E. Perdu-Durand

Metabolism of 4-hydroxynonenal, a cytotoxic product of lipid peroxidation, in rat precision-cut liver slices

4-Hydroxy-2-nonenal (HNE) is a major aldehydic product of lipid peroxidation known to exert several biological and cytotoxic effects. The in vitro metabolism of [4-(3)H]-HNE by rat precision-cut liver slices was investigated. Liver slices rapidly metabolize HNE - about 85% of 0.1 microM [4-(3)H]-HNE was degraded within 5 min of incubation. The main metabolites of HNE identified were 4-hydroxynonenoic acid (HNA), glutathione-HNE-conjugate (HNE-GSH), glutathione-1,4-dihydroxynonene-conjugate (DHN-GSH) and cysteine-HNE-conjugate (HNE-CYS). Whereas glutathione conjugation demonstrated saturation kinetics (K(m)=412.2+/-152.7 microM and V(max)=12.3+/-2.5 nmol h(-1) per milligram protein), HNA formation was linear up to 500 microM HNE in liver slices. In contrast to previous reports, no trace of the corresponding alcohol of the HNE, 1,4-dihydroxynon-2-ene was detected in the present study. Furthermore, the beta-oxidation of HNA including the formation of tritiated water was demonstrated. The identification of 4-hydroxy-9-carboxy-2-nonenoic acid and 4,9-dihydroxynonanoic acid demonstrated that omega-oxidation significantly contributes to the biotransformation of HNE in liver slices.

In vitro biotransformation of surfactants in fish. Part I: Linear alkylbenzene sulfonate (C12-LAS) and alcohol ethoxylate (C13EO8)

Developing regulatory activities (e.g., REACh, [DGEE. 2003. Directorates General Enterprise and Environment. The new EU chemicals legislation REACH. DG Enterprise, Brussels, Belgium. (http://www.europa.eu.int/comm/enterprise/reach/index_en.htm)]) will require bioaccumulation to be assessed for thousands of chemicals. Further, there is increasing pressure to reduce, refine or replace animal tests. Given this scenario, there is an urgent need to evaluate the feasibility of in vitro systems to supply data useful for bioaccumulation estimation. Subcellular and cellular hepatic systems were tested to determine the biotransformation of two surfactants: C12-2-LAS (2-phenyl dodecane p-sulfonate) and an alcohol ethoxylate C13EO8 (Octaethylene glycol monotridecyl ether). The subcellular systems tested were liver homogenates and microsomes from the common carp (Cyprinus carpio) and rainbow trout (Oncorhynchus mykiss). Cellular systems consisted of primary hepatocytes from the common carp (Cyprinus carpio) and PLHC-1 cells, hepatocarcinoma cells from the desert topminnow (Poeciliopsis lucida). All in vitro systems were exposed to radiolabeled test compounds and assayed for biotransformation using liquid scintillation and thin layer chromatographic methods. First-order kinetics were used to estimate rates of biotransformation. Bioconcentration of test materials in fish were predicted using an in vitro to in vivo metabolic rate extrapolation model linked to a mass-balance model commonly used to predict bioaccumulation in fish. Subcellular biotransformation rates for each of the surfactants were greatest with microsomes. Cellular loss rates exceeded subcellular rates, leading to lower predicted BCF values. Predicted BCFs corresponded closely to measured values in several fish species, verifying the utility of in vitro systems in refining Kow-only-based BCFs via the inclusion of biotransformation rates.

Biotransformation of pentachlorophenol, aniline and biphenyl in isolated rainbow trout (Oncorhynchus mykiss) hepatocytes: comparison with in vivo metabolism

1. The biotransformation of pentachlorophenol (PCP), aniline and biphenyl in rainbow trout (Oncorhynchus mykiss) isolated liver cells was investigated to examine if fish hepatocytes represent a suitable alternative to the in vivo approach for studying the biotransformation of chemicals. Each compound was incubated at two concentrations (10 and 60 microM) for 2 h. For comparison, the metabolic profile of these xenobiotics was also studied in urine and bile of trout orally exposed to 1.8-4.0 mg/kg wet wt of each compound. 2. In vitro as in vivo, PCP glucuronide and to a lesser extent PCP sulphate were the metabolites formed by trout from PCP. 3. Aniline was mainly metabolized to acetanilide and to a lesser extent to 2-aminophenol by isolated hepatocytes, but neither hydroxylated acetanilide nor conjugates were found in vitro whereas they were present in bile and urine of trout treated with this chemical. 4. Trout hepatocytes metabolized biphenyl to hydroxylated and dihydroxylated products and the corresponding glucuronides. These results correlated well with the metabolic profile obtained from the bile of trout exposed to this pesticide. 5. It is concluded that although hepatocytes are well suited for several types of biotransformation studies, the fact that this system may in some cases produce a different metabolic pattern than in vivo should be considered when attempting to extrapolate in vitro to in vivo data.

Cytochrome P450-dependent metabolic pathways and glucuronidation in trout liver slices.

We investigated the capacity of trout precision-cut liver slices to metabolize xenobiotics and steroids. As a first approach, liver slices were compared with freshly isolated trout hepatocytes, using 7-ethoxycoumarin (7-EC) and testosterone as substrates. Trout liver slices and freshly isolated hepatocytes had a similar capacity for conducting cytochrome P450-dependent metabolism, as indicated by the rate of oxidative metabolism of 7-EC and testosterone, and by the metabolic profile of these substrates. A lower rate of glucuronidation in slices compared with hepatocytes was observed with testosterone (50 microM), whereas the opposite situation occurred with 7-EC used at higher concentration (100 microM). In a second step, we investigated the effect of beta-naphthoflavone on 7-EC and testosterone biotransformation, using slices maintained in culture for 24 h, with or without the inducer added. The results were compared with the metabolic rates of these substrates incubated with liver slices originating from trout pretreated in vivo with beta-naphthoflavone. Cytochrome P450-mediated rates of 7-EC dealkylation and testosterone hydroxylation decreased to 38 and 55% of the control value, respectively, when incubations were performed in 24-h cultured slices instead of freshly cut slices. Exposure of the slices to 50 microM beta-naphthoflavone resulted in about 3 times higher deethylation rate of 7-EC. A similar value was obtained when treatment occurred in vivo. As demonstrated in rat by several authors, liver slices seem a useful and simple tool for studying the metabolic pathways of xenobiotics and steroids and for the assessment of inducers of the CYP1A1 family.

Influence of growth hormone on the hepatic mixed function oxidase and transferase systems of rainbow trout.

The effect of GH treatment on hepatic cytochrome P450 content, aryl hydrocarbon hydroxylase (AHH), aminopyrine-N-demethylase (AND), testosterone hydroxylase, testosterone 5α- and 5β-reductase, UDP-glucuronyl transferase (UDPGT) and glutathione S-transferase (GST) activities in immature rainbow trout were investigated. Hepatic cytochrome P450 content, AHH and GST activities were measured in both GH implanted and GH injected animals whereas other activities were assayed in GH implanted trout only.GH implants significantly decreased cytochrome P450 content at 15 days compared to the control but no significant effect was observed at 15 or 30 d when GH was injected biweekly. In both cases, AHH activity was significantly decreased by GH treatment compared to the control whereas GST remained unchanged. Compared to the control, GH implanted fish exhibited a pronounced inhibition of AND, a decreased 6β and 16β-testosterone hydroxylation, an inhibition of UDPGT with testosterone as substrate and an enhanced 17β-testosterone oxidation.

Catalytic and immunocytochemical detection of xenobiotic metabolizing enzymes in the olfactory organ of rainbow trout (Oncorhynchus mykiss)

The fish olfactory organ, which is covered with a sensory epithelium, is in direct contact with aquatic pollutants. In mammals, presence of xenobiotic metabolizing enzymes has been reported in the olfactory epithelium, but few studies have been carried out on this topic in fish. In the present study, xenobiotic metabolizing enzymes were investigated in the rainbow trout olfactory organ. Data show that cytochrome P450 reductase, p-nitrophenol hydroxylase and glutathione S-transferase specific activities were in the same range as that in the liver, whereas 7-ethoxyresorufin O-deethylase and 7-ethoxycoumarin O-deethylase specific activities were 80 and 30 times lower, respectively, in the olfactory organ compared to the liver. Immunocytochemical investigation shows that cytochrome P4501A1 was located in both the epithelial non-sensory cells and the basal cells of the sensory epithelium, as well as in goblet and microvilli cells of the non-sensory epithelium. The presence of such enzyme activities in the olfactory organ of rainbow trout addresses the question of their involvement in the detoxication/toxication of pollutants as well as in the olfactory function.

The phthalate diesters DEHP and DBP do not induce lauric acid hydroxylase activity in rainbow trout

Aquatic organisms are extensively exposed to phthalate esters. We have investigated in trout the effects of diethylhexylphthalate (DEHP) and dibutylphthalate (DBP) on xenobiotic metabolizing enzymes which have been suggested as possible environmental biomarkers. Rainbow trout (Oncorhynchus mykiss) were waterborne exposed to DEHP (1 mg/l) or DBP (0.1 or 1 mg/l) for 72 h. Another group of rainbow trout received daily for 3 days an intraperitoneal injection of 50 mg/kg of DEHP or DBP. Laurate hydroxylation, ethoxyresorufin-o-deethylation, UDP-glucuronyltransferase activity and glutathione-S-transferase activity were measured in liver and extrahepatic tissues. The phthalate esters have been found not to induce these enzymes; in particular, the results do not support the previously described induction of lauric acid hydroxylase in sea bass treated with DEHP [Comp. Biochem. Physiol. B122 (1999) 253.].

In vitro biotransformation of surfactants in fish. Part II--Alcohol ethoxylate (C16EO8) and alcohol ethoxylate sulfate (C14EO2S) to estimate bioconcentration potential.

Recent regulatory pressures (e.g., REACh, CEPA) requiring bioaccumulation assessments and the need for reduced animal use have increased the necessity for the development of in vitro-based methods to estimate bioaccumulation. Our study explored the potential use of subcellular and cellular hepatic systems to determine the biotransformation potential of two surfactants: octaethylene glycol monohexadecyl ether (C16EO8) and diethylene glycol monotetradecyl ether sulfate (C14EO2S). The subcellular systems tested were liver homogenates and microsomes from the common carp (Cyprinus carpio) and rainbow trout (Oncorhynchus mykiss). Cellular systems consisted of primary hepatocytes from the common carp (C. carpio) and PLHC-1 cells, hepatocarcinoma cells from the desert topminnow (Poeciliopsis lucida) cell line. Each in vitro system was exposed to radiolabeled test compounds and assayed for biotransformation using liquid scintillation and thin layer chromatographic methods. First-order kinetics were used to estimate rates of biotransformation. Bioconcentration of test materials in fish were predicted using an in vitro to in vivo metabolic rate extrapolation model linked to a mass-balance model commonly used to predict bioaccumulation in fish. Both subcellular and cellular tests using microsomes, liver homogenates and hepatocytes respectively showed biotransformation of the parent surfactants. Biotransformation rates were fastest for hepatocytes, followed by microsomes and homogenates. Rates were too low from homogenate tests to extrapolate to in vivo-based biotransformation rates using the extrapolation model. Trout microsomes metabolized C16EO8 faster than carp microsomes, yet rates were approximately the same for C14EO2S. Predicted BCF values incorporating in vitro biotransformation rates from hepatocytes were similar to measured in vivo or USEPAs bioconcentration model (BCFWIN) predicted values. Predicted BCF values using microsomal-based rates from trout and carp studies were only slightly less than default BCF values which assumes a linear logKow to BCF relationship with no biotransformation. However, hepatocyte-based results showed substantially decreased BCFs compared to the default BCF values. These results indicate that BCF estimates based on in vitro metabolic rates can provide reasonable estimates of in vivo BCF values, therefore, supporting the use of in vitro approaches within a tiered approach to assess bioconcentration.

Growth hormone effect on xenobiotic-metabolizing activities of rainbow trout

Abstract In recent years several studies reported the regulation by growth hormone (GH) of the expression of a variety of P450 forms in mammals. However the effect of GH on the xenobiotic-metabolizing enzymes of fish are still unknown. The aim of this work was to investigate the effects of ovine GH—a growth hormone known to be efficient in trout—on the cytochrome P450 level and on aryl hydrocarbon hydroxylase, aminopyrine-N-demethylase, glucuronyl transferase and glutathione transferase activities in trout. The GH-implanted trout (n = 50) each received a single cholesterol pellet containing ovine GH and were compared to control animals (n = 50) receiving a single cholesterol pellet without GH. After 15 days fish were killed and the liver and gills were excised for the measurement of xenobiotic-metabolizing enzyme activities. GH treatment significantly decreased the level of hepatic cytochrome P450 and the activities of cytochrome P450 dependent monooxygenases. In contrast, no significant effect of the treatment was observed on the glutathione transferase and UDP-glucuronyl transferase activities. Moreover, GH treatment had no effect on branchial phase I and phase II enzyme activities. This study provides evidence that GH level significantly affects the expression of several members of the hepatic cytochrome P450 family in trout.

Hydroxylation of pristane by isolated hepatocytes of rainbow trout: a comparison with in vivo metabolism and biotransformation by liver microsomes

Abstract In vitro models may represent a useful approach for studying the biotransformation of xenobiotics in animals. However, the value of such models can only be established by relating in vitro results with in vivo data. In fish, several investigators have compared xenobiotic metabolism in vivo with xenobiotic metabolism in subcellular liver fractions. In contrast, the biotransformation of chemicals in isolated fish hepatocytes has been scarcely studied. The objective of this work carried out in rainbow trout was to compare the metabolism of a widespread branched alkane (pristane) in isolated hepatocytes with metabolism in vivo and in liver microsomal fractions. The incubation of hepatocytes with [3H] pristane led to pristanol, pristane-diol and pristanic acid, while pristanol appeared to be the only product in microsomal preparations. In the in vivo experiment, liver contained significant amounts of pristanol and pristanic acid whereas conjugates of pristanol and pristane-diol were found in bile. Thus, the metabolism of pristane in hepatocytes suspension correlates with in vivo biotransformation better than does metabolism in microsomes.