Induction of rat hepatic and intestinal glutathione S-transferases by dietary butylated hydroxyanisole.

To obtain insight into the protection mechanism of butylated hydroxyanisole (BHA), a widely used food preservative with anticarcinogenic properties, we investigated the effects of dietary BHA on rat hepatic and intestinal glutathione S-transferase (GST) enzyme activity, and GST isozyme levels. In the proximal small intestine and liver, BHA supplementation significantly increased GST enzyme activity as compared with controls (2.3- and 1.7-fold, respectively, P less than 0.05). GST class alpha and mu contents were significantly higher only in the small intestine (1.6-2.1-fold and 1.3-1.5-fold, respectively, P less than 0.05), whereas GST class pi was significantly induced in liver (4.6-fold, P less than 0.05).     

Effects of high sucrose diet on body and liver weight and hepatic enzyme content and activity in the rat

The effect of sucrose on the induction of hepatic and peripheral insulin resistance is well-documented. Studies show that, although oral administration of glucose does not significantly decrease total hepatic microsomal cytochrome P450 content, it causes an increase in cytosolic protein and in microsomal phospholipid and fatty acid content. In this study we examined the effects of a chronic high sucrose diet (HSD) on liver enzyme activity. Male Fisher 344 weanling rats were randomly assigned to a control diet (0% sucrose by calories, n = 10) or a diet in which starch was replaced by sucrose (65% sucrose, by calories, n = 10) for 90 days. The effects of HSD on weight gain, liver weight, hepatic microsomal cytochrome P450 (CYP450) content and glutathione-S-transferase (GST) activity were measured and compared with those fed standard lab chow. A small but statistically significant decrease in body weight (g) was seen in the sucrose-fed rats after day 50. Liver GST activity (nmol/mg protein/min) at the end of 90 days was decreased in animals maintained on HSD compared to those on the control diet, (181.7 +/- 8.0, 234.7 +/- 5.5), respectively. The liver weight and total CYP450 content in the two diet groups were not significantly different. The ratios of liver weight to body weight at the end of 90 days suggested that the livers of the HSD-fed animals were larger per gram of body weight. In addition, rats on the HSD had significantly smaller amounts of liver CYP450 1A1 and 3A2 than the rats on the control diet. These results suggest that a HSD may alter the hepatic enzyme activity which may affect the metabolism of substrates for these enzyme systems.     

Influence of chronic alcohol consumption on hepatic heme and porphyrin metabolism

To study the effect of prolonged alcohol consumption on hepatic heme and porphyrin metabolism, female Wistar rats were fed for 60 days a nutritionally adequate liquid diet containing 36% of total calories as ethanol, whereas the control diet was isocaloric and contained no alcohol. Compared to pair-fed controls, the administration of the alcohol diet resulted in an increased hepatic activity of delta-aminolevulinic acid synthase by 223% (112.3 +/- 19.6 nmoles/hr/100 g b.wt. vs. 362.8 +/- 42.5; P less than 0.01), an enhanced urinary excretion of delta-aminolevulinic acid by 101% (64.8 +/- 11.8 nmoles/day vs. 130.8 +/- 22.4; P less than 0.05), and an augmented urinary output of total porphyrins by 142% (1.2 +/- 0.2 nmoles/day vs. 2.9 +/- 0.5; P less than 0.05). Concomitantly, the hepatic content of cytochrome P-450 was significantly enhanced and that of hepatic catalase activity marginally increased, whereas the hepatic iron content remained unaltered. In summary, the feeding of rats with a liquid alcohol diet for 60 days results in changes of hepatic heme and porphyrin metabolism which are associated and may be causally related with an induction of hepatic hemoproteins and subsequent derepression of hepatic delta-aminolevulinic acid synthase, whereas hepatic iron appears to play no pathogenic role.     

Effects of human diets on biotransformation enzyme activity and metabolic activation of carcinogens in rat liver

We studied the effects of a complete human diet, based on mean consumption figures in The Netherlands, the heating of food, and the presence of vegetables and fruit in the diet on the drug metabolizing capacity of the rat liver and on metabolic activation of known carcinogens. Groups of five male and five female Wistar rats were given ad lib. one of six different diets for 3 months. Each diet contained 40 energy (E)% fat, 13 E% protein, 47 E% carbohydrate and 5% fibre (w/w). The diets were as follows: a control diet of semi-synthetic materials (A); a human diet of meat, bread and eggs without processing (B); diet B heated under usual household conditions (C); a diet representing a complete human meal including (summer) vegetables and fruit (D); diets consisting of winter vegetables (E) or summer vegetables (F) with fruit. Semi-synthetic components were added to diets B-F to achieve the desired composition. There were differences between male and female rats on the effects of the different diets on hepatic enzyme activity. In female rats, but not in males, ethoxyresorufin-O-deethylase activity was increased significantly (P less than 0.05) in groups C, D and E in comparison with the controls (group A). In male rats ethoxycoumarin-O-deethylase activity was enhanced in groups D, E and F, and glutathione-S-transferase was markedly induced in group F (P less than 0.01). In males, hepatic cytochrome P-450 was significantly (P less than 0.05) increased in groups B, C and E. There was no effect on aminopyrine-N-demethylase activity and almost no effect on UDP-glucuronyltransferase activity in either sex. Microsomes from rats fed heated food (C) markedly increased the mutagenicity of benzo[a]pyrene (B[a]P) in the Ames assay using Salmonella typhimurium strain TA98, in comparison with levels using microsomes from rats fed the raw food (B). Vegetables and fruit decreased B[a]P mutagenicity. All human diets except D decreased the mutagenicity of N-nitrosodimethylamine in tester strain TA100. The results indicate that the influence of components of human diets on rat-liver drug metabolism may have quite different effects on the biotransformation of carcinogens activated by different metabolic pathways.     

Effects of Maillard browned egg albumin on drug-metabolizing enzyme systems in the rat

Adult male Sprague-Dawley rats were fed a purified diet containing 5% Maillard browned egg albumin (EA-B) or browned hydrolysed egg albumin (HEA-B) for 10 wk. Control animals were pair-fed a corresponding isocaloric, isonitrogenous non-browned egg albumin (EA-C) or hydrolysed egg albumin (HEA-C) diet. At the end of 10 wk, the rats were killed and hepatic, small intestinal and colonic microsomes and cytosol fractions were prepared by ultracentrifugation. Animals fed EA-B exhibited significantly (P less than 0.05) increased hepatic benzo[alpha]pyrene hydroxylase activity and significantly (P less than 0.05) decreased colonic aminopyrine N-demethylase activity compared to control (EA-C) animals. HEA-B-fed animals also exhibited a significant (P less than 0.05) decrease in colonic aminopyrine N-demethylase activity compared with HEA-C controls, but no significant differences were detected in hepatic or small intestinal enzyme activities in this group. These data suggest that Maillard browned protein products may modify hepatic and/or colonic drug-metabolizing enzyme system activities, and may thus contribute to alterations in the metabolism of endogenous substrates and of exogenous drugs, precarcinogens and other xenobiotics.     

Hepatic gamma-glutamyltransferase activity: its increase following chronic alcohol consumption and the role of carbohydrates

Prolonged feeding of diets containing ethanol leads to a significant increase of hepatic gamma-glutamyltransferase (GGT) activity which has been ascribed either to ethanol itself or to dietary imbalance with respect to carbohydrates. Hepatic GGT activity was therefore determined in Sprague-Dawley rats fed for five weeks liquid diets containing various amounts of protein, fat and vitamins. Compared to the normal control diet containing 47% of total calories as carbohydrates, a hypocaloric diet with 11% of total calories of the control diet as carbohydrates failed to result in major alterations of hepatic GGT activity (0.27 +/- 0.04 Units/g liver wet weight vs 0.35 +/- 0.06; N.S.). Similarly, hepatic GGT activity remained virtually unchanged under a hypercaloric carbohydrate rich diet. However, hepatic GGT activity was strikingly enhanced by a diet in which carbohydrates were replaced to the extent of 36% of total calories by ethanol to achieve a carbohydrate content of 11% (0.66 +/- 0.12 Units/g liver; P less than 0.005), indicating that alcohol itself is capable of increasing hepatic GGT activity. However, alcohol given with a high carbohydrate diet was shown to be incapable of increasing the hepatic activity of GGT. These data therefore indicate that upon chronic intake ethanol itself enhances hepatic GGT activity provided that the carbohydrate content of the diet is low, whereas such an effect could not be observed with ethanol in a high carbohydrate diet.     

Myocardial hypertrophy, cardiac beta-adrenoceptors and adenylate cyclase activity during sinoaortic denervation in dogs.

1. The long-term effects of sinoaortic denervation on the development of left ventricular hypertrophy (assessed by the measurement of the ratio (R): heart weight/total body weight and LVT: left ventricular thickness), myocardial beta-adrenergic receptivity (measured by [125I]-cyanopindolol binding and adenylate cyclase activity) and plasma catecholamine levels (measured by h.p.l.c.) were investigated in three groups of dogs: normotensive controls (group 1), dogs made hypertensive by sinoaortic denervation and evaluated 1 (group 2) and 18 months (group 3) later. 2. Noradrenaline (NA) and adrenaline (A) plasma levels were 461 +/- 54 and 85 +/- 45 pg ml-1 in controls, 861 +/- 185 and 191 +/- 23 pg ml-1 in group 2 (P less than 0.05). They were normal in group 3 (426 +/- 132 and 110 +/- 16 pg ml-1). 3. R and LVT values were significantly (P less than 0.05) higher in sinoaortic denervated dogs (R = 7.7 +/- 0.1 and 7.8 +/- 0.2; LVT = 13.6 +/- 1.3 and 14.2 +/- 0.9 mm in groups 2 and 3 respectively) than in normotensive dogs (group 1: R = 6.7 +/- 0.1, LVT = 9.3 +/- 0.8 mm). 4. In group 1, the total number of beta-adrenoceptors (Bmax) was 37 +/- 11 and 29 +/- 6 fmol mg-1 protein in the left ventricle (LV) and right auricle (RA) respectively. In group 2, Bmax was significantly lower (10 +/- 3 in LV and 13 +/- 2 fmol mg-1 protein in RA, P less than 0.05) than in group 1. There was no difference between group 1 and group 3 (37 +/- 3 fmol mg-1 prot in LV and 31 +/- 3 fmol mg-1 protein in RA). 5. The percentage of beta 1-adrenoceptors was 82 +/- 4 in LV and 75 +/- 5 in RA in group 1. It was significantly lower (P less than 0.05) in groups 2 (LV: 33 +/- 6 and RA: 33 +/- 5) and 3 (LV: 59 +/- 3 and RA: 55 +/- 4). 6. Basal values of adenylate cyclase activity in LV significantly decreased after sinoaortic denervation.(ABSTRACT TRUNCATED AT 250 WORDS)     

一氧化氮对大鼠全胃肠外营养肝脂肪变性的防护作用

目的:探讨一氧化氮(N0)在全胃肠外营养(TPN)引起的肝脂肪变性中的作用.方法:30只正常Wistar大鼠随机分为5组:A组,自由进食和水;B组,TPN;C组,TPN 精氨酸;D组,TPN N~G-硝基-L-精氨酸甲酯(N~G-nitrio-L-arginine methyl ester,L-NAME);E组,TPN 精氨酸 L-NAME.实验7天后测肝功能、肝内脂肪、肝脏NO含量及NOS活性并进行肝脏组织学检查.结果:B组肝内脂肪[甘油三酯、胆固醇(mmol·g~(-1))](39.3±2.4和 13.1±1.1)较A组(6.9±0.8和5.6±0.6)明显增加(P<0.05),D组肝内脂肪(50±6和14.1±1.7)较B组增加更显著(P<0.05),C组肝内脂肪(18±4和 7±3)较B组明显减少(P<0.05),肝内NOS活性及分布与肝内脂肪含量及分布相平行.结论:一氧化氮在TPN引起的肝脂肪变性中起防护作用.     

Phenobarbital, beta-naphthoflavone, clofibrate, and pregnenolone-16alpha-carbonitrile do not affect hepatic thyroid hormone UDP-glucuronosyl transferase activity, and thyroid gland function in mice

The effects of representative liver enzyme inducers such as clofibrate (CLO), phenobarbital (PB), pregnenolone-16alpha-carbonitrile (PCN), and beta-naphthoflavone (NF) on hepatic microsomal thyroxin (T4)- UDP-glucuronosyl transferase (UGT) and triiodothyronine (T3)- UGT activities and thyroid function were evaluated in OF-1 male mice after a 14-day po administration. CLO, PB, and PCN induced histological liver hypertrophy, increases in liver weights, in microsomal protein and cytochrome P450 contents as well as increases in specific UGT activities. Despite this, no significant changes in T4-UGT and T3-UGT activities occurred after treatment by any of these compounds. Furthermore, no significant changes in serum T4 and T3 levels were observed and thyroid histology was not affected. NF treatment induced microvacuolation of hepatocytes but did not affect any of the other tested parameters. The results show that, in contrast to the widely described effects in rats, liver enzyme inducers do not affect hepatic thyroid hormone metabolism and thyroid function in mice, suggesting that this species should be less sensitive to thyroid tumor promotion by hepatic microsomal enzyme inducers than rats.     

Increased hepatic efflux of glutathione after chronic ethanol feeding

Chronic ethanol feeding increases hepatotoxicity of drugs, such as acetaminophen, which form electrophilic metabolites. Availability of glutathione (GSH) is important in preventing liver damage from reactive metabolites. Chronic ethanol feeding has been reported to increase turnover of hepatic GSH in rats. The results of the present study show that the total hepatic efflux of GSH was increased from 5.95 +/- 0.42 nmoles/min/g liver (control) to 9.96 +/- 0.57 nmoles/min/g (P less than 0.001) in isolated perfused livers from rats 24 hr after withdrawal from chronic ethanol feeding. The increase in total efflux of GSH was due to a significant increase in sinusoidal GSH efflux from 4.76 +/- 0.49 nmoles/min/g liver in control rats to 9.07 +/- 0.47 nmoles/min/g (P less than 0.001) in ethanol-fed rats, while biliary efflux decreased slightly, 1.20 +/- 0.11 (control) vs 0.89 +/- 0.31 (ethanol). The increase in cellular efflux of GSH was similar in magnitude to the increase in hepatic GSH turnover that we reported previously. Biliary GSSG was similar in both groups of animals. Hepatic GGT activity was increased slightly, but not significantly, whereas renal GGT activity was similar in ethanol-fed rats. Hepatic GSH and GSSG levels were also similar. The increase in turnover of hepatic GSH in rats withdrawn from chronic ethanol feeding was most likely due to increased cellular efflux of GSH. This finding suggests that chronic ethanol feeding may increase cellular requirements for GSH, although the mechanism remains unknown. This alteration in GSH turnover may have important consequences for detoxification of xenobiotics or their metabolites by the liver.     

Drug metabolizing enzyme changes after chronic buthionine sulfoximine exposure modify acetaminophen disposition in rats.

This study examined the effects of prolonged exposure to buthionine sulfoximine (BSO) on 1) the overall elimination pharmacokinetics of acetaminophen; 2) the sulfate and glucuronide conjugation processes primarily responsible for acetaminophen elimination; and 3) in vitro microsomal and cytoplasmic enzyme activities in rats. Rats imbibed drinking water containing 30 mM BSO for 6 days and then received an iv injection of acetaminophen, 150 mg/kg in a propylene glycol vehicle. Exposure to BSO, a specific inhibitor of gamma-glutamylcysteine synthetase, produced marked depletion of glutathione (GSH) and resulted in induction of hepatic UDP-glucuronosyltransferase and GSH-S-transferase enzyme activities, but not cytochrome P-450. BSO pretreatment had no effect on the total or renal clearance of acetaminophen in rats. However, BSO exposure increased the partial clearance of acetaminophen to acetaminophen glucuronide by 47% (1.29 +/- 0.08 vs. 1.90 +/- 0.23 ml/min/kg; p less than 0.01) and significantly (p less than 0.02) increased the percentage of the dose recovered as the glucuronide conjugate from 17.6 +/- 2.5 to 26.5 +/- 0.6 The partial clearance of acetaminophen to acetaminophen sulfate was decreased, although not significantly, from 4.46 +/- 0.62 to 3.39 +/- 0.82 ml/min/kg. BSO treatment increased microsomal UDP-glucuronosyltransferase activity toward three xenobiotic aglycones, p-nitrophenol, 1-naphthol, and morphine by 308, 61, and 66%, respectively (p less than 0.05), but not toward testosterone or estrone. Cytosolic GSH-S-transferase activity toward 1-chloro-2,4-dinitrobenzene was increased 52% by BSO, whereas p-nitrophenol sulfotransferase activity was not altered. Cytochrome P-450 concentration and monooxygenase activity were unchanged by BSO exposure.     

Reduced hepatic uptake of propranolol in rats with acute renal failure

The effect of acute renal failure (ARF) on the hepatic uptake and metabolism of propranolol was investigated in relation to the hepatic clearance of the drug. ARF was induced by the subcutaneous injection of uranyl nitrate to rats. The uptake rate of propranolol in the isolated perfused liver was determined by the multiple-indicator dilution method and was found to decrease from 43.6 +/- 2.0 min-1 (mean +/- S.E.) in control to 29.4 +/- 1.7 min-1 in ARF (P less than 0.001). The recovery fraction of propranolol in effluent venous blood increased about twofold in ARF compared to control (P less than 0.05). The metabolic activity for propranolol was examined using the hepatic microsomal fraction prepared from control and ARF rats. There was no significant difference in the kinetics of oxidative metabolism of propranolol between two groups. These results suggest that the previously reported decrease in the hepatic clearance of propranolol in ARF is due to decreased hepatic uptake of the drug from the blood into the liver cells.     

Proteasome inhibitor lactacystin ablates liver injury induced by intestinal ischaemia-reperfusion

1. The aim of the present study was to investigate the role of proteasome in the pathogenesis of liver injury induced by intestinal ischaemia-reperfusion (I/R) and the effect of the proteasome inhibitor lactacystin on neutrophil infiltration, intracellular adhesion molecule (ICAM)-1 and nuclear factor (NF)-kappaB expression in the liver tissues of rats. 2. Thirty-two Wistar rats were randomly divided into four groups (n = 8 in each group) as follows: (i) a control, sham-operated group; (ii) an I/R group subjected to 1 h intestinal ischaemia and 4 h reperfusion; (iii) a group pretreated with 0.2 mg/kg lactacystin 1 h before intestinal I/R; and (iv) a group pretreated with 0.6 mg/kg lactacystin 1 h before intestinal I/R. Liver and intestine histology were observed. Serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT) and lactate dehydrogenase (LDH), as well as 20S proteasome activity in circulating white blood cells, were measured. Myeloperoxidase (MPO) activity in liver tissues and the immunohistochemical expression of liver NF-kappaB and ICAM-1 were assayed. In addition, a western blot of liver NF-kappaB was performed. 3. Compared with the sham-operated control group, liver and intestine injury was induced by intestinal I/R, characterized as histological damage including oedema, haemorrhage and infiltration by inflammatory cells, as well as a significant increase in serum AST (365 +/- 121 vs 546 +/- 297 IU/L, respectively; P < 0.05), ALT (65 +/- 23 vs 175 +/- 54 IU/L, respectively; P < 0.01) and LDH levels (733 +/- 383 vs 1434 +/- 890 IU/L, respectively; P < 0.05). Compared with the control group, MPO activity in the liver tissues increased significantly in the I/R group (2.05 +/- 0.69 vs 3.42 +/- 1.11 U/g, respectively; P < 0.05). Strong positive expression of liver ICAM-1 and NF-kappaB p65 was observed. 4. Compared with the intestinal I/R group, administration of 0.6 mg/kg lactacystin markedly reduced 20S proteasome activity in circulating white blood cells (15.47 +/- 4.00 vs 2.07 +/- 2.00 pmol 7-amino-4-methylcoumarin (AMC)/s per mg, respectively; P < 0.01) and ameliorated liver injury, which was demonstrated by decreased levels of serum AST (546 +/- 297 vs 367 +/- 86 IU/L, respectively; P < 0.05), ALT (175 +/- 54 vs 135 +/- 26 IU/L, respectively; P < 0.05) and LDH (1434 +/- 890 vs 742 +/- 218 IU/L, respectively; P < 0.05) and a reduced liver pathological score (2.13 +/- 0.64 vs 1.25 +/- 0.46, respectively; P < 0.01). Compared with the intestinal I/R group, MPO activity in liver tissues decreased significantly following lactacystin pretreatment (3.42 +/- 1.11 vs 2.58 +/- 0.61 U/g, respectively; P < 0.05) and the expression of liver NF-kappaB and ICAM-1 was markedly ameliorated. 5. The present study reveals that the proteasome inhibitor lactacystin ablates liver injury induced by intestinal I/R. One possible mechanism responsible for this effect is the inhibition of enhanced ICAM-1 and neutrophil infiltration by inhibition of NF-kappaB activity. The results suggest the feasibility of using proteasome inhibitor clinically in the treatment of intestinal I/R.     

Effects of beta 1- and beta 1 + beta 2-antagonists on training-induced myocardial hypertrophy and enzyme adaptation

The effects of beta 1- and beta 1 + beta 2-antagonists on the myocardial adaptation to exercise training were investigated in male Sprague-Dawley rats randomly divided into trained (treadmill, 1 hr/day, 5 days/week for 10 weeks at 27 m/min, 15% grade) without drug (TC), sedentary without drug (SC), trained treated with atenolol (TA) (10 mg/kg body wt, i.p.), trained treated with propranolol (TP, 30 mg/kg body wt, i.p.), and sedentary propranolol. Doses of both beta-antagonists were titrated to decrease the exercise heart rate by 25% compared to the controls. The heart weight and heart/body weight ratio were significantly greater in TC (1.28 +/- 0.07 g (P less than 0.01); 296 +/- 12 mg/100 g body wt (P less than 0.05) respectively) than in SC (1.09 +/- 0.04 g and 268 +/- 11 mg/100 g body wt), or in TP and TA. Myocardial mitochondrial protein was unchanged by training or beta-blockade. Citrate synthase and beta-hydroxyacyl CoA dehydrogenase activities were not altered. Carnitine palmitoyltransferase activity was increased in SP compared to SC. Training increased hexokinase activity only in TC (5.22 +/- 0.12 vs 4.26 +/- 0.23 mumol/min/g wet wt, P less than 0.01). Lactate dehydrogenase activity increased significantly (P less than 0.01) in both TC (383 +/- 14 mumol/min/g wet wt) and TA (372 +/- 14 mumol/min/g wet wt) compared to SC (276 +/- 14 mumol/min/g wet wt), but not in TP versus SP. These data indicate that (1) beta-adrenergic blockade prevents training-induced cardiac hypertrophy; (2) beta-antagonists have little effect on the myocardial oxidative capacity; and (3) while the training induction of myocardial hexokinase is inhibited by both beta 1- and beta 1 + beta 2-antagonists, myocardium may increase its ability to utilize lactate during exercise with training despite beta 1-blockade.     

Aloe-Emodin quinone pretreatment reduces acute liver injury induced by carbon tetrachloride

Aloe contains several active compounds including aloin, a C-glycoside that can be hydrolyzed in the gut to form aloe-emodin anthrone which, in turn, is auto-oxidized to the quinone aloe-emodin. On the basis of the claimed hepatoprotective activity of some antraquinones, we studied aloe-emodin in a rat model of carbon tetrachloride (CCl4) intoxication, since this xenobiotic induces acute liver damage by lipid peroxidation subsequent to free radical production. Twelve rats were treated with CCl4 (3 mg/kg) intraperitoneally and six were protected with two intraperitoneally injections of aloe-emodin (50 mg/kg; CCl4+aloe-emodin); six other rats were only aloe-emodin injected (aloe-emodin) and six were untreated (control). Histological examination of the livers showed less marked lesions in the CCl4+aloe-emodin rats than in those treated with CCl4 alone, and this was confirmed by the serum levels of L-aspartate-2-oxoglutate-aminotransferase (394+/-38.6 UI/l in CCl4, 280+/-24.47 UI/l in CCl4+aloe-emodin rats; P<0.05). We also quantified changes in hepatic albumin and tumour necrosis factor-alpha mRNAs. Albumin mRNA expression was significantly lower only in the liver of CCl4 rats (P<0.05 versus control) and was only slightly reduced in the CCl4+aloe-emodin rats. In contrast tumour necrosis factor-alpha mRNA was significantly higher (P<0.05) in the CCl4 than the control rats and almost equal in the CCl4+aloe-emodin, aloe-emodin and control groups. In conclusion, aloe-emodin appears to have some protective effect not only against hepatocyte death but also on the inflammatory response subsequent to lipid peroxidation.     

The prediction of benzo[a]pyrene clearance by rat liver and lung from enzyme kinetic data

The metabolic clearance of circulating benzo[a]pyrene (B[a]P) by liver and lung of control and 3-methylcholanthrene (3MC)-pretreated rats was predicted according to the perfusion-limited model from apparent enzyme kinetic constants determined in microsomal incubations. These predictions were tested in isolated organs perfused at normal organ flow. From microsomal incubations the apparent enzyme kinetic constants of B[a]P metabolism were determined. The apparent Km of liver microsomes was decreased 100 times by pretreatment with 3MC, while the Km of lung microsomes remained at about 0.2 microM. Maximal velocity of B[a]P metabolism was much greater in microsomes from liver than in those from lung of both control and 3MC-pretreated rats. Liver was found to have a far greater capacity for B[a]P metabolism (intrinsic free clearance) than lung. However, this large disparity was not evident in the predicted clearances. Perfused organs had B[a]P clearances very close to those predicted from the model. At normal (in vivo) organ flows, control rat lung had a clearance of 1.0 +/- 0.1 ml/min, whereas liver had a clearance of 5.9 +/- 0.2 ml/min. Corresponding clearances in organs from 3 MC-pretreated rats were 8.9 +/- 0.5 and 6.7 +/- 0.6 ml/min for lung and liver, respectively. Small discrepancies between predicted and observed values could not be explained by non-uniform distribution of B[a]P or shunting of flow. These results suggest that enzyme kinetic data can be used to assess accurately the ability of lung and liver to clear xenobiotic compounds such as B[a]P and that, despite the great disparity in their metabolic capacity, under certain conditions these two organs may function equally well in the removal of circulating compounds from the blood.     

Intestinal phase II detoxification systems: effect of low-protein diet in weanling rats

The effect of a low-protein diet (6% protein, LPD) in vivo on the in vitro activities of cytosolic and microsomal glutathione S-transferase (GST-c, GST-m) and microsomal UDP-glucuronyltransferase (UDP-GT) was studied in small intestine and liver of weanling male Wistar rats. LPD interrupted the normal curve of growth of the animals, which returned to control values after refeeding with a normal diet (27% protein, ND). Hepatic and intestinal GST-c increased in control rats in parallel with the growth curve. The microsomal enzymes did not show growth variations, except for intestinal UDP-GT activity which began to increase from the second day of ND. After 7 days of LPD there was an increase in GST-c (liver 35%, P less than 0.05; intestine 152%, P less than 0.01) and of UDP-GT (liver 58%, intestine 178%, P less than 0.05) which returned to control values after 2 days of refeeding with ND. GST-m did not show any variations in liver or intestine. The increase in GST-c, but not in GST-m, with nutritional stress suggests preferential induction of cytosolic enzymes in those enzymatic systems which are located in both positions. The increase in such enzymes after protein malnutrition could indicate an adaptive response by detoxification mechanisms, enhancing intestinal over hepatic capacity, perhaps because the intestine is the primary route of access for orally ingested xenobiotics.     

Age- and sex-related differences in activation of the carcinogen 7-hydroxymethyl-12-methylbenz[a]anthracene to an electrophilic sulfuric acid ester metabolite in rats. Possible involvement of hydroxysteroid sulfotransferase activity.

Metabolic activation of 7-hydroxymethyl-12-methylbenz[a]anthracene (HMBA) and related hydroxymethyl polycyclic aromatic hydrocarbons to electrophilic and mutagenic sulfuric acid esters has been demonstrated previously (Watabe et al., In: Xenobiotic Metabolism and Disposition (Eds. Kato R, Estabrook RW and Cayen MN), pp. 393-400. Taylor & Francis, London, 1989). In the present study, the rat hepatic sulfotransferase activity catalyzing the formation of such reactive sulfuric acid esters was inhibited strongly by dehydroepiandrosterone, a typical substrate hydroxysteroid sulfotransferases (HSSTs). Pentachlorophenol, a potent phenol sulfotransferase inhibitor, had little effect in this regard. A marked sex difference was observed for the hepatic cytosolic sulfotransferase activity for HMBA in rats. This sex difference was age-related; no significant difference was observed in preweanling rats, whereas in adult rats female rat liver showed a much higher enzyme activity. These age- and sex-related differences in the sulfonation of HMBA reflect the regulation of HMBA sulfotransferase activity by gonadal hormones as previously demonstrated with HSSTs. Thus, pretreatment with estradiol benzoate significantly enhanced the sulfotransferase activity for HMBA in both male and female rats, (P less than 0.01 and P less than 0.05 respectively), whereas testosterone propionate pretreatment decreased this activity. Castration of male rats increased the HMBA sulfotransferase activity 2- to 3-fold compared with that in control animals. By contrast, ovariectomy reduced the enzyme activity 38% in females. These results imply that rat liver HSST activity is responsible for the sulfonation of HMBA. Intraperitoneal injection of HMBA (0.25 mumol/g body wt) into infant rats produced benzylic DNA adducts in the liver which were chromatographically identical with those obtained from incubations of HMBA with deoxyguanosine and deoxyadenosine in the presence of hepatic cytosolic sulfotransferase activity. Intraperitoneal administration of sodium 7-sulfooxymethyl-12-methylbenz[a]anthracene resulted in much higher levels of these adducts and the deoxycytidine adduct in the liver DNA than did an equimolar amount of the parent hydroxymethyl hydrocarbon. The levels of hepatic benzylic DNA adducts formed from HMBA were reduced markedly by pretreatment of rats with dehydroepiandrosterone, a strong inhibitor of hepatic sulfotransferase activity for this hydrocarbon.     

Circadian rhythm of hepatic uridine phosphorylase activity and plasma concentration of uridine in mice

The activity of hepatic uridine phosphorylase (EC 2.4.2.3.) in male mice (24-29 g) maintained in standardized conditions of 12 hr light (0600-1800 hr) alternating with 12 hr darkness (1800-0600 hr), food and water ad lib., exhibited a circadian rhythm (P less than 0.0001, Cosinor analysis). The peak of enzyme activity (559 +/- 25 pmol/min/mg protein) occurred at 15 hr after light onset (HALO) with the nadir (139 +/- 25 pmol/min/mg protein) at 3 HALO when samples were taken every 4 hr. Female mice showed essentially the same pattern. A circadian rhythm (P less than 0.0001, Cosinor analysis) was also observed when the light-dark cycle was shifted (reverse cycle) so that the lights went on at 2200 hr and off at 1000 hr. Under the reverse cycle condition, there was a corresponding shift in the enzyme activity with a lag period of 3.5 hr in the time of maximum and minimum enzyme activities (i.e. the peak at 11 HALO and the nadir at 23 HALO) after a 2-week adaptation period. The lag period was reduced to 1 hr after 4 weeks of adaptation, and no further change was observed after 6 weeks of adaptation. The plasma concentration of uridine also exhibited a circadian rhythm (P less than 0.0001, Cosinor analysis) with peak concentration (10 microM) occurring at 2 HALO and a nadir (5 microM) at 14 HALO. The circadian rhythm observed in the plasma concentration of uridine is the inverse of that for uridine phosphorylase activity. These results demonstrate that hepatic uridine phosphorylase plays an important role in the regulation of the uridine level in the blood which, in turn, may be involved in the humoral control of sleep by uridine. This may also be of clinical significance in enhancing the antitumor efficacy of the 5-fluorinated pyrimidines by modulating the time of their administration.