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.

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ABSTRACT. A study was undertaken to determine the effects of incremental levels of dietary restriction (DR) in rats. Survival, growth, reproductive, and dietary intake (DI)

We have developed a limiting dilution clonal assay for determining the frequency of 6-thioguanine-resistant (TGr) lymphocytes produced in rats by in vivo exposure to genotoxic agents. Spleen lymphocytes were isolated from female Fischer 344 rats and were cultured with 1 microgram/ml of phytohemagglutinin (PHA) for 40 hr. Northern blot analysis revealed that this procedure resulted in increased hprt and beta-actin mRNA synthesis. Conditions for optimum cloning were established by culturing four PHA-primed lymphocytes/well in 96-well round-bottom microtiter plates containing a medium supplemented with interleukin-2. These cultures also contained autologous and/or TK6 feeder cells inactivated with different doses of irradiation. Lymphocyte cloning efficiencies (CEs) were highest in plates containing both irradiated TK6 cells (5 x 10(3) cells/well; 90 Gy) and irradiated autologous feeder cells (5 x 10(4) cells/well; 50 Gy). CE did not depend on the number of primed lymphocytes/well when four or fewer target cells/well were cloned. To measure the effects of chemical mutagens on the frequency of TGr lymphocytes, rats were given a single i.p. injection of 0-150 mg/kg of N-ethyl-N-nitrosourea (ENU), a direct-acting alkylating agent, or 0-50 mg/kg of cyclophosphamide (CP), an indirect acting alkylating agent. Lymphocytes were isolated, primed, and cloned at 4 weeks after CP treatment and at 1, 2, 4 and 6 weeks after ENU treatment. CE in these cultures ranged from 12% to 27%. Cultures were also established for measuring CE in the presence of 6-thioguanine (TG) and these contained 5 x 10(3) irradiated TK6 cells and 5 x 10(4) primed rat lymphocytes/well. The frequency of TGr lymphocytes was calculated by correcting the CE in the presence of TG with the CE measured in its absence. ENU exposure produced a higher frequency of TGr lymphocytes than CP, but both chemicals produced a dose-dependent increase in TGr cells. In addition, the frequency of ENU-induced TGr lymphocytes increased with time after treatment. The TGr cells are presumed to be hprt mutants, but further analysis at the DNA level is required to establish this. The dose-dependent responses obtained with both ENU and CP treatments suggest that rat lymphocytes are sensitive to direct- and indirect-acting alkylating agents administered in vivo and that the rat lymphocyte assay is a useful complement to the in vivo/in vitro mouse assay for determining the mutagenicity of environmental toxicants.

Methyleugenol (MEG), a constituent of human food, induces malignant tumors in multiple tissues of rats and mice. Although MEG forms DNA adducts and induces unscheduled DNA synthesis in rat liver, it is negative in many in vitro genetic toxicity assays. In the present study, we evaluated MEG-induced DNA damage in the rat using (1) the alkaline Comet assay, (2) the oxidative Comet assay, and (3) expression profiling of genes associated with DNA damage pathways. Male F344 rats received single oral doses of 400 or 1000 mg/kg body weight (bw) MEG and DNA damage was assessed by the Comet assay in liver, bladder, bone marrow, kidney, and lung 3 h and 24 h later. MEG failed to produce any increase in DNA damage. In addition, rats were given a single oral dose of 2000 mg/kg bw MEG, and Comet assays were performed with liver, bone marrow, and bladder 1, 3, 6, and 8 h later. With one exception (bone marrow at 8 h), no DNA damage was detected. Enzyme-modified Comet assays were conducted in parallel with standard Comet assays in liver. Whereas no MEG-induced DNA damage was detected following formamidopyrimidine DNA glycosylase digestion, digestion with endonuclease III resulted in increases in DNA damage at the 6- and 8-h sampling times. Gene expression analysis on the livers from MEG-exposed rats showed significant reduction in genes associated with DNA repair. The results indicate that MEG induces DNA damage in rat liver and that oxidative DNA damages may be partly responsible for the genotoxicity of MEG in rodents.

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N-Hydroxy-2-acetylaminofluorene (N-OH-AAF) is the proximate carcinogenic metabolite of the powerful rat liver carcinogen 2-acetylaminofluorene. In this study, transgenic Big Blue(R) rats were used to examine the relationship between in vivo mutagenicity and DNA adduct formation by N-OH-AAF in the target liver compared with that in nontarget tissues. Male rats were given one, two, or four doses of 25 mg N-OH-AAF/kg body weight by i.p. injection at 4-day intervals, and groups of treated and control rats were euthanized up to 10 weeks after beginning the dosing. Mutant frequencies were measured in the spleen lymphocyte hprt gene, and lacI mutant frequencies were determined in the liver and spleen lymphocytes. At 6 weeks after beginning the dosing, the hprt mutant frequency in spleen lymphocytes from the four-dose group was 16.5 x 10(-6) compared with 3.2 x 10(-6) in control animals. Also at 6 weeks, rats given one, two, or four doses of N-OH-AAF had lacI mutant frequencies in the liver of 97.6, 155.6, and 406.8 x 10(-6), respectively, compared with a control frequency of 25.7 x 10(-6); rats given four doses had lacI mutant frequencies in spleen lymphocytes of 55.8 x 10(-6) compared with a control frequency of 20.4 x 10(-6). Additional rats were evaluated for DNA adduct formation in the liver, spleen lymphocytes, and bone marrow by (32)P-postlabeling. Adduct analysis was conducted 1 day after one, two, and four treatments with N-OH-AAF, 5 days after one treatment, and 9 days after two treatments. N-(Deoxyguanosin-8-yl)-2-aminofluorene was the major DNA adduct identified in all the tissues examined. Adduct concentrations increased with total dose to maximum values in samples taken 1 day after two doses, and remained essentially the same after four doses. In samples taken after four doses, adduct levels were 103, 28, and 7 fmol/microg of DNA in liver, spleen lymphocytes, and bone marrow, respectively. The results indicate that the extent of both DNA adduct formation and mutant induction correlates with the organ specificity for N-OH-AAF carcinogenesis in the rat. Environ. Mol. Mutagen. 37:195-202, 2001. Published 2001 Wiley-Liss, Inc.