Mason G. Pearce

In vivo genotoxicity of furan in F344 rats at cancer bioassay doses

Furan, a potent rodent liver carcinogen, is found in many cooked food items and thus represents a human cancer risk. Mechanisms for furan carcinogenicity were investigated in male F344 rats using the in vivo Comet and micronucleus assays, combined with analysis of histopathological and gene expression changes. In addition, formamidopyrimidine DNA glycosylase (Fpg) and endonuclease III (EndoIII)-sensitive DNA damage was monitored as a measure of oxidative DNA damage. Rats were treated by gavage on four consecutive days with 2, 4, and 8mg/kg bw furan, doses that were tumorigenic in 2-year cancer bioassays, and with two higher doses, 12 and 16mg/kg. Rats were killed 3h after the last dose, a time established as producing maximum levels of DNA damage in livers of furan-treated rats. Liver Comet assays indicated that both DNA strand breaks and oxidized purines and pyrimidines increased in a near-linear dose-responsive fashion, with statistically significant increases detected at cancer bioassay doses. No DNA damage was detected in bone marrow, a non-target tissue for cancer, and peripheral blood micronucleus assays were negative. Histopathological evaluation of liver from furan-exposed animals produced evidence of inflammation, single-cell necrosis, apoptosis, and cell proliferation. In addition, genes related to apoptosis, cell-cycle checkpoints, and DNA-repair were expressed at a slightly lower level in the furan-treated livers. Although a mixed mode of action involving direct DNA binding cannot be ruled out, the data suggest that furan induces cancer in rat livers mainly through a secondary genotoxic mechanism involving oxidative stress, accompanied by inflammation, cell proliferation, and toxicity.

Confirmation of Pig-a mutation in flow cytometry-identified CD48-deficient T-lymphocytes from F344 rats

The Pig-a assay is used for monitoring somatic cell mutation in laboratory animals and humans. The assay detects haematopoietic cells deficient in glycosylphosphatidylinositol (GPI)-anchored protein surface markers using flow cytometry. However, given that synthesis of the protein markers (and the expression of their genes) is independent of the expression of the X-linked Pig-a gene and the function of its enzyme product, the deficiency of markers at the surface of the cells may be caused by a number of events (e.g. by mutation or epigenetic silencing in the marker gene itself or in any of about two dozen autosomal genes involved in the synthesis of GPI). Here we provide direct evidence that the deficiency of the GPI-anchored surface marker CD48 in rat T-cells is accompanied by mutation in the endogenous X-linked Pig-a gene. We treated male F344 rats with N-ethyl-N-nitrosourea (ENU), and established colonies from flow cytometry-identified and sorted CD48-deficient spleen T-lymphocytes. Molecular analysis confirmed that the expanded sorted cells have mutations in the Pig-a gene. The spectrum of Pig-a mutation in our model was consistent with the spectrum of ENU-induced mutation determined in other in vivo models, mostly base-pair substitutions at A:T with the mutated T on the non-transcribed strand of Pig-a genomic DNA. We also used next generation sequencing to derive a similar mutational spectrum from a pool of 64 clones developed from flow-sorted CD48-deficient lymphocytes. Our findings confirm that Pig-a assays detect what they are designed to detect-gene mutation in the Pig-a gene.

Sensitivity of the Pig-a assay for detecting gene mutation in rats exposed acutely to strong clastogens.

Clastogens are potential human carcinogens whose detection by genotoxicity assays is important for safety assessment. Although some endogenous genes are sensitive to the mutagenicity of clastogens, many genes that are used as reporters for in vivo mutation (e.g. transgenes) are not. In this study, we have compared responses in the erythrocyte Pig-a gene mutation assay with responses in a gene mutation assay that is relatively sensitive to clastogens, the lymphocyte Hprt assay, and in the reticulocyte micronucleus (MN) assay, which provides a direct measurement of clastogenicity. Male F344 rats were treated acutely with X-rays, cyclophosphamide (CP) and Cis-platin (Cis-Pt), and the frequency of micronucleated reticulocytes (MN RETs) in peripheral blood was measured 1 or 2 days later. The frequencies of CD59-deficient Pig-a mutant erythrocytes and 6-thioguanine-resistant Hprt mutant T-lymphocytes were measured at several times up to 16 weeks after the exposure. All three clastogens induced strong increases in the frequency of MN RETs, with X-rays and Cis-Pt producing near linear dose responses. The three agents also were positive in the two gene mutation assays although the assays detected them with different efficiencies. The Pig-a assay was more efficient in detecting the effect of Cis-Pt treatment, whereas the Hprt assay was more efficient for X-rays and CP. The results indicate that the erythrocyte Pig-a assay can detect the in vivo mutagenicity of clastogens although its sensitivity is variable in comparison with the lymphocyte Hprt assay.

CD48-deficient T-lymphocytes from DMBA-treated rats have de novo mutations in the endogenous Pig-a gene

A major question concerning the scientific and regulatory acceptance of the rodent red blood cell‐based Pig‐a gene mutation assay is the extent to which mutants identified by their phenotype in the assay are caused by mutations in the Pig‐a gene. In this study, we identified T‐lymphocytes deficient for the glycosylphosphatidylinositol‐anchored surface marker, CD48, in control and 7,12‐dimethylbenz[a]anthracene (DMBA)‐treated rats using a flow cytometric assay and determined the spectra of mutations in the endogenous Pig‐a gene in these cells. CD48‐deficient T‐cells were seeded by sorting at one cell per well into 96‐well plates, expanded into clones, and exons of their genomic Pig‐a were sequenced. The majority (78%) of CD48‐deficient T‐cell clones from DMBA‐treated rats had mutations in the Pig‐a gene. The spectrum of DMBA‐induced Pig‐a mutations was dominated by mutations at A:T, with the mutated A being on the nontranscribed strand and A→T transversion being the most frequent change. The spectrum of Pig‐a mutations in DMBA‐treated rats was different from the spectrum of Pig‐a mutations in N‐ethyl‐N‐nitrosourea (ENU)‐treated rats, but similar to the spectrum of DMBA mutations for another endogenous X‐linked gene, Hprt. Only 15% of CD48‐deficient mutants from control animals contained Pig‐a mutations; T‐cell biology may be responsible for a relatively large fraction of false Pig‐a mutant lymphocytes in control animals. Among the verified mutants from control rats, the most common were frameshifts and deletions. The differences in the spectra of spontaneous, DMBA‐, and ENU‐induced Pig‐a mutations suggest that the flow cytometric Pig‐a assay detects de novo mutation in the endogenous Pig‐a gene. Environ. Mol. Mutagen. 56:674–683, 2015.

Mutation analysis with random DNA identifiers (MARDI) catalogs Pig-a mutations in heterogeneous pools of CD48-deficient T cells derived from DMBA-treated rats

Identification of mutations induced by xenotoxins is a common task in the field of genetic toxicology. Mutations are often detected by clonally expanding potential mutant cells and genotyping each viable clone by Sanger sequencing. Such a “clone‐by‐clone” approach requires significant time and effort, and sometimes is even impossible to implement. Alternative techniques for efficient mutation identification would greatly benefit both basic and regulatory genetic toxicology research. Here, we report the development of Mutation Analysis with Random DNA Identifiers (MARDI), a novel high‐fidelity Next Generation Sequencing (NGS) approach that circumvents clonal expansion and directly catalogs mutations in pools of mutant cells. MARDI uses oligonucleotides carrying Random DNA Identifiers (RDIs) to tag progenitor DNA molecules before PCR amplification, enabling clustering of descendant DNA molecules and eliminating NGS‐ and PCR‐induced sequencing artifacts. When applied to the Pig‐a cDNA analysis of heterogeneous pools of CD48‐deficient T cells derived from DMBA‐treated rats, MARDI detected nearly all Pig‐a mutations that were previously identified by conventional clone‐by‐clone analysis and discovered many additional ones consistent with DMBA exposure: mostly A to T transversions, with the mutated A located on the non‐transcribed DNA strand. Environ. Mol. Mutagen. 57:114–124, 2016.

Biotransformation of acridine by Mycobacterium vanbaalenii.

Cultures of Mycobacterium vanbaalenii strain PYR-1 in a liquid medium were exposed to the toxic environmental contaminant acridine (260 microM). After incubation for 7 d, the cultures were extracted with ethyl acetate. Metabolites were purified using high-performance liquid chromatography and analyzed by mass spectrometry and 1H nuclear magnetic resonance spectroscopy. Four metabolites, 9,10-dihydroacridine, 4-hydroxyacridine, acridine cis-1 ,2-dihydrodiol, and acridin-9(10H)-one, were identified.

In Vivo Genotoxicity of Estragole in Male F344 Rats

Estragole, a naturally occurring constituent of various herbs and spices, is a rodent liver carcinogen which requires bio‐activation. To further understand the mechanisms underlying its carcinogenicity, genotoxicity was assessed in F344 rats using the comet, micronucleus (MN), and DNA adduct assays together with histopathological analysis. Oxidative damage was measured using human 8‐oxoguanine‐DNA‐N‐glycosylase (hOGG1) and EndonucleaseIII (EndoIII)‐modified comet assays. Results with estragole were compared with the structurally related genotoxic carcinogen, safrole. Groups of seven‐week‐old male F344 rats received corn oil or corn oil containing 300, 600, or 1,000 mg/kg bw estragole and 125, 250, or 450 mg/kg bw safrole by gavage at 0, 24, and 45 hr and terminated at 48 hr. Estragole‐induced dose‐dependent increases in DNA damage following EndoIII or hOGG1 digestion and without enzyme treatment in liver, the cancer target organ. No DNA damage was detected in stomach, the non‐target tissue for cancer. No elevation of MN was observed in reticulocytes sampled from peripheral blood. Comet assays, both without digestion or with either EndoIII or hOGG1 digestion, also detected DNA damage in the liver of safrole‐dosed rats. No DNA damage was detected in stomach, nor was MN elevated in peripheral blood following dosing with safrole suggesting that, as far both safrole and estragole, oxidative damage may contribute to genotoxicity. Taken together, these results implicate multiple mechanisms of estragole genotoxicity. DNA damage arises from chemical‐specific interaction and is also mediated by oxidative species. Environ. Mol. Mutagen. 56:356–365, 2015.

Spectrum of benzo[a]pyrene-induced mutations in the Pig-a gene of L5178YTk+/− cells identified with next generation sequencing

We used Sanger sequencing and next generation sequencing (NGS) for analysis of mutations in the endogenous X-linked Pig-a gene of clonally expanded L5178YTk+/- cells. The clones developed from single cells that were sorted on a flow cytometer based upon the expression pattern of the GPI-anchored marker, CD90, on their surface. CD90-deficient and CD90-proficient cells were sorted from untreated cultures and CD90-deficient cells were sorted from cultures treated with benzo[a]pyrene (B[a]P). Pig-a mutations were identified in all clones developed from CD90-deficient cells; no Pig-a mutations were found in clones of CD90-proficient cells. The spectrum of B[a]P-induced Pig-a mutations was dominated by basepair substitutions, small insertions and deletions at G:C, or at sequences rich in G:C content. We observed high concordance between Pig-a mutations determined by Sanger sequencing and by NGS, but NGS was able to identify mutations in samples that were difficult to analyze by Sanger sequencing (e.g., mixtures of two mutant clones). Overall, the NGS method is a cost and labor efficient high throughput approach for analysis of a large number of mutant clones.

In vivo mutation analysis using the ΦX174 transgenic mouse and comparisons with other transgenes and endogenous genes

The ΦX174 transgenic mouse was first developed as an in vivo Ames test, detecting base pair substitution (bps) at a single bp in a reversion assay. A forward mutational assay was also developed, which is a gain of function assay that also detects bps exclusively. Later work with both assays focused on establishing that a mutation was fixed in vivo using single-burst analysis: determining the number of mutant progeny virus from an electroporated cell by dividing the culture into aliquots before scoring mutants. We review results obtained from single-burst analysis, including testing the hypothesis that high mutant frequencies (MFs) of G:C to A:T mutation recovered by transgenic targets include significant numbers of unrepaired G:T mismatches. Comparison between the ΦX174 and lacI transgenes in mouse spleen indicates that the spontaneous bps mutation frequency per nucleotide (mf(n)) is not significantly lower for ΦX174 than for lacI; the response to ENU is also comparable. For the lacI transgene, the spontaneous bps mf(n) is highly age-dependent up to 12 weeks of age and the linear trend extrapolates at conception to a frequency close to the human bps mf(n) per generation of 1.7 × 10(-8). Unexpectedly, we found that the lacI somatic (spleen) bps mf(n) per cell division at early ages was estimated to be the same as for the human germ-line. The bps mf(n) in bone marrow for the gpt transgene is comparable to spleen for the lacI and ΦX174 transgenes. We conclude that the G:C to A:T transition is characteristic of spontaneous in vivo mutation and that the MFs measured in these transgenes at early ages reflect the expected accumulation of in vivo mutation typical of endogenous mammalian mutation rates. However, spontaneous and induced mf(n)s per nucleotide for the cII gene in spleen are 5-10 times higher than for these other transgenes.

Sex-specific dose-response analysis of genotoxicity in cyproterone acetate-treated F344 rats.

Cyproterone acetate (CPA), a synthetic hormonal drug, induces rat liver tumors in a sex-specific manner, with five-fold higher doses needed to induce liver tumors in male rats compared to females. In order to evaluate the potential of the in vivo alkaline Comet assay to predict the sex-specific carcinogenicity of CPA, CPA-induced direct DNA damage (DNA strand breaks and alkali-labile sites) were evaluated in the livers of both male and female F344 rats. In addition, secondary oxidative DNA damage was measured concurrently utilizing the human 8-oxoguanine-DNA-N-glycosylase (hOGG1) and EndonucleaseIII (EndoIII)-modified in vivo alkaline Comet assays and the reticulocyte micronucleus (MN) frequency was analyzed in peripheral blood. Groups of 5 seven-week-old male and female F344 rats received olive oil or 10, 25, 50 or 100 mg/kg bw CPA in olive oil by gavage at 0, 24, and 45 h and were sacrificed at 48 h. CPA-induced direct DNA damage in rat liver showed the same sex-specific pattern as its hepatotumorigenicity: a five-fold-higher dose of CPA was needed to induce a statistically significant increase in direct DNA damage in livers of males compared to females. However, peripheral blood MN frequency was weak in both sexes and CPA-induced oxidative DNA damage was generally greater in male than female rat livers. Taken together, our results demonstrate concordance in the sex-specificity of CPA in the in vivo alkaline Comet assay and cancer bioassay, while the induction of oxidative DNA damage by CPA was not directly correlated with its tumorigenicity.