Genome-wide mutation detection by interclonal genetic variation.

Genetic toxicology assays estimate mutation frequencies by phenotypically screening for the activation or inactivation of endogenous or exogenous reporter genes. These reporters can only detect mutations in narrow areas of the genome and their use is often restricted to certain in vitro and in vivo models. Here, we show that Interclonal Genetic Variation (ICGV) can directly identify mutations genome-wide by comparing sequencing data of single-cell clones derived from the same source or organism. Upon ethyl methanesulfonate (EMS) exposure, ICGV detected greater levels of mutation in a dose- and time-dependent manner in E. coli. In addition, ICGV was also able to identify a ∼20-fold increase in somatic mutations in T-cell clones derived from an N-ethyl-N-nitrosourea (ENU)-treated rat vs. a vehicle-treated rat. These results demonstrate that the genetic differences of single-cell clones can be used for genome-wide mutation detection.

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.

Mutagenicity of acrylamide and glycidamide in the testes of big blue mice.

Acrylamide (AA) is an industrial chemical, a by-product of fried starchy foods, and a mutagen and rodent carcinogen. It can also cause damage during spermatogenesis. In this study, we investigated whether AA and its metabolite glycidamide (GA) induce mutagenic effects in the germ cells of male mice. Male Big Blue transgenic mice were administered 1.4 or 7.0mM of AA or GA in the drinking water for up to 4 weeks. Testicular cII mutant frequency (MF) was determined 3 weeks after the last treatment, and the types of the mutations in the cII gene were analyzed by DNA sequencing. The testes cII MFs in mice treated with either the low or high exposure concentrations of AA and GA were increased significantly. There was no significant difference in the cII MFs between AA and GA at the low exposure concentration. The mutation spectra in mice treated with AA (1.4mM) or GA (both 1.4 and 7.0mM) differed significantly from those of controls, but there were no significant differences in mutation patterns between AA and GA treatments. Comparison of the mutation spectra between testes and livers showed that the spectra differed significantly between the two tissues following treatment with AA or GA, whereas the mutation spectra in the two tissues from control mice were similar. These results suggest that AA possesses mutagenic effects on testes by virtue of its metabolism to GA, possibly targeting spermatogonial stem cells, but possibly via different pathways when compared mutations in liver.

The genotoxicity of acrylamide and glycidamide in big blue rats.

Acrylamide (AA), a mutagen and rodent carcinogen, recently has been detected in fried and baked starchy foods, a finding that has prompted renewed interest in its potential for toxicity in humans. In the present study, we exposed Big Blue rats to the equivalent of approximately 5 and 10 mg/kg body weight/day of AA or its epoxide metabolite glycidamide (GA) via the drinking water, an AA treatment regimen comparable to those used to produce cancer in rats. After 2 months of dosing, the rats were euthanized and blood was taken for the micronucleus assay; spleens for the lymphocyte Hprt mutant assay; and liver, thyroid, bone marrow, testis (from males), and mammary gland (females) for the cII mutant assay. Neither AA nor GA increased the frequency of micronucleated reticulocytes. In contrast, both compounds produced small (approximately twofold to threefold above background) but significant increases in lymphocyte Hprt mutant frequency (MF, p < 0.05), with the increases having dose-related linear trends (p < 0.05 to p < 0.001). Neither compound increased the cII MF in testis, mammary gland (tumor target tissues), or liver (nontarget tissue), while both compounds induced weak positive increases in bone marrow (nontarget tissue) and thyroid (target tissue). Although the genotoxicity in tumor target tissue was weak, in combination with the responses in surrogate tissues, the results are consistent with AA being a gene mutagen in the rat via metabolism to GA.

Genotoxicity of acrylamide and its metabolite glycidamide administered in drinking water to male and female Big Blue mice.

The recent discovery of acrylamide (AA), a probable human carcinogen, in a variety of fried and baked starchy foods has drawn attention to its genotoxicity and carcinogenicity. Evidence suggests that glycidamide (GA), the epoxide metabolite of AA, is responsible for the genotoxic effects of AA. To investigate the in vivo genotoxicity of AA, groups of male and female Big Blue (BB) mice were administered 0, 100, or 500 mg/l of AA or equimolar doses of GA, in drinking water, for 3-4 weeks. Micronucleated reticulocytes (MN-RETs) were assessed in peripheral blood within 24 hr of the last treatment, and lymphocyte Hprt and liver cII mutagenesis assays were conducted 21 days following the last treatment. Further, the types of cII mutations induced by AA and GA in the liver were determined by sequence analysis. The frequency of MN-RETs was increased 1.7-3.3-fold in males treated with the high doses of AA and GA (P < or = 0.05; control frequency = 0.28%). Both doses of AA and GA produced increased lymphocyte Hprt mutant frequencies (MFs), with the high doses producing responses 16-25-fold higher than that of the respective control (P < or = 0.01; control MFs = 1.5 +/- 0.3 x 10(-6) and 2.2 +/- 0.5 x 10(-6) in females and males, respectively). Also, the high doses of AA and GA produced significant 2-2.5-fold increases in liver cII MFs (P < or = 0.05; control MFs = 26.5 +/- 3.1 x 10(-6) and 28.4 +/- 4.5 x 10(-6)). Molecular analysis of the mutants indicated that AA and GA produced similar mutation spectra and that these spectra were significantly different from that of control mutants (P < or = 0.001). The predominant types of mutations in the liver cII gene from AA- and GA-treated mice were G:C-->T:A transversions and -1/+1 frameshifts in a homopolymeric run of Gs. The results indicate that both AA and GA are genotoxic in mice. The MFs and types of mutations induced by AA and GA in the liver are consistent with AA exerting its genotoxicity in BB mice via metabolism to GA.