Exploring the molecular and functional cellular response to hydrazine via transcriptomics and DNA repair mutant cell lines.

Hydrazine is a rodent carcinogen and is classified as a probable human carcinogen by IARC. Though hydrazine is positive in both in vitro and in vivo DNA strand break (comet) assays, hydrazine was reported to be negative in an in vitro mutation Muta Mouse lung epithelial cell (FE1) test, as well as in a regulatory-compliant, in vivo Big Blue mouse mutation test. In this article, mechanistic studies explored the cellular response to hydrazine. When tested in a regulatory-compliant mouse lymphoma assay, hydrazine yielded unusual, weakly positive results. This prompted an investigation into the transcriptional response to hydrazine in FE1 cells via RNA sequencing. Amongst the changes identified was a dose-dependent increase in G2/M DNA damage checkpoint activation associated genes. Flow cytometric experiments in FE1 cells revealed that hydrazine exposure led to S-phase cell cycle arrest. Clonogenic assays in a variety of cell lines harboring key DNA repair protein deficiencies indicated that hydrazine could sensitize cells lacking homology dependent repair proteins (Brca2 and Fancg). Lastly, hprt assays with hydrazine were conducted to determine whether a lack of DNA repair could lead to mutagenicity. However, no robust, dose-dependent induction of mutations was noted. The transcriptional and cell cycle response to hydrazine, coupled with functional investigations of DNA repair-deficient cell lines support the inconsistencies noted in the genetic toxicology regulatory battery. In summary, while hydrazine may be genotoxic, transcriptional and functional processes involved in cell cycle regulation and DNA repair appear to play a nuanced role in mediating the mutagenic potential.

Use of the bacterial reverse mutation assay to predict carcinogenicity of N-nitrosamines.

Under ICH M7, impurities are assessed using the bacterial reverse mutation assay (i.e., Ames test) when predicted positive using in silico methodologies followed by expert review. N-Nitrosamines (NAs) have been of recent concern as impurities in pharmaceuticals, mainly because of their potential to be highly potent mutagenic carcinogens in rodent bioassays. The purpose of this analysis was to determine the sensitivity of the Ames assay to predict the carcinogenic outcome with curated proprietary Vitic (n = 131) and Leadscope (n = 70) databases. NAs were selected if they had corresponding rodent carcinogenicity assays. Overall, the sensitivity/specificity of the Ames assay was 93-97% and 55-86%, respectively. The sensitivity of the Ames assay was not significantly impacted by plate incorporation (84-89%) versus preincubation (82-89%). Sensitivity was not significantly different between use of rat and hamster liver induced S9 (80-93% versus 77-96%). The sensitivity of the Ames is high when using DMSO as a solvent (87-88%). Based on the analysis of these databases, the Ames assay conducted under OECD 471 guidelines is highly sensitive for detecting the carcinogenic hazards of NAs.

In vitro and in vivo mammalian mutation assays support a nonmutagenic mechanism of carcinogenicity for hydrazine.

Hydrazine has been described as a mutagenic, probable human carcinogen. It is mutagenic in in vitro systems such as bacterial reverse mutation (Ames) tests and some yeast systems, as well as in in vivo systems with drosophila. It was shown to cause chromosome damage both in vitro and in vivo but was negative in some well-validated mammalian mutation systems such as CHO HPRT assays. Importantly, there is only one in vivo gene mutation test reported, which was negative. Our objective was to determine if hydrazine is mutagenic in mammalian test systems. Thus, we conducted an in vitro gene mutation test in Muta™Mouse lung epithelial cells (FE1 cell assay) and a regulatory-compliant in vivo Big Blue® mouse test. Consistent with previous reports, an additional six-well Ames assay showed that hydrazine was mutagenic to bacteria. The FE1 cell assay was negative in conditions with and without metabolic activation when tested to cytotoxicity limits. In the Big Blue® mouse study, female mice received dosages of hydrazine up to 10.9 mg/kg via drinking water for 28 days. This dose is comparable to a dose used in a carcinogenicity study where female mice had significant increases in hepatocellular adenoma at 11.5 mg/kg. There were no increases in mutant frequency in liver and lung, two tissues sensitive to the carcinogenic effects of hydrazine in mice. Our research shows that hydrazine is not mutagenic in mammalian cells either in vitro or in vivo, indicating mutagenicity may not play a role in the carcinogenicity of hydrazine.

Advances in bumped kinase inhibitors for human and animal therapy for cryptosporidiosis.

Improvements have been made to the safety and efficacy of bumped kinase inhibitors, and they are advancing toward human and animal use for treatment of cryptosporidiosis. As the understanding of bumped kinase inhibitor pharmacodynamics for cryptosporidiosis therapy has increased, it has become clear that better compounds for efficacy do not necessarily require substantial systemic exposure. We now have a bumped kinase inhibitor with reduced systemic exposure, acceptable safety parameters, and efficacy in both the mouse and newborn calf models of cryptosporidiosis. Potential cardiotoxicity is the limiting safety parameter to monitor for this bumped kinase inhibitor. This compound is a promising pre-clinical lead for cryptosporidiosis therapy in animals and humans.