Evaluation of Rats' In Vivo Genotoxicity Induced by N-ethyl-N-nitrosourea in the RBC Pig-a, PIGRET, and gpt Assays

Abstract

The recently developed Pig-a mutation assay is based on flow cytometric enumeration of glycosylphosphatidylinositol (GPI) anchor-deficient red blood cells caused by a forward mutation in the Pig-a gene. Because the assay can be conducted in nontransgenic animals and the mutations accumulate with repeat dosing, we believe that the Pig-a assay could be integrated into repeat-dose toxicology studies and provides an alternative to transgenic rodent (TGR) mutation assays. The capacity and characteristics of the Pig-a assay relative to TGR mutation assays, however, are unclear. Here, using transgenic gpt delta mice, we compared the in vivo genotoxicity of single oral doses of N-ethyl-N-nitrosourea (ENU, 40 mg/kg), benzo[a]pyrene (BP, 100 and 200 mg/kg), and 4-nitroquinoline-1-oxide (4NQO, 50 mg/kg) in the Pig-a (peripheral blood) and gpt (bone marrow and liver) gene mutation assays. Pig-a assays were conducted at 2, 4, and 7 weeks after the treatment, while gpt assays were conducted on tissues collected at the 7-week terminal sacrifice. ENU increased both Pig-a and gpt mutant frequencies (MFs) at all sampling times, and BP increased MFs in both assays but the Pig-a MFs peaked at 2 weeks and then decreased. Although 4NQO increased gpt MFs in the liver, only weak, nonsignificant increases (two-or threefold above control) were detected in the bone marrow in both the Pig-a and the gpt assay. These findings suggest that further studies are needed to elucidate the kinetics of the Pig-a mutation assay in order to use it as an alternative to the TGR mutation assay. Environ. Mol.

Absence of in vivo mutagenicity of multi-walled carbon nanotubes in single intratracheal instillation study using F344 gpt delta rats

“…The Pig-a assay was performed as described previously [28, 29]. Blood (3 μL) was labeled with anti-rat CD59 (1 μg) and anti-rat erythroid marker (0.133 μg) antibodies.…”

Pyrene did not induce gene mutation in red blood cell Pig-a assay and PIGRET assay in rats

“…The sample preparation and the gating strategy for the RBC Pig-a assay and PIGRET assay were described previously [18][19][20][21][22]. In brief, 3 L of blood in heparin were mixed with 0.2 mL PBS, and then the cells were labeled with anti-rat CD59 (1 g) and anti-rat erythroid marker (0.133 g) antibodies.…”

“…After gating for single cells, approximately 1 × 10 6 erythroid marker (HIS49)-positive cells were analyzed for the presence of surface CD59, and the Pig-a MFs were calculated as previously described. Briefly, the FCM gating strategy to count CD59-negative cells has been described previously [18][19][20][21][22]. Before analyzing the experimental samples, all gates to count the CD59-negative RBCs and RETs were set using unstained and single-stained samples.…”

“…The enriched samples were incubated for 30 min in the dark at room temperature to label with anti-CD59 and anti-HIS49 antibodies for total RBC labeling. Finally, the cells were resuspended in 0.5 mL PBS and the Pig-a MFs using CD71-and HIS49-positive RETs were examined using a BD FACSCant TM II flow cytometer as previously described [18][19][20][21][22].…”

“…This assay was named as the PIGRET assay. Recently, a collaborative study by the Japan Health Sciences Foundation validated the single dose PIGRET assay [18][19][20][21][22]. Based on the findings from this collaborative study, it was suggested that the single dose PIGRET assay may detect the in vivo mutagenicity of test chemicals earlier than the RBC Pig-a assay.…”