Effects of hexavalent chromium on trout mitochondria.

Trout liver mitochondria were incubated in the presence of micromolar concentration of potassium dichromate (Cr(VI)) under several experimental conditions. Cr(VI) strongly inhibited both state 3 and state 4 of respiration supplemented by NAD-linked substrates; it also slightly affected the respiration of FAD-linked substrates. Evidence is provided that the respiratory inhibition induced by dichromate is partially coupled to the Cr(VI) reduction mechanism occurring in mitochondria.

Enhanced liver metabolism of mutagens and carcinogens in fish living in polluted seawater.

Specimens of the seawater fish annular seabream (Diplodus annularis) were caught from a polluted harbor area and from a clean reference area. Seawater concentrates and fish-muscle extracts were not mutagenic in the Salmonella reversion test. Liver preparations of fish from the 2 sources were comparatively assayed for microsomal mixed-function oxidases and cytosolic biochemical parameters, as well as for the ability of S12 fractions to activate promutagens or to detoxify direct-acting mutagens. A shift of the cytochrome P-450 peak from 450.3 to 448.5 was accompanied by a 4.5-fold increase in arylhydrocarbon hydroxylase activity in fish living in the polluted environment. At the same time, glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase were doubled in the cytosol of the same animals, while reduced glutathione (GSH) peroxidase and GSH S-transferase were slightly yet significantly depressed. No significant difference was recorded for other biochemical parameters, including GSH, oxidized glutathione (GSSG) reductase, NADH- and NADPH-dependent diaphorases, and DT diaphorase. In parallel, fish exposed to polluted seawater exhibited a significant and marked enhancement of the metabolic activation of the pyrolysis product Trp-P-2 and of benzo[a]pyrene-trans-7,8-diol, and at the same time were less efficient in detoxifying the antitumor compound ICR 191. Liver S12 fractions from both sources efficiently decreased the direct mutagenicity of sodium dichromate, and failed to activate benzo[a]pyrene and aflatoxin B1 to mutagenic metabolites. These results provide evidence that both biochemical parameters and the overall capacity of fish liver to activate or detoxify certain mutagens can be assumed to be sensitive indicators of exposure to mixed organic pollutants in the marine environment.

Multiple genotoxicity biomarkers in fish exposed in situ to polluted river water.

Specimens of rainbow trout (Oncorhynchus mykiss) were either kept in an aquarium under laboratory conditions or caged in the River Po (Northern Italy), upstream or downstream the confluence with the River Lambro, a small yet heavily polluted tributary. Genotoxicity biomarkers, evaluating the internal dose or early biological effects, were monitored after 7, 15 and 30 days of in situ exposure. With the exception of a slight increase of aminopyrine-N-demethylase and uridine-5'-diphospho-glucuronosyl-transferase, no significant effect was produced in fish kept upstream the River Lambro, as compared to control fish kept in the aquarium. In contrast, the responsibility of this tributary in carrying sublethal doses of both genotoxic agents and enzyme inducers into the main river was proved by the significant occurrence of early biological alterations in fish caged downstream. In fact, (a) bile extracts contained frameshift mutagens requiring metabolic activation, with a prevalence of liposoluble components after a short exposure, followed by a time-related increase of conjugated components, in minor part with glucuronic acid; (b) the monooxygenases aminopyrine-N-demethylase, uridine-5'-diphospho-glucuronosyl-transferase and, with sharp differences, arylhydrocarbon hydroxylase and 7-ethoxyresorufin-O-deethylase were enhanced in liver microsomal fractions; (c) the liver cytosolic fractions had an enhanced ability to convert 3-amino-1-methyl-5H-pyrido(4,3)indole into mutagenic metabolites in S. typhimurium; (d) cytogenetic damage was demonstrated by an increased frequency of micronuclei in peripheral blood erythrocytes.

Biomarkers of exposure and effect in flounder (Platichthys flesus) exposed to sediments of the Adriatic sea.

The modifications of several biomarkers were investigated in flounder (Platichthys flesus) when exposed in the laboratory to sediment samples collected from the Northern Adriatic Sea. Besides clean sand used as a control substrate, fish were exposed to sediments sampled offshore the delta of the Po River, the harbour of Trieste, and from the industrial harbour of Venice (Porto Marghera). After six days of exposure, the enzyme activities ethoxyresorufin-O-deethylase (EROD), UDP glucuronyltransferase (UDPGT), glutathione S-transferase (GST), glutathione reductase (GR) and glutathione peroxidase (GPx), were assayed in fish liver. In addition, the contents of reduced glutathione (GSH), nonprotein thiols (SH), total sugars and extractable lipids were also determined in hepatic tissue, as well as the number of micronucleated hepatocytes and biliary concentrations of fluorescent aromatic compounds (FAC). Despite some variability within treatment groups, differences among exposed organisms were evident and consistent with known contaminant levels of sampled areas. Microsomal activities (EROD, UD-PGT) and FAC levels were the most sensitive exposure indicators. Variations in the other biomarkers showed only tendencies which although not statistically significant were generally consistent with the contamination pattern.

Reduction of hexavalent chromium by the earthworm Eisenia foetida (Savigny).

Several metabolic systems in Eisenia foetida competent to achieve an intracellular Cr(VI) reduction are described. Evidence that earthworms act as effective scavengers of hexavalent chromium from the environment is also provided. Results suggest that E. foetida may play an important role during occasional Cr(VI) pollution of soils.

Reduction of hexavalent chromium by mitochondria: methodological implications and possible mechanisms.

The reduction of hexavalent chromium by rat liver mitochondria was studied under various experimental conditions. The occurrence of possible artifacts arising from inadequate methods for Cr(VI) detection was also tested. Erroneous results due to the presence of NAD(P)H (above 0.1 mM) could be demonstrated when the diphenylcarbazide method is employed. Modifications of this colorimetric procedure are proposed, in order to minimize artifacts occurring when Cr(VI) is detected in NAD(P)H-added mixtures. Although no interfering substances that can affect colorimetric methods are normally present in mitochondrial preparations, some methodological precautions are recommended. Mitochondria can reduce hexavalent chromium using either succinate or glutamate as electron donors. The succinate-supported reduction of Cr(VI) is active especially in the presence of ADP or an uncoupler, while reduction induced by glutamate occurs only in the presence of respiratory-chain inhibitors. In mitochondria supplemented by NAD-linked or FAD-linked substrates, different mechanisms in Cr(VI) reduction can therefore take place.

Nitrite biotransformation by mitochondria from the earthworm Eisenia foetida (Savigny).

1. Nitrite oxidase and cytochrome-c oxidase activity catalysed by cytochrome-aa3 were assayed in earthworms and rats. 2. Cytochrome-aa3 and intact mitochondria from the two species were anaerobically incubated in the presence of nitrite; the occurrence of mitochondria-induced nitrite biotransformations was evaluated by monitoring nitrite recovery in incubation medium. Possible nitric oxide production was also tested. 3. The ratio nitrite oxidase/cytochrome-c oxidase activity was much higher in earthworms than in rats. 4. Under anaerobic conditions and in the presence of respiratory substrates, earthworm mitochondria produced a time-dependent loss of nitrite in the incubation medium. On the contrary, rat mitochondria are unable to decrease environmental nitrite concentration. 5. Results support the notion that metabolic properties of earthworm mitochondria can be considered as an adaptation to chronic nitrite exposure, this toxicant being typically present in natural habitats of these worms.

Effects of ammonia on liver lysosomal functionality in Salmo gairdneri Rich.

Salmo gairdneri specimens were exposed for 4-48 hours to three different concentrations of un-ionized ammonia (UIA). An increase in lysosomal sensitivity to osmotic shock and in total proteolytic activity occurs in the liver. The amount of this increase depends upon the exposure time or upon environmental and tissue ammonia levels. On the other hand ammonia does not affect lysosome hydrolases activity in vitro.

Biochemical bases for environmental adaptation in goldfish (Carassius auratus L.): resistance to ammonia.

Goldfish specimens were exposed for 24-48 hr to 20, 40, 600, and 2500 micrograms N/liter unionized ammonia (UIA). This treatment causes, in the brain, increases in total ammonia, glutamine, lactate, and succinate and decreases in glutamate, glucidic stores, total NADH, and ATP. Most of these effects have already been reported in ammonia-treated trout. It is therefore suggested that the cerebral biochemical mechanisms of ammonia toxicity in these two species are fundamentally the same. The most important metabolic alterations, however, appear for UIA concentrations which are higher than those necessary to produce the same effects in trout, in accordance with the greater resistance of goldfish to ammonia. Some of the physiological-biochemical adaptations which are at the root of this phenomenon have been pointed out and discussed. In the liver of the ammonia-treated goldfish total ammonia, glutamine, glutamate, and succinate all increase, while lactate, glycine, and taurine decrease; liver glucidic stores remain unaltered. These results indicate a difference in the metabolic responses of goldfish and trout liver.

Xenobiotic metabolizing enzymes in rainbow trout exposed to river water.

1. Juvenile rainbow trout were exposed to river water in a flow-through system. After 15 days of exposure, hepatic biotransformation activities and related parameters were measured and compared to those of the control group organisms that were maintained in tap water under identical experimental conditions. 2. Liver somatic index (LSI), microsomal protein and cytochrome P-450 contents, benzo[a]pyrene hydroxylase (AHH), ethoxyresorufin-O-deethylase (EROD) and UDP glucuronyl transferase activities were not significantly affected. 3. Aminopyrine-N-demethylase (APD) activity showed a slight yet significant increase in exposed trout.

[Cytogenetic damage in fish (Oncorhynchus mykiss) exposed in situ to water from a polluted river]. / Danno citogenetico in pesci (Oncorhynchus mykiss) esposti in situ ad acqua di fiume inquinata.

Specimens of rainbow trout (Oncorhynchus mykiss) were exposed in situ for 7, 15, and 30 days to the water of the Po River either upstream or downstream the immission of the Lambro River, a small affluent which is highly polluted with industrial wastes. A third group of animals was maintained as control in aquarium with a flow of unchlorinated fresh water. Blood smears were obtained from each of the 90 trout specimens and examined under the microscope. The number of micronucleated erythrocytes (MNE) was registered by scoring at least 2,000 cells per animal. No increases in the number of MNE was detected in the animals exposed upstream the confluence, as compared to controls. Conversely, a significant increase in the frequency of MNE after 7 days of exposure to the water of the downstream site was observed. This frequency remained elevated, yet not significantly, after 15 and 30 days of exposure. This study demonstrates the validity of in situ exposure and the usefulness of cytogenetic end-points for environmental biomonitoring.

[Mixed-function oxidase induction as a test for the biological monitoring of water: limitations and prospects]. / Induzione delle ossigenasi a funzione mista come strumento di monitoraggio biologico delle acque: limiti e prospettive.

The induction in fish liver of some enzyme activities, and typically of microsomal mixed-function oxidases (MFO), provides the earliest biological warning signal of exposure to pollutants. Our studies provided evidence that the basal levels of cytochrome P-450 and specific MFO activities, such as arylhydrocarbon hydroxylase (AHH) and ethoxyresorufin deethylase (EROD), were strongly influenced by the diet in freshwater fish (Oncorhynchus mykiss). The response of fish liver to a known enzyme inducer, i.e., beta-naphthoflavone, was also affected by the diet, which therefore should be carefully controlled in laboratory studies. Under field conditions MFO activities were significantly enhanced in the liver of O. mykiss kept in polluted river water as well as in the liver of the seawater fish Diplodus annularis collected from a polluted harbour area, as compared to specimens of the same species collected from an unpolluted reference area.

Biochemical effects of long-term exposure to cadmium and copper on rainbow trout (Salmo gairdneri): validation of water quality criteria.

Salmo gairdneri specimens were exposed for 4 months to 1-10 micrograms/liter of Cd and 30-100 micrograms/liter of Cu. At different times from the beginning of the experiment the activity of some metal-sensitive enzymes was assayed (blood catalase and carbonic anhydrase; liver aminolevulinate dehydrase and total leucylaminopeptidase ). Biochemical indices of mitochondrial (respiratory control ratio; O2 consumption) and lysosomal function (percentage free leucylaminopeptidase activity), gill sialic acid content, and mucus lysozyme concentration were also detected. Cu (at both the levels tested) and Cd (only at the highest concentration) cause a remarkable alteration in various biochemical/physiological parameters under study. Considerations about the water quality criteria levels proposed by EIFAC /FAO for the two metals are reported.