The rat gut micronucleus assay: A good choice for alternative in vivo genetic toxicology testing strategies

Abstract

The in vivo bone marrow (BM) micronucleus assay is one of the three tests in the standard test battery to assess the genotoxic potential of a pharmaceutical candidate. In some cases, depending on results of in vitro studies, the route of administration or the degree of systemic exposure, in vivo assessment of genotoxicity in the BM alone may not be sufficient. Based on the potential for high gut exposures to orally administered compounds with low systemic exposures as well as the potential susceptibility of rapidly dividing cells of the intestinal tissues, we have developed a modified technique for evaluating micronuclei formation in both the duodenum and colon of rats based on earlier publications. Adult male Sprague Dawley rats were treated once daily for 2 days with either vehicle control or with the test articles acetyl salicylic acid (ASA), carbendazim (CAR), cyclophosphamide (CP), dimethylhydrazine (DMH), mitomycin C (MMC) or vinblastine sulfate (VIN). The duodenum, colon, and BM were harvested, processed, and analyzed for micronucleus induction. Results from these studies demonstrated differences in the susceptibility for different test compounds in the three tissues tested. When MMC and VIN were dosed by different routes at the same dose levels both compounds produced positive results in all three tissues by intraperitoneal injection but not oral administration. These studies suggest that overall the GI micronucleus assay might be a useful tool for clastogenic and aneugenic compounds that are expected to produce high sustained concentrations in the gastrointestinal tract with little systemic exposure.

Comparison of Comet assay dose‐response for ethyl methanesulfonate using freshly prepared versus cryopreserved tissues

“…Additional research into the stability of frozen, transported colon samples for use in the Comet assay may help in understanding the high control values noted in these samples. As one avenue of investigation, we intend to explore other methods for collecting colonic epithelial cells to see if other, less mechanically stressful techniques might produce samples with lower, less variable background levels of DNA damage [Coffing et al,2011]. The weaker response to EMS exposure seen in colonic epithelial cells, as evidenced by the higher LED compared with the other 3 cell types, may be due to absorption of EMS in the stomach, greatly reducing downstream exposure of colon tissue.…”

Evaluation of the Pig‐a, micronucleus, and comet assay endpoints in a 28‐day study with ethyl methanesulfonate

“…The method used for the gut micronucleus assay was previously reported [Coffing et al, ]. Briefly, on Day 29 the entire colon (from anus to cecum) was removed from the animal, flushed with PBS and inverted over a glass rod and incubated in a tube containing 0.5% trypsin/Hanks Balanced Salt Solution (HBSS) for 20 min at 30°C.…”

Report on stage III Pig‐a mutation assays using N‐ethyl‐N‐nitrosourea – comparison with other in vivo genotoxicity endpoints

“…The method used for the gut micronucleus assay was previously reported by Coffing et al [2011]. Briefly, on Day 29 the entire colon (from anus to cecum) was removed from the animal, flushed with PBS and inverted over a glass rod and incubated in a tube containing 0.5% trypsin/Hanks Balanced Salt Solution (HBSS) for 20 min at 308C.…”