Liver Micronucleus Assay Research Articles

The role of short-lived lesions in the induction of micronuclei in rat liver by ethylnitrosourea and methyl methanesulphonate: the importance of experimental design

Rats received single injections of ethylnitrosourea (ENU) or methyl methanesulphonate (MMS) at the peak of DNA synthesis after partial hepatectomy. Hepatocytes were isolated 1–4 days later, and analysed for presence of micronuclei. With both chemicals, frequencies of micronucleated hepatocytes were increased in a dose-dependent manner, but ENU proved to be both more effective (6 times; based on molar dose) and more efficient (18 times; based on total DNA alkylation) than MMS. In general, the micronucleus frequency was relatively low at 1 day after injection, then increased to reach a maximum at days 2 or 3 (depending on the dose), after which it decreased strongly in the case of MMS or remained stable in the case of ENU. The result with ENU is interpreted as a balance between loss and/or dilution of micronucleated hepatocytes and simultaneous formation of new ones. The present observations are in line with our earlier conclusion that ENU, in contrast with MMS, is able to induce persistent preclastogenic lesions in rat hepatocytes. ENU also proved to be more effective and efficient than MMS with respect to the formation of micronuclei in bone marrow cells. Our results with ENU and MMS indicate that administration of the genotoxin at the peak of DNA synthesis after partial hepatectomy, instead of before hepatectomy, increases the sensitivity of the liver micronucleus assay at least in the case of directly acting chemicals.

Genotoxicity of 1- and 2-nitropropane in the rat.

2-Nitropropane (2-NP) is a rat liver carcinogen, whilst the 1-isomer is non-carcinogenic in rodents. Although DNA repair tests in the rat liver discriminated clearly between the carcinogenic and the non-carcinogenic isomer, uniformly negative results have been published for the mouse bone marrow micronucleus test (BMMN test) with both isomers. Therefore, the latter assay did not discriminate between the carcinogenic and the non-carcinogenic isomer. To investigate whether this is due to endpoint specificity or organospecificity of 2-NP, studies were carried out in the rat in which micronucleus induction (bone marrow and liver) and unscheduled DNA synthesis (UDS) induction (liver) were measured after oral treatment with either nitropropane isomer. 2-NP induced UDS in the liver whilst the 1-isomer was negative, thus confirming the published studies. In the BMMN test, occasional small increases in the incidence of micronuclei were found for both compounds, but results were interpreted as negative after considering the control background data and the lack of reproducibility. By contrast, the liver micronucleus test revealed a clastogenic effect of 2-NP in the liver. This indicates that 2-NP induces chromosome aberrations as well as DNA repair in vivo, but it seems to act organospecifically. For 1-NP a slightly increased incidence of micronuclei was found in the liver, which was accompanied by a markedly increased mitotic index. It therefore remains questionable as to whether this increased micronucleus frequency for 1-NP is an indicator of a clastogenic effect, or whether it is caused by an increased cell proliferation induced by 1-NP. Consequently, it is too early to conclude whether the liver micronucleus assay is able to discriminate between the carcinogenic and non-carcinogenic isomer. However, the results provide further evidence that bone marrow assays are insufficient for the detection of all genotoxic carcinogens in vivo. This indicates the need for analysing a second tissue, particularly when negative bone marrow results have been obtained with in vitro genotoxins.

Quinoline: Unscheduled DNA synthesis and mitogenesis data from the rat liver in vivo

Quinoline is a specific and potent carcinogen to the rat and mouse liver. Studies are described here in which it was evaluated for its ability to initiate unscheduled DNA synthesis (UDS) in the rat liver in vivo. Although some individual animals showed indications of a marginal response the absence of clear group positive responses and the lack of an obvious dose relationship precluded the classification of quinoline as positive. The analogous NTP non-carcinogen 8-hydroxyquinoline was shown also to be devoid of UDS activity. Quinoline did, however, induce a potent mitogenic response in the rat liver between 24 and 48 hr after oral dosing of 200-500 mg/kg. Under similar conditions of test, 8-hydroxyquinoline was essentially inactive. These data represent a further instance in which mitogenicity in the liver appears to correlate better with carcinogenicity than does genotoxicity; but it may not be that simple, as discussed in the text. A single dose of quinoline was shown to act as a replacement for surgical partial hepatectomy in the liver micronucleus assay described by Tates, consistent with its potent mitogenicity.

A rat liver micronucleus assay employing 4AAF as mitogen

A rat liver micronucleus assay employing 4AAF as mitogen

A micronucleus technique for detecting clastogenic effects of mutagens/carcinogens (DEN, DMN) in hepatocytes of rat liver in vivo

A micronucleus assay in vivo has been developed that utilizes freshly isolated hepatocytes from livers of hepatectomized rats. In a small validation study, increased frequencies of micronuclei were detected in rats exposed to DEN and DMN before or after hepatectomy. The method is suitable for the detection of clastogenic effects of compounds or their metabolites that are too short-lived to reach the classical target cells used in cytogenetic studies. Because the non-hepatectomized liver shows very low levels of mitosis, the liver-micronucleus assay can also be used for the study of storage effects and the clastogenic effects of chronic exposures to mutagens/carcinogens.