P-nitrophenol Hydroxylase Activity Research Articles

CYP2E1 regulation by benzene and other small organic chemicals in rat liver and peripheral lymphocytes

The inducibility of CYP2E1 was investigated in liver and peripheral lymphocytes of rats treated with benzene (0–10 mmol/kg body weight (bw), daily for 3 days, i.p., or 0 and 5 mmol/kg bw, daily for 14 days, i.p.) or toluene (0 and 5 mmol/kg bw, daily for 3 days, i.p.) and compared with that of pyridine (5 mmol/kg bw, i.p.) or acetone (5% in drinking water) both daily for 3 days. Acute benzene treatment (5 mmol/kg bw) increased both CYP2E1 apo-protein (2-fold) and p-nitrophenol hydroxylase ( p-NPH) activity (1.4-fold) in liver, and CYP2E1 mRNA in both liver (2.2-fold) and peripheral lymphocytes (2.9-fold). The response to toluene was qualitatively similar, although smaller than that to benzene. As expected, acetone and pyridine treatments resulted in a 2- to 3-fold increase of p-NPH activity and CYP2E1 apo-protein content in liver, but not the mRNA levels. In addition, acute benzene and acetone treatments increased the 6-hydroxychlorzoxazone/chlorzoxazone metabolic ratio 1.6- and 3.1-fold, respectively. The subchronic treatment with benzene increased CYP2E1 mRNA and apo-protein from days 2 and 3 to day 14, respectively, whereas the enzyme activity increased transiently on days 3 and 5 only. These results show that acute/subacute benzene and acute toluene treatments induce CYP2E1 expression probably through a similar mechanism which might be different from that of pyridine or acetone, in that the former increase mRNA levels, both in liver and in peripheral lymphocytes, whereas the latter stabilized the apo-protein.

Anticarcinogenic effect and modification of cytochrome P450 2E1 by dietary garlic powder in diethylnitrosamine-initiated rat hepatocarcinogenesis.

The purpose of this study was to determine the effects of dietary garlic powder on diethylnitrosamine (DEN)- induced hepatocarcinogenesis and cytochrome P450 (CYP) enzymes in weaning male Sprague-Dawley rats by using the medium-term bioassay system of Ito et al. The rats were fed diets that contained 0, 0.5, 2.0 or 5.0% garlic powder for 8 weeks, beginning the diets with the intraperitoneal (i.p.) injection of DEN. The areas of placental glutathione S-transferase (GST-P) positive foci, an effective marker for DEN-initiated lesions, were significantly decreased in the rats that were fed garlic powder diets; the numbers were significantly decreased only in the 2.0 and 5.0% garlic-powder diets. The p-Nitrophenol hydroxylase (PNPH) activities and protein levels of CYP 2E1 in the hepatic microsomes of the rats that were fed the 2.0 and 5.0% garlic powder diet were much lower than those of the basal-diet groups. Pentoxyresorufin O-dealkylase (PROD) activity and CYP 2B1 protein level were not influenced by the garlic-powder diets and carcinogen treatment. Therefore, the suppression of CYP 2E1 by garlic in the diet might influence the formation of preneoplastic foci during hepatocarcinogenesis in rats that are initiated with DEN.

Role of glutathione in metabolic degradation of dichloromethane in rats

Dichloromethane (DCM) elimination and carboxyhemoglobin (COHb) generation were examined in adult female SD rats pretreated with a glutathione (GSH) depletor(s). Rats were treated with either buthionine sulfoximine (BSO; 2 mmol/kg, i.p.), diethylmaleate (DEM; 3 mmol/kg, i.p.), phorone (PHO; 1 mmol/kg, i.p.) or BSO plus PHO (BSO; 2 mmol/kg +PHO; 0.5 mmol/kg, i.p.). The hepatic GSH concentration was significantly reduced by each treatment. Decrease in hepatic GSH was maintained at least for 10 h after BSO treatment but recovered rapidly in rats treated with DEM or PHO. The hepatic p-nitrophenol hydroxylase activity was not affected by the GSH depletors at the dose used in this study. Rats were treated with an i.p. injection of DCM (3 mmol/kg) and the concentrations of DCM and the COHb levels in blood were monitored. In rats pretreated with a GSH depletor, the peak COHb level was significantly greater than that of rats treated with DCM only. The peak COHb level attained in each group of rats appeared to be inversely related to the magnitude of reduction in hepatic GSH levels. The half-life of DCM in blood was also increased in rats pretreated with the GSH depletor(s). The results indicate that the GSH-dependent metabolic reaction has an important role in the overall elimination of DCM as well as in the metabolic generation of carbon monoxide (CO) from this solvent.

Dilinoleoylphosphatidylcholine Decreases Ethanol-Induced Cytochrome P4502E1

Cytochrome P4502E1 (CYP2E1) induction by ethanol contributes to alcoholic liver disease and we found that a mixture of polyunsaturated phosphatidylcholines (PPC), which protects against alcohol-induced liver injury, also decreases CYP2E1. Since dilinoleoylphosphatidylcholine (DLPC) is the major component of PPC, we assessed here whether it is responsible for the protection of PPC by feeding rats for 8 weeks our liquid diet containing ethanol (36% of energy) or isocaloric carbohydrates, with either DLPC (1.5 g/1000 cal), PPC (3 g/1000 cal), or linoleate. CYP2E1 was assessed by Western blots and by two of its enzyme activities: the microsomal ethanol-oxidizing system (MEOS) and p-nitrophenolhydroxylase (PNP). With ethanol, CYP2E1 increased 10-fold, with corresponding rises in PNP and MEOS activities. Compared to linoleate, DLPC significantly decreased cytochrome b5, total cytochromes P450, CYP2E1 content and its corresponding activities. DLPC decreases ethanol-induced CYP2E1 and should be considered for the prevention of alcoholic liver disease.

The effects of inhalation exposure to bromo-dichloromethane on specific rat CYP isoenzymes

Several cytochrome P450 (CYP) isoenzymes may be involved in the metabolism of bromo-dichloromethane (BDCM), a drinking water disinfection byproduct. After 4-h inhalation exposures of male F344 rats to BDCM between 100 and 3200 p.p.m., hepatic microsomal methoxyresorufin demethylase (MROD), ethoxyresorufin de-ethylease (EROD) and pentoxyresorufin dealkylase (PROD) activities showed modest increases at low exposure levels and larger decreases at high exposure levels, compared with controls. Western blots for CYP1A2 and CYP2B1 showed similar trends. In addition, p-nitrophenol hydroxylase (PNP) activity was measured and Western blots for CYP2E1 were performed. CYP2E1 and CYP2B1 isoenzymes are known to metabolize BDCM (Thornton-Manning, J.R., Gao, P., Lilly, P.D., Pegram, R.A., 1993. Acute bromodichloromethane toxicity in rats pretreated with cytochrome P450 inducers and inhibitors. The Toxicologist 13: 361). When compared with a multiple gavage study of BDCM in female F344 rats (Thornton-Manning, J.R., et al., 1994. Toxicology 94, 3–18), the results of the two studies for EROD, PROD, and PNP activities were qualitatively the same; PNP activity did not change, while both PROD and EROD activities decreased at high exposures. In the current work, Western blots for CYP2E1, CYP2B1 and CYP1A2 supported the results from the PNP, PROD and MROD activities, respectively. The decreases in MROD and PROD activities and in Western blots for CYP1A2 and CYP2B1 at high exposures suggest that BDCM may be a suicide substrate for these CYP isoenzymes. Other important conclusions that can be drawn from the comparison between the current and prior work are that the liver response is similar for both sexes, and it is also similar for inhalation and gavage exposures under these conditions. Finally, the decrease in EROD activity at high doses, found in both studies, may be a further reflection of CYP1A2 activity, since little or no CYP1A1 activity is normally found in uninduced rat liver and CYP1A2 is known to metabolize ethoxyresorufin, although much more slowly than CYP1A1.

Liver subcellular fractions from rats treated by organosulfur compounds from Allium modulate mutagen activation

The effects of in vivo administration of naturally occurring organosulfur compounds (OSCs) from Allium species were studied on the activation of several mutagens. Male SPF Wistar rats were given p.o. one of either diallyl sulfide (DAS), diallyl disulfide (DADS), dipropyl sulfide (DPS) or dipropyl disulfide (DPDS) during 4 consecutive days and the ability of hepatic S9 and microsomes from treated rats to activate benzo[ a]pyrene (BaP), cyclophosphamide (CP), dimethylnitrosamine (DMN), N-nitrosopiperidine ( N-PiP) and 2-amino-1-methyl-6-phenylimidazo[4,5- b]pyridine (PhIP) was determined in the Ames test. Administration of DAS, DPS and DPDS resulted in a significant increase of the activation of BaP, CP, N-PiP and PhIP mediated by S9 and microsomes while DADS treatment only increased the mutagenicity of PhIP. In contrast, S9 from DADS-treated rats significantly inhibited the mutagenicity of N-PiP and BaP. DAS, DADS and DPS strongly inhibited DMN mutagenicity while DPDS enhanced it. To understand the mechanisms underlying these effects, the modifications of the activities of specific isozymes of CYP involved in the activation of these mutagens were studied. DAS, DPS and DPDS strongly enhanced pentoxyresorufin O-dealkylase (PROD) activity related to CYP2B and slightly increased ethoxyresorufin O-deethylase (EROD) and methoxyresorufin O-demethylase (MROD) activities related to CYP1A family. DADS exerted the same effects than other OSCs but to a lesser extent. p-Nitrophenol hydroxylase (PNPH) activity related to CYP2E1 was inhibited by DAS and DADS, whereas DPDS significantly increased this activity. Hence, the effects of OSCs on the mutagenicity of several genotoxic compounds are mediated by modification (enhancement or inhibition) of specific CYP involved in their activation.

Cytochrome P450-dependent n-dealkylation of l-deprenyl in C57BL mouse liver microsomes: effects of in vivo pretreatment with ethanol, phenobarbital, β-naphthoflavone and l-deprenyl

The monoamine oxidase inhibitor l-deprenyl [(−)-deprenyl, selegiline] is an effective therapeutic agent for improving early symptoms of idiopathic Parkinson's disease. It appears to exert this action independently of its inhibition of monoamine oxidase B (MAO-B) and some of its metabolites are thought to contribute. Cytochrome P450 (CYP) activities are known to give rise to l-deprenyl metabolites that may affect the dopaminergic system. In order to clarify the interactions of l-deprenyl with these enzymes, C57BL mice were treated with l-deprenyl, ethanol, phenobarbital or β-naphthoflavone to induce different CYP isozymes. After preincubation of l-deprenyl with liver microsomes from control or treated mice, the metabolites were analysed by a GLC method. l-deprenyl (10 mg/kg i.p. for 3 days) caused a significant decrease in total CYP levels (0.315±0.019, l-deprenyl; 0.786±0.124, control, nmol/mg protein) and CYP2E1-associated p-nitrophenol hydroxylase activity (0.92±0.04 vs. 1.17±0.06 nmol/min/mg). Both phenobarbital and ethanol increased the N-depropynylation activity towards l-deprenyl that leads to the formation of methamphetamine (4.11±0.64, phenobarbital; 4.77±1.15, ethanol; 1.77±0.34, control, nmol/min/mg). Ethanol alone increased the N-demethylation rate of l-deprenyl, that results in formation of nordeprenyl (3.99±0.68, ethanol; 1.41±0.31, control, nmol/min/mg). Moreover, the N-dealkylation pathways of deprenyl are inhibited by 4-methylpyrazole and disulfiram, two CYP2E1 inhibitors. None of the other treatments modified l-deprenyl metabolism. These findings indicate that mainly CYP2E1 and to a lesser extent CYP2B isozymes are involved in l-deprenyl metabolism. They also suggest that, by reducing CYP content, l-deprenyl treatment may impair the metabolic disposition of other drugs given in combination regimens.

Acute and subchronic evaluation of Indigofera arrecta: absence of both toxicity and modulation of selected cytochrome P450 isozymes in ddY mice.

Indigofera arrecta, an anti diabetic plant was investigated in ddY mice to determine its acute and subchronic effects, and whether it modulated hepatic cytochrome P450 (CYP) isozymes and glutathione (GSH). No mortality was observed in the acute (up to 10 g I. arrecta/kg body wt, p.o.) and subchronic (2 g I. arrecta/kg body wt, p.o. daily for 30 days) studies. The extract did not alter haematological indices, serum and tissue lipids and glutathione but lowered serum bile acids. The latter phenomenon is under further investigation. Neither the duration of pentobarbital (PB) and zoxazolamine (ZA) effects in vivo, nor CYP-dependent 7-ethoxyresorufin-O-deethylase (EROD), 7-pentoxyresorufin-O-depentylase (PROD) and p-nitrophenol hydroxylase (PNPH) activities in vitro were altered by I. arrecta. The extract was thus devoid of overt acute and subchronic toxic effects, and did not affect CYPs and GSH whose modulation may cause interactions of components in a multiple drug therapy.

Metabolism and hepatotoxicity of acetaminophen in mice fed on a liquid glucose diet

The role of dietary factors in the toxicity and metabolism of acetaminophen (APAP) was examined using male ICR mice. A 5 % ( w v ) liquid glucose solution replaced a regular lab chow and water exactly for 24 or 48 hr prior to measurement of hepatic glutathione (GSH) contents at 8 am, 2 pm, or 8 pm of a day. The characteristic temporal variation in hepatic GSH contents disappeared in mice provided with the glucose diet for 24 hr or longer. The microsomal p-nitrophenol hydroxylase and ethoxyresorufin O-deethylase activities, most responsible for metabolic activation of this drug, were examined in mice fed on the glucose diet for 24 hr. There were no differences in either enzyme activity between 8 am and 8 pm. Mice were injected with a 200 mg/kg dose of APAP, ip, 24 hr following initiation of the glucose diet supply and the plasma concentrations of this drug and its metabolites were monitored for 3 hr. There were no significant differences in plasma level of APAP or the major metabolites, APAP-glucuronide and APAP-sulfate, between the mice treated with this drug at 8 am and at 8 pm. However, the plasma concentrations of metabolites generated via the conjugation reaction with GSH were lower in mice treated with APAP at 8 pm. The APAP-induced hepatotoxicity was determined by increases in serum aspartate aminotransferase (AST), alanine aminotransferase (ALT) activities and by decreases in hepatic glucose-6-phosphatase (G-6-Pase) activity 24 hr following the treatment. Significant differences in the AST ALT and G-6-Pase activities were observed between 8 am, 2 pm and 8 pm in mice fed on the glucose solution for 24 hr. However, supply of the glucose diet for 48 hr abolished the time-dependent differences in serum AST ALT and hepatic G-6-Pase activities resulting from a dose of APAP. The results indicate that supply of a glucose diet for 24 hr may eliminate the temporal variation in hepatic GSH contents, but the glucose diet supply should continue at least for 48 hr to effectively diminish the fluctuation in the toxicity of chemicals dependent on the availability of this endogenous tripeptide.

Evaluation of rat hepatic 2E1 activity in function of age, sex and inducers: choice of an experimental model capable of testing the hepatotoxicity of low molecular weight compounds

The aim of this work on rat hepatic P450 2E1 activity was to seek the most suitable experimental model to study the role of cytochrome P450 2E1 in the metabolism of industrial chemicals. Two sets of experiments were devoted to selecting the age and sex of animals and to estimating the response of male and female rats to different inducers. In the first set, the effect of three inducers (fasting; ethanol; acetone) was studied in male rats aged 5, 7 and 9 weeks. In the second set, the effect of different inducers, namely β-naphthoflavone (BNF), phenobarbital (PB), ethanol, acetone and pyridine, on PNP and chlorzoxazone (CLZO) hydroxylase activities was studied in 7 week old male and female rats. The results demonstrate firstly that microsomal p-nitrophenol (PNP) hydroxylase activity significantly decreases in control male rats in inverse function of age, and secondly that induction by ethanol decreases with age. The PNP hydroxylase activity level of controls and the significant increases in PNP hydroxylase activity observed in 7 week old male rats show that this is the most suitable age for the second set of experiments. In this second set, it was shown that P450 1A (induced by BNF) is involved in CLZO hydroxylase activity only. PB increased the hydroxylase activities in male and female rats by about 1.5 and 1.7 times those of the controls, respectively. The effects of P450 2E1 inducers in function of sex show that male rats exhibited more significant increases in PNP and CLZO hydroxylase activities than female. The specificity of these two substrates is discussed. Neither of these two reactions was specifically catalysed by P450 2E1, but PNP may be considered as the most specific and the least sensitive substrate. In addition, the linear relationship observed between the two substrates (PNP and CLZO) showed a good correlation between their activities ( r=0.90, P<0.001). In conclusion, these results suggest the use of the 7 week old male rat as the experimental model to study the role of cytochrome P450 2E1 in the hepatotoxicity of low molecular weight industrial chemicals.

The nature of prooxidant activity of vitamin C

It was recently reported that vitamin C (500 mg/day for 6 weeks) administered as a dietary supplement to healthy humans exhibits a prooxidant, as well as an antioxidant effect in vivo. Here we show that high intakes of vitamin C (500 mg/kg b.w. for 4 days) in the rat are able to markedly induce hepatic cytochrome P4502E1-linked monooxygenases, measured as p-nitrophenol hydroxylase activity and corroborated by means of Western blot analyses. Furthermore, using Electron Paramagnetic Resonance Spectroscopy (EPR) coupled to a spin-trapping technique, we have also found that this induction generates large amounts of the anion radical Superoxide (O 2). Therefore we can conclude that the adverse prooxidant outcomes (i.e. oxidative DNA damage) associated to vitamin C supplementation, being associated to a typical reversible boosting effect (i.e. enzymatic induction), may be easily controlled by a discontinuous supply. However, since the induced P4502E1 isoforms by vitamin C are responsible for ethanol metabolism to highly reactive radicals, care should be taken even in moderate drinkers.

Differential in vivo effects of alpha-naphthoflavone and beta-naphthoflavone on CYP1A1 and CYP2E1 in rat liver, lung, heart, and kidney.

Male Sprague-Dawley rats were treated intraperitoneally with corn oil, the aryl hydrocarbon receptor (AHR) agonist beta-naphthoflavone (betaNF), or the relatively weak AHR agonist alpha-naphthoflavone (alphaNF). Animals treated with betaNF experienced a significant loss (12%) of total body mass over 5 days and a dramatic elevation of CYP1A1 mRNA in all of the organs studied. Treatment with alphaNF had no significant effect on body mass after 5 days and caused only minor increases of liver, kidney, and heart CYP1A1 mRNA. In contrast, lung CYP1A1 mRNA was increased by alphaNF treatment to levels comparable to that seen with betaNF treatment. CYP2E1 mRNA levels were also elevated in liver, lung, kidney, and heart in response to betaNF treatment, whereas alphaNF was without effect. Large increases of CYP1Al-dependent 7-ethoxyresorufin O-deethylation (EROD) activity occurred with microsomes prepared from the tissues of betaNF-treated animals. Comparatively small changes were associated with alphaNF treatment, with the exception of lung, where EROD activity was increased to approximately 60% of that with betaNF treatment. CYP2E1-dependent p-nitrophenol hydroxylase (PNP) activity was also increased by betaNF treatment in microsomes prepared from kidney (3.1-fold), whereas alphaNF was without effect. In contrast, alphaNF or betaNF treatment caused significant decreases of lung microsomal PNP (72% and 27% of corn oil control, respectively) and 7-pentoxyresorufin O-deethylation (48% and 17% of corn oil control, respectively) activities, indicating that PNP activity may be catalyzed by P450 isoforms other than CYP2E1 in rat lung. We conclude that betaNF and alphaNF have differential effects on the expression and catalytic activity of CYP1A1 and CYP2E1, depending upon the organ studied. These changes most likely occur as a result of the direct actions of these compounds as AHR agonists, in addition to secondary effects associated with AHR-mediated toxicity.

Polyenylphosphatidylcholine Opposes the Increase of Cytochrome P‐4502E1 by Ethanol and Corrects Its Iron‐Induced Decrease

Dietary iron overload damages membrane phospholipids and decreases microsomal cytochromes P-450. We wondered whether this might also pertain to cytochrome P-4502E1 (2E1) and whether polyenylphosphatidylcholine (PPC), a 94-96% pure mixture of linoleate-rich polyunsaturated phosphatidylcholines that protects against alcohol-induced liver injury, also affects 2E1, either in the presence or absence of iron. Accordingly, rats were fed for 8 weeks our standard liquid diet containing ethanol (36% of energy) or isocaloric carbohydrates, with either PPC (3 g/1000 Cal) or equivalent amounts of linoleate (as safflower oil). 2E1 was assessed by Western blots and by two of its characteristic enzyme activities: the microsomal ethanol oxidizing system (MEOS), evaluated by the conversion of ethanol to acetaldehyde (determined by head space GC), and p-nitrophenolhydroxylase (PNP) activity, measured by HPLC with UV detection of 4-nitrocatechol. With ethanol (36% of energy) replacing carbohydrates, 2E1 content increased 10-fold, with a corresponding increase in PNP and MEOS activities, but when carbonyl iron (5 g/1000 Cal) was added, the induction was significantly reduced. This iron-induced decrease was corrected by PPC. PPC is rich in linoleate, but when the latter was given as triglycerides (safflower oil), there was no effect, whereas hepatic nonheme iron content was the same in both these groups. It also was found that in the absence of iron, the ethanol-mediated induction of 2E1 and its corresponding enzyme activities were significantly less with PPC (< 0.001) than with safflower oil. In addition, in alcohol-fed animals, PPC decreased the oxidative stress (as determined by F2-isoprostanes), which reflects yet another hepatoprotective effect of PPC.

Protective effects of diallyl sulfide on N-nitrosodimethylamine-induced immunosuppression in mice

Diallyl sulfide (DAS), a flavor component of garlic that has been used as a food additive, exerts chemopreventive effects at several organ sites in rodents after administration of chemical carcinogens possibly by inhibiting carcinogen activation via cytochrome P450-mediated oxidative metabolism. In this study, we investigated the protective effect of DAS on the N-nitrosodimethylamine (NDMA)-induced immunosuppression of humoral and cellular responses in BALB/c mice and the possible mechanisms involved in this protection. We observed that oral administration of DAS prior to NDMA treatment for 14 consecutive days blocked the NDMA-induced suppression of the antibody response to a T-cell-dependent antigen, sheep erythrocytes, and the lymphoproliferative response to the T-cell and the B-cell mitogens in dose-dependent manners. Treatment of mice with DAS resulted in a significant decrease of cytochrome P450 2E1-dependent p-nitrophenol hydroxylase and NDMA demethylase activities. The results show that the protective effects of DAS against the NDMA-induced immunotoxicity may, at least in part, be due to its ability to block bioactivation of NDMA mainly by the inhibition of cytochrome P450 2E1.

Kinetic characterization of CYP2E1 inhibition in vivo and in vitro by the chloroethylenes.

Trans- and cis-1,2-dichloroethylene (DCE) isomers inhibit their own metabolism in vivo by inactivation of the metabolizing enzyme, presumably the cytochrome P450 isoform, CYP2E1. In this study, we examined cytochrome P450 isoform-specific inhibition by three chloroethylenes, cis-DCE, trans-DCE, and trichloroethylene (TCE), and evaluated several kinetic mechanisms of enzyme inhibition with physiological models of inhibition. Trans-DCE was more potent than cis-DCE, and both were much more effective than TCE in inhibiting CYP2E1. The kinetics of in vitro loss of p-nitrophenol hydroxylase (pNP-OH) activity (a marker of CYP2E1) in microsomal incubations and of the in vivo gas uptake results were most consistent with a mechanism in which inhibition of the metabolizing enzyme (CYP2E1) was presumed to be related to interaction of a reactive DCE metabolite with remaining substrate-bound, active CYP2E1. The kinetics of inhibition by TCE, a weak inhibitor in vitro, were very different from that of the dichloroethylenes. With TCE, parent compound concentrations influenced enzyme loss. Trans-DCE was a more potent inhibitor of CYP2E1 than cis-DCE based on both in vivo and in vitro studies. Quantitative differences in the inhibitory properties of the 1,2-DCE isomers may be due to the different stability of epoxides formed from bioactivation by CYP2E1. Epoxide intermediates of DCE metabolism, reacting by water addition, would yield dialdehyde, a potent cross-linking reagent.

Effects of Singly Administered Betaine on Hepatotoxicity of Chloroform in Mice

Effects of a single dose of betaine on the chloroform-induced hepatotoxicity were examined in adult male ICR mice. Administration of betaine (1000 mg/kg, ip) 1 to 7 hr prior to a chloroform challenge (0.25 ml/kg, ip) resulted in remarkable enhancement of hepatotoxicity as indicated by increases in serum sorbitol dehydrogenase (SDH), alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities. The potentiation of hepatotoxicity was most significant when mice were treated with betaine 4 hr earlier than chloroform. However, a 24 hr prior administration of betaine protected the animals from induction of the chloroform hepatotoxicity. Thus, its effect appeared to be highly dependent on the time lapse from the betaine pretreatment to the challenge of mice with chloroform. Betaine treated either 4 or 24 hr prior to sacrifice did not alter the hepatic contents of cytochrome P-450, cytochrome b 5, or NADPH cytochrome P-450 reductase activity. Accordingly the hepatic microsomal p-nitroanisole O-demethylase, aminopyrine N-demethylase, or p-nitrophenol hydroxylase activities were not influenced by the betaine pretreatment. Betaine was shown not to affect any of the enzyme activities associated with glutathione (GSH) conjugation reaction, such as glutathione S-transferases (GSTs), glutathione disulfide (GSSG) reductase and GSH peroxidase irrespective of the time of its administration. When betaine was administered to mice 2–6 hr prior to sacrifice, hepatic GSH level, but not plasma GSH, was decreased significantly. Enhancement of the chloroform hepatotoxicity by betaine correlated well with the reduction in hepatic GSH levels. Both hepatic and plasma GSH levels were elevated in mice 24 hr following the betaine treatment. The results suggest that betaine affects induction of the chloroform hepatotoxicity by modulating the availability of hepatic GSH, which appears to be associated with its role in the transsulfuration pathway in the liver.

Temporal variation in hepatotoxicity and metabolism of acetaminophen in mice

Temporal variation in metabolism and hepatotoxicity of acetaminophen (APAP) was examined using male ICR mice. Animals were injected with a single dose of APAP (400 mg/kg, i.p.) at 08:00, 14:00 or 20:00 h. APAP at this dose was markedly hepatotoxic to mice when administered at 20:00 h as determined by increases in serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities, and by decreases in hepatic glucose-6-phosphatase (G-6-Pase) activity. However, mice appeared to be entirely insensitive to an identical dose of APAP given either at 08:00 or 14:00 h. Hepatic glutathione (GSH) level was significantly higher at 08:00, but no difference in GSH levels between 14:00 and 20:00 h was observed in normal mice. APAP and its metabolites in blood were monitored using HPLC for 3 h following the treatment. There were no significant differences in the plasma concentrations of APAP, APAP-glucuronide, APAP-sulfate, or APAP-mercapturate among the mice treated with this drug at 08:00, 14:00 or 20:00 h. However, the APAP-cysteine and APAP-GSH levels measured at 1 h following the APAP treatment were significantly lower in mice treated with this analgesic either at 14:00 or 20:00 h. In vitro hepatic microsomal p-nitrophenol hydroxylase activities were not different between 08:00, 14:00 and 20:00 h. But ethoxyresorufin O-deethylase and aminopyrine N-demethylase activities measured at 14:00 h were significantly lower than those of 08:00 or 20:00 h. Thus, the greater hepatotoxicity of APAP administered at 20:00 h appears to be related to the marked decrease in hepatic GSH at this time period, whereas the simultaneous reduction in APAP activation may be responsible for the lack of hepatotoxicity in mice treated with this analgesic at 14:00 h. These results suggest that the temporal variation in hepatotoxicity and metabolism of APAP is determined by interactions of multiple factors including the hepatic GSH level and drug metabolizing activities.

Zonated expression of cytokines in rat liver: effect of chronic ethanol and the cytochrome P450 2E1 inhibitor, chlormethiazole.

The release of proinflammatory cytokines by endotoxins and during oxidative stress is considered to be an early key step in the pathogenesis of alcoholic liver disease (ALD). Ethanol-inducible cytochrome P450 2E1 (CYP2E1) has potentially pro-oxidative and toxicological properties, and its expression is restricted to the perivenous region of liver. We investigated zonal differences of cytokine expression in rat liver and how these are affected by alcohol exposure and by chlormethiazole (CMZ), a transcriptional and posttranslational inhibitor of hepatic CYP2E1. Periportal and perivenous cell lysates were obtained by the digitonin pulse technique from livers of rats treated with ethanol and CMZ for 38 days. Cytokine expression on the mRNA and protein levels was quantified using competitive polymerase chain reaction (PCR) and Western blot, respectively. Chronic ethanol treatment significantly increased the expression of CYP2E1, microsomal p-nitrophenol hydroxylase activity (indicative for CYP2E1 enzyme activity), and the expression of transforming growth factor beta1 (TGF-beta1), tumor necrosis factor alpha (TNF-alpha), and interleukin (IL)-1beta (1.4- to 4.6-fold). In contrast, ethanol caused a decrease in IL-4 expression and had no influence on IL-6 expression. CMZ treatment caused a reduction in hepatic CYP2E1 expression and in the ethanol-induced cytokine expression by 40% to 60%. Expression of IL-6, IL-2, and IL-4 mRNA occurred preferentially in the periportal region, whereas ethanol caused a pronounced increase in the perivenous expression of TGF-beta1, which was inhibited by CMZ as monitored both on the mRNA and protein levels. These results show the zonated expression of several cytokines and the counteraction of CMZ on all effects of ethanol on cytokine expression. The data further strengthen a link between increased CYP2E1 expression and enhanced cytokine expression as important events in the development of ALD.

Inducibility of Cytochromes P‐4502E1 and P‐4501A1 in the Rat Pancreas

Cytochrome P-450 (CYP) isoenzymes have been incriminated in the toxicity and carcinogenicity of various xenobiotics in different tissues, but prior measurements of their activity in pancreatic microsomes have been disappointing. We now applied new isolation methods and a highly sensitive procedure to assay for the metabolism of p-nitrophenol and 7-ethoxyresorufin, specific substrates for CYP2E1 (2E1) and CYP1A1 (1A1), respectively. 2E1 and 1A1 content was estimated with high-resolution chemiluminescent Western blots using recombinant 2E1 and 1A1 as standards. We found that p-nitrophenol hydroxylase activity was 5.07 +/- 0.66 and 1.50 +/- 0.26 pmol/ min/mg of protein in pancreatic microsomes of ethanol-fed and control rats, respectively. Chronic ethanol treatment increased 2E1 content in pancreatic microsomes 3.6-fold. Activity and content of 2E1 were also assessed in hepatic microsomes: specific activity (expressed per 2E1 content) was similar in pancreatic and hepatic microsomes. There was also an inductive effect of 3-methylcholanthrene (MC) on 1A1 in pancreatic microsomes. Pancreatic microsomal 7-ethoxyresorufin-O-dealkylation activity in MC-treated rats was 19.6 +/- 1.7 pmol/min/mg of protein, 61-fold higher than in controls. MC treatment increased the 1A1 content in pancreatic microsomes 42-fold. These results demonstrate that, in pancreatic microsomes, ethanol and MC exert striking inductive effects on 2E1 and 1A1 activities, which could play a role in the pathogenesis of pancreatitis and/or pancreatic cancer.

Heterologous expression of rat P450 2E1 in a mammalian cell line: in situ metabolism and cytotoxicity of N-nitrosodimethylamine.

GM0637, a human fibroblast cell line, was transfected with pCMV2E1, an expression vector containing the full length cDNA for rat cytochrome P450 2E1 (P450 2E1), and with pCMVneo, which contained vector alone, and the selected clones were designated GM2E1 and GMneo, respectively. Western blot analysis showed that GM2E1, but not GMneo, expressed a protein that reacted with anti-human P450 2E1 antibody. The 7-ethoxycoumarin O-deethylase,p-nitrophenol hydroxylase, and N-nitrosodimethylamine (NDMA) demethylase activities of the P450 in these cells were measured in monolayer cell cultures without preparing microsomes. Exposure of the GM2E1 cells to NDMA for 4 days caused severe decreases in cell viability, as determined by crystal violet uptake, and showed a sigmoidal dose-response curve with a median lethal dose of 17 microM. In contrast, the viability of GMneo cells was not altered by NDMA even at concentrations up to 10 mM. Time- and concentration-dependent methylation of DNA, RNA and protein by [14C]NDMA was only observed in cells expressing P450 2E1. Inhibitors of P450 2E1 activity such as ethanol, 4-methylpyrazole, and isoniazid caused a 90% decrease in the methylation of cellular macromolecules and also completely protected the cells against NDMA-mediated toxicity. The cytotoxicity due to exposure to NDMA was partially inhibited by antioxidants such as N-acetylcysteine, ascorbic acid, butylated hydroxyanisole and N-t-butyl-alpha-phenylnitrone but was not potentiated upon glutathione depletion. These results document the ability of rat P450 2E1 to metabolize NDMA to toxic reactive intermediates and demonstrate that this cell line provides a useful model for studying the mechanisms of metabolism-mediated toxicity and carcinogenesis.