Spectrum of Pig-a mutations in T lymphocytes of rats treated with procarbazine

Abstract

Procarbazine is a primary component of antineoplastic combination chemotherapy often used for the treatment of Hodgkin's lymphoma. It is believed that cytostatic and cytotoxic properties of procarbazine are mediated via its interaction with genomic DNA. Procarbazine is a carcinogen in animal models; it is classified as Group 2A compound by IARC. Also it is known as an in vitro and in vivo mutagen and genotoxicant. However, the molecular mechanism by which procarbazine induces mutations is not thoroughly understood and the spectrum of procarbazine-induced in vivo mutations is described insufficiently. We employed flow cytometry-based erythrocyte and T lymphocyte assays in order to quantify the frequencies of cells deficient in glycosylphosphatidyl inositol-anchored surface markers CD59 and CD48 (presumed mutants in the endogenous X-linked Pig-a gene) in rats. The rats were treated once daily with 100 mg/kg procarbazine HCl for 3 days. In addition, we sorted mutant-phenotype spleen T cells and immediately analysed their Pig-a gene using next generation sequencing of dual-indexed multiplex libraries and error-correcting data filtering. More than 100-fold increase in the frequencies of CD59-deficient RBCs was observed at Day 29 after the last administration, and a 10-fold increase in the frequency of CD48-deficient T cells was observed at Days 45 to 50. Sequencing revealed that, in T cells from procarbazine-treated rats, mutations in the Pig-a gene occurred predominantly at A:T basepairs when A was located on the non-transcribed DNA strand. A→T transversion was the most common mutation. Our results suggest that, at least for the transcribed X-linked Pig-a gene, in vivo methyl guanine adducts are not the major contributors to mutations induced by procarbazine.

Sequencing Chemically Induced Mutations in the Mutamouse Lacz Reporter Gene Identifies Human Cancer Mutational Signatures

“…ENU induced T>A mutations consistent with alkylation of thymine, specifically O 2 -and O 4 -ethyl thymine 50, 51 . We found that PRC induced T>A mutations and, to a lesser extent T>C mutations, which is consistent with the pattern of mutations that was observed in an endogenous gene 52 . The mutation spectrum of TEM was significantly different from controls, but there were no significant changes in specific SNV types.…”

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

“…Likewise, the maximal increases in mean RBC CD59− frequencies were~105.9-fold and~374.4-fold over background in the 3-day and 28-day studies, respectively. The strong Pig-a responses described herein for PCZ in the 3-day study are greater than earlier positive Pig-a findings for similar treatments in CD-1 mice treated orally with 37.5-150 mg/kg/day PCZ (Phonethepswath et al, 2013) and in Sprague-Dawley rats exposed intraperitoneally to 100 mg/kg/day PCZ (Revollo et al, 2017).…”

“…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.…”

Analysis of mutation in the rat Pig‐a assay: II. Studies with bone marrow granulocytes

“…While the magnetic enrichment did not eliminate all CD48 + BMGs, it made the population of CD48‐deficient BMGs more prominent, as can be seen in Figure D. Mutant BMGs (from the “CD11B‐mutants” gate) from Rats #15, 16, 19, and 20 were sorted in small pools of 25 cells per well for sequencing of the Pig‐a gene using a previously described protocol, Mutation Analysis with Multiplexed Libraries (MAML; Revollo et al, , ). Wild‐type BMGs (from the gate “WT‐granulocytes” in Fig.…”

“…For instance, Miura et al (, ) found that the average mutant frequency of proaerolysin‐resistant T‐cells isolated from rat spleens was 2.3 × 10 −6 . Previous studies demonstrated that the spontaneous mutant frequency of CD48‐deficient Pig‐a mutant T‐cells in rats was 3.5 ± 2 × 10 −6 and 10.5 ± 5.5 × 10 −6 (Dobrovolsky et al, ; Revollo et al, ). For in vitro Pig‐a assays, the background frequency of Pig‐a mutants was 20–30 × 10 −6 for L5178Y Tk +/− cells (Wang et al, ) and 0.76 × 10 −6 for TK6 cells (Kruger et al, ).…”