New Approach Methods to Assess the Enteropathogenic Potential of Strains of the Bacillus cereus Group, including Bacillus thuringiensis.

Bacillus cereus (Bc) is a wide group of Gram-positive and spore-forming bacteria, known to be the etiological agents of various human infections, primarily food poisoning. The Bc group includes enteropathogenic strains able to germinate in the digestive tract and to produce enterotoxins such as Nhe, Hbl, and CytK. One species of the group, Bacillus thuringiensis (Bt), has the unique feature of producing insecticidal crystals during sporulation, making it an important alternative to chemical pesticides to protect crops from insect pest larvae. Nevertheless, several studies have suggested a link between the ingestion of pesticide strains and human cases of food poisoning, calling their safety into question. Consequently, reliable tools for virulence assessment are worth developing to aid decision making in pesticide regulation. Here, we propose complementary approaches based on two biological models, the human intestinal Caco-2 cell line and the insect Drosophila melanogaster, to assess and rank the enteric virulence potency of Bt strains in comparison with other Bc group members. Using a dataset of 48 Bacillus spp. strains, we showed that some Bc group strains, including Bt, were able to induce cytotoxicity in Caco-2 cells with concomitant release of IL-8 cytokine, a landmark of pro-inflammatory response. In the D. melanogaster model, we were able to sort a panel of 39 strains into four different classes of virulence, ranging from no virulence to strong virulence. Importantly, for the most virulent strains, mortality was associated with a loss of intestinal barrier integrity. Interestingly, although strains can share a common toxinotype, they display different degrees of virulence, suggesting the existence of specific mechanisms of virulence expression in vivo in the intestine.

Hydrogen inhalation: in vivo rat genotoxicity tests.

Preclinical and clinical studies have shown that molecular hydrogen (H2) has anti-oxidant, anti-inflammatory, and anti-apoptotic properties. Safety data are available in the literature and acute toxicity has been tested in isolated cells and laboratory animals. We have evaluates the genotoxicity of H2 in vivo in rats after 72 h exposure, following the International Council for Harmonization guidelines ICH S2 (R1). The study was conducted on three groups of male Wistar rats: a negative control group, a positive control group receiving methyl methanesulfonate, and a H2-treated group receiving a 3.1% H2 gas mixture for 72 h. Alkaline comet, formamidopyrimidine DNA glycosylase (Fpg)-modified comet and bone marrow micronucleus assays were performed. H2 exposure increased neither comet-tail DNA intensity (DNA damage) nor frequency of "hedgehogs" in blood, liver, lungs, or bronchoalveolar lavage fluid. No increase in Fpg-sensitive sites in lungs, no induction of micronucleus formation, and no imbalance of immature erythrocyte to total erythrocyte ratio (IME%) was observed in rats exposed to H2. The ICH S2 (R1) test-battery revealed no in vivo genotoxicity in Wistar rats after 72 h inhalation of a mixture containing 3.1% H2.

Toxic responses of metabolites produced by Ostreopsis cf. ovata on a panel of cell types.

Blooms of the dinoflagellate Ostreopsis cf. ovata are regularly associated with human intoxications that are attributed to ovatoxins (OVTXs), the main toxic compounds produced by this organism and close analogs to palytoxin (PlTX). Unlike for PlTX, information on OVTXs'toxicity are scarce due to the absence of commercial standards. Extracts from two cultures of Mediterranean strains of O. cf. ovata (MCCV54 and MCCV55), two fractions containing or not OVTXs (prepared from the MCCV54 extract) and OVTX-a and -d (isolated from the MCCV55 extract) were generated. These chemical samples and PlTX were tested on a panel of cell types from several organs and tissues (skin, intestine, lung, liver and nervous system). The MCCV55 extract, containing a 2-fold higher amount of OVTXs than MCCV54 extract, was shown to be more cytotoxic on all the cell lines and more prone to increase interleukin-8 (IL-8) release in keratinocytes. The fraction containing OVTXs was also cytotoxic on the cell lines tested but induced IL-8 release only in liver cells. Unexpectedly, the cell lines tested showed the same sensitivity to the fraction that does not contain OVTXs. With this fraction, a pro-inflammatory effect was shown both in lung and liver cells. The level of cytotoxicity was similar for OVTX-a and -d, except on intestinal and skin cells where a weak difference of toxicity was observed. Among the 3 toxins, only PlTX induced a pro-inflammatory effect mostly on keratinocytes. These results suggest that the ubiquitous Na+/K+ ATPase target of PlTX is likely shared with OVTX-a and -d, although the differences in pro-inflammatory effect must be explained by other mechanisms.

Investigation of the Genotoxic Potential of the Marine Toxin C17-SAMT Using the In Vivo Comet and Micronucleus Assays.

The contaminant responsible for the atypical toxicity reported in mussels from Bizerte Lagoon (Northern Tunisia) during the last decade has been characterized as C17-sphinganine analog mycotoxin (C17-SAMT). This neurotoxin showed common mouse toxic symptoms, including flaccid paralysis and severe dyspnea, followed by rapid death. For hazard assessment on human health, in this work we aimed to evaluate the in vivo genotoxic effects of this marine biotoxin using the classical alkaline and modified Fpg comet assays performed to detect DNA breaks and alkali-labile sites as well as oxidized bases. The micronucleus assay was used on bone marrow to detect chromosome and genome damage. C17-SAMT induces a statistically insignificant increase in DNA tail intensity at all doses in the duodenum, and in the spleen contrary to the liver, the percentage of tail DNA increased significantly at the mid dose of 300 µg/kg b.w/d. C17-SAMT did not affect the number of micronuclei in the bone marrow. Microscopic observations of the liver showed an increase in the number of mitosis and hepatocytes' cytoplasm clarification. At this level of study, we confirm that C17-SAMT induced DNA damage in the liver but there was no evidence of effects causing DNA oxidation or chromosome and genome damage.