Determination of lipid peroxides in invertebrates tissues using the Fe(III) xylenol orange complex formation.

Reactive oxygen species (ROS) are subproducts of the oxidative metabolism known to initiate chain reactions with polyunsaturated fatty acids that generate lipid peroxides (LPO). The objective of this work was to adapt the ferrous oxidation/xylenol orange (FOX) assay to measure LPO in invertebrate tissues i.e.: from polychaeta (Laeonereis acuta) and crab (Chasmagnathus granulata) species. Whole polychaetes were homogenized in methanol 100%, being determined the optimal sample volume and the time required for color development. It was tested five sample volumes (8-30 microl), following color development up to 215 min. Absorbance stabilization was observed after 90 min, being linearly related with sample volume. A similar procedure was adopted for crab tissues (anterior gills, posterior gills, and hepatopancreas). Differences between species and between organs of the same species were observed when analyzed nonspecific absorbance increments after adding the standard cumene hydroperoxide (CHP). In polychaeta and crab anterior gills tissue, absorbance increments were lower (21-25%) than samples without tissue extracts (blanks) that received CHP. In crab posterior gills and hepatopancreas, the nonspecific increment was almost negligible. Correction formulae are given to account for these differences and simplified protocols for each tissue and species are also included. Great differences in the lipid peroxides content was detected between worms (127.05 +/- 19.32 nmoles CHP/g of wet tissue) respect to anterior gills, posterior gills, and hepatopancreas from the crab species (52.65 +/- 3.59, 30.54 +/- 4.73, and 48.51 +/- 8.78 nmoles CHP/g of wet tissue, respectively).

Effect of microcystin on ion regulation and antioxidant system in gills of the estuarine crab Chasmagnathus granulatus (Decapoda, Grapsidae).

The objective of this work was to evaluate mechanisms of microcystin toxicity on crustacean species. Adult male crabs of Chasmagnathus granulatus (13.97+/-0.35 g) acclimated to low salinity (2 per thousand ) were injected with saline (control) or Microcystis aeruginosa aqueous extract (39.2 microg/l) at 24 h intervals for 48 h. After the exposure period, the anterior and posterior gills were dissected, measuring Na(+),K(+)-ATPase and glutathione-S-transferase (GST) activity. Total oxyradical scavenging capacity (TOSC) and lipid peroxides (LPO) content were also determined. Na(+),K(+)-ATPase activity in anterior gills was significantly lower in crabs injected with toxin than in control crabs, while no significant difference in the enzyme activity was detected in posterior gills. Both sodium and chloride concentration in the hemolymph were not affected by toxin exposure. Significant changes in GST activity were detected in posterior gills, with higher values being observed in the toxin-injected crabs. Crabs exposed to microcystin also showed a significant increase in the TOSC value against peroxyl radicals, for both anterior and posterior gills. Lipid peroxides level did not change in both gill types after exposure to the toxin. The increased levels of TOSC suggest the occurrence of a crab response against oxidative stress induced by toxin injection, which prevents lipid peroxidation.

Effects of extracts from the cyanobacterium Microcystis aeruginosa on ion regulation and gill Na+,K+-ATPase and K+-dependent phosphatase activities of the estuarine crab Chasmagnathus granulata (Decapoda, Grapsidae).

Recent discoveries indicate that microcystins affect enzymes, such as Na(+),K(+)-ATPase, involved in ion regulation of aquatic animals, through K(+)-dependent phosphatase inhibition. In vitro studies showed the inhibitory effect of Microcystis aeruginosa extracts on Na(+),K(+)-ATPase and K(+)-dependent phosphatase activities in gills of Chasmagnathus granulata (Decapoda, Grapsidae). Extracts of M. aeruginosa were prepared from lyophilized or cultures cells of the cyanobacterium. For lyophilized cells, IC(50) values were estimated as 0.46 microg/L (95% confidence interval [CI]=0.40-0.52 microg/L) and 1.31 microg/L (95% CI=1.14-1.51 microg/L) for Na(+),K(+)-ATPase and K(+)-dependent phosphatase, respectively. However, extracts prepared from cultured cells presented a much lower inhibitory potency against both enzymes. Gas chromatography revealed long-chain fatty acids in the lyophilized cell extracts, indicating that they are in part responsible for the enzyme inhibition. In vivo studies showed that the toxin inhibited Na(+),K(+)-ATPase activity in anterior gills, whereas an increased augmented activity of glutathione-S-transferase was observed in both kind of gills, indicating that the crab has increased its ability to conjugate the toxin. No significant differences in hemolymph sodium or chloride concentration were detected. This result is in agreement with the lack of effects of microcystin on Na(+),K(+)-ATPase activity of posterior (osmoregulating) gills.