Integrated In Vivo Genotoxicity Assessment of Procarbazine Hydrochloride Demonstrates Induction of Pig‐a and LacZ Mutations, and Micronuclei, in MutaMouse Hematopoietic Cells

Abstract

Procarbazine hydrochloride (PCH) is a DNA‐reactive hematopoietic carcinogen with potent and well‐characterized clastogenic activity. However, there is a paucity of in vivo mutagenesis data for PCH, and in vitro assays often fail to detect the genotoxic effects of PCH due to the complexity of its metabolic activation. We comprehensively evaluated the in vivo genotoxicity of PCH on hematopoietic cells of male MutaMouse transgenic rodents using a study design that facilitated assessments of micronuclei and Pig‐a mutation in circulating erythrocytes, and lacZ mutant frequencies in bone marrow. Mice were orally exposed to PCH (0, 6.25, 12.5, and 25 mg/kg/day) for 28 consecutive days. Blood samples collected 2 days after cessation of treatment exhibited significant dose‐related induction of micronuclei in both immature and mature erythrocytes. Bone marrow and blood collected 3 and 70 days after cessation of treatment also showed significantly elevated mutant frequencies in both the lacZ and Pig‐a assays even at the lowest dose tested. PCH‐induced lacZ and Pig‐a (immature and mature erythrocytes) mutant frequencies were highly correlated, with R 2 values ≥0.956, with the exception of lacZ vs. Pig ‐ a mutants in mature erythrocytes at the 70‐day time point (R 2 = 0.902). These results show that PCH is genotoxic in vivo and demonstrate that the complex metabolism and resulting genotoxicity of PCH is best evaluated in intact animal models. Our results further support the concept that multiple biomarkers of genotoxicity, especially hematopoietic cell genotoxicity, can be readily combined into one study provided that adequate attention is given to manifestation times. Environ. Mol. Mutagen. 60:505–512, 2019. © 2018 Her Majesty the Queen in Right of Canada

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

“…Mutation spectra were generated from plaques collected during experiments aimed at evaluating the induction of mutations in the bone marrow of MutaMouse males exposed to either BaP, ENU, PRC, or TEM using the lacZ assay 5 . Mutant frequencies were previously reported for BaP 8 , PRC 47 and TEM 48 , while mutant frequencies are reported here for the first time for ENU. All of the exposures caused increases in mutant frequencies relative to vehicle-matched controls (Supplementary Table 2), and the results were highly significant (P < 0.0001) for BaP (122.9-fold), PRC (9.7-fold), and ENU (7.2-fold).…”

“…All doses were selected based on pilot studies conducted to identify the maximum tolerated dose as per TG 488 guidance. The BaP 8 , PRC 47 and TEM 48 data are the same presented their respective reference. Matched controls received the solvent (olive oil or water) by oral gavage during the same period.…”

The 28 + 28 day design is an effective sampling time for analyzing mutant frequencies in rapidly proliferating tissues of MutaMouse animals

“…A noteworthy exception is female germ cells since it is not possible to collect a sufficient number of eggs to obtain enough DNA for conducting the TGR assay (Yauk et al 2005 ). While the majority of TGR models detect mostly point mutations and short indels (Lambert et al 2005 ), the TGR assay can be easily integrated with the MN assay and other genotoxic endpoints (Hori et al 2019 ; Lemieux et al 2011 ; Long et al 2018 ; Maurice et al 2019 ) for the simultaneous analysis of both mutations and chromosomal events.…”

Chemically induced mutations in a MutaMouse reporter gene inform mechanisms underlying human cancer mutational signatures

“…Mutation patterns were generated from plaques collected during experiments aimed at evaluating the induction of mutations in the bone marrow of MutaMouse males exposed to either BaP, ENU, PRC, or TEM using the lacZ assay 5 . Mutant frequencies were previously reported for BaP 8 , PRC 47 , and TEM 48 . All of the exposures caused increases in mutant frequencies relative to vehicle-matched controls (Supplementary Table 2 ), and the results were highly significant ( P < 0.0001) for BaP (122.9-fold), PRC (9.7-fold), and ENU (7.2-fold).…”

“…All doses were selected based on pilot studies conducted to identify the maximum tolerated dose as per TG 488 guidance. The BaP 8 , PRC 47 , and TEM 48 data are the same as presented in the respective reference. Matched controls received the solvent (olive oil or water) by oral gavage during the same period.…”