P XV.4 4-Chloro-o-phenylenediamine a 26-week oral (in feed) mutagenicity study in Big Blue™” mice (original) (raw)
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Specific mutational spectrum of dimethylnitrosamine in the lacI transgene of Big Blue® C57BL/6 mice
Mutagenesis, 1998
Dimethylnitrosamine (DMN) produces tumors in mice predominantly in the liver, but also in the kidney and lung. It forms C^-methylguanine adducts in DNA, which induce G:C-»A:T transitions. We have analyzed the spectra of spontaneous and DMN-induced mutations in the lacl transgene of the Big Blue* mouse (C57BL/6). In both cases, mutations in the liver, kidney and lung were predominantly base substitutions, among which G:C->A:T transitions were the most frequent In contrast, a high incidence of short deletions (2-23 bp) was only found in the liver of treated mice. The deletions often occurred at direct repeat sequences. Single-base deletion incidence was also higher in the liver than in the kidney and lung. These results imply that accumulation of DNA lesions or their repair in liver is different from other organs. Spontaneous and induced base substitutions and deletions appeared to be randomly distributed in the lac I gene and an apparent hotspot was not observed, except for a 4 bp deletion of a (TGGC) 3 sequence at positions 621-632. The present data demonstrate, for the first time, that DMN induces short deletions especially in the liver, although the mechanism involved needs further investigation.
Acta Pharmacologica et Toxicologica, 1981
The effects of 3-methylcholanthrene (MC) pretreatment on metabolism and mutagenic activation of dimethylnitrosamine (DMN) were studied with liver subfractions from two strains of mice differing genetically with respect to aromatic hydrocarbon responsiveness. Both mutagenic activation and DMN N-demethylase activity segregated with aryl hydrocarbon (benzo[a]pyrene) hydroxylase activity as a dominant trait in appropriate crosses between C57BL/6J (&Ahb) and DBA/2J (AhdAhd) mice. DMN metabolism and mutagenicity were increased by MC-pretreatment in responsive Ahb Ahb and Ahb Ahd mice, but not in nonresponsive AhdAhd mice. This indicates the involvement of the A h locus in the genetic regulation of these activities in mice. Deuteration of DMN reduced mutagenicity and DMN N-demethylase activity by approximately 90 and 50 percent, respectively.
Environmental Mutagenesis, 1982
The cytotoxicity and mutagenicity of dimethylnitrosamine (DMN) was determined in the CHO/HGPRT system. Metabolic activation of the promutagen was achieved by use of a liver homogenate supernatant (S9) prepared from Aroclor 1254-induced Sprague-Dawley rats. The cytotoxic and mutagenic effects of DMN were enhanced by the inclusion of calcium chloride in the incubation mix, and this enhancement was dependent on the presence of sodium phosphate. Under conditions that yielded maximal activity (10 mM calcium chloride, 10 mM magnesium chloride, 50 mM sodium phosphate), an apparent calcium phospate precipitate was observed. DMN activity increased with increasing amounts of S9 protein over the range 0.3–3.0 mg/ml in the S9 mix and appeared to plateau at higher concentrations. The mutagenicity of DMN can be described as 110 mutants/106 cells per mM DMN per mg/ml S9 protein per hour.
Evaluation of mutagenic mode of action in Big Blue mice fed methylphenidate for 24 weeks
Mutation Research/Genetic Toxicology and Environmental Mutagenesis, 2009
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 ≤ 0.01) was found in mice fed the 2000 and 4000 ppm doses of MPH; however, there was no significant difference in the food consumption by any of the MPH-treated groups. The average liver cII MF in control animals was 20 ± 4.5 × 10 −6 where as in MPH-treated animals, the average cII MFs ranged between 20 ± 2.5 and 32 ± 6.7 × 10 −6 . None of the cII MFs in livers from any of the MPH treatment was significantly higher than the concurrent controls at either 4, 12 or 24 weeks. Further, there was no significant increase in either the Hprt MF or the micronucleus frequency at any time point in the treated animals. These results suggest that MPH is not mutagenic in mice and that the induction of tumors as previously reported in the liver is probably through a nongenotoxic mode of action.
Mutation Research/Genetic Toxicology and Environmental Mutagenesis, 1997
w x Ž. Feeding of 2-amino-1-methyl-6-phenylimidazo 4,5-b pyridine PhIP to F344 rats induces colon tumors specifically in male rats. Mutant frequencies and mutational spectra of the lacI transgene were studied in male and female Big Blue w transgenic rats after feeding 400 ppm of PhIP in the diet for 60 days. Mutant frequencies in the colon mucosa were increased 20-25 times compared with those of the control rats, being 661.4 " 33.3 = 10 y6 and 718.2 " 16.9 = 10 y6 in males and females, respectively. No significant differences in types and distribution of the mutations were detected between males and females. One-base deletions were the most frequent mutation, including the characteristic guanine deletion at 5 X-GGGA-3 X which is also seen in the Apc gene of rat colon cancers induced by PhIP. Comparison of the lacI mutations in the rat colon with those previously identified in the mouse colon showed that the rate of G to T transversions was significantly higher in the mouse. This is the first report stating that there exist differences in the mutation specificity on the same gene, among mammalian species. However, the characteristic guanine deletion was recovered in both the mouse and the rat. These findings do not offer a mechanistic explanation of the gender specificity of PhIP-induced colon cancer in rats, though the universality of the guanine deletion suggests that this alteration may prove a useful indicator of human exposure. q 1997 Elsevier Science B.V.
Biochemical Pharmacology, 2001
Oxazepam (OX), a widely used benzodiazepine anxiolytic, phenobarbital (PHE), a drug used for convulsive disorders, and Wyeth 14,643 (WY; [4-chloro-6-(2,3-xylidino)-2-pyrimidinylthio]acetic acid), a hypolipidemic agent, are all hepatocarcinogenic in B6C3F1 mice. They have been classified as "non-genotoxic" carcinogens since they are non-DNA reactive in in vitro assays and are either negative or weakly positive in Salmonella typhimurium (Ames assay). Male B6C3F1 Big Blue ® transgenic mice were fed 2500 ppm of OX or PHE or 500 ppm of WY in their diet, while a control group of mice received diet alone for 180 days. The mutant frequency (MF) of cII in the control mice, after correction for clonality, was 6.2 Ϯ 2.8 ϫ 10 Ϫ5. The MF values for mice fed OX, PHE, and WY were 10.0 Ϯ 3.6 ϫ 10 Ϫ5 (P Ͻ 0.05), 7.9 Ϯ 1.3 ϫ 10 Ϫ5 (P ϭ 0.1) and 17.4 Ϯ 4.2 ϫ 10 Ϫ5 (P Ͻ 0.01), respectively. The mutation spectrum (MS) at cII from the PHE-fed mice was significantly different (P Ͻ 0.05) from that of the control mice even though the MF was not, whereas the MS spectra of mice fed OX (P ϭ 0.4) and WY (P ϭ 0.7) were not significantly different. The PHE-derived spectrum differed from the spontaneous spectrum in the lower occurrence of G:CϾC:G transversions (17 vs 1.6%) and the higher incidence of A:TϾT:A transversions (3.4 vs 9.5%). Prior to correction for clonal expansion, each treated group exhibited a high incidence of frameshift mutations at the homopolymeric run of guanines at bp 179-184 (OX 21%, PHE 21%, WY 16% of the total mutations); this was not the case with the control group (6%). Even after clonal correction, more than 10% of the mutations were frameshifts in the treated mice, while 5% were frameshifts in the control mice. Despite this hypersensitive region of the gene, our findings suggest that the cII locus is less sensitive than the lacI locus to mutation induction by non-DNA reactive carcinogens.
Mutation Research Letters, 1993
2-Acetylaminofluorene (2-AAF) was administered at Ievels of 0, 300 and 600 ppm in the diet for 28 days to female transgenic micc bearing the lacl genein a Iambda vector (Big Blue® mice). The Iambda vector was excised from liver DNA and packaged in vitro into bacteriophage particles which were allowed to infect E. coli bacteria, forming plaques on agar plates. Approximately 10 5 plaques wcre screened per animal for the appearance of a bluc colour, indicative of mutations in the lac/ gcnc which had resulted in an inactive gene product. Background mutation rate was 2.7 x 10-5 (pooled results of two animals, 8 mutant plaques/289 530 plaques). At 300 ppm in the diet, the rate of 3.5 X 10-5 (8/236 300) was not significantly increased over background. At 600 ppm in the dict, the rate increased approximately 3 fold to 7.7 x 10-5 (17 /221240). In comparison to the usual single or 5-day carcinogen exposure regimes, the 4-week exposure protocol allowed the use of much lower dose Ievels 00-1000 fold lower). Overt toxicity could thus be avoided. The daily doses used were somewhat higher than those required in 2-year carcinogenicity studies with 2•AAF. Transgenie animal technology is currently opening up new possibilities in biomedical research. One application of particular interest to toxicology is the devclopment of in vivo mutation assays as quantitative tools to assess thc mutagenic potency of chemicals. One such assay uscs transgenic mice bearing the /ac/ genc (ßig Bluc®
Cancer Letters, 1999
Gender differences and organ speci®city of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP)-induced mutagenesis were examined with the new gptD transgenic mouse (TA new transgenic mouse mutagenesis test system using Spi 2 and 6-thioguanine selections (Environ. Mol. Mutagen. 28 (1996) 465±470). In this mouse model, two distinct selections are employed to ef®ciently detect different types of mutations, i.e 6-thioguanine (6-TG) selection for point mutations and Spi 2 selection for deletions, respectively. In both selections, the highest mutant frequencies were observed in colon, followed by in spleen and liver. No increases in mutations were observed in testis, brain and bone marrow in PhIP-treated male mice. No signi®cant differences in 6-TG and Spi 2 mutant frequencies were observed in colon and liver between male and female treated mice. The correlation between PhIP-induced mutagenesis and carcinogenesis in colon is discussed. q
The use of transgenic mice for short-term, in vivo mutagenicity testing
Gene Analysis Techniques, 1990
In order to develop a short-term, in vivo assay to study the mutagenic effects of chemical exposure, transgenic mice were generated using a lambda shuttle vector containing a lacZ target gene. Following exposure to mutagens, this target can be rescued efficiently from genomic DNA prepared from tissues of the treated mice using restriction minus, in vitro lambda phage packaging extract and restriction minus Escherichia coli plating cultures. Mutations in the target gene appear as colorless plaques on a background of blue plaques when plated on indicator agar. Spontaneous background levels were ~1 x 10 -5 in each of three mouse lineages analyzed. Exposure of lambda transgenic mice to N-ethyl-N-nitrosourea resulted in as much as a 14-fold induction in detected mutations over background levels. The assay is currently being modified to incorporate lacl as the target for ease of mutation detection as well as in vivo excision properties of the Lambda ZAP vector, facilitating sequence analysis of mutant plaques.