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.

Evaluation of mutagenic mode of action in Big Blue mice fed methylphenidate for 24 weeks.

Methylphenidate hydrochloride (MPH), a widely prescribed pediatric drug for attention deficit hyperactivity disorder, induced liver adenocarcinomas in B6C3F1 mice exposed to 500 ppm in feed for 2 years (Dunnick and Hailey (1995) [14]). In order to determine if the induction of liver tumors was by a mutagenic mode of action, groups of male Big Blue (BB) mice (B6C3F1 background) were fed diets containing 50-4000 ppm MPH for 4, 12, or 24 weeks. At sacrifice, the livers were removed and the cII mutant frequency (MF) and spectrum of cII mutations were determined. In addition, the frequencies of micronucleated reticulocytes (MN-RETs) and normochromatic erythrocytes (MN-NCEs) were measured in peripheral blood erythrocytes as was the Hprt MF in splenic lymphocytes. Food consumption and body weight gain/loss were recorded weekly for each animal. The levels of MPH and RA were determined immediately before sacrifice in the serum of mice fed MPH for 24 weeks. A significant loss in body weights (p

DNA adduct formation and induction of micronuclei and mutations in B6C3F1/Tk mice treated neonatally with acrylamide or glycidamide.

Acrylamide, a food contaminant, is carcinogenic in experimental animals, with both genotoxic and nongenotoxic pathways being proposed. To obtain information regarding mechanisms of acrylamide tumorigenesis, we compared the extent of DNA adduct formation and induction of micronuclei and mutations in mice treated neonatally with acrylamide and its electrophilic metabolite glycidamide. Male and female B6C3F1/Tk mice were treated intraperitoneally on postnatal days (PNDs) 1, 8 and 15 or PNDs 1-8 with 0.14 or 0.70 mmol acrylamide or glycidamide per kg body weight per day. One day after the final dose, B6C3F1/Tk(+/+) mice were killed to measure DNA adduct levels and peripheral blood micronuclei. Three weeks after the last treatment, B6C3F1/Tk(+/-) mice were killed to assess the Hprt and Tk mutant frequencies in spleen lymphocytes. The levels of N7-(2-carbamoyl-2-hydroxyethyl)guanine, the major glycidamide-DNA adduct, decreased in the order 0.70 mmol glycidamide > 0.70 mmol acrylamide > 0.14 mmol glycidamide approximately 0.14 mmol acrylamide. Only glycidamide increased the frequency of micronucleated reticulocytes and normochromatic erythrocytes. In mice treated on PNDs 1, 8 and 15, the Hprt mutant frequency was increased by 0.70 mmol glycidamide. In mice dosed on PNDs 1-8, 0.70 mmol glycidamide caused extensive mortality; each of the other treatments increased the Tk mutant frequency, whereas acrylamide increased the Hprt mutant frequency. These data suggest that the mutagenic response in neonatal mice treated on PNDs 1, 8 and 15 is due to glycidamide, whereas mutations resulting from dosing on PNDs 1-8 are due to another mechanism.

Interference of cell cycle progression by zidovudine and lamivudine in NIH 3T3 cells.

Zidovudine (3'-azido-3'-deoxythymidine; AZT) and lamivudine [(-)2',3'-dideoxy-3'-thiacytidine; 3TC] are nucleoside reverse transcriptase inhibitors used to treat and prevent human immunodeficiency virus-1 infections. In short-term incubations (<48 h), AZT, but not 3TC, has been shown to interfere with cell cycle progression. In the present study, we examined if these alterations persist during long-term incubations in which cells were exposed to AZT (0-1000 microM) or 3TC (0-500 microM) in continuous culture for up to 5 weeks. In addition, we investigated the reversibility of these effects upon removal of the drugs. Both drugs caused concentration- and time-dependent decreases in the number of viable cells, with the effect being more pronounced with AZT. There was only a slight increase in the number of viable cells treated with AZT for 5 weeks and then allowed a 1-week recovery period; cell viability in cells treated with 3TC returned to control levels during the recovery period. The decrease in viable cells was not due to apoptotic or necrotic cell death, but rather was associated with S and G2/M phase cell cycle arrest. Western blot analysis indicated that AZT treatment caused a decrease in checkpoint kinase 1 (Chk1) and checkpoint kinase 2 (Chk2) at all time points. Cyclin-dependent kinase 1 was decreased at later time points, while cyclin A was increased at early times. These data indicate that AZT and, to a lesser extent, 3TC interfere with cell growth by slowing cell cycle progression and that checkpoint proteins Chk1 and Chk2 may play an important role in this delay.

Pharmacokinetics, dose-range, and mutagenicity studies of methylphenidate hydrochloride in B6C3F1 mice.

Methylphenidate hydrochloride (MPH) is one of the most frequently prescribed pediatric drugs for the treatment of attention deficit hyperactivity disorder. In a recent study, increased hepatic adenomas were observed in B6C3F1 mice treated with MPH in their diet. To evaluate the reactive metabolite, ritalinic acid (RA) of MPH and its mode of action in mice, we conducted extensive investigations on the pharmacokinetics (PK) and genotoxicity of the drug in B6C3F1 mice. For the PK study, male B6C3F1 mice were gavaged once with 3 mg/kg body weight (BW) of MPH and groups of mice were sacrificed at various time points (0.25-24 hr) for serum analysis of MPH and RA concentrations. Groups of male B6C3F1 mice were fed diets containing 0, 250, 500, 1,000, 2,000, or 4,000 ppm of MPH for 28 days to determine the appropriate doses for 24-week transgenic mutation studies. Also, the micronucleus frequencies (MN-RETs and MN-NCEs), and the lymphocyte Hprt mutants were determined in peripheral blood and splenic lymphocytes, respectively. Mice fed 4,000 ppm of MPH lost significant BW compared to control mice (P < 0.01). There was a significant increase in the average liver weights whereas kidneys, seminal vesicle, testes, thymus, and urinary bladder weights of mice fed higher doses of MPH were significantly lower than the control group (P < or = 0.05). There was no significant increase in either the Hprt mutant frequency or the micronucleus frequency in the treated animals. These results indicated that although MPH induced liver hypertrophy in mice, no genotoxicity was observed.

Transplacental drug transfer and frequency of Tk and Hprt lymphocyte mutants and peripheral blood micronuclei in mice treated transplacentally with zidovudine and lamivudine.

In previous studies, we have shown that zidovudine (3'-azido-3'-deoxythymidine; AZT), but not lamivudine [(-)2',3'-dideoxy-3'-thiacytidine; 3TC], is genotoxic when administered to neonatal mice, and that 3TC when coadministered with AZT does not alter the responses observed with AZT alone (Von Tungeln et al. [2002] Carcinogenesis 23:1427-1432). We now have investigated the transplacental transfer of these drugs and the induction of mutants and micronuclei in the neonatal offspring. From gestational day 12 until parturition, female C57BL/6N and C57BL/6N/Tk(+/-) mice, which had been mated to male C3H/HeNMTV mice, were treated daily by gavage with AZT, 3TC, or a combination of AZT and 3TC. In both dams and fetuses, AZT was found at much higher levels than its metabolites, AZT 5'-glucuronide and 3'-azido-3'-deoxythymidine. In the neonates, AZT and the mixture of AZT and 3TC caused a decrease in the percentage of reticulocytes (RETs) and an increase in the percentage of micronucleated RETs and micronucleated normochromatic erythrocytes. When assessed 3 weeks after birth, AZT and the combination of AZT and 3TC increased the thymidine kinase (Tk) mutant frequency in male mice; at 5 weeks, 3TC increased the Tk mutant frequency in female mice. The increase in Tk mutants in mice treated with AZT and the mixture of AZT and 3TC was associated with loss of the wild-type (Tk(+)) allele (loss of heterozygosity; LOH) and a pattern of discontinuous LOH. These data indicate that AZT, 3TC, and the combination of AZT and 3TC are transplacental mutagens and that the increase in mutants resulting from AZT is due mainly to large-scale genetic alterations.

Micronucleated erythrocyte frequency in control and azidothymidine-treated Tk+/+, Tk+/- and Tk-/- mice.

The first step in the activation of the anti-retroviral nucleoside analogue azidothymidine (AZT) involves its conversion to a 5'-monophosphate. In this study, we have evaluated the role of cytosolic thymidine kinase (Tk), the major enzyme involved in phosphorylating thymidine and its analogues, in the nuclear DNA damage produced by AZT in neonatal mice. Tk+/+, Tk+/- and Tk-/- mice were treated intraperitoneally with 200 mg/kg/day of AZT on postnatal days 1 through 8, and micronuclei were measured in peripheral blood 24 h after the last dose. AZT treatment increased the micronucleus (MN) frequencies to similar extents in both the reticulocytes (RETs) and normochromatic erythrocytes (NCEs) of Tk+/+ and Tk+/- mice; AZT did not increase the frequency of micronucleated RETs (MN-RETs) or micronucleated NCEs (MN-NCEs) in Tk-/- mice. Unexpectedly, neonatal Tk-/- mice treated with the vehicle had significantly elevated MN frequencies for both RETs and NCEs relative to Tk+/+ and Tk+/- mice (e.g., approximately 3.4% MN-RETs and approximately 4.8% MN-NCEs in Tk-/- mice versus approximately 0.7 and approximately 0.6% MN-RETs and MN-NCEs in neonatal Tk+/+ mice). Additional assays performed on untreated Tk-/- mice showed that elevated spontaneous MN frequencies persisted until at least 20 weeks of age, which approaches the average lifespan of Tk-/- mice. These results indicate that metabolism by Tk is necessary for the genotoxicity of AZT in neonatal mice; however, the genotoxicity of AZT is not altered by reducing the Tk gene dose by half. The elevated spontaneous MN frequencies in Tk-/- mice suggest the presence of an endogenous genotoxic activity in these mice.

Alterations in gene expression profiles and the DNA-damage response in ionizing radiation-exposed TK6 cells.

Identifying genes that are differentially expressed in response to DNA damage may help elucidate markers for genetic damage and provide insight into the cellular responses to specific genotoxic agents. We utilized cDNA microarrays to develop gene expression profiles for ionizing radiation-exposed human lymphoblastoid TK6 cells. In order to relate changes in the expression profiles to biological responses, the effects of ionizing radiation on cell viability, cloning efficiency, and micronucleus formation were measured. TK6 cells were exposed to 0.5, 1, 5, 10, and 20 Gy ionizing radiation and cultured for 4 or 24 hr. A significant (P < 0.0001) decrease in cloning efficiency was observed at all doses at 4 and 24 hr after exposure. Flow cytometry revealed significant decreases in cell viability at 24 hr in cells exposed to 5 (P < 0.001), 10 (P < 0.0001), and 20 Gy (P < 0.0001). An increase in micronucleus frequency occurred at both 4 and 24 hr at 0.5 and 1 Gy; however, insufficient binucleated cells were present for analysis at the higher doses. Gene expression profiles were developed from mRNA isolated from cells exposed to 5, 10, and 20 Gy using a 350 gene human cDNA array platform. Overall, more genes were differentially expressed at 24-hr than at the 4-hr time point. The genes upregulated (> 1.5-fold) or downregulated (< 0.67-fold) at 4 hr were those primarily involved in the cessation of the cell cycle, cellular detoxification pathways, DNA repair, and apoptosis. At 24 hr, glutathione-associated genes were induced in addition to genes involved in apoptosis. Genes involved in cell cycle progression and mitosis were downregulated at 24 hr. Real-time quantitative PCR was used to confirm the microarray results and to evaluate expression levels of selected genes at the low doses (0.5 and 1.0 Gy). The expression profiles reflect the cellular and molecular responses to ionizing radiation related to the recognition of DNA damage, a halt in progression through the cell cycle, activation of DNA-repair pathways, and the promotion of apoptosis.

Genotoxicity of malachite green and leucomalachite green in female Big Blue B6C3F1 mice.

Malachite green, a triphenylmethane dye used in aquaculture as an antifungal agent, is rapidly reduced in vivo to leucomalachite green. Previous studies in which female B6C3F1 mice were fed malachite green produced relatively high levels of liver DNA adducts after 28 days, but no significant induction of liver tumors was detected in a 2-year feeding study. Comparable experiments conducted with leucomalachite green resulted in relatively low levels of liver DNA adducts but a dose-responsive induction of liver tumors. In the present study, we fed transgenic female Big Blue B6C3F1 mice with 450 ppm malachite green and 204 and 408 ppm leucomalachite green (the high doses used in the tumor bioassays) and evaluated genotoxicity after 4 and 16 weeks of treatment. Neither malachite green nor leucomalachite green increased the peripheral blood micronucleus frequency or Hprt lymphocyte mutant frequency at either time point; however, the 16-week treatment with 408 ppm leucomalachite green did increase the liver cII mutant frequency. Similar increases in liver cII mutant frequency were not seen in the mice treated for 16 weeks with malachite green or in female Big Blue rats treated with a comparable dose of leucomalachite green for 16 weeks in a previous study [Mutat. Res. 547 (2004) 5]. These results indicate that leucomalachite green is an in vivo mutagen in transgenic female mouse liver and that the mutagenicities of malachite green and leucomalachite green correlate with their tumorigenicities in mice and rats. The lack of increased micronucleus frequencies and lymphocyte Hprt mutants in female mice treated with leucomalachite green suggests that its genotoxicity is targeted to the tissue at risk for tumor induction.

Mutagenicity of food-derived carcinogens and the effect of antioxidant vitamins.

The food-derived heterocyclic amines (HCAs) 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ), 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) are mutagenic in the Ames test and produce tumors in laboratory animals, including monkeys. These HCAs have also been shown to induce gene mutations in vivo. To assess the antimutagenic effects of dietary antioxidant vitamins, beta-carotene, ascorbic acid (vitamin C), and alpha-tocopherol (vitamin E), on food-borne mutagenes/carcinogens, we evaluated the mutagenic activity of the compounds alone or combined with antioxidant vitamins. We utilized the rat lymphocyte mutation assay at the hypoxanthine guanine phosphoribosyl transferase (Hprt) locus. Female Fischer 344 rats treated with different doses (0, 2.5, 5.0, 25.0, and 50.0 mg/kg) of the carcinogens were sacrificed 5 wk after mutagen treatment. Although IQ and MeIQ slightly increased mutation frequency (MF) at some doses, a significant (P < 0.0009) increase in MF was found in animals exposed to MeIQx at 25 mg/kg. PhIP was the most mutagenic of the HCAs, with increases (P < 0.0001) in MF detected at all dose levels compared with controls. Because PhIP was the most mutagenic, it was selected for studies using the dietary antioxidant vitamins. Addition of antioxidant vitamins, singly or in a mixture, caused a significant (P < 0.0001) decrease in PhIP-induced Hprt MF. Vitamin E was the most effective at decreasing Hprt MF. In addition, we determined whether carcinogen metabolism would be affected by ingestion of vitamins. The activities of endogenous detoxification enzymes, glutathione S-transferase and glutathione peroxidase (GPx), were thus examined. Intake of beta-carotene and vitamin C without the carcinogen resulted in an increase (P < 0.05) in GPx activity. Also a modest increase in GPx activity was seen in animals that received the antioxidant mixture alone. Although the mechanisms of action of the antioxidants remain to be determined, the results indicate that dietary-derived HCA treatment induced MF in rat lymphocytes and suggest that antioxidants in food or taken as supplements could, in part, counteract such mutagenic activities.

Detection of mutation in transgenic CHO cells using green fluorescent protein as a reporter.

A novel approach was developed for rapidly estimating the frequency of specific mutations in genetically engineered Chinese hamster ovary (CHO) cells. We designed double-transgenic CHO cell lines that contain a transgene consisting of the sequence coding for green fluorescent protein under the control of a tetracycline (Tet) responsive promoter and a second transgene coding for the constitutively expressed Tet repressor. Cultures of these CHO cells were treated with gamma-radiation, N-methyl-N-nitrosourea or methyl methanesulfonate, and the fluorescence of individual cells from both control and treated cultures was measured by flow cytometry. The treatments increased the number of highly fluorescent cells, those with presumed mutations in the Tet-repressor gene. Mutant cells from gamma-radiation-exposed cultures were isolated by fluorescence-activated cell sorting, cultured, and individual clones expanded. A PCR-based analysis indicated that the highly fluorescent expanded cells had lost the transgene coding for the Tet repressor, suggesting that the system mainly detects large genetic alterations. A similar approach may be useful for making high-throughput in vivo models for mutation detection.