In vivo assessment of <i>Pig‐a</i> gene mutation—recent developments and assay validation

Dertinger, Stephen D.; Heflich, Robert H.

doi:10.1002/em.20685

Cited by 28 publications

(14 citation statements)

References 29 publications

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“…Multi-laboratory trials initiated by Litron Laboratories [19], the Japanese Research Group [20], and the HESI initiative [5] have contributed to establishing protocols for the assay, testing the inter-laboratory reproducibility of the assay, and in expanding the number of agents tested.…”

Section: State Of Validationmentioning

confidence: 99%

The in vivo Pig-a assay: A report of the International Workshop On Genotoxicity Testing (IWGT) Workgroup

Gollapudi¹,

Lynch

Heflich

et al. 2015

Mutation Research/Genetic Toxicology and Environmental Mutagene

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146

151

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Section: State Of Validationmentioning

confidence: 99%

The in vivo Pig-a assay: A report of the International Workshop On Genotoxicity Testing (IWGT) Workgroup

Gollapudi¹,

Lynch

Heflich

et al. 2015

Mutation Research/Genetic Toxicology and Environmental Mutagene

Self Cite

146

151

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“…BaP is one of the five standard mutagens included in Stage III of an international validation study on the rat Pig-a gene mutation assay [Dertinger and Heflich, 2011]. Stage III studies involve the use of standardized reagents and a core protocol, including a 28-day daily dosing regime, three predetermined test agent doses, and mandatory sampling times.…”

Section: Introductionmentioning

confidence: 99%

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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“…In recent years, a novel in vivo gene mutation assay has been developed that uses the phosphatidylinositol glycan, class A gene (Pig-a in rodents, PIG-A in humans) as an endogenous reporter (Dertinger and Heflich, 2011;Dobrovolsky et al, 2010a;Peruzzi et al, 2010). The principle of the assay is that the pathway for glycosylphosphatidylinositol (GPI) anchor synthesis requires the Pig-a gene product; thus the absence of GPI-anchored proteins on the surface of mammalian cells (usually erythrocytes) can be used as a reporter of Pig-a mutation.…”

Section: Introductionmentioning

confidence: 99%

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

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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In vivo assessment of Pig‐a gene mutation—recent developments and assay validation

Cited by 28 publications

References 29 publications

The in vivo Pig-a assay: A report of the International Workshop On Genotoxicity Testing (IWGT) Workgroup

The in vivo Pig-a assay: A report of the International Workshop On Genotoxicity Testing (IWGT) Workgroup

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

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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