The <i>in vivo</i> Pig-a gene mutation assay is applied to study the genotoxicity of procarbazine hydrochloride in Sprague-Dawley rats

Abstract

-The Pig-a gene is well-known to encode a key enzyme essential in the biosynthesis of glycosylphosphatidylinositol (GPI), which attaches CD molecules (Cluster differentiation), such as CD55 and CD59, to red blood cells (RBCs) membranes. In this study, the blood was marked with the special antigen CD45-PE (Phycoerythrin) to separate the erythrocytes from the leukocytes, then the Pig-a mutant frequency (MF) of RBCs could be investigated without pivotal antigen CD59-FITC (Fluorescein isothiocyanate), and the optimal ENU concentration was determined to be 100 mg/kg/day. The optimal cell number to count and the stability of specimens were determined. At last, the in vivo Pig-a gene mutation assay was utilized to detect the potential genotoxicity of Cis-Dichlorodiammineplatinum (DP), Procarbazine Hydrochloride (PH), and Triptolide (TP). The results indicated that the Pig-a gene mutation obviously occurred in the PH treatment group. In the DP treatment group, an irregular shape with a slightly serrated border in erythrocytes was observed in the blood smear. However, no obvious Pig-a gene mutations were detected in the DP treatment and TP treatment groups under the experimental conditions. In conclusion, this primary in vivo Pig-a gene mutation assay will provide an easy protocol to use in screening potential genotoxic compounds.

Assessment of the Pig‐a, micronucleus, and comet assay endpoints in rats treated by acute or repeated dosing protocols with procarbazine hydrochloride and ethyl carbamate

“…Based on this mode of action, the mutagenicity of PCZ has been reliably detectable in many in vivo tests, but it has been negative using in vitro genotoxicity assays due to the complex metabolism of the drug and deficiencies in the metabolic activation systems used for in vitro testing (Bronzetti et al, 1979;Suter, 1987;Myhr, 1991;Suzuki et al, 1999). In our previous in vivo Pig-a study (Pu et al, 2016), PCZ produced weak positive results in limited testing, where we detected a maximal~2.9-fold increase in mutant erythrocytes. In this study, the leading phenotypic indicator of the Pig-a mutation, the mean RET CD59− frequency, exhibited maximal~75.6-fold and 124.6-fold increases higher than the controls in PCZtreated rats in the 3-day and 28-day studies, respectively.…”

“…Although the two test chemicals in our study, PCZ and EC, have been investigated previously in similar integrated studies as well as assays measuring single genotoxicity endpoints, including Pig‐a gene mutation (Phonethepswath et al, ; Bemis et al, ; Labash et al, ; Stankowski et al, ; Pu et al, ; Revollo et al, ), the data reported herein allow comparisons between the responses for multiple genotoxicity endpoints as a function of two commonly employed treatment regimens (short‐term, 3‐day and subchronic, 28‐day studies). In addition to conducting multiple genotoxicity assays, analyses were made to collect data for hematology, clinical chemistry, organ weight, and other general toxicity indicators.…”