Ethanol-pretreated Rats Research Articles

Acute Alcohol Intoxication Aggravates Brain Injury Caused by Intracerebral Hemorrhage in Rats

Alcohol intoxication is associated with worse intracerebral hemorrhage (ICH) outcome, indicating the important role of alcohol in ICH pathogenesis. We intended to investigate the effects of ethanol pretreatment on the severity of ICH-induced brain injury in rats. At 1 hour after intraperitoneal injection of ethanol (3 g/kg), 0.2 U bacterial collagenase was infused into the striatum of male Sprague-Dawley rats to induce ICH. Accumulative mortality rate, body weight changes, and motorsensory and neurological abnormalities were evaluated. The hemorrhagic volume, hematoma expansion, and water content were measured by Drabkin's method, morphometric assay, and dry/wet method, respectively. Blood-brain barrier disruption was assessed using Evans blue assay. Oxidative stress was evaluated by the enzymatic activity of glutathione peroxidase, oxidation of hydroethidine, and the production of malondialdehyde. Cerebral blood flow perfusion volume and hypo-/hyperperfusion neuroimaging were examined by magnetic resonance imaging. Ethanol pretreatment aggravates the hematoma hemolysis, hemorrhagic volume, hematoma expansion, brain edema, blood-brain barrier disruption, microglial activation, elevated oxidative stress, and neuroinflammation in the hemorrhagic striatum. The summation effect of these consequences is the major cause of marked neurological impairment and higher mortality rate (64%) in ethanol-pretreated rats with ICH. This is a novel model to evaluate the effects of high-dose alcohol administration on experimental ICH rats. The present study may provide clues for making novel strategies in the management of patients with ICH who overconsume alcoholic drinks before the attack.

Expression of Aquaporin-4 Augments Cytotoxic Brain Edema after Traumatic Brain Injury during Acute Ethanol Exposure

We previously reported that ethanol consumption affects morbidity and mortality after traumatic brain injury (TBI) by accelerating brain edema via oxidative stress after TBI. Aquaporin-4 (AQP4), a water channel, is involved in brain edema formation. In this study, we found that acute ethanol administration increased AQP4 expression after TBI, leading to severe brain edema in rats. Rats were pretreated with ethanol (3 g/kg) or dl-buthionine-(S,R)-sulfoximine (BSO; 100 mg/kg), an oxidative stressor, before TBI. Acetazolamide, an AQP4 inhibitor, was administered to ethanol-pretreated rats 3 or 12 hours after TBI. Brain edema was increased 24 hours after TBI in both the ethanol- and BSO-pretreated groups. Ethanol pretreatment induced lipid peroxidation 24 hours after TBI. Transcription factors, NF-κB and hypoxia-inducible factor-1α, were activated 3 and 24 hours after TBI in the BSO- and ethanol-pretreated groups, respectively. In the ethanol-pretreated group, AQP4 was accumulated, particularly in astrocyte end feet, 24 hours after TBI. Acetazolamide treatment improved the survival rate to 100% and decreased brain edema and AQP4 in ethanol-pretreated rats. These findings suggest that ethanol induces up-regulation of AQP4, leading to brain edema. The accumulation of AQP4 may play an important role in the augmentation of brain edema after TBI under ethanol consumption.

Protective effect of Cassia fistula Linn. on diethylnitrosamine induced hepatocellular damage and oxidative stress in ethanol pretreated rats.

Diethylnitrosamine (DEN), found in many commonly consumed foods, is widely reported to induce cancer in animals and humans. The aim of the present study was to investigate the hepatoprotective and antioxidant activities of the leaf extract of the medicinal plant Cassia fistula Linn. against diethylnitrosamine induced liver injury in ethanol pretreated rats. Albino Wistar rats, pretreated with ethanol for 15 days, were administered a single dose of DEN. Thirty days after DEN administration, hepatotocellular damage was observed histologically, along with elevated levels of serum AST, ALT, ALP, LDH, γ-GT and bilirubin and a simultaneous fall in the levels of the marker enzymes in the liver tissue. Liver oxidative stress was confirmed by elevated levels of lipid peroxidation (LPO) and a decrease in enzymic and non-enzymic antioxidants activities. Oral administration of the ethanolic leaf extract (ELE) of Cassia fistula for 30 days to ethanol + DEN treated rats significantly improved the above alterations in the markers of hepatotoxicity and oxidative stress, resulting in the reversal of most of the parameters studied and were comparable to the standard hepatoprotective drug silymarin.

Protective effect of Cassia fistula Linn. on diethylnitrosamine induced hepatocellular damage and oxidative stress in ethanol pretreated rats

Diethylnitrosamine (DEN), found in many commonly consumed foods, is widely reported to induce cancer in animals and humans. The aim of the present study was to investigate the hepatoprotective and antioxidant activities of the leaf extract of the medicinal plant Cassia fistula Linn. against diethylnitrosamine induced liver injury in ethanol pretreated rats. Albino Wistar rats, pretreated with ethanol for 15 days, were administered a single dose of DEN. Thirty days after DEN administration, hepatotocellular damage was observed histologically, along with elevated levels of serum AST, ALT, ALP, LDH, γ-GT and bilirubin and a simultaneous fall in the levels of the marker enzymes in the liver tissue. Liver oxidative stress was confirmed by elevated levels of lipid peroxidation (LPO) and a decrease in enzymic and non-enzymic antioxidants activities. Oral administration of the ethanolic leaf extract (ELE) of Cassia fistula for 30 days to ethanol + DEN treated rats significantly improved the above alterations in the markers of hepatotoxicity and oxidative stress, resulting in the reversal of most of the parameters studied and were comparable to the standard hepatoprotective drug silymarin.

Sensitization of Ventral Tegmental Area Dopamine Neurons to the Stimulating Effects of Ethanol

Previous studies indicated that chronic alcohol drinking increased the sensitivity of the posterior ventral tegmental area (p-VTA) to the reinforcing effects of ethanol. The current study tested the hypothesis that local exposure of the p-VTA to ethanol would increase the sensitivity of dopamine (DA) neurons to the stimulating effects of ethanol. Experiment 1 examined the stimulating effects of ethanol in the p-VTA after a 7-day ethanol pretreatment in the p-VTA. Adult female Wistar rats were pretreated with microinjections of 200 mg% ethanol or artificial cerebrospinal fluid (aCSF) into the p-VTA once a day for 7 days. On the eighth day, rats received a challenge injection of ethanol (100, 200, or 300 mg%) or aCSF into the p-VTA, and extracellular DA levels were measured in the nucleus accumbens (NAc) shell with microdialysis. Experiment 2 examined the stimulating effects of ethanol (200 mg%) after a 3- or 5-day ethanol (200 mg%) pretreatment in the p-VTA. Experiment 3 examined the stimulating effects of ethanol (200 mg%) 7 days after the last of the 7-day ethanol (200 mg%) pretreatments in the p-VTA. Experiment 1: in both aCSF- and ethanol-pretreated rats, the challenge microinjection of ethanol dose-dependently increased DA release in the NAc shell, with significantly greater increases in ethanol-pretreated groups. Experiment 2: the 5-day, but not 3-day, ethanol pretreatment protocol increased the response of p-VTA dopamine neurons to the ethanol challenge. Experiment 3: the increased stimulating effects of ethanol were still evident after 7 days. The results indicate that repeated local ethanol exposure of the p-VTA produced neuroadaptations in DA neurons projecting to the NAc shell, resulting in a persistent increase in the sensitivity of these neurons to the stimulating effects of ethanol.

Expression of constitutive and inducible cytochrome P450 2E1 in rat brain

Studies initiated to investigate the expression of cytochrome P450 2E1 (CYP2E1) in rat brain demonstrated low but detectable protein and mRNA expression in control rat brain. Though mRNA and protein expression of CYP2E1 in brain was several fold lower as compared to liver, relatively high activity of N-nitrosodimethylamine demethylase (NDMA-d) was observed in control rat brain microsomes. Like liver, pretreatment with CYP2E1 inducers such as ethanol or pyrazole or acetone significantly increased the activity of brain microsomal NDMA-d. Kinetic studies also showed an increase in the Vmax and affinity (Km) of the substrate towards the brain enzyme due to increased expression of CYP2E1 in microsomes of brain isolated from ethanol pretreated rats. In vitro studies using organic inhibitors, specific for CYP2E1 and anti-CYP2E1 significantly inhibited the brain NDMA-d activity indicating that like liver, NDMA-d activity in rat brain is catalyzed by CYP2E1. Olfactory lobes exhibited the highest CYP2E1 expression and catalytic activity in control rats. Furthermore, several fold increase in the mRNA expression and activity of CYP2E1 in cerebellum and hippocampus while a relatively small increase in the olfactory lobes and no significant change in other brain regions following ethanol pretreatment have indicated that CYP2E1 induction maybe involved in selective sensitivity of these brain areas to ethanol induced free radical damage and neuronal degeneration.

Biogenic amines in striatum of rats that had been treated with ethanol, and their brains later stored in different temperatures.

The purpose of this study was to investigate how ethanol pretreatment and storage temperatures of brain striatum affect levels of biogenic amines in this tissue. Adult Wistar male rats were injected with 25% ethanol (5.0 g/kg i.p.) while the control rats were administered i.p. with the same volume of saline. Two hours later the rats were decapitated, their brains removed, and the striatum separated. Each striatum was divided into three parts: one part was immediately frozen on dry ice and kept at -70 degrees C; a second fragment was kept in a household refrigerator (+4 degrees C); and the third fragment was kept at +22 degrees C. Twenty-four hours later, levels of DA, DOPAC, HVA, 3-MT, 5-HT, and 5-HIAA in the striatum were assayed by HPLC/ED. Immediately after decapitation; ethanol levels were assayed in the serum of ethanol-pretreated and saline-pretreated rats using gas chromatography. Our results indicate that levels of striatal DA, DOPAC, and HVA in saline-pretreated rats decreased significantly when the storage temperature of the striatum was raised from -70 degrees C, through +4 degrees C, to +22 degrees C, while levels of striatal 5-HT and 5-HIAA remained constant within the temperature range tested and levels of 3-MT fluctuated. In ethanol-pretreated rats, striatal levels of DOPAC, HVA, and 5-HIAA were increased in all three storage temperatures, while levels of DA, 5-HT, and 3-MT were decreased in those temperatures. Those decreases were most profound in striatal samples kept at +22 degrees C. We conclude that concern about possible interactions between drugs and biogenic amines should be exercised.

Cytochrome P450 2E1 dependent catalytic activity and lipid peroxidation in rat blood lymphocytes

To investigate the similarities in the catalytic activity of blood lymphocyte P450 2E1 in blood lymphocyte with the liver isoenzyme, NADPH dependent lipid peroxidation and activity of N-nitrosodimethyamine demethylase (NDMA-d) was studied in rat blood lymphocytes. Blood lymphocytes were found to catalyse NADPH dependent (basal) lipid peroxidation and demethylation of N-nitrosodimethylamine (NDMA). Pretreatment with ethanol or pyrazole or acetone resulted in significant increase in the NADPH dependent lipid peroxidation and the activity of NDMA-d in blood lymphocytes and liver microsomes. In vitro addition of CCl 4 to the blood lymphocytes isolated from control or ethanol pretreated rats resulted in an increase in the NADPH dependent lipid peroxidation. Significant inhibition of the basal and CCl 4 supported NADPH dependent lipid peroxidation and NDMA-d activity in blood lymphocytes isolated from control or ethanol pretreated rats by dimethyl formamide or dimethyl sulfoxide or hexane, solvents known to inhibit P450 2E1 catalysed reactions in liver and anti- P450 2E1, have indicated the role of P450 2E1 in the NADPH dependent lipid peroxidation in rat blood lymphocytes. The data indicating similarities in the NADPH dependent lipid peroxidation and NDMA-d activity in blood lymphocyte with the liver microsome have provided evidence that blood lymphocyte P450 2E1 could be used as a surrogate to monitor and predict hepatic levels of the enzyme.

A Specific Hydroxysteroid UGT Is Responsible for the Conjugation of Aliphatic Alcohols in Rats: An Estimation of the Importance of Glucuronidation Versus Oxidation

UDP-glucuronosyltransferase (UGT) activity for aliphatic alcohols was determined in microsomal liver fractions of Wistar rats. The rats were pretreated with inducers of cytochrome P450 and UGTs [phenobarbital (PB), β-naphtoflavone (βNF), and ethanol (10%)], and inhibition experiments with aliphatic alcohols and specific substrates for UGTs were performed to characterize the UGT form(s) responsible for the glucuronidation of aliphatic alcohols. Several UGT isoforms were purified from liver microsomes of low-androsterone-conjugating activity (LA), controls, and ethanol-pretreated rats by chromatofocusing and affinity chromatography. The results show aliphatic alcohols to be specific substrates for 17β-hydroxysteroid UGT with considerable glucuronidation rates. This elimination pathway for aliphatic alcohols is not inducible by the tested inducers. Compared with kinetic data of oxidation, glucuronidation is probably the main elimination pathway for alcohols with a longer chain length than C3, especially when oxidation pathways are inhibited by the presence of proportionately high ethanol concentrations.

Role of CYP3A in Ethanol-Mediated Increases in Acetaminophen Hepatotoxicity

CYP2E is considered the only form of cytochrome P450 responsible for ethanol-mediated increases in acetaminophen hepatotoxicity. However, in experimental systems used for investigating ethanol-mediated increases in acetaminophen hepatotoxicity, animals are withdrawn from ethanol for 16 to 24 hr before the administration of acetaminophen to ensure the clearance of ethanol from the circulation. In rats, CYP2E has been shown to decrease to control levels after this time period of withdrawal from ethanol. We have previously shown in cultured human and rat hepatocytes, and in intact rats, that ethanol induces CYP3A in addition to CYP2E. To determine if there might be a role for CYP3A in ethanol-mediated APAP hepatotoxicity in addition to the recognized role for CYP2E, we investigated the effect of triacetyloleandomycin (TAO) on acetaminophen hepatotoxicity in ethanol-pretreated rats, as well as the effect of 11 hr withdrawal from ethanol on hepatic levels of CYP3A and CYP2E. TAO was dissolved in saline instead of dimethylsulfoxide, the solvent most usually employed, since dimethylsulfoxide inhibits CYP2E. Rats were administered 6.3% ethanol as part of the Lieber–DeCarli diet for 7 days, followed by replacement of the liquid diet with water for 11 hr. This 11-hr withdrawal from ethanol resulted in a decrease in hepatic levels of ethanol-induced CYP2E; however, considerable induction was still evident. There was no significant decrease in CYP3A. TAO completely prevented the histologically observed liver damage from acetaminophen in ethanol-pretreated rats, but did not prevent the increase in serum levels of AST. In ethanol-pretreated rats, exposure to APAP in the absence of TAO was associated with a 75% decrease in CYP3A, compared to animals exposed to APAP in the presence of TAO. These results suggest that CYP3A may have been suicidally inactivated by acetaminophen in the absence of TAO. Our findings suggest that CYP3A has a major role in ethanol-mediated increases in acetaminophen hepatotoxicity.

Prooxidant and antioxidant hepatic factors in rats chronically fed an ethanol regimen and treated with an acute dose of lindane

While acute lindane treatment and chronic ethanol feeding to rats have been associated with hepatic oxidative stress, the possible roles of these stresses in the pathogenesis of hepatic lesions reported in acute lindane intoxication and in those observed in some models of chronic alcoholism have not been established. Our previous studies in rats chronically fed ethanol regimens and then treated with a single intraperitoneal (ip) dose of lindane (20 mg/kg) showed that while lindane per se was invariably associated with hepatic oxidative stress, chronic ethanol feeding only produced this stress when the dietary level of vitamin E was relatively low. Chronic ethanol pretreatment did not significantly affect the lindane-associated oxidative stress, and neither chronic ethanol feeding not acute lindane, single or in combination, produced any histologic and biochemical evidence of liver damage. In the present experiment, the acute dose of lindane was increased to 40 mg/kg, and we have studied a larger number of prooxidant and antioxidant hepatic factors. Male Wistar rats (115.5 ± 5.4g) were fed ad lib for 11 weeks a calorically well-balanced and nutritionally adequate basal diet, or the same basal diet plus a 32% ethanol/25% sucrose solution, also ad lib, and were then injected ip with a single dose of lindane or with equivalent amounts of corn oil. The results indicated that acute lindane treatment to naive rats increased practically all the prooxidant hepatic factors examined (cytochromes P450 and b 5, NADPH cytochrome c reductase, NADPH oxidase), as well as the generation of microsomal superoxide radical and thiobarbituric acid reactive substances of liver homogenates, but did not modify any of the antioxidant hepatic factors studied. Conversely, the chronic administration of ethanol alone did not significantly affect the prooxidant hepatic factors but reduced some of the antioxidants (i.e., the activities of GSH-Px and the contents of α-tocopherol and ubiquinols 9 and 10). Although chronic ethanol pretreatment further increased the superoxide generation induced by lindane per se, it did not increase but generally reduced the effects of lindane per se on the other prooxidant factors studied. Furthermore, although acute lindane administration to ethanol-pretreated rats was associated with decreases in GSH and catalase (not affected by ethanol or lindane treatment alone), it did not substantially modify the reducing effects of ethanol feeding per se on GSH-Px, α-tocopherol, and ubiquinols. Once again, neither chronic ethanol feeding nor lindane treatment, single or in combination, was associated with any evidence of liver damage.

Comparative metabolism of bis(2-methoxyethyl) ether by rat and human hepatic microsomes: formation of 2-methoxyethanol

Rat hepatic microsomes catalysed the NADPH-dependent cleavage of the central ether linkage of bis(2-methoxyethyl) ether (diglyme) yielding 2-methoxyethanol (2ME). Microsomes isolated from phenobarbital- or ethanol-pretreated rats exhibited an increased capacity to cleave diglyme to 2ME. This ethanol-induced increase in 2ME formation was not observed if incubations contained the cytochrome P450IIE1 inhibitor isoniazid. Pretreatment of rats with diglyme significantly increased microsomal P-450 levels, P-450-associated enzyme activities and the conversion of diglyme to 2ME. Following the diglyme pretreatment, an almost 30-fold increase in pentoxyresorufin dealkylase activity ( P450IIB1/2) was evident in rat hepatic microsomes. Human hepatic microsomes also catalysed the NADPH-dependent cleavage of diglyme to 2ME. The formation of 2ME from diglyme correlated with the aniline hydroxylase activity ( P450IIE1) levels measured in human hepatic microsomes. Studies using microsomes isolated from a cell line transfected with specific human P-450 cDNAs indicate that human CYP2E1 catalyses the conversion of diglyme to 2ME. These results suggest that the central ether linkage of diglyme is cleaved by rat and human P-450 and the specific involvement of hepatic P450IIE1 in this process is implicated.

Zonation of acetaminophen metabolism and cytochrome P450 2E1-mediated toxicity studied in isolated periportal and perivenous hepatocytes

To study the mechanism of centrilobular damage developing in the centrilobular region after high doses of acetaminophen (APAP), its metabolism and toxicity were compared in periportal and perivenous hepatocytes isolated by digitonin/collagenase perfusion. Contrary to earlier reports, based on perfusions, no evidence for a periportal dominance of APAP sulfation could be observed. Glucuronidation, the dominant pathway of conjugation at high (5 mM) APAP concentration, was faster in perivenous cells. During primary culture, prolonged exposure (⩾ 24 hr) to 5 mM APAP damaged perivenous cells, with a higher P450 2E1 level than periportal cells. When cells were isolated from ethanol-pretreated rats, to induce P450 2E1 levels specifically in the perivenous region, perivenous hepatocytes exhibited enhanced APAP vulnerability and extensive glutathione depletion. In contrast, corresponding periportal cells retained good viability. Isoniazid, an inhibitor of cytochrome P450 2E1, protected cells against APAP toxicity and prevented glutathione depletion. Induction of P450 2E1 also caused a 3-fold increase in the covalent binding of reactive intermediates from [ 14C]APAP, and this increase was mainly confined to perivenous cells. These results indicate that in rat liver there is only slight perivenous zonation of APAP conjugation and suggest that zone-specific APAP activation, mediated by the regional expression of ethanol-inducible cytochrome P450 2E1, is responsible for the characteristic centrilobular liver damage elicited by APAP.

Glutathione homeostasis in rats chronically treated with ethanol. Evidence for an increased hepatic GSH export in vivo.

The influence of chronic ethanol feeding to rats on the hepatic glutathione (GSH and GSSG) system (synthesis, catabolism, export) and on the GSH and GSSG concentrations in extrahepatic tissues was investigated. Histological examination of livers from ethanol pretreated rats revealed a minor dilatation of the hepatic sinusoids. After ethanol administration the distribution pattern of gamma-glutamyltranspeptidase (enzymehistochemistry) was nearly unchanged, but the hepatic activity of this enzyme was increased. The ethanol pretreatment led to a decrease in hepatic GSH content. The hepatic activity of the GSSG-reductase were increased after ethanol treatment whereas the activities of the GSH synthesizing enzymes (gamma-glutamyl-cysteinyl-synthetase and GSH-synthetase) were not affected. A strong increase in sinusoidal GSH export was found in the ethanol-pretreated rats. The GSH- and GSSG concentrations of brain, lung, kidney and skeletal muscle were unchanged. It can be concluded that the ethanol-induced alteration of the hepatic GSH metabolism is caused mainly by changes of the sinusoidal membrane of the hepatocytes (direct effect of ethanol on the sinusoidal GSH carrier) leading to an increased GSH export into plasma. This effect should not due to an increased extrahepatic requirement for GSH.

Morphological and biochemical analyses of trichloroethylene hepatotoxicity: Differences in ethanol- and phenobarbital-pretreated rats

The histological and biochemical differences between ethanol- and phenobarbital (PB)-potentiated hepatotoxicity of trichloroethylene (TRI) in Wistar strain male rats were investigated. Both ethanol (2 g in daily liquid diet for 3 weeks) and PB (80 mg/kg/day for 4 days, ip) pretreatments enhanced TRI (inhalation exposures of 500 ppm for 8 hr, 2000 ppm for 2 or 8 hr, and 8000 ppm for 2 hr)-induced hepatic damage as judged by increases in plasma transaminase activities. Livers from PB-treated rats exposed to TRI displayed centrilobular necrosis, whereas livers from ethanol-treated rats exposed to TRI were characterized by ballooning degeneration mainly in midzonal areas. TRI exposure decreased the in vitro metabolism of TRI, high- K m benzene aromatic hydroxylase (BAH) activity, and cytochrome P450 content in livers of PB-treated rats with severe hepatic damage. In ethanol-treated rats, TRI exposure increased both the in vitro metabolism of TRI and the low- K m BAH activity but did not cause an apparent decrease in cytochrome P450 content even in animals with severe hepatic damage. These results suggest that TRI caused necrosis of centrilobular hepatocytes in PB-pretreated rats, which was accompanied by loss of xenobiotic metabolizing functions, whereas ballooning degeneration of hepatocytes mainly in midzonal areas occurred in ethanol-pretreated rats without loss of xenobiotic metabolizing functions.

Metabolism of N-nitrosodimethyl- and N-nitrosodiethylamine by rat hepatocytes: effects of pretreatment with ethanol

Hepatocytes were isolated from the livers of ethanol-pretreated rats, and the relationship between the generation of CO2 and the loss of N-nitrosodimethylamine (NDMA) and N-nitrosodiethylamine (NDEA) from the incubation mixtures was examined. The evolution of CO2 by hepatocytes isolated from untreated, control rats was compared with the evolution of CO2 by hepatocytes isolated from rats treated with 10% EtOH in their drinking water. The CO2 generated from either NDMA or NDEA represented only a fraction of the parent compound that was metabolized during the incubation period. Therefore, the measurement of CO2 evolution as an indication of the metabolism of these simple dialkylnitrosamines is inadequate, and the actual loss of the parent compound must be measured directly when utilizing isolated hepatocytes as a model system to study the metabolism of nitrosamines. The liver microsomal metabolism of NDMA and NDEA was also examined. Pretreatment of the rats with ethanol resulted in a marked increase in the microsomal metabolism of NDMA but had a relatively small effect on NDEA metabolism. Phenobarbital pretreatment did not result in any increase in NDMA metabolism whereas there was a very significant (6-fold) increase in NDEA metabolism. These results suggest that different isozymes of cytochrome P-450 may be primarily responsible for the metabolism of the two nitrosamines. The inhibition patterns observed when an antibody inhibitory to cytochrome P-450j was added to microsomes derived from control and ethanol- and phenobarbital-pretreated rats conclusively demonstrate that NDMA and NDEA are preferentially metabolized by distinct isozymes of cytochrome P-450.

Influence of ethanol induction on the metabolic activation of genotoxic agents by isolated rat hepatocytes.

The effects of ethanol-feeding to rats, over a 6-week period, on the activation of genotoxic compounds of different chemical classes, requiring metabolic conversion to exert their mutagenic activity, were studied in isolated rat hepatocytes. The influence of such treatment on cytochrome P-450 content and N-acetylation in isolated hepatocytes was also investigated. Benzidine (BZ), dimethylnitrosamine (DMN), diethylnitrosamine (DEN), isoniazid (INH) and cyclophosphamide (CP) were more effectively activated to products mutagenic towards Salmonella typhimurium by hepatocytes from ethanol-pretreated rats than by hepatocytes from controls. The mutagenic potency of 2-aminofluorene (2-AF) and 2-acetylaminofluorene (2-AAF) was not influenced by ethanol pretreatment. Ethanol consumption was found to be associated with increased cytochrome P-450 content and enhanced N-acetylation in the isolated hepatocytes. Our results support the hypothesis that an alteration of the hepatic drug-metabolizing system may be responsible for the ethanol-induced increase in susceptibility to certain genotoxic compounds.

Tolerance towards nitroglycerin, induced in vivo, is correlated to a reduced cGMP response and an alteration in cGMP turnover

Rats were made tolerant towards nitroglycerin (GTN) by subcutaneous injections of 50 mg/kg GTN, 3 times daily for 3 consecutive days. The effects of a test dose of GTN on tension and on the cGMP response were studied in aortic preparations. The activities of the enzymes regulating cGMP turnover were also investigated. In noradrenaline (2.5 μM)-contracted tissue from GTN-treated animals, the relaxant response towards the test dose of GTN (44 μM) was reduced by about 75% as compared to the ethanol-treated control rats. The cGMP-elevating action of GTN was reduced by 55%, while no significant effect on the cAMP level could be detected. The cyclic GMP-phosphodiesterase (G-PDE) activity was increased from 770 pmol/min × mg prot (ethanol-pretreated rats) to 1340 pmol/min × mg prot (GTN-pretreated animals). The guanylate cyclase activity, stimulated with 1 mM nitroprusside, was determined with both MnGTP and MgGTP as substrate and found to be reduced by about 75% in aortic homogenates from the GTN-pretreated animals. A slight depression in cGMP-dependent protein kinase activity was detected in aortas from GTN-treated animals. However, this depression seemed to be due to an increased breakdown of the activator (cGMP) since the inclusion of 2.5 mM theophylline in the assay solution abolished the difference. These results strongly support the suggestion that cGMP acts as a mediator of GTN-induced vascular smooth muscle relaxation. The tolerance towards the pharmacological action of GTN after repeated exposure could well be explained by the reduced formation and increased rate of breakdown of cGMP as demonstrated in this study.

Enflurane, isoflurane, and methoxyflurane metabolism in rat hepatic microsomes from ethanol-treated animals.

The effects of ethanol on the metabolism of enflurane, isoflurane, and methoxyflurane were investigated to determine if alterations in biotransformation of these agents occur as a result of this treatment. In vitro incubations of hepatic microsomes from rats pretreated with 10 days' ethanol vapor inhalation revealed a fourfold increase in inorganic fluoride from enflurane when compared with incubations of microsomes from unpretreated rats and from phenobarbital-pretreated rats. Isoflurane, while metabolized to a lesser extent than enflurane, showed a similar stimulation of metabolism. Methoxyflurane, while metabolized to a greater extent than either enflurane or isoflurane, had lesser fluoride production by the microsomes from ethanol-pretreated rats than microsomes from phenobarbital-pretreated rats, but greater fluoride production than that found in microsomes from unpretreated rats. Ethanol pretreatment did not alter the levels of cytochrome P-450 which is the enzyme responsible for such metabolism. This suggests that the altered metabolism involves either a specific P-450 isozyme or an unidentified enzyme.

Effect of chronic ethanol administration on bromobenzene liver toxicity in the rat

Female Sprague-Dawley rats were pair-fed a nutritionally adequate liquid diet containing either ethanol or isocaloric carbohydrate for 3 weeks. In vitro studies showed that chronic ethanol pretreatment preferentially increased the liver microsomal biotransformation of bromobenzene to p-bromophenol (via the toxic 3,4-epoxide) rather than to o-bromophenol (via the nontoxic 2,3-epoxide) and could thus potentiate bromobenzene hepatotoxicity. Bromobenzene administration (500 mg/kg body weight, ip), after an overnight fast, was associated in ethanol-pretreated rats with greater and accelerated liver glutathione depletion and subsequent decrease in liver cytochrome P-450 content than in controls. As assessed histologically and by determination of the rise in activities of serum enzyme markers of liver necrosis, chronic ethanol pretreatment, however, mainly resulted in earlier onset and resolution of bromobenzene-induced liver necrosis, with only a mild increase in the maximal severity of liver lesions. These results suggest that the twofold increase in liver microsomal bromobenzene 3,4-epoxidation by ethanol, being much less than that seen after phenobarbital pretreatment in our animal model and in that of others, is apparently not sufficient to markedly affect the severity of bromobenzene-induced liver toxicity.