Report on stage III Pig‐a mutation assays using N‐ethyl‐N‐nitrosourea – comparison with other in vivo genotoxicity endpoints

Cammerer, Zoryana; Bhalli, Javed A.; Cao, Xuefei; Coffing, Stephanie L.; Dickinson, Donna; Dobo, Krista L.; Dobrovolsky, Vasily N.; Engel, Maria; Fiedler, Ronald D.; Gunther, William C.; Heflich, Robert H.; Pearce, Mason G.; Shaddock, Joseph G.; Shutsky, Thomas; Thiffeault, Catherine J.; Schuler, Maik

doi:10.1002/em.20686

Cited by 38 publications

(35 citation statements)

References 19 publications

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“…With regard to relative weight gain, F344 rats treated with 150 mg/kg/day BaP had only 5.6% of the weight gain of the vehicle control, while similarly treated Han Wistar rats gained 20% of the control in this study and 10% of the control in the study conducted by the reference laboratory [Dertinger et al, 2010]. Note that Pig-a mutant frequencies and MN frequencies were very similar for F344 and Han Wistar (and Sprague Dawley) rats in Stage III assays conducted with Nethyl-N-nitrosourea [Cammerer et al, 2011].…”

Section: Discussionmentioning

confidence: 54%

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

Bhalli

Shaddock

Pearce

et al. 2011

Environ and Mol Mutagen

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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.

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Section: Discussionmentioning

confidence: 54%

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

Bhalli

Shaddock

Pearce

et al. 2011

Environ and Mol Mutagen

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“…high degree of correlation has been consistent among the Stage II and Stage III interlaboratory collaborators Cammerer et al, 2011;Dertinger et al, 2011b;Lynch et al, 2011;Shi et al, 2011], and demonstrates the transferability of the assay across test sites.…”

Section: Discussionmentioning

confidence: 56%

Integration of Pig‐a, micronucleus, chromosome aberration, and comet assay endpoints in a 28‐day rodent toxicity study with 4‐nitroquinoline‐1‐oxide

Stankowski

Roberts

Chen

et al. 2011

Environ and Mol Mutagen

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As part of the Stage III Pig-a multilaboratory validation trial, we examined the induction of CD59-negative reticulocytes and total red blood cells (RET(CD59-) and RBC(CD59-) , respectively) in male Sprague Dawley(®) rats treated with 4-nitroquinoline-1-oxide (4NQO), for 28 consecutive days by oral gavage, at doses of 1.25, 2.50, 3.75, 5.00, and 7.50 mg kg(-1) day(-1) (the high dose group was sacrificed on Day 15 due to excessive morbidity/mortality). Animals also were evaluated for: micronucleated reticulocytes (mnRET) by flow cytometry; DNA damage in peripheral blood, liver, and stomach using the Comet assay; and chromosome aberrations (CAb) in peripheral blood lymphocytes (PBL). All endpoints were analyzed at two or more timepoints where possible. Mortality, body and organ weights, food consumption, and clinical pathology also were evaluated, and demonstrated that the maximum tolerated dose was achieved at 5.00 mg kg(-1) day(-1) . The largest increases observed for the genetic toxicology endpoints (fold-increase compared to control, where significant; all at 5.00 mg kg(-1) day(-1) on Day 29) were: RET(CD59-) (21X), RBC(CD59-) (9.0X), and mnRET (2.0X). In contrast, no significant increases were observed for the CAb or Comet response, in any tissue analyzed, at any timepoint. Because 4NQO is a well known mutagen, clastogen, and carcinogen, the lack of response for these latter endpoints was unexpected. These results emphasize the extreme care that must betaken in dose and endpoint selection when incorporating genotoxicity endpoints into routine toxicity studies as has been recommended or is under consideration by various regulatory and industrial bodies.

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“…The potent gene mutagen, ENU, was shown in several earlier studies to produce large increases in Piga mutant frequency in dosed rats (e.g., Cammerer et al, 2011;Dertinger et al, 2010;Miura et al, 2009). In this study, we compared the kinetics of Pig-a mutant frequency and micronucleus induction in rats after four weekly doses of ENU.…”

Section: Discussionmentioning

confidence: 99%

Effective use of the Pig-a gene mutation assay for mutagenicity screening: measuring CD59-deficient red blood cells in rats treated with genotoxic chemicals

et al. 2012

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-The Pig-a gene mutation assay using perpherial blood erythrocytes is being investigated as a screening tool for assessing mutagenicity in vivo. In this study, we evaluated two distinct approaches for performing the Pig-a assay in rats. We used antibodies to CD45 or the erythroid marker HIS49 to identify red blood cells (RBCs), and then monitored the kinetics of Pig-a mutant frequency, as measured by the frequency of CD59-deficient RBCs, in rats treated with the genotoxic chemicals, N-ethyl-N-nitrosourea, cyclophosphamide, 4-nitroquinoline-1-oxide, and ethylmethanesulfonate. In some instances, micronucleus frequency also was measured in the same animals. Time-and dose-related increases in Pig-a mutant frequency were found in all the chemical-treated groups, except for the groups treated with cyclophosphamide, which was a potent inducer of micronuclei. The two different approaches we employed were comparable for measuring induced mutant frequencies, but our historical data showed that the mean background frequencies for the CD45/CD59 method and the HIS49/CD59 method were 12.7 × 10 -6 and 5.5 ×10 -6 , respectively. The relatively low, stable background mutant frequency associated with the HIS49/CD59 method indicates that it may have greater power for discriminating weak induced responses. These results suggest that the HIS49/CD59 method is a promising tool for measuring Pig-a mutant RBCs. In addition, differences in their manifestation kinetics and in their relative sensitivity for detecting different test compounds suggest that the combination of the Pig-a assay and the micronucleus assay may be effective in identifying in vivo genotoxicity.

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Report on stage III Pig‐a mutation assays using N‐ethyl‐N‐nitrosourea – comparison with other in vivo genotoxicity endpoints

Cited by 38 publications

References 19 publications

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

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

Integration of Pig‐a, micronucleus, chromosome aberration, and comet assay endpoints in a 28‐day rodent toxicity study with 4‐nitroquinoline‐1‐oxide

Effective use of the <i>Pig-a</i> gene mutation assay for mutagenicity screening: measuring CD59-deficient red blood cells in rats treated with genotoxic chemicals

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