Hepatic Biotransformation Enzymes Research Articles

Phenanthrene and nitrite effects on juvenile sea bass, Dicentrarchus labrax, using hepatic biotransformation enzymes, biliary fluorescence, and micronuclei as biomarkers

Aquatic organisms may absorb organic compounds mainly from water and by ingestion of contaminated food. The toxicity of such compounds may be intensified by the presence of certain inorganic compounds such as nitrite (NO2–). In order to evaluate the effect of phenanthrene (PHE), a polycyclic aromatic hydrocarbon, in the presence and absence of NO2–, juvenile sea bass, Dicentrarchus labrax L, were exposed to PHE i.p. and to NO2– in water, and several endpoints were measured at days 1, 3, and 6 of exposure. Sea bass exposed to PHE exhibited lower hepatic 7-ethoxyresorufin O-deethylase (EROD) activity as compared to the control group. The activity of the phase II enzyme, glutathione S-transferase (GST), was similar in all the groups of fish. The concentration of PHE metabolites, determined as fluorescent aromatic compounds, was nearly 14 times higher both in the presence and absence of NO2–, showing that even at low EROD activities this PAH is metabolized. The presence of micronuclei was observed to be significantly higher only in sea bass treated with PHE alone, suggesting that different PHE metabolites, without genotoxic properties, were formed in the presence of NO2–.

Induction of Mouse UDP-Glucuronosyltransferase mRNA Expression in Liver and Intestine by Activators of Aryl-Hydrocarbon Receptor, Constitutive Androstane Receptor, Pregnane X Receptor, Peroxisome Proliferator-Activated Receptor α, and Nuclear Factor Erythroid 2-Related Factor 2

UDP-glucuronosyltransferases (UGTs) catalyze the addition of UDP-glucuronic acid to endo- and xenobiotics, enhancing their water solubility and elimination. Many exogenous compounds, such as microsomal enzyme inducers (MEIs), alter gene expression through xenobiotic-responsive transcription factors, namely, the aryl hydrocarbon receptor (AhR), constitutive androstane receptor (CAR), pregnane X receptor (PXR), peroxisome proliferator-activated receptor alpha (PPARalpha), and nuclear factor erythroid 2-related factor 2 (Nrf2). These transcription factors regulate xenobiotic-inducible expression of hepatic and intestinal biotransformation enzymes and transporters. The purpose of this study was to determine hepatic and intestinal inducibility of mouse Ugt mRNA by MEIs. Male C57BL/6 mice were treated for four consecutive days with activators of AhR [2,3,7,8-tetrachlorodibenzodioxin (TCDD), polychlorinated biphenyl 126, and beta-naphthoflavone], CAR [1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP), phenobarbital, and diallyl sulfide], PXR [pregnenolone-16alpha-carbonitrile (PCN), spironolactone, and dexamethasone], PPARalpha (clofibrate, ciprofibrate, and diethylhexylphthalate), and Nrf2 (oltipraz, ethoxyquin, and butylated hydroxyanisole), respectively. Ugt1a1 mRNA expression in liver was induced by activators of all five transcription factor pathways, Ugt1a5 by Nrf2 activators, Ugt1a6 by all the pathways except CAR, and Ugt1a9 by all the pathways except Nrf2. Ugt2b35 mRNA in liver was induced by AhR activators and Ugt2b36 by CAR and PPARalpha activators. Throughout the small and large intestine, the AhR ligand TCDD increased Ugt1a6 and Ugt1a7 mRNA. In small intestine, the PXR activator PCN increased Ugt1a1, Ugt1a6, Ugt1a7, Ugt2b34, and Ugt2b35 mRNA in the duodenum. In conclusion, chemical activation of AhR, CAR, PXR, PPARalpha, and Nrf2 in mouse results in induction of distinct Ugt gene sets in liver and intestine, predominantly the Ugt1a isoforms.

Endocrine alteration and other biochemical responses in juvenile turbot exposed to the Prestige fuel oil

Juvenile turbot ( Scophthalmus maximus) were exposed to different concentrations of the Prestige fuel oil through the diet. The effects on hepatic biotransformation enzymes, namely, 7-ethoxyresorufin O-deethylase (EROD), catalase, and phase II activities – UDP-glucuronosyltransferases (UGT) and cytosolic sulfotransferases (SULT) –, were monitored after 42-day exposure. Additionally, potential alterations on the endocrine system of juvenile turbot were assessed by measuring circulating levels of testosterone and 17β-estradiol in plasma, together with gonadal P450 aromatase activity and the glucuronidation of testosterone by liver microsomal fractions. Fluorescent aromatic compounds (FACs) in bile were determined as an indicator of exposure to polycyclic aromatic hydrocarbons (PAHs). EROD activity increased in a dose dependent manner, and a positive correlation between levels of FACs in bile and EROD activity was observed. A significant increase in UGT activity was observed in fish exposed to medium and high fuel doses, whereas SULT cytosolic activity was not responsive to exposure. Exposure to the Prestige fuel oil sharply reduced circulating levels of testosterone in plasma. The obtained results suggest the ability of the Prestige fuel oil to alter hepatic biotransformation enzymes and to disrupt endogenous hormone levels in juvenile turbot, with unknown consequences in terms of sexual differentiation and reproduction.

Dicrocoeliosis of Old Mouflon Ewes - Effect on Biotransformation Enzymes and Metabolism of Anthelmintics In Vitro

Parasitic infections can modify the host's ability to metabolize drugs by altering the biotransformation en- zymes. Mouflon (Ovis musimon), the wild sheep, is very sensitive to parasitic diseases. In the present study, in vitro ac- tivities of thirteen hepatic biotransformation enzymes and the in vitro metabolism of albendazole and flubendazole were compared in old mouflon ewes, either non-infected or infected with lancet flukes (Dicrocoelium dendriticum). The most pronounced decline in 6�� testosteron hydroxylase, glutathione-S-transferase and UDP-glucuronosyl transferase activities was observed in Dicrocoelium-infected animals. In addition, dicrocoeliosis caused a decrease in both albendazole and flubendazole phase I hepatic metabolism. However, the changes were very slight therefore any undesirable alterations in albendazole and flubendazole pharmacokinetics are not expected. Decreased activities of 6�� testosterone hydroxylase, glutathione-S-transferase and UDP-glucuronosyl transferase can impair drug elimination from the infected animals. This should be taken into consideration in pharmacotherapy or pharmacoprophylaxis of mouflons and sheep.

Activities of biotransformation enzymes in pheasant ( Phasianus colchicus) and their modulation by in vivo administration of mebendazole and flubendazole

Basal activities of certain pheasant hepatic and intestinal biotransformation enzymes and modulation of their activities by anthelmintics flubendazole (FLBZ) and mebendazole (MBZ) were investigated in subcellular fractions that were prepared from liver and small intestine of control and FLBZ or MBZ treated birds. Several oxidation, reduction and conjugation enzyme activities were assessed. In the liver, treatment of pheasants by FLBZ or MBZ caused very slight or no changes in monooxygenase activities and conjugation enzymes. More significative changes were detected in small intestine. Metyrapone and daunorubicin reductase activities were increased by both substances in the liver. This is the first evidence that certain benzimidazoles modulate reductases of carbonyl group. With respect to the relatively slight extent of the changes caused by FLBZ or MBZ we can assume that repeated administration of therapeutic doses of both FLBZ and MBZ has probably no serious influence on pheasant biotransformation enzyme system.

Hepatic metabolism, phase I and II biotransformation enzymes in Atlantic salmon ( Salmo Salar, L) during a 12 week feeding period with graded levels of the synthetic antioxidant, ethoxyquin

The synthetic antioxidant ethoxyquin (EQ) is a widely used additive in animal feeds, including farmed fish feed. The use of EQ as food additive is prohibited and it is also undesirable in farmed meat and fish products. The possible negative aspects of EQ in fish feeds, such as modulation of hepatic detoxifying enzymes and possible effects through “carry-over” to edible parts of fish are not known. In addition, the subsequent consequences for human consumers have not been previously studied. In the present work, the alteration in gene and protein expression patterns, and catalytic activities of phase I and II hepatic biotransformation enzymes due to prolonged exposure to graded levels of dietary EQ in the range of 11–1800 mg EQ/kg feed were studied. The kinetics of parent EQ and its major metabolite, ethoxyquin dimer (EQDM) was also studied. In general two weeks seem to be the critical point in the entire toxicological response of salmon to dietary consumed EQ. Biotransformation of EQ to EQDM is shown to be a rapid process. However, the decrease in biotransformation rate results in the accumulation of EQ metabolites, high concentration of which was postulated to alter translation and post-translational modification of CYP3A, GST and UDPGT at feeding day 14 and 42, with subsequent decreases in the biotransformation of consumed EQ. Decrease in the biotransformation of consumed EQ produced the retention of un-metabolized EQ rather than metabolites in salmon liver. This may be considered as undesirable effect, since it could lead to the transport and accumulation in other organs and edible tissues. It may also cause a new wave of biotransformation with formation of metabolites inhibiting detoxifying enzymes. In general, these processes may prolong the excretion of dietary EQ from the fish body and produce EQ-derived residues in the ready-to-consume salmon or fish products. These EQ residues may have higher toxicological effects for human consumers than the parent compound and therefore need to be studied in more detail.

Hepatic Biotransformation and Metabolite Profile during a 2-Week Depuration Period in Atlantic Salmon Fed Graded Levels of the Synthetic Antioxidant, Ethoxyquin

The synthetic antioxidant ethoxyquin (EQ) is increasingly used in animal feeds and has been candidate for carcinogenicity testing. EQ has the potential for toxicological and adverse health effects for both fish and fish consumers through "carryover" processes. The toxicological aspects of EQ have not been systematically investigated. The present study was performed to investigate the hepatic metabolism, metabolite characterization, and toxicological aspects of EQ in salmon during a 2-week depuration after a 12-week feeding period with 18 mg (low), 107 mg (medium), and 1800 mg/kg feed (high). The alteration in gene expressions and catalytic activities of hepatic biotransformation enzymes were studied using real-time polymerase chain reaction with specific primer pairs and by kinetics of two identified hepatic metabolites. Analysis of EQ metabolism was performed using high performance liquid chromatography (HPLC) method and showed the detection of four compounds of which two were quantified, parent EQ and EQ dimer (EQDM). Two metabolites were identified as de-ethylated EQ (DEQ) and quinone imine, but these were not quantified. The concentration of the quantified EQ-related compounds in the liver at day 0 showed a positive linear relationship with measured dietary EQ (R2= 0.86 and 0.92 for parent EQ and EQDM, respectively). While the low-EQ-feeding group showed a time-specific increase of aryl hydrocarbon receptor (AhR) mRNA expression, the medium-dose group showed decreased AhR mRNA at depuration day 7. Expression of CYP1A1 was decreased during the depuration period. Consumption of dietary EQ produced the expression of CYP3A, glutathione S-transferase (GST), and uridine diphosphate glucuronosyl-transferase (UDPGT) mRNA during the depuration period. A similar pattern of effect was observed for both CYP3A and phase II genes and supports our previous postulation of common regulation of these enzymes by the same inducer, namely EQ metabolites. The increase of CYP3A, UDPGT, and GST gene expressions at day 7 was in accordance with the low concentration of DEQ. The low concentration of putative DEQ may induce the CYP3A with subsequent increase in the biotransformation of EQ into DEQ. The increase in UDPGT may seem to be a synchronizing mechanism required for the excretion of DEQ. The biotransformation of dietary EQ is proven by simultaneous induction of both phase I and II detoxification system in the liver of Atlantic salmon. Therefore, the apparent low concentration of putative DEQ may account for the induced phase I and II detoxifying enzymes at least during depuration. This speculated hypothesis is currently a subject for systematic investigation in our laboratory using in vitro and genomic approaches.

Modulation of porcine biotransformation enzymes by anthelmintic therapy with fenbendazole and flubendazole

Fenbendazole (FEN) and flubendazole (FLU) are benzimidazole anthelmintics often used in pig management for the control of nematodoses. The in vivo study presented here was designed to test the influence of FLU and FEN on cytochrome P4501A and other cytochrome P450 (CYP) isoforms, UDP-glucuronosyl transferase and several carbonyl reducing enzymes. The results indicated that FEN (in a single therapeutic dose as well as in repeated therapeutic doses) caused significant induction of pig CYP1A, while FLU did not show an inductive effect towards this isoform. Some of the other hepatic and intestinal biotransformation enzymes that were assayed were moderately influenced by FEN or FLU. Strong CYP1A induction following FEN therapy in pigs may negatively affect the efficacy and pharmacokinetics of FEN itself or other simultaneously or consecutively administered drugs. From the perspective of biotransformation enzyme modulation, FLU would appear to be a more convenient anthelmintic therapy of pigs than FEN.

Phytochemical-induced changes in gene expression of carcinogen-metabolizing enzymes in cultured human primary hepatocytes

The naturally occurring compounds curcumin (CUR), 3,3′-diindolylmethane (DIM), isoxanthohumol (IXN), 8-prenylnaringenin (8PN), phenethyl isothiocyanate (PEITC) and sulforaphane (SFN) protect animals against chemically induced tumours. Putative chemoprotective mechanisms include modulated expression of hepatic biotransformation enzymes. However, few, if any, studies have used human primary cells as test models.The present study investigated the effects of these phytochemicals on the expression of four carcinogenesis-relevant enzymes — cytochrome P450 (CYP)1A1 and 1A2, NAD(P)H:quinone oxidoreductase (NQO1) and glutathione S-transferase A1 (GSTA1) — in primary cultures of freshly isolated human hepatocytes.Quantitative RT-PCR analyses demonstrated that CYP1A1 was up-regulated by PEITC and DIM in a dose-dependent manner. CYP1A2 transcription was significantly activated following DIM, IXN, 8PN and PEITC treatments. DIM exhibited a remarkably effective induction response of CYP1A1 (474-, 239- and 87-fold at 50, 25 and 10 μM, respectively) and CYP1A2 (113-, 70- and 31-fold at 50, 25 and 10 μM, respectively), that was semiquantitatively reflected in protein levels. NQO1 expression responded to PEITC (11 × at 25 μM), DIM (4.5 × at 50 μM) and SFN (5 × at 10 μM) treatments. No significant effects on GSTA1 transcription were seen.The findings show novel and unexpected effects of these phytochemicals on the expression of human hepatic biotransformation enzymes that play key roles in chemical-induced carcinogenesis.

Long-term exposure effects in vitellogenin, sex hormones, and biotransformation enzymes in female carp in relation to a sewage treatment works

In the present study, the estrogenic response in the reproductive cycle of female carp, Cyprinus carpio, in relation to a sewage treatment works (STWs) was measured as: alterations in plasmatic and hepatic vitellogenin (VTG) content, plasma levels of testosterone (T) and estradiol (E2), and effects on key enzymes of phases I and II biotransformation, ethoxyresorufin O-deethylase (EROD) and glutathione S-transferase (GST). Low plasma VTG content in females collected 4 km downstream of an effluent discharge suggested possible estrogenic disturbances; however, hepatic VTG content and sex hormone levels did not mirror this. In fact, E2 and T largely fluctuated among fish, with individual variations even greater than among sampling stations or periods. This suggests that at moderately polluted sites, biological variations of female carp and genetic particularities are prevalent. Hepatic biotransformation enzymes, such as EROD, increased with increasing water temperature while, in contrast, GST was maximal at the lowest water temperature.

BIS-1,7-(2-HYDROXYPHENYL)-HEPTA-1,6-DIENE-3,5-DIONE (A CURCUMIN ANALOG) AMELIORATES DMH-INDUCED HEPATIC OXIDATIVE STRESS DURING COLON CARCINOGENESIS

The protective effect of a curcumin analog [bis-1,7-(2-hydroxyphenyl)-hepta-1,6-diene-3,5-dione] was investigated on hepatic lipid peroxidation (LPO) and antioxidant status during 1,2-dimethylhydrazine-induced colon carcinogenesis in male Wistar rats. The effects were compared with that of curcumin, a known antioxidant and anticarcinogen. Colon cancer was induced by sub-cutaneous injection of DMH at a dosage of 20 mg/kg body weight (15 doses, at 1-week intervals). DMH administered rats developed gross tumours in the colon. Enhanced lipid peroxidation in the liver of colon tumour bearing rats was accompanied by a significant decrease in the activities of glutathione peroxidase (GPx), glutathione S-transferase (GST), superoxide dismutase (SOD) and catalase (CAT). Intragastric administration of curcumin (80 mg/kg body weight) and curcumin analog (80 mg/kg body weight) to DMH-injected rats significantly reduced the number and size of tumour in the colon, lowered lipid peroxidation and enhanced the activities of GPx, GST, SOD and CAT in the liver. We speculate that the curcumin analog used in the present study exerts chemoprevention against cancer development at extrahepatic sites by modulating hepatic biotransformation enzymes and antioxidant status. The effect is comparable with that of curcumin. This shows that the hydroxyl group in the aromatic ring is responsible for the protective effect rather than the methoxy group.

Factors influencing activities of biotransformation enzymes, concentrations and compositional patterns of organochlorine contaminants in members of a marine food web

The accumulation of polychlorinated biphenyls (PCBs; 34 congeners), ∑DDT ( p, p′-DDT, o, p′-DDT, p, p′-DDD, o, p′-DDD and p, p′-DDE), chlordanes (∑CHL; trans-chlordane, cis-chlordane, trans-nonachlor, cis-nonachlor and oxychlordane), hexachlorocyclohexanes (∑HCH; α-, β- and γ-isomers), hexachlorobenzene (HCB) and mirex was investigated in members of a marine food web from the Hvaler and Torbjørnskjær archipelago, south–eastern Norway. The species studied were bullrout ( Myoxocephalus scorpius), cod ( Gadus morhua), herring gull ( Larus argentatus) and harbour seal ( Phoca vitulina). Furthermore, hepatic biotransformation enzyme activities (ethoxyresorufin- O-deethylase (EROD), pentoxyresorufin- O-depentylase (PROD) and glutathione S-transferase (GST)) were measured in all species. The objectives of the study were to investigate factors causing intraspecies variation in activities of biotransformation enzymes, as well as in concentrations and compositional patterns of the organochlorines (OCs). High correlations between EROD and PROD activities were found in all species, suggesting a single, common catalyst, CYP1A, and render the PROD assay questionable as biomarker for CYP2B inducers in marine wildlife. Furthermore, GST activities are shown to be dependent on biological factors, such as age (in harbour seal) and sex (in bullrout). In fish, the OC concentrations vary between the sexes, likely due to differences in fat deposition strategies and possibly sex dimorphism. In seals, concentrations and compositional patterns of the OCs vary with age, owing to selective transfer from mother to pup in utero and mainly through lactation, but likely also due to age specific xenobiotic metabolising capacity.

Lycopene, an antioxidant carotenoid modulates glutathione-dependent hepatic biotransformation enzymes during experimental gastric carcinogenesis

We investigated the effects of lycopene on hepatic oxidant-antioxidant status during N-methyl-N′-nitro-N-nitrosoguanidine and saturated sodium chloride (S-NaCl)-induced gastric carcinogenesis. The extent of lipid peroxidation and the levels of reduced glutathione (GSH) and activities of the GSH-dependent enzymes glutathione peroxidase (GPx), glutathione S-transferase (GST) and glutathione reductase (GR) were used to monitor the peroxidative balance. Enhanced lipid peroxidation in the livers of tumor-bearing animals was accompanied by significant decreases in the activities of GSH, GPx, GST and GR. Administration of lycopene significantly lowered the levels of lipid peroxides and enhanced GSH levels and activities of GSH-dependent enzymes. Our results suggest that lycopene blocks experimental gastric carcinogenesis by upregulating GSH-dependent hepatic detoxification systems thereby protecting against carcinogen-induced oxidative damage.

Effects of prochloraz and nonylphenol diethoxylate on hepatic biotransformation enzymes in trout: a comparative in vitro/in vivo-assessment using cultured hepatocytes

The suitability of cultured rainbow trout hepatocytes as a model system for the assessment of xenobiotic effects on hepatic biotransformation enzymes in fish was examined. Two model water pollutants, the imidazole fungicide prochloraz and the alkylphenolic compound nonylphenol diethoxylate (NP2EO), were investigated in a comparative in vitro/in vivo approach. Biotransformation enzymes were measured in cultured rainbow trout hepatocytes following exposure to xenobiotics in vitro, or in the liver of juvenile rainbow trout ( Oncorhynchus mykiss) exposed in vivo. The patterns of biochemical responses to the model pollutants were generally similar between in vitro and in vivo investigations. Levels of cytochrome P4501A (CYP1A) protein and the catalytic activity of the CYP1A-dependent enzyme 7-ethoxyresorufin- O-deethylase (EROD) were induced in vitro after 24 h of exposure to 1.0 μM prochloraz. In vitro, higher prochloraz concentrations induced only the levels of CYP1A above control levels, but not EROD activity. In vivo exposure of juvenile trout to 0.27 μM prochloraz resulted in an induction of CYP1A and EROD after 7 and 14 days, while 0.027 μM prochloraz had no effects. In vitro, the 6β- and 16β-hydroxylation of testosterone was significantly decreased by 1.0–3.0 μM prochloraz, while in vivo these variables were significantly inhibited after exposure to 0.27 μM prochloraz for 7 and 14 days. NP2EO did not affect EROD activity in vitro. In vivo, EROD activity and CYP1A remained unchanged following 7 days of exposure to 0.32 or 1.30 μM NP2EO. NP2EO (15–50 μM) inhibited the 16β-hydroxylation and glucuronidation of testosterone in vitro. In vivo, 7 days of exposure to 0.32 or 1.30 μM NP2EO resulted in a significant inhibition of the 6β- and 16β-hydroxylation of testosterone. The good qualitative correspondence between in vitro and in vivo results indicates that studies using trout hepatocytes allow the identification of biochemical targets of xenobiotic effects in fish liver. However, more research is needed before quantitative predictions, e.g. of effective concentrations, can be made from in vitro investigations.

Hepatic biotransformation enzymes of fish exposed to non-point source pollution in small streams

The response of hepatic phase I and phase IIbiotransformation enzymes in brown trout(Salmo trutta f. fario) and stone loach(Barbatula barbatula) to differentpollution exposure scenarios was investigatedduring a 5-year-study. Phase I enzymes wereassessed by means of7-ethoxyresorufin-O-deethylase (EROD) activityand cytochrome P4501A (CYP1A) protein level,and phase II enzymes were measured by thecatalytic activity of glutathione-S-transferase(GST). Fish were exposed in the laboratory totap water (controls) or under semi-fieldconditions in so-called bypass systems to waterfrom two streams, with one of the streams(Krahenbach) receiving contaminants mainlyfrom non-point sources (for instance, surfacerun-off), and the second stream (Korsch)receiving contaminants from both non-pointsources and point sources (sewage plants). Inaddition to the fish in the bypass systems,feral fish from the streams were sampled. Thequestions addressed by the study were: (1) isit possible to discriminate the study sites onthe basis of hepatic biotransformation indices?(2) are the metabolic enzyme responses of thetwo species, brown trout and stone loach,comparable? (3) are the metabolic enzymeresponses of fish from the bypass systemscomparable to those of feral fishes? HepaticEROD activities were elevated in both browntrout and stone loach exposed in the bypasssystems compared to the laboratory control. Adiscrimination of the two field sites relativeto pollution intensity was possible by hepaticEROD activities of brown trout only, whereasbiotransformation enzymes of stone loach showedno consistent differences between the stream.Hepatic GST levels of both speciesdid not discriminate between the streams orbetween streams and laboratory. The enzymeresponses measured in fishes from the bypasssystems were comparable to those of feralfishes living in the streams. The findings ofthis study point to critical factors for thebiological monitoring of low-level pollutionwith complex chemical mixtures, including theselection of appropriate test species,monitoring endpoints, and sampling strategies.

Effects of primary- and secondary-treated bleached kraft mill effluents on the immune system and physiological parameters of roach

The present study was designed to examine, whether, effluents from a modern pulp and paper mill using elemental chlorine-free/total chlorine-free (ECF/TCF) bleaching, exert effects on the immune system of fish and, in addition, to relate these findings to physiological parameters known to be affected by bleached kraft-mill effluents (BKME). Roach ( Rutilus rutilus) were exposed in laboratory conditions to primary- or secondary-treated effluent from a pulp and paper mill. In order to study their capability to respond to foreign antigens they were immunised with bovine γ-globulin (BGG) prior to exposure. The number of anti-BGG antibody-secreting cells (ASC) and the number of immunoglobulin-secreting cells (ISC) in the spleen and blood as well as the level of anti-BGG specific antibodies and concentration of plasma immunoglobulin (IgM) were studied. Phagocytosis and migration of granulocytes of the head kidney were also determined. In addition to the immunological parameters, the activity of hepatic biotransformation enzymes, the carbohydrate metabolism and osmoregulation were examined. Exposure of roach for 21 days to BKME affected several immunological parameters. Both effluents, primary- and secondary-treated, impaired the immunoreactivity of the fish. Sex-related differences in the immune responses were evident in many parameters e.g. in the number of blood ISC and splenic ASC. Sex also had effects on cortisol levels and in the induction of 7-ethoxyresorufin O-deethylase (EROD). These results demonstrate that both primary- and secondary-treated effluent from a pulp and paper mill using ECF/TCF bleaching have effects on fish immune functions. Further, these findings suggest that steroids may contribute to immunomodulation in fish.

Potency of Semecarpus anacardium Linn. Nut milk extract against aflatoxin B1-induced hepatocarcinogenesis: reflection on microsomal biotransformation enzymes

The effect of Semecarpus anacardium Linn. nut milk extract on host detoxification system in aflatoxin B1induced hepatocellular carcinoma, which is a vital mechanism in cancer treatment, was studied in male albino rats. Oral administration of nut extract (200 mg kg−1body weight per day for 14 days) is found to be highly effective in inducing phase I and phase II biotransformation enzymes. The obtained results have shown an overall decrease of liver microsomal cytochrome P450, cytochrome b5, NADPH-cytochrome P450 reductase, NADH-cytochrome b5 reductase, and aniline hydroxylase with a subsequent decrease of phase II enzymes, glutathione-S-transferase and UDP-glucuronyl transferase in cancer-bearing animals. The Semecarpus anacardium nut extract affords anticancer activity by enhancing both phase I and phase II enzymes to near normal levels. We propose that, much of the anticarcinogenic potency of Semecarpus anacardium nut extract on aflatoxin B1-induced hepatocarcinogenesis is mediated through the induction of hepatic biotransformation enzymes.

Seasonal variations in the activity of selected hepatic biotransformation and antioxidant enzymes in eelpout ( Zoarces viviparus)

The eelpout Zoarces viviparus is used in Sweden for environmental monitoring of pollutant effects in fish. The activities of several hepatic biotransformation and antioxidant enzymes were monitored during the period of 12 months from August 1996 to July 1997 using newly caught feral eelpout from Kattegatt, off the west coast of Sweden. In females, the enzyme activities showed significant seasonal variations; CYP1A-activity, measured as ethoxyresorufin- O-deethylase (EROD), was lowest during the spawning season in August, possibly due to high estrogen levels. UDP-glucuronosyl transferase activity was lowest in November, while the glutathione- S-transferase activity markedly decreased during the winter. On the other hand, catalase activity peaked in April. The number of male fish was limited and the seasonal variations less clear. However, in both sexes, glutathione reductase and peroxidase activities were highest in the autumn and in May, respectively. The results are important for the ability to distinguish between pollutant effects and natural variations in the activity of selected enzymes in eelpout.

Garlic exerts hepatoprotective effects during 4‐nitroquinoline 1‐oxide‐induced oral carcinogenesis in rats

The modifying effects of garlic (Allium sativum Linn.) on hepatic lipid peroxidation and antioxidant status during 4-nitroquinoline 1-oxide (4NQO)-induced oral carcinogenesis were investigated in male Wistar rats. Enhanced lipid peroxidation in the liver of tumour-bearing animals was accompanied by significant decreases in the activities of glutathione peroxidase (GPx), glutathione S-transferase (GST) and reduced glutathione (GSH) levels. Administration of aqueous garlic extract significantly lowered lipid peroxidation and enhanced the levels of hepatic antioxidants and detoxifying enzymes. The results of the present study suggest that garlic exerts its hepatoprotective effects by influencing hepatic biotransformation enzymes and antioxidants.

Garlic and neem leaf extracts enhance hepatic glutathione and glutathione dependent enzymes during N‐methyl‐N′‐nitro‐N‐nitrosoguanidine (MNNG)‐induced gastric carcinogenesis in rats

The protective effect of garlic (Allium sativum L.) and neem leaf (Azadirachta indica A. Juss.) was investigated on hepatic lipid peroxidation and antioxidant status during N-methyl-N′-nitro-N-nitrosoguanidine (MNNG)-induced gastric carcinogenesis in male Wistar rats. Enhanced lipid peroxidation in the liver of tumour-bearing animals was accompanied by significant decreases in the activities of glutathione peroxidase (GPx), glutathione-S-transferase (GST), gamma-glutamyl transpeptidase (GGT) and reduced glutathione (GSH) levels. Administration of garlic and neem leaf extracts significantly lowered lipid peroxidation and enhanced the hepatic levels of glutathione and glutathione dependent enzymes. We speculate that garlic and neem leaf significantly alter cancer development at extrahepatic sites by influencing hepatic biotransformation enzymes and antioxidants. Copyright © 2000 John Wiley & Sons, Ltd.