Absence of carcinogenic and anticarcinogenic effects of annatto in the rat liver medium-term assay.

Annatto (Bixa orellana L.) is a natural food colorant extensively used in many processed foods, especially dairy products. The lower cost of production and the low toxicity, make annatto a very attractive and convenient pigment in substitution to the many synthetic colorants. In the present study we investigate the carcinogenic and anticarcinogenic effects of dietary annatto in Wistar rat liver using the preneoplastic glutathione S-transferase (GST-P) foci and DNA damage biomarkers. Annatto, containing 5% bixin, was administered in the diet at concentrations of 20, 200, and 1000 ppm (0.07; 0.80 and 4.23 bixin/kg body wt/day, respectively), continuously during 2 weeks before, or 8 weeks after DEN treatment (200 mg/kg body wt, i.p.), to evaluate its effect on the liver-carcinogenesis medium-term bioassay. The comet assay was used to investigate the modifying potential of annatto on DEN (20 mg/kg body wt)-induced DNA damage. The results showed that annatto was neither genotoxic nor carcinogenic at the highest concentration tested (1000 ppm). No protective effects were also observed in both GST-P foci development and comet assays. In conclusion, in such experimental conditions, annatto shows no hepatocarcinogenic effect or modifying potential against DEN-induced DNA damage and preneoplastic foci in the rat liver.

Antigenotoxicity of trans-dehydrocrotonin, a clerodane diterpene from Croton cajucara.

The antigenotoxic action of three doses of trans-dehydrocrotonin (t-DCTN), the active ingredient obtained from the bark extract of Croton cajucara, a plant native to the Amazon, was determined in Swiss mice in vivo. Mice were submitted to acute intraperitoneal and gavage treatments, then their bone marrow cells were subsequently analyzed by micronucleus (MN) and chromosome aberration (CA) assays. Comparisons were performed between the three doses of t-DCTN and the negative-control group. Statistical analysis indicated that doses of 50 and 75 % of the LD(50), via intraperitoneal treatment or gavage injection, were antimutagenic with regard to cyclophosphamide. However, the dose of 25 % of the LD(50) was only antimutagenic when administered by gavage. Based on these observations, it can be suggested that gavage is the most effective method of administering t-DCTN. In addition, t-DCTN showed no cytotoxic effects in the bone marrow cells regardless of the route of exposure.

Investigation of genotoxic activity of trans-dehydrocrotonin, a clerodane diterpene from Croton cajucara.

The genotoxic action of three doses of trans-dehydrocrotonin (t-DCTN), an active ingredient obtained from the bark extracts of an Amazon native plant, Croton cajucara, were examined in Swiss mouse bone marrow cells in vivo, submitted to acute intraperitoneal treatment, by micronucleus (MN) and chromosomal aberration (CA) tests. The statistical tests (Anova and Tukey) made to compare the results obtained in each of the three doses of t-DCTN with the negative-control group showed that the frequencies of MN and mitotic index were equal to the negative-control and that the frequencies of CA were lower than that observed in the negative-control. Therefore, based on our results it can be said that t-DCTN is not genotoxic nor cytotoxic to mouse bone marrow cells, submitted to acute intraperitoneal treatment in vivo. Teratogenesis Carcinog. Mutagen. 19:377-384, 1999.

Recombinant human interleukin-1-mediated killing of Schistosoma mansoni primary sporocysts in Biomphalaria glabrata.

Previous work has indicated that injection of recombinant-human interleukin (rhIL)-1beta in Schistosoma mansoni-infected M-line Biomphalaria glabrata resulted in a significant reduction in the number of cercariae shed. The purpose of the present work was to determine if primary sporocysts were killed following rhIL-1beta injection in susceptible snails and, if so, to determine if killing was the direct result of hemocyte activity. Counting of primary sporocysts indicated a 50% reduction in the number surviving at 3 days PE in snails from 2 susceptible strains following injection. Histological analysis indicated that killing occurred with little-to-no observable hemocyte/parasite contact, whereas short-term culture of primary sporocysts with cell-free plasma (hemolymph) from injected snails rapidly initiated killing in vitro. Because levels of a snail IL-1-like molecule (SnaIL-1) drop significantly following schistosome exposure in M-line snails, because resistant snails maintain higher SnaIL-1 levels following infection, and because rhIL-1beta upregulates hemocyte cytotoxic mechanisms, these data support the contention that SnaIL-1 plays a role in determining resistance in B. glabrata. These data also indicate that schistosome death may be separated from parasite encapsulation by hemocytes and that an as yet unidentified humoral killing mechanism/factor may exist in B. glabrata. Lastly, these data further support the hypothesis that cytokine-like molecules are important, functionally conserved immunodefense mediators in both vertebrates and invertebrates.

Hemocytes of schistosome-resistant and -susceptible Biomphalaria glabrata recognize different antigens on the surface of Schistosoma mansoni sporocysts.

A cytoadherence assay was used to determine whether antibodies to plasma and hemocyte components of Schistosoma mansoni-susceptible (M-line) and -resistant (10-R2, 13-16-R1) strains of Biomphalaria glabrata affected attachment of hemocytes to chemically fixed schistosome sporocysts differentially. Experiments used purified, intact IgG and purified Fab fragments of each antibody. Indirect fluorescent antibody tests confirmed that the intact purified IgG to plasma and hemocytes from the 3 strains of snails bound to sporocysts. There was no qualitative difference in fluorescence among any of the antibodies to snail components, although the various antibodies affected adherence of hemocytes to the parasite differentially. Adherence assays revealed that antibodies to plasma components inhibited binding of hemocytes from the snail strain to which the antibody was generated. Additionally, antibody to plasma from resistant strains of B. glabrata inhibited binding of hemocytes from the homologous and heterologous resistant strains but not hemocytes from susceptible snails. Antibody to hemocytes from M-line snails did not inhibit binding of hemocytes from any of the snail strains. Since results of assays using intact IgG correlated well with assays using their Fab fragment counterparts, it was concluded that hemocytes from the 3 snail strains utilize specific antigen-binding sites on sporocysts. Results of these assays also indicate that, with regard to the mechanism of hemocyte binding to sporocysts, M-line and 13-16-R1 snails are more dissimilar than M-line and 10-R2 or 10-R2 and 13-16-R1 B. glabrata.