Repeated treatment with furazolidone induces multiple cytochrome p450-related activities in chicken liver, but not in rat liver.

The nitrofuran antimicrobial agent, furazolidone (FZ), is still used in veterinary medicine in some countries in the Middle and Far Eastern countries. The present study aimed to investigate the effects of successive bolus doses of FZ and its metabolite 3-amino-2-oxazolidinone (AOZ) on cytochrome P450 (CYP)-related activities in the livers of rats and chickens. Female Wistar rats and white Leghorn chickens were orally administered FZ once a day for 4 consecutive days. FZ-treated chickens showed an increase in multiple CYP-related activities, however, rats treated with FZ did not show these changes. In chickens, treatment with FZ also induced production of microsomal CYP2C6-like apoprotein. The present study demonstrated that FZ caused a multiple-type induction of CYP-related activities in chickens, but not in rats.

[Effects of dietary fat level on the xenobiotic metabolism enzymes activity and antioxidant enzymes in rats].

Male Wistar rats received fat-free diet or diets containing 5, 10 and 30% of fat (sunflower oil + lard, 1:1) for 4 weeks. The direct relationship between dietary fat level and ethoxyresorufin O-dealkylase activity of CYP1A1, methoxyresorufin O-dealkylase activity of CYP1A2, pentoxyresorufin O-dealkylase activity of CYP2B1 and testosterone 6beta-hydroxylase activity of CYP3A was found. Activities of key enzymes of phase II xenobiotic metabolism (total activity of glutathione transferase, activity of UDP-glucuronosyle transferase) and antioxidant enzymes (catalase, glutathione peroxidase, glutathione reductase, paraoxonase-1 and heme oxygenase-1) also increased with higher dietary fat level.

Cytochrome P450 system as a toxicity biomarker of industrial wastewater in rat tissues.

In the present study the effect of laboratory exposure to wastewaters from Aligarh (AWW) and Saharanpur (SWW) on the activities of cytochrome P450 (CYP450) isozymes like ethoxyresorufin-o-deethylase (EROD), pentoxyresorufin-o-deethylase (PROD) and N-nitrosodimethylamine demethylase (NDMA-d) were investigated in the liver and kidney of rats. The industrial wastewater samples from Saharanpur city of northern India resulted 12, 3.5 and 1.5-fold rise in the EROD (CYP1A1), PROD (CYP2B1) and NDMA-d (CYP2E1) activity, respectively, in the liver of treated animals. Renal EROD and PROD activities were found to be enhanced by around 5 and 7-folds, respectively, as a result of SWW treatment. On the other hand, Aligarh samples showed significant inhibition in these test CYP450 enzymes both in hepatic as well as renal tissues. Strong induction of CYP1A1 (>10-fold) suggests that EROD can serve as a potent biomarker of SWW in the liver of treated animal. However, PROD and EROD can also act as fairly good biomarkers in case of renal tissue. Marked elevation of EROD activity in SWW treated animals strongly suggests the overwhelming levels of EROD inducers in the Saharanpur sample while a meagre amount of inducers accompanied with significant levels of inhibitors in the Aligarh sample.

In vivo indirect measurement of cytochrome P450-associated activities in freshwater gastropod molluscs.

The cytochrome P450 (CYP) system is widely distributed across phyla and plays a key role in the metabolism of xenobiotic compounds. However, most studies on CYP system were developed on vertebrates and among invertebrates, gastropod molluscs are rarely used. In this context, ethoxycoumarin-O-deethylase (ECOD), ethoxyresorufin-O-deethylase (EROD), and pentoxyresorufin-O-dealkylase (PROD) activities, which are indirect measurements of CYP system, were characterized in two freshwater gastropod molluscs, Potamopyrgus antipodarum, and Valvata piscinalis, to ascertain their potential interest as biomarkers of exposure to chemicals. Activities were measured using an in vivo non lethal method based on the measurement of formed product (resorufin or hydroxycoumarin). This in vivo assay allowed to measure the three activities in P. antipodarum and two of them (ECOD and PROD) in V. piscinalis. The detection of activities and the optimization of experimental design were carried out first and allowed to measure the selected activities for one individual. The modulation of the detected activities was secondly assessed using a polycyclic aromatic hydrocarbon (Benzo(a)pyrene). Based on this non destructive measurement, effect of BaP exposure could be detected on ECOD and EROD activity in P. antipodarum, as well on PROD activity of V. piscinalis after 96 h of exposure. Such an in vivo assay must be further developed to be valuably used to screen the exposure of gastropod species to CYP inducer chemicals and its consequences in terms of fitness of the organisms and of the population.

Induction of hepatic enzymes and oxidative stress in Chinese rare minnow (Gobiocypris rarus) exposed to waterborne hexabromocyclododecane (HBCDD).

The objective of this study was to evaluate the sub-lethal toxicity of hexabromocyclododecane (HBCDD) in fish. Adult Chinese rare minnows as in vivo models were exposed to waterborne HBCDD from 1 to 500 microg/l for 14, 28 and 42 days. Hepatic CYP1A1 (ethoxyresorufin-O-deethylase, EROD) and CYP2B1 (pentaoxyresorufin-O-depentylase, PROD) activities were measured. At the same time, molecular biomarkers of oxidative stress were also assayed in the brain, including reactive oxygen species (ROS), lipid peroxidation products (thiobarbituric acid-reactive substances, TBARS), DNA damage and protein carbonyl, as well as superoxide dismutase (SOD) activity and glutathione (GSH) content. DNA damage was evaluated using the Comet assay on erythrocytes. Besides, the content of HBCDD in whole fish was determined after 42 days exposure. The results show that HBCDD could induce EROD and PROD at 500 microg/l after 28 days exposure, and at 100 to 500 microg/l after 42 days exposure (P<0.05), respectively. ROS formation in fish brain was observed to be increased in both time- and dose-dependent manner due to HBCDD exposure. The significant increases in TBARS and protein carbonyl contents occurred in fish brain after 28 and 42 days exposure (P<0.05). Significant DNA damage in erythrocytes by Comet assay was also found in the 100-500 microg/l exposure groups (P<0.05) after 42 days exposure. Moreover, significant depletion in brain GSH content occurred in all treated groups (P<0.05) and apparent inhibition in SOD activity in brain was observed in the groups of 10-500 microg/l concentrations during 42 days exposure. The results demonstrate that increasing duration of HBCDD exposure induced EROD and PROD activities, caused excess ROS formation, finally resulted in oxidative damage to lipids, proteins and DNA and decreased antioxidant capacities in fish. Chemical analysis of HBCDD in whole fish showed accumulation up to 654 microg/g wet weight.

Decrease of cholesterol in mouse melanoma causes secretion of lysosomal enzymes.

We examined the change in the subcellular distribution of a lysosomal enzyme, beta-glucuronidase (beta-G), caused by decreased cholesterol levels in mouse melanoma cells using an HMG-CoA reductase inhibitor, lovastatin and lipoprotein-deficient serum (LDS). There was a decrease in the cholesterol content of the cells and increased secretion of the mature form of beta-G located in lysosomes, as documented by Percoll density gradient fractionation, digitonin permeabilization and immunoprecipitation. Furthermore, another lysosomal enzyme, cathepsin H, was found to be released in the medium from cells treated with lovastatin. Both the precursor and mature forms of cathepsin H were detected in the medium of treated cells. Next, when cells were treated with LDS without lovastatin, concomitantly with the decrease in the levels of cholesterol and beta-G activity in the cells, beta-G activity in the medium increased. Also, the ratio of beta-G (3.2-fold) released in the medium from cells treated with Dulbecco's modified Eagle medium (D-MEM) containing lovastatin and LDS was higher than that (2.3-fold) on treatment with D-MEM containing LDS without lovastatin. From these results, it was suggested that the exocytosis of mature enzymes from lysosomes into the medium or mis-sorting of the lysosomal precursor forms to the medium was caused by the lovastatin- and/or LDS-induced decrease in the cholesterol content of the cells, although the mechanism of secretion by lysosomal enzymes differed somewhat.

Characterization of metabolites and cytochrome P450 isoforms involved in the microsomal metabolism of aconitine.

Aconitine, a major Aconitum alkaloid, is well known for its high toxicity that induces severe arrhythmias leading to death. The current study investigated the metabolism of aconitine and the effects of selective cytochrome P450 (CYP) inhibitors on the metabolism of aconitine in rat liver microsomes. The metabolites were separated and assayed by liquid chromatography-ion trap mass spectrometry (LC/MS(n)) and further identified by comparison of their mass spectra and chromatographic behaviors with reference substances. Various selective inhibitors of CYP were used to identify the isoforms of CYP, that involved in the metabolism of aconitine. A total of at least six metabolites were found and characterized in rat liver microsomal incubations. Result showed that the inhibitor of CYP 3A had an inhibitory effect on aconitine metabolism in a concentration-dependant manner, the inhibitor of CYP1A1/2 had a modest inhibitory effect, whereas inhibitors of CYP2B1/2, 2D and 2E1 had no obvious inhibitory effects on aconitine metabolism. Aconitine might be metabolized by CYP 3A and CYP1A1/2 isoforms in rat liver microsome.

Modification by curcumin of mutagenic activation of carcinogenic N-nitrosamines by extrahepatic cytochromes P-450 2B1 and 2E1 in rats.

To elucidate the mechanism underlying suppression by curcumin of esophageal carcinogenesis induced by NMBA, we evaluated the CYP level and mutagenic activation of environmental carcinogens, by immunoblot analyses and Ames preincubation test, respectively, and bilirubin, 4-nitrophenol and testosterone UDPGT activities in F344 rats treated with curcumin and/or NMBA. No significant alterations in the hepatic levels of constitutive CYP proteins, mutagenic activation by liver S9 or hepatic UDPGT activities were produced by subcutaneous treatment with 0.5 mg/kg NMBA for 5 weeks and/or feeding of 0.05% and 0.2% curcumin for 6 weeks. In contrast, gavage of 0.2% curcumin decreased esophageal CYP2B1 and 2E1 by up to 60%, compared with vehicle control. Similarly, intragastric treatment with 270 mg/kg curcumin decreased esophageal and gastric CYP2B1 and CYP2E1, but not in lung, kidney or intestine. Conversely, large intestinal CYP2B1 was 2.8-fold higher in the treated rats than in control rats. Mutagenic activities of NOC, including NMBA, in the presence of esophagus and stomach S9 were markedly decreased in the treated rats, whereas those in the presence of large intestine S9 were 2.2-3.0-fold above control. These results show that modifying effects of curcumin on esophageal carcinogenesis can be attributed to a decrease in metabolic activation of NMBA by esophageal CYP2B1 during the initiation phase, without the contribution of metabolic activation and inactivation by liver. Further, the present findings suggest the potential of curcumin for modification of gastric and intestinal carcinogenesis initiated with NOC.

Temporal kinetics and concentration-response relationships for induction of CYP1A, CYP2B, and CYP3A in primary cultures of beagle dog hepatocytes.

Compared to other species, little information is available on the xenobiotic-induced regulation of cytochrome P450 enzymes in the beagle dog. Dogs are widely used in the pharmaceutical industry for many study types, including those that will impact decisions on compound progression. The purpose of this study was (1) to determine the temporal kinetics of drug-induced changes in canine CYP1A, CYP2B, and CYP3A mRNA and enzymatic activity, and (2) to characterize concentration-response relationships for CYP1A2, CYP2B11, and CYP3A12 using primary cultures of canine hepatocytes treated with beta-naphthoflavone (BNF), phenobarbital (PB), and rifampin (RIF), respectively. CYP1A1 and CYP1A2 mRNA exhibited maximal expression (12,700-fold and 206-fold, respectively) after 36 h of treatment with BNF. PB treatment, but not RIF treatment, caused maximal induction of CYP2B11 mRNA (149-fold) after 48 h of treatment. CYP3A12 and CYP3A26 mRNA levels were increased maximally after 72 h of treatment with PB and RIF (CYP3A12, 35-fold and 18-fold, and CYP3A26, 72-fold and 22-fold with PB and RIF treatment, respectively). Concentration-response relationships for BNF induced 7-ethoxyresorufin O-dealkylation (EROD) (EC(50) = 7.8 +/- 4.2 microM), PB induced 7-benzyloxyresorufin O-dealkylation (BROD) (EC(50) = 123 +/- 30 microM), and PB and RIF induced testosterone 6beta-hydroxylation (EC(50) = 132 +/- 28 microM and 0.98 +/- 0.16 microM) resembled the relationship for human CYP induction compared to that of rodent. Interestingly, RIF had no effect on CYP2B11 expression, which represents a species difference overlooked in previous investigations. Overall, the induction of dog CYP1A, CYP2B, and CYP3A exhibits characteristics that are intermediate to those of rodent and human.

Thyroid lesions and dioxin accumulation in the livers of jungle crows (Corvus macrorhynchos) in urban and suburban Tokyo.

Wild jungle crows (Corvus macrorhynchos) captured from three different areas of Tokyo were examined to evaluate environmental contamination of dioxins. In addition to the pathologic examination of their whole body, accumulation of dioxins, mRNA expression of the aryl hydrocarbon receptor (AhR), and pentoxyresorufin-O-depenthylase (PROD) activity in the liver were determined. Marked histopathologic changes were observed in the thyroid glands, especially in the crows from the urban downtown area. Levels of dioxins and their toxic equivalents (TEQs) and AhR mRNA expression in the livers of the crows from the urban area were higher than those from the suburban area. There was a high correlation between the levels of TEQs and PROD activity. The results of the present study demonstrated that jungle crows possess AhR-mediated toxicologic pathways similar to those of mammals and suggest the possibility that the thyroidal changes observed in the adult crows from the urban areas are one of the toxic manifestations resulting from exposure to dioxins and other environmental chemicals.

Ecotoxicological risks associated with land treatment of petrochemical wastes. II. Effects on hepatic phase I and phase II detoxification enzymes in cotton rats.

The purpose of this study was to evaluate possible exposure and resultant hepatic effects of petrochemical waste on cotton rats (Sigmodon hispidus) living on landfarmed sites. Male and female cotton rats were collected in summer, fall, and winter from four landfarm sites and four ecologically similar reference sites. Hepatic methoxyresorufin O-deethylase (MROD) activity was significantly induced in male and female rats collected from landfarms compared to rats collected from reference sites. In contrast, changes in ethoxyresorufin O-deethylase (EROD) activity were inconsistent due to season, sex, and treatment variation. A significant decrease in EROD and MROD activity was found in cotton rats held for 48 h prior to sacrifice compared to rats euthanized on the day of capture. These results indicate that when using hepatic EROD and MROD activities as biochemical markers of exposure to aryl hydrocarbon receptor agonists, animals should be euthanized as quickly as possible after capture. The cotton rats collected from one landfarm unit exhibited a pattern of consistent elevation of EROD, MROD, and pent-oxyresorufin O-deethylase (PROD) activity. This unit also had a pattern of elevated CYP1A2 protein expression determined by Western blotting. There were no consistent alterations from contaminant exposure on hepatic glutathione S-transferase (GST) activity, glutathione levels, or CYP1A1 protein. Hepatic EROD and MROD activities varied considerably between seasons and sex of rats. In conclusion, consistent induction of hepatic EROD and MROD activities in cotton rats was found in three out of four sampled landfarm sites compared to the rats collected from the reference sites, indicating exposure to contaminants-likely polyaromatic hydrocarbons.

Dose-response modeling of cytochrome p450 induction in rats by octamethylcyclotetrasiloxane.

Inhalation of octamethylcyclotetrasiloxane (D4) induces CYP2B1/2 protein and causes liver enlargement. We have developed a pharmacodynamic (PD) extension to a physiologically based pharmacokinetic (PBPK) model to characterize these dose-response behaviors. The PD model simulates interactions of D4 with a putative receptor, leading to increased production of cytochrome P450 2B1/2. Induction was modeled with a Hill equation with dissociation constant, Kd, and Hill coefficient, N. Both a 1- and a 5-compartment liver model were evaluated. The PBPK model provided excellent simulations of tissue D4 and hepatic CYP2B1/2 protein concentrations following 6 h/day, 5-day inhalation exposures to 0, 1, 7, 30, 70, 150, 300, 500, 700, or 900 ppm D4. Either the 1- or 5-compartment liver model could accurately simulate increases in CYP2B1/2 protein in the liver. With a 1-compartment liver, Kd and N were 0.67 microM (free liver concentration) and 1.9, respectively. The 5-compartment model used higher N-values (approximately 4.0) and varied Kd between compartments. The fitted 5-compartment model parameters were Kd = 0.67 microM in the midzonal compartment with geometric differences in Kd between compartments of 2.9. On the basis of unbound (free) plasma concentrations, D4 appeared to be a higher potency inducer than phenobarbital (PB). Dose-response curves for increased liver weights had N/mS 1.0 and Kd/mS 3.4 microM, very different values from those for enzyme induction. Exposure concentration leading to a 0.1% increase in CYP2B1/2 protein predicted by the 1- and 5-compartment models were 2.1 ppm and 5.1 ppm, respectively. The 1- and 5-compartment liver models provided very similar fits to the whole liver induction data, excluding the lowest dose, but the 5-compartment liver model had the additional advantage of simultaneously describing the regional induction of CYP2B1/2.

Probing enhanced cytochrome P450 2B1/2 activity in rat hepatocyte spheroids through confocal laser scanning microscopy.

Cytochrome P450 (CYP450) enzymes are essential for xenobiotic metabolism. Although CYP450s are found in many tissues, CYP2B1/2 are primarily expressed in the rat liver. The constitutive expression in vivo of CYP2B1/2 is low but it is induced in the presence of various drugs such as phenobarbital (PB). In this study, CYP2B1/2 activity in cultured hepatocytes was assessed in situ with the introduction of a fluorogenic substrate, pentoxyresorufin. The product of 7-pentoxyresorufin-O-dealkylation (PROD), which is catalyzed specifically by CYP2B1/2, was detected using confocal laser scanning microscopy (CLSM). Primary hepatocytes cultured as monolayers on collagen-coated surfaces exhibited background PROD activity and minimal PB inducibility after 4 days in culture. In contrast, rat hepatocytes organized in compacted aggregates, or spheroids, exhibited higher levels of PROD activity and retained their ability for PB induction. The results from the CLSM analysis were verified by RT-PCR and Western immunoblotting analysis. Furthermore, CLSM in conjunction with image processing techniques and three-dimensional reconstruction revealed the localization of enhanced PROD activity in the center of spheroids. The results support the use of CLSM as a powerful tool for investigating CYP2B1/2 activity in cultured rat hepatocytes.

Expression and localization of the CYP2B subfamily predominantly in neurones of rat brain.

Despite the very small amounts of cytochrome P450 (P450, CYP) enzymes expressed in different areas and cell populations of the brain as compared with the liver, there is significant evidence for their specific involvement in brain development, function and plasticity. Nevertheless, the current discussion about occurrence and importance of cerebral cytochrome P450s is determined by inconsistent interpretations of their function in general and with respect to single isoforms. Continuing a series of publications about brain P450 isoforms, we now present evidence for the constitutive expression of CYP2B1 and CYP2B2 mRNAs in rat brain. Immunocytochemical and non-radioactive in situ hybridization studies revealed the same expression pattern throughout the brain predominantly in neuronal populations, but to some extent in astrocytes of corpus callosum and olfactory bulb. The well known testosterone-metabolizing capacity and the presence of CYP2B isoforms shown in steroid hormone-sensitive areas and neurones (e.g. hippocampus) clarify the significance of isoforms like CYP2B1 and CYP2B2 for impairment of steroid hormone actions by P450 inducing environmental substances. We argue that cerebral P450 isoforms which are induced by xenobiotics and are able to metabolize these as well as endogenous substrates help us to understand fundamental aspects of brain's functioning.

The effect of fetal hypoxia on adrenocortical function in the 7-day-old rat.

Fetal hypoxia in late gestation is a common cause of postnatal morbidity. The purpose of the present study was to evaluate adrenal function in vivo and in vitro in 7-d-old rat pups previously exposed to normoxia or hypoxia (12% O2) during the last 2-3 d of gestation. Seven-day-old rats exposed to fetal hypoxia had a small, but significant decrease in plasma aldosterone despite no decreases in plasma ACTH or renin activity. There was a small (approx 20%) but significant decrease in the aldosterone and corticosterone response to cAMP in vitro in dispersed cells from 7-d-old pups exposed to fetal hypoxia. The aldosterone, corticosterone, and cAMP response to ACTH, however, was not altered by prior fetal hypoxia. There was also no effect of fetal hypoxia on steroidogenic enzyme expression or zonal dimension in 7-d-old rats. We conclude that fetal hypoxia in late gestation results in a subtle decrease in cAMP-stimulated steroidogenesis. Fetal hypoxia appears to have minimal effects on subsequent adrenal function in the neonatal rat.

Modulation of carbon tetrachloride-induced lipid peroxidation and xenobiotic-metabolizing enzymes in rats fed browned yam flour diet.

The modulatory effect of browned yam flour diet, a dietary, staple in south-western Nigeria, on carbon tetrachloride (CCl4)-mediated lipid peroxidation and on the activities of liver microsomal and cytosolic enzymes was studied in male rats. Browned yam flour diet fed at the level of 25% and 50% to rats for 5 weeks significantly reduced the lipid peroxidation induced by CCl4 (0.5 ml/kg/wk) administered two weeks after starting the animals with the diets. The diets elicited 62% and 79% reductions in CCl4-mediated peroxidation, respectively, in the absence of exogenously added oxidants. The activities of microsomal aniline hydroxylase (AH), aminopyrine-N-demethylase (APD), pentoxyresorufin-O-dealkylase (PROD) and cytosolic GSH S-transferase (GST) were increased when rats were fed the 25% or 50% browned yam flour diets. Browned yam flour fed at the level of 25% to rats decreased the CCl4-mediated reduction in the activities of microsomal AH, APD, PROD and GST by 64%, 28%, 58% and 25%, respectively, and by 82%, 48%, 83% and 55% when rats were fed with 50% of the diet. The results suggest that browned yam flour diet could protect against chemically-mediated lipid peroxidation and tissue damage possibly by scavenging chemically generated reactive species and enhancing carcinogen-detoxifying system.

Induction of hepatic cytochromes P450 in dogs exposed to a chronic low dose of polychlorinated biphenyls.

Induction of cytochrome P450 isoforms, specifically CYP1A1, and their catalytic activities are potential biomarkers of environmental contamination by polychlorinated biphenyls (PCBs). In this study, dogs were exposed to 25 ppm or 5 ppm Aroclor 1248 (PCB mixture) daily in their diet for 10 or 20 weeks, respectively. Relative to controls, hepatic microsomes from dogs dosed with PCBs had higher levels of CYP1A1 detected in immunoblots and higher levels of EROD activity, but low levels of induction for CYP2B and PROD activity. Concentrations of 96 PCB congeners in serum and liver were evaluated using capillary chromatography. Results showed that all dogs exposed to PCB mixtures had higher levels of PCB in serum and liver. Dogs preferentially sequestered highly chlorinated PCB congeners in liver relative to serum. With these experiments, we demonstrated that EROD activity was a potentially sensitive marker of PCB exposure at 5 and 25 ppm. Furthermore, CYP1A1 and EROD activity were maximally induced in dogs consuming dietary concentrations only 2.5 times the maximal permissible level for human food (FDA). The value of CYP1A1 induction as a biomarker of PCB exposure was tenuous because neither CYP1A1 levels nor EROD activity correlated with total PCB body burden. However, a small subset of congeners were identified in liver that may strongly influence EROD and PROD induction. Finally, two dogs in the 25 ppm dose group were fasted for 48 h. After 24 h of fasting, several new congeners appeared in the serum and remained in the serum for the remainder of the fast. The fast caused a 293% increase in PCB concentration in serum. This increase has strong implications regarding mobilization of toxic PCBs in wildlife during fasting (e.g., migration, hibernation).

Effect of 3-methylcholanthrene administration on expression of cytochrome P-450 isoforms induced by phenobarbital in rat hepatocytes.

The effects of an inducer on expression of cytochrome P-450 (P-450) isoforms induced antecedently by another inducer are unknown. Thus, we examined the amount of phenobarbital (PB)-inducible P-450 isoforms (P-450 2B1/2B2) in hepatocytes from rats injected first with PB and then with 3-methylcholanthrene (MC) (PB+MC-treated animals) by quantitative immunohistochemistry. In addition, expression of P-450 2B2 mRNA was examined by in situ hybridization. In PB-treated animals, P-450 2B1/2B2 content increased in perivenular and midzonal hepatocytes. In PB+MC-treated animals, however, the PB-induced increase in 2B1/2B2 content was suppressed in perivenular hepatocytes but promoted in midzonal hepatocytes. The hybridization signal for P-450 2B2 mRNA appeared almost exclusively in perivenular hepatocytes after 24 hr of PB injection and disappeared after 48 hr of injection. In PB+MC-treated animals, however, strong hybridization signal was observed in midzonal and perivenular hepatocytes after 48 hr of PB injection. The promotion of the increase in P-450 2B1/2B2 content in midzonal hepatocytes in PB+MC-treated animals probably corresponds to the strong hybridization signal, whereas there appeared to be a divergence between the intensity of the signal and the content in perivenular hepatocytes. The results indicate that MC administration drastically influences the pattern of expression of P-450 isoforms induced by PB in perivenular and midzonal hepatocytes.

Cytochromes P450, P420 & mixed-function oxidases as biomarkers of polychlorinated biphenyl (PCB) exposure in harbour seals (Phoca vitulina).

Hepatic microsomal cytochrome P450, EROD and ECOD activity were investigated as biomarkers of PCB exposure in harbour seals (Phoca vitulina). Due to the difficulty of obtaining undegraded seal liver samples, standard spectrophotometric methodology was adapted to investigate P420 (degraded P450) as a PCB biomarker with partially degraded samples. Total PCB burdens in both blubber and liver had positive correlations with P450, P420 and MFO activity levels. The use of P420 biomarkers in this study supports the inclusion of samples from by-caught marine mammals for future biomonitoring studies. P450 isozymes CYP1A (P4501A) and CYP2B (P4502B) in conjunction with MFO activity were investigated as "specific" biomarkers of PCB exposure. They were found to reliably reflect levels of [MC] and [PB]-type PCB exposure in harbour seal liver.

Long-term maintenance of drug-metabolizing enzyme activities in rat hepatocytes after cryopreservation.

Recent studies have demonstrated that freshly isolated adult hepatocytes from various species can be hypothermically preserved for a short period or cryopreserved for a prolonged period before seeding in primary culture. This study was designed to determine whether rat hepatocytes could be maintained functional for a prolonged period after either hypothermic preservation or cryopreservation. Cold storage was carried out in University of Wisconsin solution (UW) and freezing in Leibovitz medium added with 10% fetal calf serum and 16% dimethyl sulfoxide. Rat hepatocytes were then set up either in pure conventional culture or in coculture with rat liver epithelial cells. Various functions were measured over 4- and 15-day periods, i.e., albumin secretion rate, deethylation of ethoxyresorufin and phenacetin, dealkylation of pentoxyresorufin, glucuronidation and sulfoconjugation of paracetamol, and N-acetylation of procainamide. No major differences were observed between unfrozen, frozen, and UW-preserved cells. While in pure culture all the functions tested were markedly decreased after 3 or 4 days, they remained high over the 15-day period in coculture, being either maintained or increased after 7-12 days compared to initial values. These results clearly demonstrate that when maintained under suitable culture conditions, rat hepatocytes can fully recover after hypothermic preservation or cryopreservation and therefore represent a suitable in vitro model system for pharmacotoxicological studies.