Maija Pesonen

Differential induction of cytochrome P-450-dependent monooxygenase, epoxide hydrolase, glutathione transferase and UDP glucuronosyl transferase activities in the liver of the rainbow trout by β-naphthoflavone or clophen A50

After administration of beta-naphthoflavone and Clophen A50 to juvenile rainbow trout, activities of hepatic cytochrome P-450-dependent deethylation of 7-ethoxyresorufin was increased 172- and 49-fold, respectively. Glutathione transferase activity towards 1-chloro 2,4 dinitrobenzene and UDP glucuronosyltransferase activities towards p-nitrophenol, 1-naphthol and testosterone were increased 1.4 to 3.0-fold by beta-naphthoflavone or Clophen A50. However, significant increases of the rate of glucuronidation of 1-naphthol by Clophen A50 and of testosterone by both Clophen A50 and beta-naphthoflavone were only determined when the activities were measured in digitonin activated microsomes. Epoxide hydrolase activity was not affected by beta-naphthoflavone or Clophen A50. The time course of induction of the various xenobiotic metabolizing enzymes exhibited different patterns. 7-Ethoxyresorufin-O-deethylase activity reached peak values 3 and 7 days after the administration of beta-naphthoflavone and Clophen A50, respectively. The rate of induction of glutathione transferase activity and UDP glucuronosyltransferase activities towards p-nitrophenol and 1-naphthol were relatively slow and did not reach distinct peak levels. These activities were still on maximum levels 4-6 weeks after the treatment. Glucuronidation of testosterone reached peak values 1 week after treatment with both beta-naphthoflavone and Clophen A50. The dissimilar patterns of induction of the cytochrome P-450-dependent activities and the various conjugation activities may indicate that these xenobiotic metabolizing enzymes are differently regulated in the rainbow trout liver.

Comparative analysis of estrogenic activity in sewage treatment plant effluents involving three in vitro assays and chemical analysis of steroids

In this study, we assessed and compared the suitability of three in vitro screening tools for the measurement of estrogenic activity in sewage treatment plant effluents (STPEs). These assays were the yeast estrogen screen (YES), production of zona radiata proteins (ZRPs) in trout hepatocytes, and the induction of reporter gene expression in the transfected rainbow trout gonad cell line RTG-2. Data obtained with the YES were additionally compared with calculated estrogenicity, based on steroid analysis data of the effluents. For comparison purposes, the response of the in vitro systems toward the estrogenic chemicals beta-estradiol, ethinyl estradiol, bisphenol-A, nonylphenol, and octylphenol was assessed. All three assays showed sensitivities in the same order of magnitude in response to the steroid compounds tested, with ZRP production being the least sensitive. Regarding the estrogenic environmental chemicals tested, the RTG-2 assay was more than an order of magnitude more sensitive than the other two assays. Despite their different sensitivities toward selected test chemicals, the three in vitro systems indicated estrogenic activity in the same concentration range for the tested STPEs. Calculated estrogenicity (chemical analysis) and measured estrogenicity (YES) were of the same order of magnitude for the STPEs tested. The present study indicates that all three in vitro systems, with the yeast-based system being the easiest and most robust, are applicable for the screening of estrogenic activity in effluent samples.

Alkaline comet assay in rainbow trout hepatocytes

The alkaline comet assay was performed to measure DNA integrity in fish hepatocytes. Primary cultures of rainbow trout hepatocytes were exposed to two known genotoxic compounds, hydrogen peroxide (H(2)O(2)) and benzo[a]pyrene (B[a]P), and to organic extracts of river sediments. The DNA damage in the form of single-strand breaks was monitored following the formation of DNA comets after alkaline electrophoresis. Exposure of the hepatocytes to H(2)O(2) for 2 hr increased strand breaks in a dose-related manner at the concentration range reported previously in studies with mammalian hepatocytes. B[a]P treatment led to a significant increase in strand breaks at the concentrations ranging from 0.1 to 10 muM after 4 hr of exposure. After 48 hr of exposure to B[a]P, the level of DNA strand breaks was lower than that of the control. The organic extracts obtained from river sediments significantly increased DNA strand breaks in trout hepatocytes, indicating the presence of genotoxic compounds in the sediment. The results show that the alkaline comet assay is a promising method by which to study the genotoxic potential of xenobiotics found in the aquatic environment.

Expression of P4501A1 in a primary culture of rainbow trout hepatocytes exposed to β-naphthoflavone or 2,3,7,8-tetrachlorodibenzo-p-dioxin

Primary cultures of rainbow trout hepatocytes were used to study the expression of CYP1A1 mRNA, its protein product (P4501A1), and catalytic activities (7-ethoxy-resorufin-O-deethylase, EROD) during a 96-h period after exposure of cells to beta-naphthoflavone (BNF) or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Hepatocytes were isolated from immature rainbow trout by a two-step perfusion method and incubated at 10 degrees C. Cells were exposed to the inducers for 48 h after 24 h of preculturing. The EROD activity of BNF-treated hepatocytes was higher than that of the control hepatocytes 12 h after addition of the inducer. Activities peaked after 48 h and had declined to control levels after 72 h. EROD activity in TCDD-treated cells was significantly elevated after 12 h, but in contrast to BNF-exposure, activities continued to increase during the experimental period. The content of P4501A1 protein, measured with an indirect ELISA technique (using anti-codP4501A1 IgG), increased linearly during the first 12 h and remained constant thereafter. In TCDD-exposed cells the immunochemically determined P4501A1 levels changed in parallel with EROD activity. CYP1A1 mRNA levels, determined by Northern blot and slot-blot analyses (using the trout P4501A1 cDNA pSg15 probe), were hardly detectable in control cells. In BNF- and TCDD-treated cells a 2.8-kb mRNA band was detected by the probe 6 h before the protein and catalytic activities became detectable. The elevated levels of CYP1A1 mRNA were sustained more effectively by TCDD than by BNF. In addition, a second mRNA band at 1.9 kb was seen. The results suggest that transcriptional activation is probably the prime factor even though post-transcriptional events may be involved in the regulation of P4501A1 induction by PAHs in rainbow trout hepatocytes.

Environmental contaminants and biochemical responses in flatfish from the Hvaler Archipelago in Norway.

The levels of several environmental contaminants, including selected polyaromatic hydrocarbons (PAH), organochlorines (DDT/DDE, hexachlorobenzene), 15 polychlorinated biphenyl (PCB) congeners, and polychlorinated dibenzofurans and dibenzo-p-dioxins, PCDF/PCDD), and heavy metals (Cd, Hg, Pb, and As) were analyzed in muscle and liver of three different flatfish species (dab,Limanda limanda; flounder,Platichthys flesus; plaice,Pleuronectes platessa) caught by gill netting at different sites in the Hvaler Archipelago. Indices of biochemical effects in liver S9-fractions were studied by measuring cytochrome P450-dependent monooxygenase and UDP-glucuronyl transferase activities, and by immunoquantitating cytochrome P450 1A1 using an indirect enzyme-linked immunosorbent assay (ELISA). Only low levels of PCDD/PCDF, Cd, and Pb were observed, whereas PCB levels were significantly elevated in fish from the inner sites of the Archipelago compared to a reference site. The contaminant gradient toward the Glomma estuary was correlated with increased cytochrome P450 1A1 activity, measured as 7-ethoxyresorufin O-deethylase (EROD), and with immunoquantitated P450 1A1. In contrast, fish from the site at Idefjorden, although containing elevated contaminant levels, did not show elevated EROD activity, but apparently elevated P450 1A1 protein. These findings may reflect different pollution histories of the sites, and indicate the applicability of biochemical effect indices (i.e., EROD and P450 1A1 immunoquantitation) to monitoring studies. The integrated chemical-biochemical approach employed in this study can obviously be expanded to give fruitful information about cause-effect relationships in other contaminant situations.

Fish primary hepatocyte culture; an important model for xenobiotic metabolism and toxicity studies

Abstract The aquatic environment is affected by numerous chemical contaminants. There is an increasing need to identify these chemicals and evaluate their potential toxicity towards aquatic life. In this review, we present the technique of primary cell culture of fish hepatocytes as one adjunct model for the ecotoxicological evaluation of the potential hazards of xenobiotics in the aquatic environment. The isolation of hepatocytes involves the perfusion of the liver with a Ca 2+ -free balanced saline solution followed by a second perfusion with the digesting enzyme collagenase. Hepatocytes or parenchymal cells can be separated from cell debris and from non-parenchymal cells by low-speed centrifugation. Fish hepatocytes have been shown to retain many important functions in primary cell culture which is a prerequisite for obtaining results relevant to the in-vivo situation. Thus, fish primary hepatocytes have been used to study the metabolism of xenobotics, the formation of toxic products, chemical-induced DNA damage, the induction of enzymes, peroxisome proliferation, and effects on hormone-signalling systems, etc. From the results, the mechanisms involved in chemical-induced toxicity can be determined in more detail than would be possible in in-vivo studies.

Glucocorticoid-mediated potentiation of P450 induction in primary culture of rainbow trout hepatocytes

Induction of 7-ethoxyresorufin O-deethylase activity (a cytochrome P450IA-dependent activity) by beta-naphthoflavone (0.36 microM) is increased 2-3-fold by dexamethasone or cortisol (10(-9)-10(-7) M) in rainbow trout hepatocyte cultures. This potentiation does not seem to be a time-dependent process and could be a classical glucocorticoid receptor-mediated event resulting in enhanced transcriptional activation of the CYP1A, as previously shown in mammals. Since glucocorticoid levels can increase in fish exposed to pollutants, such steroids may interfere with the induction response to xenobiotics.

Comparison of xenobiotic biotransformation enzymes in kidney and liver of rainbow trout (Salmo gairdneri)

The microsomal cytochrome P-450 content in kidney of rainbow trout (Salmo gairdneri) was approximately 5-fold lower than the content in liver. The renal ethoxycoumarin- and ethoxyresorufin-O-deethylase activities calculated on a per-cytochrome P-450 basis were, however, found to be about 10-fold higher than the hepatic activities. The patterns of time-dependent increase and subsequent decrease of microsomal cytochrome P-450-dependent monooxygenase activities after a single injection of beta-naphthoflavone (BNF) were similar in the kidney and liver. The microsomal ethoxyresorufin- and ethoxycoumarin-O-deethylase activities were maximally induced in liver (120- and 10-fold, respectively) by a single BNF injection (50 mg/kg body wt), whereas in kidney the maximal levels of induction (135- and 21-fold, respectively) were reached after three injections with BNF. The induction of cytochrome P-450 systems was associated with synthesis of a new microsomal protein of 58,000 Da in both kidney and liver. UDP-glucuronosyl transferase activity toward p-nitrophenol was about 8-fold lower in kidney than in liver. A significant 2.5-fold elevation in microsomal UDP-glucuronosyltransferase activity was found in the kidney 14 days after a single injection with BNF (50 mg/kg). In the liver, a 2-fold increase of this activity was seen 3 days after the treatment. The results indicate that the rainbow trout kidney in addition to the liver is of great importance in biotransformation of lipophilic xenobiotics.

Toxic effects of bleached and unbleached paper mill effluents in primary cultures of rainbow trout hepatocytes.

Toxic effects of unbleached (sulfate or sulfite) and bleached (sulfate) paper mill effluents were studied in a primary culture of rainbow trout liver cells. The effluents and control water from a clean area were extracted with diethyl ether and added to the cultures dissolved in dimethyl sulfoxide. Plasma membrane integrity was studied by measuring lactate dehydrogenase (LDH) leakage. The cellular content of glutathione (GSH) was used as an indicator of oxidative stress and the formation of reactive intermediates. Dose-response studies indicated that unbleached effluents contained more potent toxic substances than bleached effluents. Both unbleached and bleached effluents contained organic diethyl ether-extractable substances which increased cytochrome P450-dependent 7-ethoxyresorufin-O-deethylase (EROD) activities. The inducing effects were seen at concentrations substantially lower than those decreasing GSH content and increasing LDH leakage. Possible EROD inducing substances in bleached effluents are chlorinated organic compounds. Inducing compounds in unbleached effluents are yet to be identified. Furthermore, at higher concentrations the effluents contained substances that inhibited the cytochrome P450 system. The results show that the trout primary hepatocyte cultures afford a convenient in vitro method for screening cytochrome P450 inducing components extracted from industrial effluents to investigate mechanisms by which wastewaters cause injury in cells.

Unbleached pulp mill effluents affect cytochrome P450 monooxygenase enzyme activities

Abstract The polysubstrate monooxygenase system has been shown to be highly responsive to chemical pollution. The present study summarizes the enzyme based biomonitoring of the waste waters released by a pulp mill producing unbleached pulp and paperboard. Cytochrome P4501A enzyme activities of feral and caged fish, as well as cultures of fish hepatocytes, were tested. The 7-ethoxyresorufin-O-deethylase (EROD) activity was clearly induced in fish hepatocytes exposed to biotreated unbleached pulp mill effluent fractions in vitro. The effluent increased EROD activities also in feral perch, compared with controls. Caging experiments showed similar effects to those seen in feral fish: however, the maximal induction coefficients observed were higher. Unbleached effluents contain compounds that are able to affect the P4501A activities in fish.