotspots in the TP53 (p53) gene in human lung cancer (data extracted from the IARC TP53 database) and mutations we identified in gpt delta mice showed that G to A transitions centered in CGT and CGG trinucleotides were mutation hotspots on both TP53 genes in human lung cancers and gpt genes in transgenic mice that inhaled diesel exhaust. The carcinogenic potency (TD50 value) of genotoxic carcinogen was shown to be correlated with the in vivo mutagenicity (total dose per increased mutant frequency). These results suggest that the mutations identified in transgenic rodents can help identify environmental mutagens that cause cancer.
Project description:In this study, we employ high-density oligonucleotide microarrays to characterize the MutaMouse FE1 cell line at various stages of cell growth, in primary MutaMouse lung epithelial cell cultures, and in whole lung. Global transcriptional analysis and real-time RT-PCR was applied to (1) further define the cellular origin of the FE1 cell line and its responses under different culture conditions (media and substratum), (2) provide insight into the transcriptional differences in cellular processes between FE1 cultures compared to whole lung tissues, more specifically in toxicological response, and (3) preliminarily examine FE1 culture response to exposure of benzo(a)pyrene compared to whole animals. Total RNA samples from 3 cell culture types (50% FE1, %100 FE1, and Primary lung) or MutaMouse lung were labeled with Cyanine 5-CTP, and universal reference total RNA (Stratagene, CA, USA) was labeled with Cyanine 3-CTP (Perkin Elmer Life Sciences, Woodbridge, ON, Canada) using Agilent linear Amplification kits (Agilent Tech. Inc. Mississauga, ON, Canada) following the manufacturer's instruction. Briefly, double-stranded cDNA was synthesized using MMLV-RT with T7 promoter primer, starting with 5 ug total RNA. Cyanine-labeled cRNA targets were in vitro transcribed using T7 RNA polymerase. The synthesized cRNA was precipitated by LiCl and fragmented at 60 degrees C for 30 min with fragmentation solution. Cy5- sample cRNA and Cy3- reference cRNA were hybridized to Agilent mouse development microarrays (containing ~20,000 unique 60 mer oligonucleotides; Agilent Tech. Inc. Mississauga, ON, Canada) at 60 degrees C overnight with Agilent hybridization solution and washed according to manufacturer's instruction. Arrays were scanned on a VersArray ChipReader (BioRad Laboratories Ltd., Waterloo, Ontario, Canada), and data were acquired with ImaGene 5.5 (BioDiscovery, Inc. CA, USA). Present calls were determined as signals that were greater than the mean plus three times the standard deviation of the average of the negative control spots.