No full-text available
To read the full-text of this research,
you can request a copy directly from the authors.
Xenobiotic metabolizing enzyme systems in test fish. III. Comparative studies of liver cytosolic glutathione S-transferases
Abstract
The reduced glutathione (GSH)-conjugating capacities of the livers of trout, carp, zebra fish, guppy, and bluegill have been evaluated with several substrates preferentially metabolized by the major GSH-transferase isozymes. Carp and zebra fish lack GSH-transferase activity with 1,2-epoxy-3-(p-nitrophenoxy)propane. Guppy is characterized by the highest activities with 1-chloro-2,4-dinitrobenzene and the absence of activity toward trans-4-phenyl-3-buten-2-one. Enzyme activities with the other substrates demonstrate quantitative interspecific differences which presumably are of minor relevance to the metabolism and toxicity of chemicals.
... Relatively little research has been performed investigating the activities of piscine GSTs, particularly for sunfish. Donnarumma et al. (1988), in comparative studies of hepatic teleost GSTs, estimated the activity of bluegill GSTs to CDNB to be approximately 350 nmol/mg/minute. The results of our study show that mean sunfish GST activities toward CDNB range from 75 to 350 nmol/mg/minute, which is the basal level activity seen in most fish species (George 1994) (Figures 10A and 11A). ...
... Assessing the risks of environmental pollution needs essentially knowledge about the effects of toxic substances on a broad spectrum of organismic reactions. Therefore it is desirable to identify endpoints of high sensitivity and different biological expressiveness as biomarkers in toxicity testing, which may be detected on a sublethal level, for example in fish: irregularities during embryonic development (LANDNER et al., 1985;WESTERNHAGEN, 1988;SCHULTE and NAGEL, 1994;OBEREMM et al., 1997), changes in fish behaviour (WEBER and SPIELER, 1994;BAGANZ et al., 1998) or alterations in activity of detoxication enzymes (FUNARI et al., 1986;DONNARUMMA et al., 1988;FÖRLIN and CELANDER, 1993). Depending on the testsystem used, response on different levels are received, ranging from genetic, biochemical or physiological effects with their possible manifestation on the whole organism, its population or abundances in the investigated ecosystem. ...
Activities of two detoxication enzyme systems, microsomal and soluble glutathione S-transferases (m-/sGST, EC 2.5.1.18), and Se-dependent glutathione peroxidase (GP-X, EC 1.11.1.9) were monitored during zebrafish (Danio rerio) development. Starting with the 2–4 cell stage of ontogenesis successively up to adult fish, the presence of these detoxication enzymes could be demonstrated. Activities increased slowly during development and showed elevations corresponding to increased stress conditions. Plain increase occured after hatching, possibly caused by the change of the surrounding micromilieu. The juveniles showed highest activities, certainly provoked by new substances as from the food. Enzyme activities of adults revealed moderate levels probably due to a dilution of highly concentrated detoxication activities in the ready developed organs, especially the liver, in the whole body homogenate.
... It has reported that zebrafish expressed metabolic enzymes such as cytochrome P450 isoforms or conjugation enzymes (GST, UGTs and SULTs) similar to mammals' [7][8][9][10][11][12], in addition, many studies including those from our laboratory, have demonstrated that a variety of fish species, especially zebrafish, possess similar types of metabolic capability as that observed in mammalian species [13][14][15][16][17][18][19][20][21][22][23][24][25]. For example, step-wise deglycosylation and hydroxylation metabolites of notoginsenoside R 1 , ginsenoside Rg 1 and ginsenoside Rb 1 , and deglycosylation metabolites of icariin, epimedin A and epimedin C by adult zebrafish were found [23,25]. ...
The study aimed to investigate the potential of zebrafish in imitating mammal phase I metabolism of natural compounds. Three diterpenoid quinones from Radix Salvia miltiorrhiza, namely tanshinone IIA (TIIA), cryptotanshinone (Cry) and tanshinone I (TI) were selected as model compounds, and their metabolites mediated by zebrafish were characterized using a high-performance liquid chromatography coupled ion-trap mass spectrometry (HPLC/IT-MSn) method with electrospray ionization in positive mode. The separation was performed with a Zorbax C-18 column using a binary gradient elution of 0.05% formic acid acetonitrile/0.05% formic acid water. According to the MS spectra and after comparison with reference standards and literature reports, hydroxylation, dehydrogenation or D-ring hydrolysis metabolites of TIIA and Cry but not of TI were characterized, which coincided with those reported using regular in vivo or in vitro metabolic analysis methods, thus verifying that zebrafish can successfully imitate mammalian phase I metabolism which instills further confidence in using zebrafish as a novel and prospective metabolism model.
... The variety of isoenzymes represents the wide range of di!erent compounds which can be conjugated. Detoxication of aquatic contaminants by conjugation to glutathione is well documented in "sh (Donnarumma et al., 1988;George et al., 1989;Wiegand et al., 2000). In contrast, information about detoxication in amphibians is rare. ...
Using ring-(14)C-labeled isoproturon (1 microg/L), the uptake into spawn and tadpoles of Bombina bombina and Bombina variegata was investigated. Two percent of the applied radioactivity was found per gram fresh weight in the embryo after 24h. Results indicate that the jelly mass of the spawn does not act as a sufficient physical barrier for protection against the uptake and influence of isoproturon (IPU) on the embryo. In vivo metabolism of ring-14C-labeled IPU by the cytochrome P-450 system was analyzed in tadpoles. Different metabolites of IPU, such as N-demethylated and C-hydroxylated derivatives, and the olefinic metabolite were detected. In tadpoles of B. variegata, the activity of microsomal and soluble glutathione-S-transferase (sGSTs) toward different model substrates was measured after treatment with IPU. Activities of sGST increased corresponding to elevated stress by IPU dependent on exposure time and dose. Compared to the pure active ingredient IPU, the commercial phenyl-urea herbicide Tolkan Flo, consisting of IPU and an emulsifier, also caused significantly elevated enzymatic response.
Abstract In recent years, zebrafish has emerged as a useful animal model for biomedical research. The deciphering of the zebrafish genome has revealed that many of the enzymes involved in the metabolism of endogenous compounds and xenobiotics are conserved between zebrafish and humans. This review summarizes the information currently available concerning the zebrafish cytosolic sulfotransferases (SULTs), a group of phase II enzymes that have been proposed to be involved in the regulation and homeostasis of key endogenous compounds and the detoxification of xenobiotics. To date, 20 zebrafish SULTs that fall into six major SULT gene families have been identified. Of the 20 SULTs 18 have been cloned, expressed, purified and characterized. These zebrafish SULTs were shown to exhibit differential substrate specificities for endogenous compounds such as monoamine transmitters, steroid/thyroid hormones and bile salts, as well as xenobiotics including environmental toxicants and drugs. These findings provide a foundation for using zebrafish as a model for investigating further the physiological, pharmacological, and toxicological involvement of the SULTs.
In this study the acute toxicity of two carbamate pesticides, Carbaryl and Aldicarb, to the Guppy and the Zebrafish has been separately assessed with 96-h toxicity tests, performed using a semistatic procedure according to EEC guidelines. There was no significant difference in the toxicity of Aldicarb and Carbaryl to the Zebrafish, but Aldicarb was ∼ 4 times more toxic than Carbaryl to the Guppy. Moreover, the toxicity of both carbamates was higher to the Guppy than to the Zebrafish. Values for the 96-h LC50 obtained with Aldicarb were 52.9 μmol/l for the Zebrafish and 3.5 μmol/l for the Guppy. LC50 values obtained with Carbaryl were 46.0 μmol/l for the Zebrafish and 12.5 μmol/l for the Guppy. Analysis of test substance concentrations in the test system demonstrated, for each substance, a loss during each 24-h exposure period. In spite of the variability observed, the losses of Carbaryl appeared independent of time, whereas the losses of Aldicarb were higher during the initial exposure days. In addition, there was an indication that the rate of loss observed with both substances was greater in the presence of the Guppy than in the presence of the Zebrafish.
In fish, glutathione S-transferases are very important in the detoxification of water-borne pollutants. Carp, which often live and thrive under extreme conditions of water pollution, have been recommended by the European Community as an ideal species to test for the effect of various chemical substances often found in the aqueous environment. In this paper we describe the purification and kinetic characterization of the three major forms of glutathione S-transferase in carp liver cytosol. The main step for purification was affinity chromatography on S-hexylglutathione-Sepharose 6B and the isoenzymes have been separated by isoelectric focusing at pH 7.7, 7.3, 7.1. Values of Km and kcat/Km for both reduced glutathione and 1-chloro-2,4-dinitrobenzene as substrates have been determined, and the isoenzymes characterized with several substrates and inhibitors. J. Exp. Zool. 284:130–136, 1999.
Biotransformation of a series of organophosphorus compounds by the 9,000-g supernatant of rainbow trout (Oncorhynchus mykiss) liver was tested in an in vitro system fortified either with NADPH-generating cofactors or with reduced glutathione (GSH). Elimination rate constants for both systems were calculated from linear decay curves when substrate concentrations were used that were considerably lower than the Km values of the concerned enzymatic reactions. The results reveal a large variation in both the oxidative and the glutathione-mediated biotransformation rate of the organophosphorus compounds. Half-lives ranged from 25 to 1,216 min in the NADPH system and from 18 to 381 min in the GSH system. Elimination rate constants in the GSH system were related to Hammett σ constants or reactivity toward 4-nitrobenzylpyridine, which substantiates the assumption that electrophilicity is the controlling variable for the reaction with GSH within this particular class of compounds. A remarkable analogy was observed between compounds that were metabolized relatively quickly by glutathione S-transferases and compounds that showed a reduced bioconcentration factor in guppies. A significant improvement of the relationship between the bio-concentration factor in guppies and the octanol/water partition coefficient was obtained when the rate constant with GSH was introduced in this relationship. Such an improvement was not obtained with the rate constants from the oxidative system. These observations are discussed in view of the differences in the activities of the involved enzyme systems in the test species and in view of the possible relevance of the different biotransformation pathways for the in vivo situation.
The 96-h LC50 value of the organophosphorus pesticide diazinon (O,O-Diethyl-O-2-isopropyl-4-methyl-6-pyrimidyl thiophosphate) was about 0.8 for the guppy and about 8 for the zebra fish. Both the guppy and the zebra fish metabolized diazinon. The detected metabolite was 2-isopropyl-6-methyl-4-pyrimidinol (pyrimidinol). Diazoxon (O,O-diethyl-O-2-isopropyl-4-methyl-6-pyrimidylphos-phate) could not be found as a metabolite.After short exposures (up to 48 h) to low sublethal diazinon concentrations the body concentration of pyrimidinol was linearly dependent on the body concentration of diazinon. The ratio of pyrimidinol to diazinon body concentrations was about 5 times higher in the guppy than in the zebra fish. In both species however, the pyrimidinol level reached a maximum at concentrations corresponding to to of the LC50 value and these levels were remarkably similar. Kinetic experiments at diazinon concentrations of 0.1 and 0.4 ppm pesticide in water showed that an apparent steady state was reached after 24 to 48 h. After longer exposures an increased accumulation of diazinon was observed concomitantly with the inhibition of pyrimidinol formation.The inhibition of the brain acetylcholinesterase reached 73% in the guppy and 83% in the zebra fish. The maximum of the inhibition was already reached when the concentration of diazinon in water was 25% of the LC50 value.The factor of bioconcentration (BCF) was dependent on the level of the pesticide in the water and varied between 39 (in the guppy, at a water concentration of 0.1 ppm) and more than 300 (in the zebra fish, at a water concentration of 4 ppm).The results indicate that the metabolism plays a key role in the bioaccumulation and in the species specific toxicity of diazinon in fish.
An exhaustive literature survey has been carried out to collect comparative data of acute toxicity of organic chemicals to species recommended by OECD for toxicity testing. These species are: trout (Salmo gairdneri), bluegill (Lepomis macrochirus), guppy (Poecilia reticulata), carp (Cyprinus carpio), fathead min-now (Pimephales promelas), red killifish (Oryzias latipes), golden orfe (Leuciscus idus) and zebra fish (Brachydanio rerio). Comparative information was available only among the first five species. Among the 200 collected compounds, 24 (most of which were phosphoesters), showed a marked species-dependent toxicity, with 96 h LC50 ratios between two species ranging from 10 to about 300. With reference to the action of the selective toxicants, fathead minnow was the most resistant species in practically all the available comparisons. No dependence of the selective toxicity on the n-octanol/water partition coefficient of the chemicals could be observed.
Liver and gills of roach (Rutilus rutilus) and silver carp (Hypophthalmichthys molitrix) were examined for glutathione S-transferases (GSTs) contents and their substrate specificity and capacity to biotransform microcystin-LR (MC-LR). GSTs and other glutathione (GSH) affine proteins were purified using a GSH-agarose matrix and separated by anionic chromatography (AEC). Substrate specificities were determined photometrical for 1-chloro-2,4-dinitrobenzene (CDNB), 1,2-dichloro-4-nitrobenzene (DCNB), 4-nitrobenzyl chloride (pNBC) and ethacrynic acid (ETHA). Biotransformation rate of MC-LR was determined by high performance liquid chromatography (HPLC). Roach exhibited different hepatic and branchial GST activities for used substrates (DNB, pNBC and DCNB) compared to silver carp but not for ethacrynic acid. It suggests that, both fish species have similar amount of pi and/or alpha class, which were the dominant GST classes in liver and gills. Gills of both fish species contained a higher number of GST isoenzymes, but with lower activities and ability of MC-LR biotransformation than livers. GST isoenzymes from roach had higher activity to biotransform MC-LR (conversion rate ranging up to 268 ng MC-LR min(-1) mL(-1) hepatic enzyme) than that isolated from silver carp. Without any prior contact to MC-LR or another GST inducer, roach seems to be better equipped for microcystin biotransformation than silver carp.
1. The initial slopes of the substrate-activity curves of several hydrolases were determined in the microsomal and cytosolic fractions of the liver of several fish recommended by OECD for the regulatory testing of chemicals. 2. Inter-species differences ranged within a factor of 7-17 for the esterases and reached a factor of 60 for the amidase. Guppy and carp appeared endowed with hydrolase activities which, overall, are much higher than zebra fish, trout and golden orfe. 3. The comparison with the rat liver microsomal hydrolases strongly suggests that fish are endowed with similar or higher levels of A-esterase and with much less B-esterase/amidase activities.
The in vitro hepatic metabolism of diazinon, as well as the sensitivity of the brain acetylcholine esterase, to diazoxon inhibitory action have been studied in order to explain the different toxicity of diazinon to Oncorhynchus mykiss (rainbow trout), Poecilia reticulata (guppy), Brachydanio rerio (zebra fish) and Cyprinus carpio (carp). In spite of a very sensitive acetylcholine esterase the carp is very resistant to diazinon toxicity because of its very low rate of bioactivation and relatively high activity of detoxicating enzymes. The trout is very sensitive towards diazinon in spite of its low activity of bioactivation, because of its lack of detoxicating enzymes and a very sensitive acetylcholine esterase. Diazinon is very toxic for the guppy, because this fish combines a relatively sensitive acetylcholine esterase with a high rate of bioactivation. The zebra fish has the most insensitive acetylcholine esterase, associated with a limited activation rate, thus resulting a rather resistant species. The results obtained indicate that diazinon toxicity differences among the fish species studied can largely be explained in relation to metabolic balances in the liver and with the features of the target enzyme.
The zebrafish (Danio rerio) is now the pre-eminent vertebrate model system for clarification of the roles of specific genes and signaling pathways in development. The zebrafish genome will be completely sequenced within the next 1-2 years. Together with the substantial historical database regarding basic developmental biology, toxicology, and gene transfer, the rich foundation of molecular genetic and genomic data makes zebrafish a powerful model system for clarifying mechanisms in toxicity. In contrast to the highly advanced knowledge base on molecular developmental genetics in zebrafish, our database regarding infectious and noninfectious diseases and pathologic lesions in zebrafish lags far behind the information available on most other domestic mammalian and avian species, particularly rodents. Currently, minimal data are available regarding spontaneous neoplasm rates or spontaneous aging lesions in any of the commonly used wild-type or mutant lines of zebrafish. Therefore, to fully utilize the potential of zebrafish as an animal model for understanding human development, disease, and toxicology we must greatly advance our knowledge on zebrafish diseases and pathology.
ResearchGate has not been able to resolve any references for this publication.