The detection and evaluation of aneugenic chemicals.

Although aneuploidy makes a significant contribution to both somatic and inherited disease the mechanisms by which environmental chemicals may induce numerical chromosome aberrations are only poorly defined. The European Union Project was aimed to further our understanding of those chemical interactions with the components of the mitotic and meiotic cell division cycle which may lead to aneuploidy and to characterise the parameters such as cellular metabolism which may influence the activity of aneugenic chemicals. C-mitosis can be induced by the highly lipophilic polychlorinated biphenyl and the completion of mitosis and cleavage can be modified by agents which deplete cellular levels of reduced glutathione. Modifications of the fidelity of chromosome segregation were produced by inhibiting the functioning of topoisomerase II during chromatid separation. In contrast, the modification of centromere integrity resulted in chromosome breakage as opposed to disturbance of segregation. Modifiers of tubulin assembly and centriolar functioning in somatic cells such as acrylamide, vinblastine and diazepam reproduced their activity in rodent bone marrow and male germ cells. The analysis of chromosome malsegregation in Aspergillus nidulans by a structurally related series of halogenated hydrocarbons was used to develop a QSAR model which had high predictive value for the results of fungal tests for previously untested related chemicals. Metabolic studies of potential aneugens in genetically engineered human lymphoblastoid cells demonstrated the detoxification of the aneugenic activity of chloral hydrate and the activation of 2,3-dichlorobutane, 1,1,2-trichloroethane and trichloroethylene by Phase I biotransforming enzymes. Cell transformation studies in Syrian hamster dermal cultures using a panel of 22 reference and or potential aneugens indicated that 15 of the 22 produced positive results following single exposures. Five of the aneugens which were negative following single exposures produced positive results where cultures were continuously exposed for up to 6 weeks to low concentrations following a single non-transforming exposure to the mutagen dimethyl sulphate. The transformation studies indicate that a significant proportion of chemical aneugens are potential complete carcinogens and/or co-carcinogens. To optimise the enumeration of chromosomes following exposure to potential chemical aneugens whole chromosome paints and centromere specific probes suitable for use in fluorescence in situ hybridisation (FISH) were developed for the rat, mouse and Chinese hamster and selected human probes evaluated for their suitability for routine use. Molecular chromosome probes were used to develop protocols for enumerating chromosomes in metaphase cells and centromeres and micronuclei in interphase cells. The analysis of segregation of specific centromeres in binucleate cells following cytochalasin B treatment was shown to be a potentially valuable system for characterising non-disjunction following chemical exposure. Whole chromosome paints and centromere specific probes were used to demonstrate the presence of dose-response thresholds following treatment with a reference panel of spindle inhibiting chemicals. These data indicate that the FISH technology is suitable for evaluating the relative hazards of low-dose exposures to aneugenic chemicals.

Antagonists to cholinergic receptors increase the frequency of binuclear V79 Chinese hamster cells. A mechanism for induction of aneuploidy.

V79 Chinese hamster cells were found to produce significant amounts of acetylcholine. Asynchronously growing V79 cells were treated with five different antagonists to cholinergic receptors: atropine and scopolamine, which are inhibitors of muscarinic receptors, and mecamylamine, d-tubocurarine and alpha-bungarotoxin, which are inhibitors of nicotinic receptors. All compounds caused a slight but significant increase of the frequency of binuclear interphase cells and also of the frequency of cells in late telophase and early G1 that had not completed cleavage. In addition, hemicholinium-3, a specific choline uptake antagonist, inhibited cleavage. Taken together, it seems reasonable to hypothesize that acetylcholine and its receptors take part in the regulation of cleavage in these cells. As binuclear cells are prone to aberrant spindle functions in following mitoses, inhibition of cleavage may constitute a risk for generation of cells with highly aberrant chromosome numbers.

The action of carbaryl and its metabolite alpha-naphthol on mitosis in V79 Chinese hamster fibroblasts. Indications of the involvement of some cholinester in cell division.

Carbaryl (1-naphthyl-N-methylcarbamate) is a spindle-disturbing agent. A number of effects probably contribute to this activity. Carbaryl efficiently lowers the intracellular level of glutathione, thus endangering the integrity of the spindle. Carbaryl also causes lipid peroxidation. With anti-oxidant pretreatment, the frequency of c-mitotic cells is lowered which indicates that lipid peroxidation is a partial cause of the spindle-disturbing activity. At high concentrations of carbaryl, monopolar configurations in combination with cleavage are frequent and when tested, alpha-naphthol, which is thought to be formed from carbaryl in significant amounts at these concentrations, also gave monopolar configurations but with significantly lower frequencies of concomitant cleavage. Carbaryl inhibits cholinesterases and when tested, another cholinesterase inhibitor, diisopropylfluorophosphate, in combination with alpha-naphthol also increased the frequency of monopolar configuration in combination with cleavage. We therefore propose the involvement of some cholinester in the process of cell division.