Ecotoxicology and environmental safety toxicity of pyrethroid cypermethrin on the freshwater snail Chilina parchappii: Lethal and sublethal effects.

The aim of the present work was to study the effect of the pyrethroid cypermethrin (CYP) on the non-target freshwater snail Chilina parchappi. Initially, the sensitivity of adult snails to CYP was evaluated via the 96-h LC50 test. Then, snails were exposed to subtethal CYP concentrations (0.1 and 10 mg/l) for 1, 4 and 10 days and the digestive glands were dissected for biomarkers analyses. Enzymatic activity of catalase (CAT), glutathione peroxidase (GPx) and glutathione-S-transferase (GST), as well as total glutathione reduced (GSH) levels, were determined. Histological analyses of morphology, intracellular accumulation of lipofucsins and neutral lipids accumulation in the digestive gland were also evaluated. As compared to other molluscs, C. parchappi showed high resistance to CYP exposure evidenced by the 96-h LC50 value (44.59 mg/l). Snails exposed to sublethal CYP concentrations showed a statistically significant increase (p < 0.01) in GST (79-116%) and GPx (45-190%) activities with respect to controls. However, CAT activity showed a tendency to decrease with CYP treatment but was not statistically significantly different compared to control. Only high CYP concentration caused a statistically significant increase (p < 0.01) in GSH content (95-196%). There was evidence of structural changes in the digestive gland of snails exposed to CYP, showing a dose-dependent response. In exposed snails, some of the main symptoms included a reduction in the thickness of the epithelium, vacuolisation of the digestive cells and an increase in the number of excretory cells. Accumulation of lipofuscins (933-1006%) and neutral lipids (403%) were statistically significantly higher (p < 0.05) in snails exposed to CYP compared to control. This study showed that C. parchappii is quite tolerant to CYP exposure and that at sublethal concentrations, GSH metabolism could play a protective role against the pesticide harm in snails. Therefore, it would be interesting to study the response of this organism to other environmental stressors to assess its potential use in monitoring programs.

Lectin histochemistry for detecting cadmium-induced changes in the glycosylation pattern of rat placenta.

Cadmium (Cd) is an industrial and environmental pollutant that produces toxic effects on gametogenesis, pre- and post-implantation embryos, and the placenta. Because the effects of acute Cd intoxication on the placenta are not well understood, we investigated changes in its glycosylated components in Cd treated dams at days 4, 7, 10 and 15 of gestation using lectin histochemistry. CdCl2 was administered to pregnant rats; control animals received sterile normal saline. Placentas were processed for DBA, Con A, SBA, PNA, UEA-I, RCA-I and WGA lectin histochemistry to evaluate changes in the carbohydrate pattern of the placenta that might modify cell interactions and contribute to embryonic alterations. Lectin binding was analyzed in the yolk sac; trophoblast giant cells; trophoblast I, II and III; spongiotrophoblast cells and endovascular trophoblast cells in the chorioallantoic placenta. Our lectin binding patterns showed that Cd caused alteration of SBA and DBA labeling of trophoblast-derived cells, which suggested increased expressions of α and ß GalNAc. Cd also caused decreased UEA-1 binding affinity, which indicated fewer α-L-Fuc residues in placentas of Cd treated dams. The nonreactivity in trophoblast I of the control placentas incubated with Con-A contrasted with the labeling in placentas of experimental dams, which indicated increased expression of terminal α-D-Man, and α-D-Glc residues. We found that Cd altered the reactivity of placenta to several lectins, which indicated modification of the glycotype presented by the fetal component of the placenta. We report that Cd exerts a deleterious effect on the glycosylation pattern of the placenta.

Effect of a single dose of cadmium on pregnant Wistar rats and their offspring.

Cadmium (Cd) is a well-known toxicant targeting many organs, among them placenta. This heavy metal also has embryonary and foetal toxicity. This study was undertaken to analyse the effect of a single Cd dose administered at 4, 7, 10 or 15 days of gestation on the offspring of pregnant rats sacrificed at 20 days of gestation. Cadmium chloride was administered subcutaneously at 10 mg/kg body weight to Wistar pregnant dams; control animals received a proportionate volume of sterile normal saline by the same route. Maternal uteri, livers, kidneys and lungs, and foetuses were examined at necropsy. Samples of maternal organs and whole foetuses were collected for histopathologic examination, determination of Cd levels and staining by the Alizarin red S technique. Results revealed a clear embryotoxic and a teratogenic effect of this heavy metal, the former as a significant increase in the number of resorptions, and the latter as significant decrease of the gestational sac weight, and the size and weight of foetuses of Cd-treated dams as well as induced malformations in skull bones, vertebrae and thoracic, and pelvian limbs. The deleterious effects found were similar to those previously reported for other animal models suggesting a high conservation of the pathogenic mechanisms of Cd. Additionally, many of the addressed aspects showed a slight dependence on the time of administration of the toxic that might be due to the accumulation of the metal in different organs, as we were able to demonstrate by the analysis of its concentration.

Effects of sub-lethal exposure of rats to the herbicide glyphosate in drinking water: glutathione transferase enzyme activities, levels of reduced glutathione and lipid peroxidation in liver, kidneys and small intestine.

Glyphosate (GLP), the active ingredient of many weed killing formulations, is a broad spectrum herbicide compound. Wistar rats were exposed during 30 or 90 days to the highest level (0.7 mg/L) of GLP allowed in water for human consumption (US EPA, 2011) and a 10-fold higher concentration (7 mg/L). The low levels of exposure to the herbicide did not produce histomorphological changes. The production of TBARS was similar or tended to be lower compared to control animals not exposed to the herbicide. In rats exposed to GLP, increased levels of reduced glutathione (GSH) and enhanced glutathione peroxidase (GPx) activity may act as a protective mechanism against possible detrimental effects of the herbicide. Overall, this work showed certain biochemical modifications, even at 3-20-fold lower doses of GLP than the oral reference dose of 2mg/kg/day (US EPA, 1993). The toxicological significance of these findings remains to be clarified.

Characteristics of an environmental strain, Enterococcus faecalis CECT7121, and its effects as additive on craft dry-fermented sausages.

Lactic acid bacteria are the most adequate microorganisms for natural preservation of food. In the present work, the strain of Enterococcus faecalis CECT7121 was employed in the manufacture of craft dry-fermented sausages and its performance as a biopreservative was analysed. This strain is devoid of the genes for haemolysin and gelatinase and does not produce biogenic amines. It is sensitive to almost all the antibiotics tested and opsonophagocytic assays showed that it is devoid of a capsule. This strain had a high LD50 (10(11)CFU ml(-1)) in mice. No statistical differences were found between control and sausages inoculated with E. faecalis CECT7121 regarding the production of lactic acid, pH variation over time, reaching a minimum pH value of 5.1, and sensory analysis in both series. Sausages inoculated with E. faecalis CECT7121 had lower viable counts of Enterobacteriaceae, Staphylococcus aureus and other Gram-positive cocci at the end of fermentation and 7 days and no viable enterobacteria and S. aureus were recovered at the end of drying. E. faecalis CECT7121 did not affect the growth of Lactobacillus spp. but it displaced the autochthonous populations of enterococci. E. faecalis CECT7121 was recovered in each time point as assessed by its inhibitory activity on Listeria monocytogenes and S. aureus. These results would indicate that the addition of E. faecalis CECT7121 during the manufacture of craft dry-fermented sausages offers an interesting alternative for biopreservation.

Arrested development of Ostertagia ostertagi: effect of the exposure of infective larvae to natural spring conditions of the humid pampa (Argentina).

The aim of this study was to determine the effect of environmental conditions and the time of exposure to the conditions required for Ostertagia ostertagi to become inhibited in development at the early fourth larval stage in the host. Two comparable experiments were conducted from September to January, experiment I in 1997-1998 and experiment II in 1999-2000. Twenty-thousand third-stage larvae (L3), freshly obtained from coprocultures, were spread in different parasite-free grass plots at the beginning of September, October and November in each experiment and exposed to environmental conditions throughout spring and early summer. Duplicate plots for each exposure period were grazed for 3 days by two dewormed tracer calves after 1, 2, 3, 4 weeks of exposure during the corresponding month, and the remaining plots were grazed for 3 days at monthly intervals until the end of the experimental period. For each month in both experiments, control animals were inoculated orally with 20,000 L3 newly recovered from coprocultures (week 0 animals; infection controls). The control and tracer calves were sacrificed and their parasite burdens analysed. The time required to obtain greater than 50% inhibited larvae (IeL4) in the tracer animals during September and October was 3 weeks, whereas during November around 60% of the parasites were inhibited after one week of exposure. During the period tested, greater than 50% inhibition was found in concurrence with a photoperiod ranging between 13 and 14 h. The highest proportion of IeL4 (75% average) in the animals was found concomitant with a 14 h 43 min photoperiod. A high correlation between the percentage of inhibition and day length was established (0.870 p < 0.001 and 0.815 p < 0.001 for experiment I and II, respectively). In both years, the capacity for developmental arrest was lost by the end of December, when the photoperiod begins to decrease, suggesting either a disappearance of the induction stimulus, or that an excess of the stimulus could block the mechanism of inhibition. The induction time was extended 2 weeks in all months tested when the coprocultures were maintained in the dark (experiment II), suggesting that accumulation of the light stimulus contributes to shortening of the induction time. The data presented here would suggest that photoperiod is a key environmental factor for the induction of hypobiosis.

Oxidative stress generated by diesel seawater contamination in the digestive gland of the Antarctic limpet Nacella concinna.

The aim of this work was to investigate the activity of antioxidant enzymes and oxidative damage in the digestive gland of the limpet Nacella concinna, and their suitability as biomarkers for hydrocarbon pollution in Antarctic coasts. Three groups of 30 individuals each were kept in seawater containing 0%, 0.05% or 0.1% diesel. Superoxide dismutase, catalase, glutathione S transferase and glutathione peroxidase activities, as well as lipid peroxidation and protein oxidation were studied in 18 animals of each group after 24, 48 and 168 h of exposure. The activity levels of most enzymes were increased by diesel in a dose-dependent manner. Glutathione peroxidase showed the most clear effect; its activity significantly increased in the 0.1% diesel group respect to the control. Lipid peroxidation and protein oxidation were significantly increased by diesel after 168 h. Both variables were higher in the group exposed to the lowest dose.

Liver sulphoxidative metabolism of albendazole in rat: enantioselectivity and effect of methimazole.

The aim of the present work was to evaluate the effects of methimazole (MTZ) on the enantioselective sulphoxidation of albendazole (ABZ) by rat liver microsomes and tissue slices. Albendazole sulphoxide (ABZSO) was the metabolite recovered after the incubation with ABZ in both liver preparations. MTZ significantly reduced ABZSO production both in microsomes and slices. ABZSO production decreased as a function of MTZ concentration. The sulphoxidation reaction performed by rat liver explants in the presence of MTZ was 65% lower than that observed in controls. The reduction in the production of ABZSO in the presence of MTZ was mainly due to a lower production of (+) ABZSO. The results reported further contribute to the understanding of the enantioselective metabolism of ABZ. In addition, the work presented provides information on the comparison of two different liver tissue preparations for the evaluation of xenobiotic metabolism.

Histopathology of the digestive gland of an Antarctic limpet exposed to cadmium.

Histopathological alterations induced experimentally with cadmium (Cd) in Antarctic limpets (Nacella concinna), exposed for different times and concentrations were compared to controls. At the light microscope level, samples exposed to the contaminant for short periods (6, 12 and 24 h) at two different concentrations (0.25 and 0.5 mg l(-1)) showed no alterations compared to controls. After 48 h of exposure at a 0.5 mg l(-1) Cd concentration, vacuolisation of the basophilic cells was observed. After 72 h exposure, there was a marked loss of all the digestive gland structure, with cell autolysis and loss of basophilia.

The anthelmintic albendazole affects in vivo the dynamics and the detyrosination-tyrosination cycle of rat brain microtubules.

Albendazole (ABZ) is an anthelmintic benzimidazole drug widely used in human and veterinary medicine. ABZ has binding affinity to both mammalian and helminth parasite tubulin. In the current work, we have performed in vitro assays and in vivo experiments in which rats were given ABZ orally to better characterize the action of the drug on the polymerization of rat brain microtubules and on the detyrosination/tyrosination cycle that occurs on the COOH-terminal end of alpha-tubulin. The results showed that ABZ inhibits brain microtubule polymerization in vitro, and significantly delayed microtubule assembly in vivo. The tyrosination reaction cycle was not affected in vitro; however, in rats to which the drug was administered orally, the levels of in vitro tyrosination were reduced when compared to the controls with mock treatment. These results suggest that this apparent inhibition would be due to a decrease in the amount of substrate caused by the depolymerizing effect of ABZ and the subsequent tyrosination in the intact brain with endogenous tyrosine. In conclusion, ABZ strongly affects tubulin dynamics both in vivo and in vitro. The outcome of these experiments is a contribution to the understanding of the molecular mechanisms involved in the antimicrotubular action of benzimidazole compounds.

Turnover of phosphatidyl choline and changes in enzymatic activity in cell membranes of the CNS during early myelination.

Subfractions of fraction A, which floats on top on 0.8 M sucrose in the classical density gradient used for the isolation of brain subcellular fractions (A1 corresponding in adult rats to myelin and A2 which corresponds to the myelin-like fraction), were studied in comparison to other brain subcellular fractions in 5 day old rats and at different stages of development, up to 60 days of age. Variations in the enzymatic activity of acetylcholinesterase, non-specific cholinesterase and 2',3'-Cyclic nucleotide phosphohydrolase, changes in lipid and protein composition and turnover of phosphatidyl choline were investigated. Results indicate that fractions A1 and A2 obtained prior to the beginning of myelination could be composed of fragments of the oligodendroglial cell plasma membranes, and that both fractions undergo substantial changes in chemical composition, enzymatic activity and in turnover of phosphatidyl choline during maturation. In vivo experiments at short times, using radioactive choline as a precursor of phosphatidylcholine suggest that membrane fragments isolated in fraction A2 are precursors of those isolated in fraction A1 at all ages.

Incorporation of CDP-choline (methyl-14C) into lecithin or adult rat liver mitochondria and microsomes.

Incorporation of CDP-choline (Methyl-14C) into liver microsomes and mitochondria was studied in vitro. Labelling of a total homogenate and subsequent separation of subcellular fractions showed that radioactivity was present in both microsomes and mitochondria, the highest specific activity being attained by microsomes. Chase experiments disclosed any transfer of label from one fraction to the other. Individual incubation of microsomes and mitochondria indicated that both fractions were able to incorporate label into phosphatidyl choline independently. The addition of increasing amounts of microsomes to mitochondria enhanced incorporation in proportion to the amount of microsomes added. If 10% contamination of mitochondria with microsomes is assumed, extrapolation of the curve back to zero contamination shows that mitochondria seem to have an autonomous capacity to incorporate the precursor into phosphatidyl choline.

Incorporation of CDP-choline (methyl-14C) into lecithin or adult rat liver mitochondria and microsomes

Incorporation of CDP-choline (Methyl-14C) into liver microsomes and mitochondria was studied in vitro. Labelling of a total homogenate and subsequent separation of subcellular fractions showed that radioactivity was present in both microsomes and mitochondria, the highest specific activity being attained by microsomes. Chase experiments disclosed any transfer of label from one fraction to the other. Individual incubation of microsomes and mitochondria indicated that both fractions were able to incorporate label into phosphatidyl choline independently. The addition of increasing amounts of microsomes to mitochondria enhanced incorporation in proportion to the amount of microsomes added. If 10

contamination of mitochondria with microsomes is assumed, extrapolation of the curve back to zero contamination shows that mitochondria seem to have an autonomous capacity to incorporate the precursor into phosphatidyl choline.

Incorporation of CDP-choline (methyl-14C) into lecithin or adult rat liver mitochondria and microsomes.

Incorporation of CDP-choline (Methyl-14C) into liver microsomes and mitochondria was studied in vitro. Labelling of a total homogenate and subsequent separation of subcellular fractions showed that radioactivity was present in both microsomes and mitochondria, the highest specific activity being attained by microsomes. Chase experiments disclosed any transfer of label from one fraction to the other. Individual incubation of microsomes and mitochondria indicated that both fractions were able to incorporate label into phosphatidyl choline independently. The addition of increasing amounts of microsomes to mitochondria enhanced incorporation in proportion to the amount of microsomes added. If 10

contamination of mitochondria with microsomes is assumed, extrapolation of the curve back to zero contamination shows that mitochondria seem to have an autonomous capacity to incorporate the precursor into phosphatidyl choline.