Analysis of mutations and bone marrow micronuclei in Big Blue rats fed leucomalachite green

Leucomalachite green (LMG) is the major metabolite of malachite green (MG), a triphenylmethane dye that has been used widely as an antifungal agent in the fish industry. Concern over MG and LMG is due to the potential for consumer exposure, suggestive evidence of tumor promotion in rodent liver, and suspicion of carcinogenicity based on structure-activity relationships. In order to evaluate the risks associated with exposure to LMG, female Big Blue rats were fed up to 543 ppm LMG; groups of these rats were killed after 4, 16, or 32 weeks of exposure and evaluated for genotoxicity. We previously reported that this treatment resulted in a dose-dependent induction of liver DNA adducts, and that the liver lacI mutant frequency (MF) was increased, but only in rats fed 543 ppm LMG for 16 weeks. In the present study, we report the results from lymphocyte Hprt mutant assays and bone marrow micronucleus assays performed on these same rats. In addition, we have determined the types of lacI mutations induced in the rats fed 543 ppm LMG for 16 weeks and the rats fed control diet. No significant increases in the frequency of micronuclei or Hprt mutants were observed for any of the doses or time points assayed. Molecular analysis of 80 liver lacI mutants from rats fed 543 ppm LMG for 16 weeks revealed that 21% (17/80) were clonal in origin and that most (55/63) of the independent mutations were base pair substitutions. The predominant type of mutation was G:C --> A:T transition (31/63) and the majority (68%) of these involved CpG sites. When corrected for clonality, the 16-week lacI mutation frequency (36 +/- 10) x 10(-6) in treated rats was not significantly different from the clonally corrected control frequency (17 +/- 9 x 10(-6); P = 0.06). Furthermore, the lacI mutational spectrum in treated rats was not significantly different from that found for control rats (P = 0.09). Taken together, these data indicate that the DNA adducts produced by LMG in female rats do not result in detectable levels of genotoxicity, and that the increase in lacI MF observed previously in the liver of treated rats may be due to the disproportionate expansion of spontaneous lacI mutations.     

Molecular analysis of mitochondrial DNA mutations from bleomycin-treated rats

In our previous studies, we have shown the mutagenicity of bleomycin (BLM) at the nuclear hprt locus. In the present study we have analyzed mutagenic effects of BLM in mitochondrial DNA (mtDNA) using short extension-PCR (SE-PCR) method for detection of low-copy deletions. Fisher 344 rats were treated with a single dose of BLM and total DNA preparations from splenic lymphocytes were processed in SE-PCR assay. Spontaneous deletions were typically flanked by direct repeats (78.5%), while the in BLM-treated group, direct repeats were found in only 46.6% of breakpoints. The ratio between deletions based on direct repeats and random sequence deletions changed from 3.67 in control group to 0.87 in BLM-treated animals, which corresponds to an approximate 1.7-fold increase in the deletion mutation frequency. Furthermore, 62.5% of deletions not flanked by direct repeats in the treated group contained cleavage sites for BLM. The localization of breakpoints was not entirely random. We have found four clusters containing deletions from both groups indicative of deletion hot spots. The results indicate that BLM exposure may be associated with the induction of mtDNA mutations, and suggest the utility of SE-PCR method for evaluating drug-induced genotoxicity.     

Accumulation of point mutations in mitochondrial DNA of aging mice

Mitochondrial DNA (mtDNA) exists in a highly genotoxic environment created by exposure to reactive oxygen species, somewhat deficient DNA repair, and the relatively low fidelity of polymerase gamma. Given the severity of the environment, it was anticipated that mutation accumulation in the mtDNA of aging animals should exceed that of nuclear genes by several orders of magnitude. We have analyzed fragments amplified from the D-loop region of mtDNA from 2 to 22-month-old mice. The amplified 432 bp fragments were cloned into plasmid vectors, and plasmid DNAs from individual clones were purified and sequenced. None of 110 fragments from young mice contained a mutation, while 9 of 87 clones originating from old animals contained base substitutions (chi square = 11.9, P<0.001). The estimated mutation frequency in mtDNA from old mice was 11.6+/-2.7 or 25.4+/-7.8 per 10(5) nucleotides (depending on assumptions of clonality), which exceeds existing estimates for mutation frequencies for nuclear genes by approximately 1000-fold. Our data suggest that at 22 months of age, which roughly corresponds to 3/4 of the mouse natural life span, most mtDNA molecules carry multiple point mutations.     

Hprt mutant frequency and molecular analysis of Hprt mutations in rats treated with mutagenic carcinogens

Much of the progress in the field of cancer research has come from the increased understanding of the molecular events associated with the initiation and accumulation of mutational events associated with carcinogenesis. Genetic toxicologists have developed a number of in vitro and in vivo non-mammalian and mammalian systems to predict those genetic events required to induce the cancer process. Several model rodent systems have been proposed that have the ability to detect and quantify in vivo somatic mutation in endogenous genes and transgenes and relate the nature of the mutation to the specific type of chemical damage. One such system, the rat lymphocyte hypoxanthine guanine phosphoribosyl transferase (Hprt) assay is described in this review. Data are presented that describe mutant induction and mutational spectra in N-ethyl-N-nitrosourea (ENU), 7,12-dimethylbenzo[a]anthracene (DMBA) and thiotepa (TEPA) treated rats.     

Metabolism of daidzein by fecal bacteria in rats

Daidzein (4',7-dihydroxyisoflavone), a soy phytoestrogen, is a weakly estrogenic compound that may have potential health benefits. Biotransformation of daidzein by the human gut microflora after ingestion converts it to either the highly estrogenic metabolite equol or to nonestrogenic metabolites. We investigated the metabolism of daidzein by colonic microflora of rats. Fecal samples, obtained before and after rats were exposed to daidzein at 250 or 1000 parts per million, were incubated in brain-heart infusion (BHI) broth with daidzein under anaerobic conditions. Samples were removed from the cultures daily and analyzed by high-performance liquid chromatography (HPLC) and mass spectrometry. The fecal bacteria of all rats, regardless of prior daidzein exposure, metabolized the added daidzein to dihydrodaidzein. Both compounds disappeared rapidly from BHI cultures incubated for more than 24 h, but no other daidzein metabolites were detected. Only daidzein and dihydrodaidzein were found in a direct analysis of the feces of rats that had consumed daidzein in their diets. Unlike the fecal bacteria of humans and monkeys, the rat flora rapidly metabolized daidzein to aliphatic compounds that could not be detected by HPLC or mass spectral analysis.