Federico Melodia

Ammonia toxicity mechanism in fish: studies on rainbow trout (Salmo gairdneri Rich).

Abstract Salmo gairdneri specimens were exposed for 4–48 hr to various environmental un-ionized ammonia (UIA) concentrations. In the brain of NH3-treated trout glutamate, GABA, total sugars, ATP, NADH, and succinate decrease, while tissue NH3 content, glutamine, and lactate increase. In the liver total sugars, succinate, and lactate decrease, while tissue NH3, glutamine, and the taurine/glycine ratio increase. Such variations depend upon exposure time and environmental UIA level. Ammonia toxicity mechanism in fish is discussed and compared with the one proposed in mammals. Further data obtained from overturned specimens suggest that in these animals a cerebral hypoxic phenomenon also occurs.

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.

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.

Xenobiotic metabolizing enzymes in uninduced and induced rainbow trout (Oncorhynchus mykiss): Effects of diets and food deprivation

Abstract 1. Untreated and β-naphtoflavone (BNF)-treated trout ( Oncorhynchus mykiss ) were maintained for 15–21 days under 3 different feeding regimens or under starvation. 2. Possible diet effects were studied by measuring the following 13 hepatic parameters: aminopyrine demethylase (APDM), ethoxyresorufin-O-deethylase (EROD), benzo(a)pyrene hydroxylase (AHH), UDP glucuronyltransferase (UDPGT), glutathione reductase (GR), glutathione-S-transferase (GST), glutathione peroxidase (GPx), glucose 6-phosphate dehydrogenase (G6PD) and 6-phosphogluconate dehydrogenase (6PGD) enzyme activities; the content of total cytochrome P-450 (P-450); proteic and non-proteic SH groups (SHP, SHNP) and liver somatic index (LSI). 3. The results show that some feeding regimens affect a large number of these parameters in both uninduced and BNF-treated fish. 4. Hence, caution should be imposed in the use of the biochemical markers as a water-quality monitoring tool. 5. In particular, it is advisable to maintain fish without feeding during short-term experiments, especially because trout exhibit an excellent biochemical response to inducer even after a 21-day starvation period. 6. Conversely, when fish are to be necessarily fed (long-term experiments), diet must be carefully chosen and controlled by preliminary tests because some commercial fish pellets contain both inducers and inhibitors of several microsomal/cytosolic enzymes.

Protective effect of fish mucus against Cr(VI) pollution

Abstract Skin mucus from Salmo gairdneri contains some molecules (probably protein-bound sulfhydryl groups) able to reduce hexavalent chromium (Cr-VI). Results suggest also that fish slime layer can markedly decrease the Cr(VI) penetration-rate in animal tissues. For these reasons, mucus is regarded as a protective barrier against Cr(VI) pollution, especially during occasional and short lasting exposures to metal.

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.

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.

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.

Biochemical effects of long term exposure to Cr,Cd,Ni on rainbow trout (Salmo gairdneri Rich.) : Influence of sex and season

Abstract Salmo gairdneri specimens were exposed for 6 months to low concentrations of Cr,Cd,Ni, and their mixture. Treated animals, particularly males, showed an impairment in glucidic metabo - lism, a modification in lysosomal function, and alterations in gill sialic acid content. Some of these alterations persist in males after 3 month recovery, with only exception for Ni - treated animals. A connection between sex, seasonal cycle, and biochemical response to metal pollution was observed.

Hepatic and biliary biomarkers in rainbow trout injected with sediment extracts from the River Po (Italy)

Abstract Immature rainbow trout (Oncorhyncus mykiss) were injected i.p. with varying doses of extracts of sediments collected from the River Po (Northern Italy), upstream and downstream the immission of a heavily contaminated tributary (River Lambro). Six days after treatment, metabolic biomarkers were monitored in fish liver and bile. Microsomal enzyme activities, including arylhydrocarbon hydroxylase, ethoxyresorufin O-deethylase, aminopyrine N-demethylase and uridine-5′-diphosphoglucuronyltransferase, were induced by exposure to the polluted sediment to a moderate yet statistically significant extent, with dose-related effects. The cytosolic enzymes glutathione reductase, glutathione peroxidase and glutathione S-transferase were not affected by injection of sediment extracts, whereas both upstream and downstream specimens produced a depletion of nonprotein sulfhydryl groups. Elimination of fluorescent metabolites occured in the bile of trout injected with the polluted sediment extract, whereas organic extracts of bile were devoid of mutagenic activity in strain TA98 of S. typhimurium. In spite of some positive responses, the method used appears to be less sensitive in revealing the toxicological impact of pollution than either exposure of rainbow trout larvae to river sediment or in situ exposure of the same fish species in river water.