Relationships between exposure, cell loss and proliferation, and manifestation of Hprt mutant T cells following treatment of preweanling, weanling, and adult male mice with N-ethyl-N-nitrosourea

Azidothymidine (AZT) is a nucleoside reverse transcriptase inhibitor (NRTI) that is used for reducing mother-to-child transmission of human immunodeficiency virus I. Combinations of AZT and 3'-thiacytidine (3TC) are even more effective than AZT alone. AZT, however, is a mutagen and carcinogen in rodent models and 3TC can increase the genotoxicity of AZT. Since p53 plays a key role in human and mouse tumorigenesis, p53-haplodeficient mice are currently being evaluated as a model for assessing the carcinogenicity of perinatal exposure to NRTIs. In the present study, male C57BL/6 p53(+/+) and p53(-/-) mice were mated with C3H p53(+/+) females; the pregnant females were treated on gestation day 12 through parturition with 40, 80, and 160 mg/kg of AZT or a combination of 160 mg/kg AZT and 100 mg/kg 3TC (AZT-3TC); the p53(+/+) and p53(+/-) offspring were treated daily after birth through postnatal day (PND) 28. The frequencies of micronucleated reticulocytes (MN-RETs) and micronucleated normochromatic erythrocytes (MN-NCEs) were determined on PND1, PND10, and PND28; the frequency of Hprt mutant lymphocytes was measured on PND28. The frequencies of MN-RETs and MN-NCEs were increased in treated animals at all time points; there were no differences in the responses of p53(+/+) and p53(+/-) animals treated with identical doses of NRTIs. After correction for clonal expansion, both AZT and AZT-3TC treatments induced small but significant increases in the frequency of Hprt mutant lymphocytes in p53(+/-) mice, but not in p53(+/+) mice. The data indicate that p53 haplodeficiency affects the genotoxicity of NRTIs; thus, p53(+/-) mice may be a sensitive model for evaluating the carcinogenicity of perinatal exposure to NRTIs.

Section: Discussionsupporting

confidence: 55%

Section: Discussionmentioning

confidence: 99%

Frequency of Hprt mutant lymphocytes and micronucleated erythrocytes in p53‐haplodeficient mice treated perinatally with AZT and AZT in combination with 3TC

Dobrovolsky

Shaddock

Mittelstaedt

et al. 2007

“…Both the Hprt and Pig-a genes are located on the X-chromosome, and mutations measured in lymphocytes and in RETs/RBCs for both genes are hypothesized to be induced in bone marrow progenitor cells [Jansen et al, 1996;Miura et al, 2009], although lymphocyte and RET/ RBC mutations may be induced in progenitor cells of different lineages. The Day 56 sampling time (4 weeks following a 28-day treatment) represents a reasonable time for measuring Hprt lymphocyte mutants in rodents [Aidoo et al, 1993;Smith et al, 1995;Walker et al, 1999]; Piga lymphocyte mutant manifestation is not known, but assumed to be similar to that of Hprt. Because of the similar kinetics of their manifestation (4 to 6 weeks), Hprt lymphocyte mutant frequencies are probably best compared with Pig-a total RBC mutant frequencies.…”

Section: Discussionmentioning

confidence: 99%

Report on stage III Pig‐a mutation assays using benzo[a]pyrene

Bhalli

Shaddock

Pearce

et al. 2011

Genotoxicity assays were conducted on rats treated with benzo[a]pyrene (BaP) as part of Stage III of a validation study on the Pig-a gene mutation assay. Assays were performed at the U.S. FDA-NCTR and Bayer-Germany. Starting on Day 1, groups of five 6- to 7-week-old male Fischer 344 (F344, used at FDA-NCTR) and Han Wistar rats (Bayer) were given 28 daily doses of 0, 37.5, 75, or 150 mg/kg BaP; blood was sampled on Days -1, 4, 15, 29, and 56. Pig-a mutant frequencies were determined on Days -1, 15, 29, and 56 in total red blood cells (RBCs) and reticulocytes (RETs) as RBC(CD59-) and RET(CD59-) frequencies; percent micronucleated-RETs (%MN-RET) were measured on Days 4 and 29. RBC(CD59-) and RET(CD59-) frequencies increased in a dose- and time-dependent manner, producing significant increases by Day 29 in both rat models. The responses for RETs were stronger than those for RBCs, and the responses in F344 rats were stronger than in Han Wistar rats. BaP also produced significant increases in %MN-RET frequency at Days 4 and 29, with the responses being greater in F344 than Han Wistar rats. The overall findings were consistent with those of the reference laboratory using Han Wistar rats. Finally, mutation assays performed on splenocytes from Day 56 F344 rats indicated that BaP mutant frequencies were three to fivefold higher for the Hprt gene than the Pig-a gene. The results indicate that the Pig-a RET and RBC assays are reproducible, transferable, and show promise for integrating gene mutation into 28-day repeat-dose studies.

“…Mutation assays that utilize the X-linked Hprt gene, which codes for a nonessential enzyme in the purine salvage pathway, have been developed to detect, quantify, isolate, and sequence mutants in peripheral T-lymphocytes from humans, monkeys, mice, and rats [reviewed in Casciano et al, 1999;Albertini, 2001]. The rodent lymphocyte Hprt mutation assay is a valuable and sensitive tool for investigating the mutagenic potency and specificity of alkylating agents and potential environmental carcinogens in the whole animal [Jones et al, 1987;Casciano et al, 1999;Walker et al, 1999b], permitting important direct comparisons between mutations in the same gene in experimental animals and people. Because mutations in the Hprt reporter gene reflect changes in genes critical to carcinogenesis [Wang et al, 1999;Noori and Hou, 2001], studies of the impact of potential carcinogens upon the mutant frequency in this reporter gene are informative for evaluating cancer risk in exposed humans.…”

Section: Introductionmentioning

confidence: 99%

Hprt mutant frequencies in splenic T‐cells of male F344 rats exposed by inhalation to propylene

Walker

Seilkop

Scott

et al. 2004

Propylene is a major industrial intermediate and atmospheric pollutant to which humans are exposed by inhalation. In this study, 6-week-old male F344 rates were exposed to 0, 200, 2000, or 10,000 ppm propylene by inhalation for 4 weeks (6 h/day, 5 days/week), and mutant frequencies were determined in the Hprt gene of splenic T-lymphocytes. Twenty milligrams of cyclophosphamide monohydrate (CPP)/kg bw, given on the penultimate day of propylene exposure, was used as a positive control mutagen. Rats (n = 8/group) were necropsied for isolation of T-cells 8 weeks after the last dose, a sampling time that produced peak spleen Hprt mutant frequencies (Mfs) in a preliminary mutant manifestation study using CCP treatment. Hprt Mfs were measured via the T-cell cloning assay, which was performed without knowledge of the animal treatment groups. Mean Hprt Mfs were significantly increased over control values (mean Mf = 5.24 +/- 1.55 (SD) x 10(-6)) in CPP-treated rats (10.37 +/- 4.30 x 10(-6), P = 0.007). However, Hprt Mfs in propylene-exposed rats were not significantly increased over background, with mean Mfs of 4.90 +/- 1.84 x 10(-6) (P = 0.152), 5.05 +/- 3.70 x 10(-6) (P = 0.895), and 5.95 +/- 2.49 x10(-6) (P = 0.500) for animals exposed to 200, 2000, or 10,000 ppm propylene, respectively. No significant increase in F344 rat or B6C3F1 mouse cancer incidence was reported in the National Toxicology Program carcinogenicity studies of propylene across this same exposure range. Taken together, these findings support the conclusion that inhalation exposure of rats to propylene does not cause mutations or cancer.