Pirjo Lindström-Seppä

Biotransformation and other toxicological and physiological responses in rainbow trout (Salmo gairdneri Richardson) caged in a lake receiving effluents of pulp and paper industry

Abstract Hatchery reared immature rainbow trout ( Salmo gairdneri Richardson) were transferred to eight field stations at the southern Lake Saimaa (SE Finland). They were caged in the receiving water body of effluents from a mill producing chlorine bleached kraft pulp and printing paper. The controls were caged upstream to the sewer. The microsomal cytochrome P -450 content, NADPH cytochrome c reductase and monooxygenase enzyme activities as well as conjugation reactions with glucuronic acid and glutathione were studied in the liver, kidney and gills. Conjugated metabolites of chlorophenolics in the bile and some physiological blood parameters were investigated. The caging in polluted water increased the monooxygenase enzyme activities. Compared to control trout, up to 7 times induction of liver 7-ethoxyresorufin O -deethylase (EROD) activity was observed at the nearest caging station, 3 km from the effluent outlet. Activity of EROD was the best indicator for the induction caused by pulp and paper mill effluent, however, pentoxyresorufin O -dealkylase was also responsive. Analysis of conjugated chlorinated phenolics in the bile showed highest concentrations at the caging station nearest to the mill and displayed good distance related decreases. The levels of conjugated toxicants found in control fish indicated a low contamination of the whole southern Lake Saimaa.

Seasonal variation of the xenobiotic metabolizing enzyme activities in the liver of male and female vendace (Coregonus albula L.)

Abstract The activities of xenobiotic metabolizing enzymes measured in the liver of freshwater vendace (Coregonus albula L.) varied with the seasons. The determinations were made at a constant temperature (18°C) and at the environmental (water) temperature, in which the fish were caught. The monooxygenase activities (benzo[a]pyrene, 7-ethoxycoumarin and 7-ethoxyresorufin as substrates) decreased gradually in the autumn before spawning and increased thereafter. The activities in the male vendace were higher than in the female fish. Slight thermal compensation was seen during water cooling in the autumn from temperatures of 10 to 1°C when benzo[a]pyrene or 7-ethoxyresorufin were used as substrates. UDP-glucuronosyltransferase activity, p-nitrophenol as substrate, increased slowly before spawning and just after the event the activity rose to much higher levels. It did not show any thermal compensation during water cooling in the autumn. The amounts of reduced glutathione, the cosubstrate of the enzyme glutathione S-transferase, varied, being highest in the spring and lowest in the autumn. When studying the xenobiotic metabolizing enzyme activities of fish the sexual maturation and reproduction cycle have to be taken into consideration.

Extrahepatic xenobiotic metabolism in North-European freshwater fish

Abstract 1. 1. Cytochrome P450 and monooxygenase activity and glucuronidation were measured in the gills, intestine, heart and kidney as well as in the liver of vendace, perch and roach and rainbow trout for comparison. 2. 2. Cytochrome P450 content and the specific monooxygenase activities were always highest in the liver. In the rainbow trout and vendace the renal 7-ethoxycoumarin-O-deethylase and in the roach the renal 3,4-benzpyrene hydroxylase activity was about half of that in liver. The activities in gills were relatively high in roach and vendace. In rainbow trout, perch and roach the intestinal activities were easily detectable. 3. 3. In all fish high UDPglucuronosyltransferase activities were found in the liver and intestine. In vendace and roach both the gills and the kidney showed, however, even higher specific activities. 4. 4. The rainbow trout showed higher hepatic and intestinal biotransformation activities than the other species.

Biotransformation and other physiological responses in whitefish caged in a lake receiving pulp and paper mill effluents

Hepatic monooxygenase (MO) and conjugation enzyme activities, metabolites of chlorinated phenolics in the bile, and blood ionoregulatory parameters were studied in juvenile whitefish (Coregonus muksun Pallas and C. muksun X Coregonus peled Gmelin hybrid) held in cages downstream from a mill producing chlorine-bleached kraft pulp and printing paper. MO activities, measured as benzo[a]pyrene hydroxylase, 7-ethoxycoumarin O-deethylase, and 7-ethoxyresorufin O-deethylase, were significantly induced in whitefish caged about 5 km from the effluent outlet. The highest mean increase detected was 17 times the control value. In the nearest caging station (3 km) the induction was lower, indicating inhibition or toxicity caused by the effluent. The levels of bile metabolites of chlorinated phenolics showed highest concentrations at the nearest station and decreased levels at more distant locations over the whole water area studied (15 km). Bile metabolites in whitefish exposed in control areas confirmed low-level background pollution of the lake system due to chlorinated phenolics. Observations on blood ionic concentrations suggest that whitefish were able to regulate their hydromineral balance despite the environmental pollution affecting physiology of biotransformation.

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.

Uptake of waterborne 3,3′,4,4′-tetrachlorobiphenyl and organ and cell-specific induction of cytochrome P4501A in adult and larval fathead minnow Pimephales promelas

Female fathead minnows (Pimephales promelas) were exposed to water-borne [14C]-3,3′,4,4′-tetrachlorobiphenyl (TCB) on days 0 and 5, for 24 h each time, in water with 0, 0.29, 2.9 or 29 mg TCB/kg total fish mass. Between treatments, fish were kept in clean water with normal feeding and light conditions. Liver, ovary and skeletal muscle were collected at 2 h, on day 5 prior to the second dosing and on day 12. Initially, TCB uptake was greatest in liver, but after 5 days the content in ovaries (ca. 5 μg TCB/g wet weight) was similar to that in liver (ca. 4 μg/g wet weight). TCB concentration in muscle was 10% of that in liver or ovary. Tissue concentrations of TCB were similar at 5 days after the first dose and 6 days after the second dose. Immunoblot analysis with monoclonal antibody 1-12-3 (specific for cytochromes P4501A; CYP1A) showed that CYP1A content in liver homogenates increased with increasing content of TCB in the liver, to about 5 pmol scup CYP1A equivalents per mg protein at 1.8 μg TCB/g liver. Between 1.8 and 4 μg TCB/g liver, the CYP1A content increased another 10-fold. Ethoxyresorufin O-deethylase (EROD) activity in liver homogenates also increased with increasing content of TCB in liver. However, EROD activity was suppressed at TCB concentrations greater than 1.8 μg TCB/g liver, consistent with other results showing that 3,3′,4,4′-TCB can inhibit or inactivate CYP1A. Immunohistochemical staining with MAb 1-12-3 showed induction in liver and in multiple extrahepatic organs; analysis with a second anti-scup CYP1A1 MAb (1-71-3) confirmed the patterns of induction. A strong CYP1A staining signal was detectable only in the highest dose group, and induction in epithelial cells, including hepatocytes, was weaker than that in endothelium. This suggests that the CYP1A seen in immunoblots of liver homogenates could reflect a substantial contribution from endothelial cell P450. There was mild staining indicating CYP1A induction in endothelium of embryos hatched from eggs of exposed fish. Analysis of induction in fathead minnows is relevant to their use in evaluating chemicals that may be activated or detoxified by CYP1A enzymes. The results further illustrate the utility of immunohistochemistry to evaluate CYP1A induction as a marker of exposure.

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.

Role of gut in xenobiotic metabolism

The gastrointestinal tract forms the first line of defense in the body against the main load of xenobiotics. The gastrointestinal mucosa has several mechanisms through which the xenobiotics are modified. The monooxygenase activities in most species are relatively low in the mucosa as compared to the liver, but conjugation, for example, via glucuronide formation proceeds efficiently. UDP-glucuronosyltransferase activities can exceed those in the liver. Glutathione S-transferase activity is also high. The biotransformation activities are readily inducible in the mucosa and this is, at least partly, responsible for the oral-aboral gradient seen in enzyme activities. In rainbow trout glutathione S-transferase is, however, significantly higher at the aboral third than in two oral segments, although in rats the intestinal glutathione S-transferase shows a clear oral-aboral gradient. The gradient is independent of the presence of microflora at least in the case of carboxylesterase and glutathione S-transferase. A similar gradient can also be found from the gut lumen, in both germ-free and specific pathogen-free rats. The cells in the middle of the villi appear to be most responsive under the influence of inducers. The readily occurring induction in the mucosa provides a suitable model for studies on biological effects to defined compounds and mixtures.

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.