农药叶枯灵对大鼠离体灌流肝的影响研究

进行了叶枯灵的大鼠肝脏离体循环式灌流肝实验研究,结果表明,灌流肝正常功能至少可维持2小时,叶枯灵在500uM 时,对大鼠灌流肝有毒作用,LDH 酶活性及 K~+浓度对评价叶枯灵的有毒作用是敏感的指标。本文建立了大鼠离体灌流肝的模型,适用于化合物的毒性及代谢的研究。     

MATERNAL ETHANOL CONSUMPTION INDUCES TRANSIENT COMPENSATORY HYPERPLASIA OF DEVELOPING CARDIAC TISSUE IN THE NEONATAL RAT

The effect of continuous exposure to ethanol in utero and postpartumon growth and cell division in developing cardiac tissue wasstudied in neonatal Fischer rats. Pregnant and lactating femaleswere maintained on three dietary regimens; a control group fedrat chow ad libirum, an experimental group receiving an ethanol-containing(6% by volume) liquid diet, and a pair-fed control group, whichreceived an isocaloric amount of control liquid diet. At days1, 5, and 10 postpartum, five litters of pups from each controland experimental group were sacrificed and the body weights,heart weights, heart-to-body weight ratios, and mitotic frequencyof the ventricular myocardium were measured. When compared toeither group of controls, pups continuously exposed to dietaryethanol expressed significantly (P < 0.01) lower body weights.Pups maintained by the pair-fed females had significantly (P< 0.01) lower body weights at days 5 and 10 than pups maintainedby the chow-fed females, indicating a pair-fed effect of suboptimalnutrition of the model. As the pups developed, the heart weightsof pups maintained by the chow-fed females became progressivelygreater (P < 0.01) than the heart weights of pups maintainedby the pair-fed and ethanol-fed females, which expressed noweight difference. The reduction of heart weight present inthe ethanol-fed and pair-fed pups represents a pair-fed effectof suboptimal nutrition and not an obvious effect of exposureto dietary ethanol. The ratio of heart weight to body weightand mitotic frequency were significantly greater (P < 0.01)in 1- to 5-day-old pups exposed to ethanol. Following day 5,these parameters decreased and approached the control values.This indicates that growth of cardiac tissue is not suppressedin the 1- to 5-day-old rat pups exposed continuously to dietaryethanol. These observations further suggest the presence ofa mechanism intrinsic to the heart which can provide stage-dependentprotection from the adverse effects of ethanol during earlydevelopment. The decline in heart weight to body weight ratiosand mitotic frequency in pups of ethanol-fed females also suggeststhat ethanol may initiate suppression of the growth of cardiactissue or may incur stage-dependent injury during the laterstages of development. The possible mechanism of this stage-dependentprotection during early neonatal development is an increasedmitotic activity of the cardiac myocytes.     

CYTOCHROME P-450 2E1 IN RAT LIVER, KIDNEY AND LUNG MICROSOMES AFTER CHRONIC ADMINISTRATION OF ETHANOL EITHER ORALLY OR BY INHALATION

In this study, microsomal cytochrome P-450 2E1 (CYP2E1) contentsand activities were tested in liver, kidney and lung from Wistarrats after the following treatments (1) oral administrationof a 10% ethanol solution for 4 weeks; (2) pair fed controls;(3) oral administration of a 5% acetone solution for 1 week;(4) inhalation of ethanol vapour for 4 weeks. CYP2E1 activitywas measured using chlorzoxazone as substrate and CYP2E1 contentwas measured using Western blot analysis. In addition, the cellulardistribution of CYP2E1 was studied in liver, lung and kidneyby immunohistochemistry. Basal liver CYP2E1 was 10–20times lower in lung and kidney than in liver. Inhalation wasclearly the most efficient way of inducing CYP2E1, probablydue to the continuous and high alcohol exposure. Among the organstested, lung appeared to be the tissue least sensitive to inductioneven after ethanol inhalation, suggesting the absence of localinduction. After ethanol intoxication, immunostaining was increasedin the centrilobular region of the liver, in the alveolar cellsof the lung and in the proximal convoluted tube of the kidney.The CYP2E1 activities decreased to control values in the threetissues tested, within 24 h after cessation of intoxication.     

INFLUENCE OF ETHANOL ADMINISTRATION ON THE ACTIVITY AND COMPARTMENTATION OF RAT LIVER MONOAMINE OXIDASES

Single-dose ethanol administration to rats caused inhibitionof liver mitochondrial monoamine oxidases (MAO) A and B, andan increase in susceptibility of MAO A (but not MAO B) to limitedproteolysis. Chronic ethanol feeding resulted in a less distinctalteration in catalytic activity and susceptibility to proteolysisof mitochondrial MAO, but increased the amount of soluble MAO.The sensitivity of membrane-bound MAO to inhibitors (imipramineand chlorpromazine), action of which depends on their lipophilicityand/or hydrophobicity, remained unchanged, compared with controls.Increased amounts of soluble MAO seen after chronic ethanolfeeding probably reflect an impairment of insertion of newlysynthesized enzyme molecules into the outer mitochondrial membrane,rather than solubilization of MAO from it.     

FURTHER EVIDENCE AGAINST INHIBITION OF SYNTHESIS OF RAT LIVER TRYPTOPHAN PYRROLASE BY CHRONIC ETHANOL ADMINISTRATION

The decrease in rat liver tryptophan pyrrolase activity causedby chronic ethanol administration has previously been shownto be reversed by addition in vitro of NAD⁺ or NADP⁺ This reversalis now shown to be unaffected by pyrazole, thus suggesting non-involvementof alcohol dehydrogenase in the reversal. By contrast, whenpyrrolase synthesis is inhibited, the decreased activity cannotbe reversed by NAD⁺ or NADP⁺ in vitro. These results providefurther evidence for involvement of NADH and NADPH in the pyrrolaseinhibition by chronic ethanol administration and against thesuggested inhibition of synthesis of the apoenzyme.     

ADDITIONAL PROOF OF REDUCTION OF ETHANOL ABSORPTION FROM RAT INTESTINE IN VIVO BY HIGH ACETALDEHYDE CONCENTRATIONS

We investigated intestinal ethanol absorption in rats pretreatedwith saline, cyanamide, 4-methylpyrazole and cyanamide + 4-methylpyrazole.The value of the absorption rate constant in the cyanamide-pretreatedgroup with high acetaldehyde levels was the lowest among thefour groups, but there were no significant differences amongthe remaining groups. We found that high acetaldehyde concentrationitself clearly reduces intestinal ethanol absorption.     

ALTERATION OF ACETALDEHYDE METABOLISM IN CARBON TETRACHLORIDE-INTOXICATED RAT LIVER: ANALYSIS USING LIVER PERFUSION SYSTEM

The hepatic metabolism of acetaldehyde in carbon tetrachloride(CCl₄)-intoxicated rats was studied using a non-recirculatinghaemoglobin-free liver-perfusion system. Acetaldehyde uptakeby the liver from acutely CCl₄-treated animals (4.16 mmol/kg,i.p.) at 24 hr after the treatment was not significantly altered,whereas that by the liver from chronically CCl₄-treated animals(2.08 mmol/kg,i.p., twice a week, for 8–12 weeks) wasdecreased by approximately 50% when it was determined in thepresence of 0.01–5 mM acetaldehyde. In liver from ratschronically intoxicated with CCl₄, the following important biochemicalchanges were observed: (1) The activity of low K_m aldehyde dehydrogenase(ALDH) in hepatic mitochondria was decreased by approximately75%. (2) The basal levels of the lactate/pyruvate (cytosolic[NADH]/[NAD⁺]) ratio as well as the ß-hydroxybutyrate/acetoacetate(mitochondrial [NADH]/[NAD⁺]) ratio were elevated by more than2-fold. (3) Mitochondrial NADH oxidation was also reduced byapproximately 35% of the control level. (4) The basal levelof hepatic oxygen uptake was attenuated by approximately 50%,and the infusion of acetaldehyde (0.01–5.0 mM) causeda further decrease in the uptake. (5) The rate of ethanol productionfrom acetaldehyde by the catalytic action of alcohol dehydrogenasewas found to be unaltered when low concentrations of acetaldehyde(0.01–0.2 mM) were used, whereas a significant suppressionof the rate of ethanol production was detected in the presenceof high concentrations of acetaldehyde (0.6–5 mM). Thesedata suggest that the changes in activity of the lowK_m mitochondrialacetaldehyde dehydrogenase and those in mitochondrial NADH oxidationcoupled with mitochondrial respiration may, at least in part,play important roles in the decreased hepatic acetaldehyde metabolismobserved in chronically CCl₄-treated rats.     

EFFECT OF CHRONIC ETHANOL CONSUMPTION ON GLYCOSYLATION PROCESSES IN RAT LIVER MICROSOMES AND GOLGI APPARATUS

Previous studies have demonstrated that acute ethanol intoxicationaffects various steps of protein glycosylation at the levelof rat liver endoplasmic reticulum and Golgi apparatus. Theaim of this investigation was to demonstrate whether chronicethanol intake can induce definitive changes of liver glycoproteinprocessing. Rats were given ethanol by liquid diet for 8 weeks.At the end of this period the triglyceride levels in liver homogenateand microsomes were significantly higher than in controls. Isolatedhepatocytes prelabelled with [³H]Na palmitate and [¹⁴C]glucosamineshowed a significant storage of the lipid and carbohydrate radioactivityin microsomes and Golgi apparatus and a significant impairmentof labelled glycolipoprotein secretion. Changes of the glycosylationsteps were observed both in endoplasmic reticulum and in Golgiapparatus: in the former the levels of dolichyl phosphate, whichis rate-limiting for the synthesis of glycoprotein, showed asignificant reduction; in the latter the activity of the mainenzymes responsible for the terminal glycosylation process wassignificantly decreased. These data suggest that an impairmentof glycoprotein maturation may be involved in the pathogenesisof liver injury induced by chronic ethanol intake.     

COMPARISON OF THE ACUTE EFFECTS OF ETHANOL ON LIVER AND SKELETAL MUSCLE PROTEIN SYNTHESIS IN THE RAT

(1) The acute effects of ethanol on protein synthesis by liverand skeletal muscle were investigated in young (95–100g) rats. Rats were injected intraperitoneally with ethanol,75 mmol/kg body wt; controls were injected with isovolumetric0.15 M NaCl. After 140 min rates of protein synthesis were measuredby injection of a large dose of L[4³H]phenylalanine and at 150min rats were killed. (2) Fractional rates of protein synthesisin control animals were approximately four to five times greaterin liver than muscle. Absolute rates were, however, comparablein liver and skeletal muscle. Ethanol reduced the fractionalrate of liver protein synthesis by 5–20%; the responsefor muscle was relatively greater (25–30%). The decreasein the amount of protein synthesised by muscle was also greaterthan that by liver. (3) After 150 min, plasma gamma-glutamyltransferase, alanine aminotransferase, alkaline phosphatase,lactate dehydro-genase and creatine kinase activities were alldecreased by 25–60%. Aspartate aminotransferase activitywas increased by 42%, though this was not statistically significant.(4) Increased plasma glucose and triglycerides in ethanol-dosedrats indicated that limitations in substrate supply were notmediating factors in reducing protein synthesis. Ethanol wasalso able to exert its effects in the presence of elevated insulinlevels. A direct effect of ethanol, or its metabolites, on proteinsynthesis, is therefore implied.     

INCREASE IN RAT LIVER TRYPTOPHAN OXYGENASE ACTIVITY AFTER ACUTE ETHANOL TREATMENT: DEPENDENCE ON FEEDING STATE AND ROUTE OF ETHANOL ADMINISTRATION

A time course and dose-response study of the effects of acuteethanol administration on the activlty of liver tryptophan oxygenasewas carried out in fed and starved rats. Ethanol doses rangingfrom 0.5 to 5.0 g/kg body weight were given either intragastricallyor intraperitoneally. In a detailed time curve for an oral 3.3g/kg dose of ethanol in starved rats, the total activity oftryptophan oxygenase was increased 1.5-fold 2 hr after ethanoladministration and the activity of the holoenzyme (haem-saturatedform) increased one hour later, thus indicating that increasedhaem-saturation is not the critical stimulus for accumulationof the enzyme after acute ethanol ingestion. Both the totaland holoenzyme activities reached a peak, whlch was4.5–5.5times the control activity, 5–6 hr after ethanol treatment.The increase in the enzyme activities was not directly dependenton the dose of ethanol, but rather the peak blood ethanol concentration,which had to reach a critical level to trigger enzyme activation.In fed rats when ethanol was given orally, this critical bloodethanol level was not achieved even with the highest ethanoldose and no significant increases in tryptophan oxygenax activitieswere seen. 4-Methylpyrazole (75 mg/kg) inhibited the ethanol-inducedincrease in the total tryptophan oxygenase actlvity by 30%,but did not affect that in the holoenzyme activity. Cyanamide(5 mg/kg) did not affect the ethanol-induced increases in eithertotal enzyme or holoenzyme activities. t-Butanol (1.0 g/kg)caused increases of the same order of magnitude as ethanol did.It was concluded that 70% of the increase in total tryptophanoxygenase activity after acute ethanol ingestion is probablycaused by an action of ethanol itself. while 30% may be causedby ethanol metabolism. However, the increase in haem-saturationwas apparently caused by an action of ethanol itself.     

DISCREPANCY BETWEEN THE DIFFERENT SUBCELLULAR ACTIVITIES OF RAT LIVER CATALASE AND SUPEROXIDE DISMUTASES IN RESPONSE TO ACUTE ETHANOL ADMINISTRATION

After acute ethanol administration (2.3 g/kg) peroxisomal andextra-peroxisomal catalase activities were affected in oppositedirections. As long as ethanol was present, peroxisomal catalaseactivity was enhanced, but unaffected by amino-triazole (AT)injection. Extra-peroxisomal catalase activity was decreasedduring the 24hr following ethanol administration and was alsoinhibited after AT injection. Whereas mitochondrial manganese-dependentsuperoxide dismutase (Mn-SOD) was unaffected, the cytosolicCu,Zn-SOD activity was decreased. The time-course of this decreasesuggests that acute ethanol administration affects primarilyextra-peroxisomal catalase activity, thus rendering cytosolicsuperoxide dismutase more exposed to oxygen derivatives.     

ETHANOL AND PHOSPHATIDYLETHANOL REDUCE THE BINDING OF [3H]INOSITOL 1,4,5-TRISPHOSPHATE TO RAT CEREBELLAR MEMBRANES

In this study, we have analysed the effect of ethanol and phosphatidylethanol,a unique phospholipid formed only in the presence of ethanol,on the binding of [³H]inositol 1,4,5-trisphosphate to rat cerebellarmembranes. Rats were intraperitoneally injected daily with 3g of ethanol/kg body weight for different periods of time. Repeatedadministration of ethanol induced a reduction in the bindingcapacity (B_max) without affecting the affinity constant (K_d).A significant 32% reduction was observed after 21 days of exposure(from control B_max values of 25±3 pmol/mg and K_d valuesof 9±2 nM). In an in-vitro assay, phosphatidylethanol(500 µM) and phosphatidic acid (500 µM), but noother phospholipids tested, induced a reduction in B_max (39%and 43%, respectively). The observed effect displayed by phosphatidylethanolwas not due to its degradation to phosphatidic acid or otherphospholipids. The results emphasize the importance of examiningphosphatidylethanol (PEth) as a possible mediator of the effectsof ethanol on cellular processes. However, the role of PEthin the observed effect of long-term ethanol exposure still needsfurther consideration.     

A COMPARATIVE STUDY OF THE EFFECTS OF ( + )-CATECHIN AND 3-PALMITOYL-( + )-CATECHIN ON ALCOHOLIC FATTY LIVER IN THE RAT

Administration of a liquid diet containing 36% of the totalcalories as ethanol to male Wistar rats for three weeks causeda threefold increase in total hepatic lipid, a tenfold increasein hepatic triglycerides, a twofold increase in hepatic unesterfiedfatty acids, and elevated the serum activities of the liverenzymes alanine aminotransferase and glutamate dehydrogenase,when compared with control data from animals pair-fed an isocaloricamount of glucose instead of ethanol. Simultaneous administrationof either ( + )-catechin (200mg/kg by gastric tube) or 3-palmitoyl-(+ )-catechin (l00mg/kg daily by gastric tube) to ethanol treatedanimals caused significant reductions in the extent of fat accumulationand hepatocellular damage as assessed by both biochemical andhistological means. 3-Palmitoyl-( + )-catechin was more effectivethan ( + )-catechin, even though it was administered at onlyhalf the dose of that of the former. Pharmacokinetic studiesin which [U-¹⁴C] labelled 3-palmitoyl-( +)-catechin and (+ )-catechinwere given orally (150mg/kg each) to male rats showed that 3-palmitoyl-(+ )-catechin achieved a higher peak tissue concentration andremained in the liver for a longer period than (+ )-catechin.These properties of 3-palmitoyl-( + )-catechin are thought tobe due to its greater lipid solubility, thereby explaining itsgreater potency in the chronic fatty liver studies.