Monitoring genotoxicity in freshwater microcrustaceans: A new application of the micronucleus assay.

We have applied the micronucleus (MN) assay to the measurement of genotoxicity in microcrustaceans. Daphnids (Daphnia magna) and Copepods (Acanthocyclops robustus) were collected in situ and acclimated in the lab for 24h. The MN assay was successful with the Daphnids but not with the Copepods. Adult Daphnids were exposed to sublethal concentrations of metals (Cu, Zn, Cd) or insecticide (deltamethrin) for 2 and 7d. Dose-dependent induction of MN was observed after 2 d exposure, with 2-fold induction at the highest doses for each chemical tested. The advantages and ecological relevance of using Daphnids in genotoxicity assessment are highlighted. The Daphnid assay may be a reliable test for aquatic genotoxicity hazard/risk assessment and a useful alternative to studies of amphibians.

Comparative study of toxic effects of zearalenone and its two major metabolites alpha-zearalenol and beta-zearalenol on cultured human Caco-2 cells.

Zearalenone (ZEN) is a fusarotoxin converted predominantly into alpha-zearalenol (alpha-Zol) and beta-zearalenol (beta-Zol) by hepatic hydroxysteroid dehydrogenases. The feeding of naturally contaminated grains with ZEN was associated with hyperestrogenic and adverse effects on humans and animals. There is a lack of information on the attribution of the toxic effects of these toxins. One wonders if these effects are due to the parent molecule (ZEN) or to its major metabolites (alpha-Zol and beta-Zol). Using human Caco-2 cells, we looked for the molecular mechanisms of toxicity of ZEN, alpha-Zol, and beta-Zol. Toxicity effects were studied by MTT viability assay and oxidative stress induction by measuring malondialdehyde (MDA) generation. To check whether the oxidative stress induction was associated to DNA lesions, we looked for DNA fragmentation by means of the Comet and the diphenylamine assays. To specify cell death pathway, we investigated caspase-3 activation, confirmed by poly(ADP-ribose) polymerase cleavage and by Bcl-2 depletion. Our results clearly demonstrated that ZEN as well as its two metabolites presented variable toxic effects. They induced cell death and an increase in MDA generation. These effects were associated to DNA fragmentation as well as caspase-3 activation. The observed toxic effects seem to be relieved by the metabolism of ZEN into alpha-Zol and beta-Zol.

Zearalenone induces chromosome aberrations in mouse bone marrow: preventive effect of 17beta-estradiol, progesterone and Vitamin E.

The cytogenetic effect of zearalenone (ZEN), a non-steroidal estrogenic mycotoxin, was evaluated in vivo, in mouse bone marrow cells, by assessing the percentage of cells bearing different chromosome aberrations. The studies included different conditions for animal treatment, as follows: (1) single intraperitoneal (ip) injection, (2) repeated ip injections, (3) pre-treatment for 24h with Vitamin E (Vit E), and (4) pre-treatment for 4h with 17beta-estradiol (17beta-Est) or progesterone (Prog). ZEN induced different types of chromosome aberrations, which was concentration-dependent (2-20 mg/kg bw). These doses corresponded to 0.4-4% of the LD50 in the mouse. Interestingly, when the dose of ZEN (40 mg/kg) was fractionated into four equivalent doses (4 x 10 mg/kg bw), into three doses (15 + 10 + 15 mg/kg bw), or into two equivalent doses (2 x 20 mg/kg bw), given every 24 h, the percentage of chromosome aberrations increased significantly. This finding suggests that ZEN proceeds by reversible binding on receptors that could become saturated, and that it damages the chromosomes in a 'hit and go' manner. Furthermore, pre-treatment of animals with 17beta-estradiol or progesterone significantly decreased the percentage of chromosome aberrations, suggesting that (i) these hormones bind to the same cytoplasmic receptors transported into the nucleus to elicit DNA damage, (ii) they may play a role in preventing chromosome aberrations induced by ZEN. Similarly, Vit E prevented these chromosome aberrations indicating that Vit E, previously reported to prevent most of the toxic effects induced by ZEN, may also bind to the same receptors.

Cytotoxicity, inhibition of DNA and protein syntheses and oxidative damage in cultured cells exposed to zearalenone.

Mycotoxins are toxic metabolites of various fungi commonly found in feed and foodstuff and can cause very serious health problems in animals as well as in humans. Zearalenone (ZEN), a mycotoxin produced by various Fusarium species has several adverse effects. Indeed, ZEN has strong estrogenic activity associated with hyperestrogenism and several physiological alterations of the reproductive tract. Moreover, ZEN was shown to be hepatotoxic, haematotoxic, immunotoxic and genotoxic. The exact mechanism of ZEN toxicity is not completely established. The observed strong estrogenic effect of ZEN resulting from its competition with 17beta-estradiol in the binding to estrogen receptors is generally considered to underline most toxic effects of ZEN, but estrogenic activity alone cannot explain the diverse and apparent adverse effects. The objective of the present study was to determine the involvement of other possible mechanisms in ZEN induced toxicity. Cytotoxicity, cell cycle perturbation, inhibition of protein and DNA synthesis as well as the presumed later marker of oxidative stress, malondialdehyde, were monitored in Vero and Caco-2 cells exposed to ZEN. Our results showed that ZEN reduces cell viability correlated to cell cycle perturbation, inhibits protein and DNA syntheses and increases MDA formation in both cell lines in concentration-dependant manner. We assumed that cytotoxicity and oxidative damage are additional mechanisms of ZEN mediated toxicity.