Negative Micronuclei Research Articles

Evalution of benomyl and carbendazim in the vivo aneuploidy/micronucleus assay in BDF1 mouse bone marrow

Benomyl and its active metabolite carbendazim were investigated in BDF1 mouse bone marrow to establish whether micronuclei induced by these fungicides are caused by clastogenic or aneugenic events. Micronuclei were evaluated for kinetochores using immunofluorescent antikinetochore antibodies. Kinetochore positive (K +) micronuclei are likely to arise from chromosome loss since they presumably contain intact kinetochores and are indicative of aneuploidy. Conversely, kinetochore negative (K −) micronuclei are most likely to contain acentric chromosome fragments arising primarily from clastogenic damage. Benomyl and carbendazim were administered as single oral doses of 0.3, 8.6 or 17.2 mmol/kg (for benomyl, equivalent to 100, 2500 or 5000 mg/kg; for carbendazim, equivalent to 66, 1646 or 3293 mg/kg). Both compounds were positive in the micronucleus test at doses of 8.6 and 17.2 mmol/kg, and an average of 82% (benomyl)_and 87% (carbendazim) of the total micronucleated polychromatic erythrocytes were K +. No effects were seen with either fungicide at 0.3 mmol/kg. These results are analogous to findings with known aneugens such as vincristine but are in contrast to results with classical such as cyclophosphamide. Thus, benomyl and carbendazim induced micronuclei in mmouse bone marrow cells primarily throygh an aneugenic mechanism.

Role of oxygen radicals in the chromosomal loss and breakage induced by the quinone-forming compounds, hydroquinone and tert-butylhydroquinone.

The mechanisms by which two quinone-forming compounds, hydroquinone (HQ) and tert-butyl-hydroquinone (tBHQ), induce chromosomal loss and breakage in a prostaglandin H synthase-containing V79 cell line have been investigated using the cytokinesis-block micronucleus assay with CREST antibody staining. Increased frequencies of CREST-positive micronuclei (indicating chromosome loss) and CREST-negative micronuclei (indicating chromosome breakage) were observed following exposure of cells to HQ and tBHQ. The formation of micronuclei by HQ, but not tBHQ, was dependent on arachidonic acid supplementation, indicating activation by prostaglandin H synthase. Since the oxidation of hydroquinones can result in the generation of oxygen radicals, the contribution of oxygen radicals to the formation of chromosomal alterations induced by HQ and tBHQ was investigated. In the presence of a superoxide-generating system consisting of hypoxanthine and xanthine oxidase, a significant increase in micronucleated cells was observed. These induced micronuclei consisted exclusively of CREST-negative micronuclei and their formation was completely inhibited by pretreatment with catalase. Catalase also significantly inhibited the CREST-negative micronuclei induced by HQ and tBHQ. In addition, glutathione treatment inhibited both CREST-positive and negative micronuclei induced by these phenolic compounds. These results indicate that both chromosome loss and breakage are induced by these two quinone-forming agents. Reactive oxygen species contribute to the chromosomal breakage induced by HQ and tBHQ but the observed chromosomal loss appears to result from other mechanisms such as an interference of quinone metabolites with spindle formation.

Micronuclei in neonatal lymphocytes treated with the topoisomerase II inhibitors amsacrine and etoposide

It has been suggested that the enzyme topoisomerase II may be important in chromosome segregation due to the role played by the enzyme in decatenating the intertwined DNA molecules that result from DNA replication. Inhibition of the enzyme has been found by some workers to inhibit chromatid separation in mammalian cells, while others have reported that the passage of cells through mitosis is unaffected. Inhibition of the enzyme with topoisomerase II inhibiting drugs also results in the formation of micronuclei as a consequence of DNA damage. We have used the micronucleus assay with CREST staining to investigate whether the micronuclei formed in neonatal lymphocytes after inhibition of topoisomerase II are formed from whole chromosomes, implying non-disjunction, or acentric fragments. We found that treatment with both amsacrine and etoposide caused a dose-related increase in the number of CREST negative micronuclei, with only a very small increase in the number of CREST positive micronuclei at high concentrations of the compounds. Although we cannot conclude from our experiments that treatment with topoisomerase II inhibitors does not affect the segregation of neonatal lymphocytes, the production of CREST negative micronuclei suggests that segregation abnormalities are less important than other mechanisms which may cause cytotoxicity from exposure to these compounds.

Induction of kinetochore positive and negative micronuclei in mouse bone marrow cells by salicylazosulfapyridine and sulfapyridine

Salicylazosulfapyridine (SASP) and its major metabolite sulfapyridine (SP) have been shown to induce chromosomal damage in vivo. Both chemicals were tested in the micronucleus (MN)/kinetochore (KC) staining test to gain insight into the question of whether chromosomal breakage, aneuploidy-inducing events, or both were important to the observed production of MN in bone marrow cells of mice. In this test, both SASP and SP were shown to be strong inducers of kinetochore positive (KC +) MN. Although small increases in kinetochore negative (KC −) MN were also observed in SP treated mice, as well as in mice receiving the highest dose of SASP tested, the results suggest that both chemicals induce predominantly aneuploidogenic type damage.

Negative micronucleus tests on caprolactam and benzoin in ICR/JCL male mice

Male ICR/JCL mice were given a single intraperitoneal injection of 125, 250, and 500 mg/kg of caprolactam (CAP), or 250, 500, and 1000 mg/kg of benzoin (ZOIN). Bone marrow preparations were made 24, 30, and 48 h after treatment with the maximum dose, and 30 h after treatment with the other doses. The slides were coded before microscopic examination. No significant increase was found in the incidence of micronucleated polychromatic erythrocytes after treatment with either CAP or ZOIN.

Induction of kinetochore positive and negative micronuclei in V79 cells by the alkylating agent diethylsulphate.

The induction by diethylsulphate of micronuclei derived from acentric fragments or from whole chromosomes was studied in Chinese hamster V79 cells using autoantibodies from the serum of a scleroderma patient (CREST-syndrome) to detect centromere--kinetochore structures. Centromere-containing micronuclei appeared early after treatment and plateaued both earlier and at lower level than centromere-lacking micronuclei. The frequency of centromere-containing micronuclei was in good agreement with that of mitotic chromosome displacement, suggesting that a high proportion of displaced chromosomes were transmitted to the cytoplasm of one of the two daughter cells, where they gave rise to micronuclei. On the contrary, centromere-lacking micronuclei were more frequent than what could be expected from chromosome fragments observed in mitotic stages.