Integrated In Vivo Genotoxicity Assessment of Procarbazine Hydrochloride Demonstrates Induction of Pig-a and LacZ Mutations, and Micronuclei, in MutaMouse Hematopoietic Cells
- DOI
- Language of the publication
- English
- Date
- 2018-12-28
- Type
- Article
- Author(s)
- Maurice, Clotilde
- Dertinger, Stephen D.
- Yauk, Carole L.
- Marchetti, Francesco
- Publisher
- Wiley
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 R2 values ≥0.956, with the exception of lacZ vs. Pig-a mutants in mature erythrocytes at the 70-day time point (R2 = 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.
Plain language summary
Health Canada is responsible for evaluating the hazards posed by new and existing chemicals, including assessment of a chemical’s ability to damage genetic material (i.e., genotoxicity). Genetic damage can manifest as abnormalities in chromosome integrity (i.e., clastogenicity) or changes in the DNA sequence (i.e., mutations), both of which are enablers of cancer, as well as other types of disease. Health Canada also supports the development and validation of test guidelines for the Organisation for the Economic Co-operation and Development (OECD) that are routinely used for assessing chemicals for genotoxicity. In this study, Health Canada scientists together with scientists from a US biotechnology company (Litron Laboratories) have used procarbazine, a prototypical chemotherapeutic agent, to demonstrate the feasibility of integrating multiple endpoints of genotoxicity within the same experimental design, thereby reducing the number of laboratory animals used for testing. The results showed that procarbazine was a strong inducer of both clastogenicity and mutagenicity in the mouse hematopoietic cells (all types of blood cells). Furthermore, the study showed that the two mutagenic assays that were employed (the lacZ assay with an established OECD test guideline, and the Pig-a assay with an OECD test guideline in development) produced similar results indicating that both assays are effective at detecting the mutagenic activity of chemicals. Overall, these results provide an example of how multiple endpoints of genotoxicity can be integrated into a single study design to obtain a comprehensive understanding of the mechanism by which chemicals can damage the genetic material and provide support data for the development on an OECD test guideline for the Pig-a assay.
Subject
- Health,
- Health and safety