Sirpa Huuskonen

Hepatic cytochrome P4501A and other biotransformation activities in perch (Perca fluviatilis): the effects of unbleached pulp mill effluents

Abstract The present study describes the use of enzyme based biomonitoring of the unbleached bio-treated pulp and paper mill effluent. The waste waters of a sulphite mill, producing unbleached semipulp and cellular board, were released into an inland water system. Cytochrome P450 related as well as conjugation enzyme activities were analyzed from the livers of perch (Perca fluviatilis) during different seasons. Part of the material was investigated by measuring the relative content of P4501A protein. Further, physiological indices such as condition factor (CF), liver somatic index (LSI), and gonado somatic index (GSI) were calculated. Exposure to the unbleached sulphite mill effluent elevated 7-ethoxyresorufin O-deethylase (EROD) and 7-pentoxyresorufin O-dealkylase (PROD) activities in feral perch in the contaminated area in summer, compared to fish caught from an upstream control site. EROD activity was reflected in the enhanced amounts of P4501A (or CYP1A) protein found in fish downstream to the discharge sewer. Assay of the P4501A protein content suggested that the effluent contained agents which, in addition to causing enzyme induction, also suppressed catalytic EROD activities. Among conjugation enzyme activities, UDP-glucuronosyltransferase (UDP-GT) showed a slight statistical significance in winter. The LSI and CF were slightly increased in perch inhabiting the polluted waters. Based on the current findings it is suggested that unbleached sulphite effluents can cause some changes in xenobiotic metabolism and in physiological parameters of perch.

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.

Effects of non-ortho-substituted polychlorinated biphenyls (congeners 77 and 126) on cytochrome p4501a and conjugation activities in rainbow trout (Oncorhynchus mykiss)☆☆☆

Abstract Immature rainbow trout ( Oncorhynchus mykiss ) in two separate experiments received a single intraperitoneal injection of 0.1, 1 and 5 mg/kg of either 3,3′,4,4′-tetra- or 3,3′,4,4′,5-pentachlorobiphenyl (IUPAC congeners 77 and 126, respectively). The experiments were run at water temperatures of 6 °C and 4 °C. Fish were killed 6 days after the injection. Biotransformation enzyme activities and cytochrome P4501A (CYP1A) amount and occurrence in different tissues were assayed. Congeners 77 and 126 strongly induced 7-ethoxyresorufin O-deethylase (EROD) and benzo(α)pyrene hydroxylase (AHH) activities in liver and kidney of rainbow trout. The induction of these cytochrome P4501A dependent monooxygenases was dose-related especially with congener 77 in the kidney. However, in the liver the highest dose of both congeners and in kidney the highest dose of congener 126 did not increase the catalytic monooxygenase activities as much as would have been expected based on the responses obtained with the lower doses. This may be because the monooxygenase activities already had attained their maximal induction capacity at 1 mg/kg dose of each congener. The PCB residues in liver were also determined and found to be highest after 5 mg/kg injections (610 μg/kg wet weight with congener 77 and 220 μg/kg with congener 126). When cytochrome P4501A protein content was measured, the induction of cytochrome P4501A was still on the increase even in those cases where catalytic activity failed to show any further induction. Immunohistochemical samples from liver, kidney and intestine showed cytochrome P4501A staining which strongly correlated with cytochrome P4501A in microsomes. Such observations suggest that the amount and occurrence of P4501A in the tissues can express the induction even when catalytic activities seem to be suppressed. With respect to enzymes mediating conjugation reactions, hepatic and renal UDP-glucuronosyltransferase (UDP-GT) activities showed elevated levels especially with the 1 and 5 mg/kg doses of both congeners. Glutathione S -transferase (GST) activities did not show such a clear trend. Congeners 77 and 126 preferentially affected the P4501A enzymes but to some extent also conjugation activities.

Histopathology of Feral Fish from a PCB-Contaminated Freshwater Lake

Abstract The purpose of this study was to evaluate the potential toxic effects of chronic sublethal polychlorinated biphenyl (PCB) exposure on feral fish, using histopathology as an endpoint. Histopathological study of bream (Abramis brama) and asp (Aspius aspius) living in a PCB-polluted freshwater lake revealed abnormal cellular changes in the renal corpuscle of both species. Dilation of glomerular capillaries (DGC), mesangial edema (ME), an adhesion between visceral and parietal layers of Bowmans capsule (ABC), and filling of Bowmans space (FBS), were highly prevalent features in lake fish. The prevalence of each of these lesions was significantly lower, or totally absent in fish caught from reference locations. Cellular alterations in liver, gill, gonads, spleen, and intestine were all linked to seasonal changes. The results suggest that some of the observed histopathological changes in renal glomeruli, particularly DGC and ME, could possibly indicate a prolonged chemical stress caused by PCBs and related compounds. It is also possible that chronic PCB exposure may have suppressed and weakened the immuno systems of exposed fish making them more vulnerable to secondary parasitic infections.

A fish hepatoma cell line (PLHC-1) as a tool to study cytotoxicity and CYP1A induction properties of cellulose and wood chip extracts

Cytotoxicity and CYP1A induction properties of celluloses and wood chips were studied with a teleost liver cell line, PLHC-1. Cells were exposed to acetone extracts of celluloses produced using new bleaching techniques (elemental chlorine free, ECF; totally chlorine free, TCF) in two sulphate mills or without any bleaching (unbleached, UB) in a sulphite mill. In another set of exposures, celluloses (ECF and TCF bleached) and wood chips (from pine and birch) were collected from a sulphate mill, extracted with acetone, and the extracts used to treat the cells. After exposure, O-deethylation of 7-ethoxyresorufin (EROD, a measure of cytochrome P4501A (CYP1A) catalytic activity), and total protein content, a measure of cytotoxicity, were assayed. The presence of the CYP1A protein in the exposed cells was assessed by immunoblotting. The cellulose and wood chip extracts were able to cause both cytotoxicity and EROD induction in the PLHC-1 cells. In the exposures conducted with the material from three different mills, the celluloses made of birch were more cytotoxic and more potent inducers of EROD activity than were the celluloses of pine. Further, UB celluloses increased EROD activity and caused cytotoxicity at lower doses than material bleached with modern bleaching techniques. In the exposures made with material from one single mill, there were no clear trends between the celluloses made of pine or birch. Wood chips of pine, however, were more cytotoxic than wood chips of birch. Especially with pine wood chips, cytotoxicity interfered with the induction of EROD activity, thus complicating the evaluation of CYP1A induction. CYP1A protein content was not detected in cells exposed to extracts of celluloses or wood chips, possibly due to low amounts of protein available for the assay. Wood and pulp processing, like bleaching, may change the chemical composition of the raw material in a way that reduces the potency for biological effects of the final product, cellulose. This could explain why both UB celluloses and wood chips were more potent in the cells than ECF or TCF bleached celluloses. In this study the PLHC-1 cell line showed its potential for use in evaluating the biological activity existing in pulp and paper mill products and raw materials. The identity and source of the compounds that were able to affect the PLHC-1 cell line remain to be determined.

Responses of biotransformation and antioxidant enzymes in Lemna minor and Oncorhynchus mykiss exposed simultaneously to hexachlorobenzene

Effect of hexachlorobenzene (HCB), a persistent environmental contaminant, exposure on the enzymes of oxidative biotransformation and antioxidant system of the aquatic plant L. minor and fish were studied simultaneously. Furthermore, the effects of fish - plant interactions in a HCB polluted environment on the responses of hepatic biotransformation and antioxidant enzymes of fish were also investigated. The peroxidase and glutathione S-transfemse (GST) activity in HCB exposed plants were significantly high following 1, 2 and 7 days of the exposure. In general, the activity of antioxidant enzymes in plants also increased following exposure to HCB. Exposure to either HCB or plants in uncontaminated water did not cause any major changes in the hepatic microsomal monooxygenase and antioxidant enzyme activities in fish. However, significant inhibition in liver monooxygenase enzyme activities and increase in the antioxidant enzyme activities were observed in fish that were exposed to HCB in the presence of L. minor. Such observations suggests that interactions between fish and plants in a HCB contaminated environment may affect fish hepatic biotransformation and antioxidants enzymes.

Comparison of two bioassays, a fish liver cell line (PLHC-1) and a midge (Chironomus riparius), in monitoring freshwater sediments

Abstract Two bioassays, a fish hepatoma cell line (PLHC-1) and a midge ( Chironomus riparius ), were used to monitor surficial sediments from Lake Vortsjarv and River Narva, Estonia. L. Vortsjarv is polluted with mainly polycyclic aromatic hydrocarbons (PAHs) but R. Narva possesses complex contamination (PAHs, heavy metals, sulphates, chlorides). The PLHC-1 cells were exposed to the extracted lipid soluble compounds (PAH fraction) of the sediments, after which the cytotoxicity (total protein content) and cytochrome P4501A (CYP1A) inducibility (7-ethoxyresorufin O -deethylase activity (EROD) and CYP1A protein content) were measured. The midges were grown in whole sediments after which the midge growth (larval growth and survival as endpoints) or the emergence (larval survival and adult emergence) were tested. Contents of selected PAHs and heavy metals in the sediments were also evaluated. In the PLHC-1 screening experiments, most of the sediments from R. Narva were more toxic and caused higher EROD activities at lower doses than the sediments from L. Vortsjarv. The most polluted sediment in R. Narva (total PAH content 744 ng g −1 dry weight sediment) gave 15 mg dry sediment ml −1 as the ED 50 for induction of EROD activity in the cells exposed for 3 days. In the midge growth test, larvae seemed to grow better in the sediments from L. Vortsjarv than from R. Narva and the mortality was somewhat higher in two areas in R. Narva than in other study areas. Adult emergence did not show such clear trends between these two watersheds, though emergence was accelerated in some sediments. A sediment from a point source of pollution (accidental release of asphalt) from R. Narva was also studied and found to be the most toxic for the PLHC-1 cells and the most potent inducer of CYP1A (ED 50 s 0.59 and 0.56 mg dry sediment ml −1 ). None of the midge larvae survived in this sediment. In a time-course study, the highest EROD activity in the PLHC-1 cells was reached at lower doses of PAH fraction after 24-h exposure than after 48 or 72 h, suggesting metabolism of PAHs in the cultures. Further, CYP1A induction was still seen as elevated amounts of CYP1A protein in cases where catalytic EROD activity was decreased at higher doses of PAH fraction. Overall, the PLHC-1 bioassays were shown to be sensitive methods for detecting PAH pollution. The midge bioassays reflected better the bioavailability and the in situ effects of the complex mixture of compounds in the sediments.

Biomonitoring of South Estonian waters by means of xenobiotic metabolism of rainbow trout (Oncorhynchus mykiss) liver

Abstract The aquatic pollution in waters of Tartu County (South Estonia) was monitored by measuring biotransformation enzyme activities in rainbow trout ( Oncorhynchus mykiss ) liver. Juvenile fish were caged for 3 weeks in winter and in spring at selected study areas: Lake Vortsjarv, the second largest lake in Estonia, representing an agricultural and recreational area as well as an important fishing site, and River Suur Emajogi, whose main source of pollution is in the form of improperly treated urban sewage from the town of Tartu. To obtain an overview of the degree of contamination, the contents of polycyclic aromatic hydrocarbons were determined in selected samples of fish muscle. During both seasons, cytochrome P 450 -dependent monooxygenase (MO) activities in rainbow trout liver showed the highest values in the fishing harbor area in L. Vortsjarv. During winter, elevated MO activities were measured in fish in R. Suur Emajogi up to 3 km downstream from the town of Tartu, compared with the values from the upstream reference area. In the spring, MO activities at the same site did not reach such high levels as earlier. At that time, the contaminants in R. Suur Emajogi were much more diluted than in winter. This was due to the much higher runoffs and possibly causes the lack of induction. Conjugation enzyme activities did not show any significant changes in any of the studies conducted.

Biotransformation and glutathione homeostasis in rainbow trout Exposed to chemical and physical stress

Abstract Glutathione, together with biotransformation and antioxidative enzymes, plays an important role in protecting the cells against damage caused by free radicals, peroxides, oxidizing metabolites and xenobiotics introduced by environmental stress. Pollution of the environment causes many long-term physiological effects on fish. Furthermore, in mammals exercise increases oxidative stress (increase of reactive oxygen species) which overloads the defence mechanisms. Induction of the glutathione system could show whether physical stress also affects defence mechanisms of fish. In the present series of studies we have investigated the biotransformation and glutathione homeostasis of rainbow trout exposed to chemical and physical stress. Juvenile rainbow trout ( Oncorhynchus mykiss ) were exposed to waterborne hexachlorobenzene (HCB; 2 μg/litre) together with duck-weed ( Lemna minor ) using different combinations of fish, plants and HCB. In another experiment, fish were held in different water flows (none, medium, high). Hepatic mono-oxygenase (EROD) and conjugation (GST) enzyme activities, as well as antioxidant systems (GPX, GR, tGSH) were measured. In the HCB-exposed fish-plant group EROD activities in fish were suppressed compared with the uncontaminated group. It is probable that through digested plants the fish could have been exposed to possible plant-derived HCB metabolites or secondary metabolites. Fish of different physical activity showed slightly elevated EROD activities three weeks from the beginning of the experiment. After the following three-week period EROD activities had decreased to the starting level. Three weeks from the beginning GST activities were at their highest level. The elevation of mono-oxygenase activities may have caused increased production of reactive intermediates which were further metabolized through activated glutathione system. Elevated total glutathione contents in studied tissues showed the potency of these fish to resist oxidative stress.

Time-course studies of the biotransformation enzymes in control rainbow trout when adjusting to new habitats

Abstract Hepatic monooxygenase enzyme activities and relative cytochrome P4501A protein content were measured to evaluate the time-course alterations in rainbow trout after change in living habitat. Fish were transferred from one fish farm to the tanks of another hatchery and/or into cages kept in a lake. In the new habitats, cytochrome P450-dependent enzyme activities in rainbow trout decreased, and were at their lowest levels after two or three weeks in the summer. Later the activities partly reversed. The immunodetection of cytochrome P4501A protein expressed a similar trend as for catalytic monooxygenase activities.