Evaluation of phenolphthalein, diazepam and quinacrine dihydrochloride in the in vitro mammalian cell micronucleus test in Chinese hamster ovary (CHO) and TK6 cells.

The in vitro micronucleus assay has been extensively used as an in vitro screening tool to identify test articles that might have aneugenic or clastogenic potential. Currently, the Organization for Economic Co-operation and Development (OECD) is working towards a final version of the guideline for the conduct of the in vitro mammalian cell micronucleus Test (MNvit). A few questions regarding appropriate cytotoxicity measurements and cytotoxicity limits to use remain to be answered. In order to resolve the remaining questions, we compared the induction of micronuclei at the top dose (50-60% cytotoxicity) determined by either Relative Cell Counts (RCC), Relative Increase in Cell Counts (RICC), Relative Population Doublings (RPD), or Cytokinesis-Blocked Proliferating Index (CBPI) using weak and strong inducers of micronuclei in both the presence and absence of cytochalasin B (CYB) in Chinese hamster ovary (CHO) and human lymphoblastoid TK6 cells. In order to assess extensive dose-response relationships, we selected expected weak (diazepam, phenolphthalein, quinacrine dihydrochloride dihydrate) and strong (cytosine arabinoside, mitomycin C, vinblastine sulphate) inducers of micronuclei with a variety of different mechanisms of action for testing. The results clearly demonstrated that all six compounds produced positive responses using either cytotoxicity measurement. The outcome from these studies further supports the cytotoxicity measurements and cytotoxicity limits proposed in the draft OECD guideline.

Further evaluation of a flow cytometric in vitro micronucleus assay in CHO-K1 cells: a reliable platform that detects micronuclei and discriminates apoptotic bodies.

The in vitro micronucleus (MN) assay is widely used to assess genotoxic potential of the test substances by measuring frequency of MN in cultured mammalian cells. Traditionally, MN frequency has been determined by microscopy. In recent years, a flow cytometric method for enumeration of MN has been developed, which significantly shortens analysis time and enhances assay throughput. However, a major concern has been raised that the MN results obtained from flow cytometry can be impacted by chromatin bodies produced during apoptosis or necrosis. In this work, we further evaluated this flow cytometry-based in vitro MN assay with CHO-K(1) cells in a 24-well platform. Our results showed that the MN frequency determined using the flow cytometric method was highly correlated with the microscopy results. Importantly, several non-genotoxic apoptosis inducers or cytotoxins that have been previously reported to produce 'artificial positives' in various in vitro genotoxicity tests were evaluated in this system. As a result, these non-genotoxic cytotoxins did not produce false-positive MN response in the flow cytometric system in CHO-K(1) cells when cytotoxicity was <50 +/- 10%. Moreover, a total of 21 compounds were evaluated in this work, including direct or indirect clastogens, aneugens and non-genotoxic chemicals. A sensitivity of 83.3% and a specificity of 100% were obtained from the compounds we tested. Finally, significant increase of incidents in the hypodiploid region, an aneugenic signature, was confirmed in our evaluation. In conclusion, the flow cytometric in vitro MN assay is a reliable method that can be used to detect clastogenic or aneugenic potential of the test substances in CHO-K(1) cells.

Genotoxicity of 10 cigarette smoke condensates in four test systems: comparisons between assays and condensates.

The particulate fraction of cigarette smoke, cigarette smoke condensate (CSC), is genotoxic in many short-term in vitro tests and is carcinogenic in rodents. However, no study has evaluated a series of CSCs prepared from a diverse set of cigarettes and produced with different smoking machine regimens in several short-term genotoxicity tests. Here we report on the genotoxicity of 10 CSCs prepared from commercial cigarettes that ranged from ultra-low tar per cigarette (< or =6.5 mg) to full flavor (>14.5 mg) as determined by the Federal Trade Commission (FTC) smoking regimen, a reference cigarette blended to be representative of a U.S. FTC-regimen low-tar cigarette, and experimental cigarettes constructed of single tobacco types. CSCs were tested in the presence of rat liver S9 in the Salmonella plate-incorporation assay using frameshift strains TA98 and YG1041; in micronucleus and comet assays in L5178Y/Tk(+/-) 7.3.2C mouse lymphoma cells, and in CHO-K(1) cells for chromosome aberrations. All 10 CSCs were mutagenic in both strains of Salmonella, and the rank order of their mutagenic potencies was similar. Their mutagenic potencies in Salmonella spanned 7-fold when expressed as rev/mug CSC but 158-fold when expressed as rev/mg nicotine; the range of genotoxic potencies of the CSCs in the other assays was similar regardless of how the data were expressed. All 10 CSCs induced micronuclei with a 3-fold range in their potency. All but one CSC induced DNA damage over a 20-fold range, and all but one CSC induced chromosome aberrations over a 4-fold range. There was no relation among the genotoxic potencies of the CSCs across the assays, and a qualitative advantage of the addition of the other assays to the Salmonella assay was not supported by our findings. Although consideration of nicotine levels may improve the relevance of the quantitative data obtained in the Salmonella and possibly comet assays, compensatory smoking habits and other factors may make the data from the assays used here have qualitative but not quantitative value in assessing risk of cigarette types and cigarette smoking to human health.