Selective increase in cytochrome P450 content in hepatic microsomes from rats with ventromedial hypothalamic lesions

Changes in the components of hepatic microsomal electron transport systems and in drug hydroxylase activities were investigated in ventromedial hypothalamus (VMH) lesioned obese rats. Eight weeks after electrolysis of the bilateral VMH, the content of cytochrome P450 per mg microsomal protein (0.79 +/- 0.07 nmol/mg protein) was significantly higher (P less than 0.02) than that in the sham-operated rats (0.59 +/- 0.02). Cytochrome P450 per whole liver in the VMH-lesioned obese rats had also significantly increased (87 +/- 9 nmol vs 56 +/- 3, P less than 0.02). No significant differences were found in the cytochrome b5 contents, and the activities of NADPH- and NADH-cytochrome c reductases between the VMH-lesioned obese and sham-operated rats. The demethylation activities of aminopyrine (1.04 +/- 0.02 nmol/mg protein/min vs 0.94 +/- 0.02, P less than 0.05) and p-nitroanisole (0.96 +/- 0.02 vs 0.89 +/- 0.02 , p less than 0.02) and the aniline hydroxylase activity (0.22 +/- 0.01 vs 0.16 +/- 0.01, P less than 0.01) were enhanced, but 7-ethoxycoumarin O-deethylase activity was unchanged in the VMH-lesioned obese rats. These results indicate a selective increase in the content of cytochrome P450 among the components of the P450-dependent mixed function oxidase system in the liver of VMH-lesioned obese rats. Our observations suggest that drug metabolism may be enhanced in the hypothalamic obesity.     

Leptin administration exacerbates thioacetamide-induced liver fibrosis in mice

AIM: To investigate the effects of leptin administration on liver fibrosis induced by thioacetamide (TAA). METHODS: Twenty-four male C57Bl/6 mice were randomly allocated into four groups, which were intra-peritoneally given saline (2 mL/kg), leptin (1 mg/kg), TAA (200 mg/kg), TAA (200 mg/kg) plus leptin (1 mg/kg) respectively, thrice a week. All mice were killed after 4 wk. The changes in biochemical markers, such as the levels of alanine aminot-ransferase (ALT) and aspartate aminotransferase (AST) in serum and superoxide dismutase (SOD), malondialdehyde (MDA) in liver were determined. For histological analysis, liver tissues were fixed with 10% buffered formalin, embedded with paraffin. Hematoxylin-eosin (HE) staining and picric acid-Sirius red dyeing were performed. The level of α1(I) procollagen mRNA in liver tissues was analyzed by RT-PCR. RESULTS: Apparent liver fibrosis was found in TAA group and TAA plus leptin group. Compared to saline group, the levels of ALT and AST in serum and MDA in liver increased in TAA group (205.67±27.69 U/L vs50.67±10.46 U/L, 177.50±23.65 U/L vs 76.33±12.27 U/L, 2.60±0.18 nmol/mg pro vs 1.91±0.14 nmol/mg pro, P<0.01) and in TAA plus leptin group (256.17±22.50 U/L vs 50.67±10.46 U/L, 234.17±27.37 U/L vs 76.33±12.27 U/L, 2.97±0.19 nmol/mg pro vs 1.91±0.14 nmol/mg pro,P<0.01). The level of SOD in livers decreased (51.80±8.36 U/mg pro vs 81.52±11.40 U/mg pro, 35.78±6.11 U/mg pro vs 81.52± 11.40 U/mg pro, P<0.01) and the level of α1(I) procollagen mRNA in liver tissues also increased (0.28±0.04 vs 0.11± 0.02, 0.54±0.07 vs 0.11±0.02, P<0.01). But no significant changes were found in leptin group and saline group. Compared to TAA group, ALT, AST, MDA, and α1(I) procollagen mRNA and grade of liver fibrosis in TAA plus leptin group increased (256.17±22.50 U/L vs 205.67± 27.69 U/L, P<0.05; 234.17±27.37 U/L vs 177.50±23.65 U/L, P<0.05; 2.97±0.19 nmol/mg pro vs 2.60±0.18 nmol/mg pro,P<0.05; 0.54±0.07 vs 0.28±0.04, P<0.01; 3.17 vs 2.00, P<0.05), and the level of SOD in liver decreased (35.78±6.11 U/mg pro vs 51.80±8.36 U/mg pro, P<0.05). There were similar changes in the degree of type I collagen deposition confirmed by picric acid-Sirius red dyeing. CONCLUSION: Leptin can exacerbate the degree of TAA-induced liver fibrosis in mice. Leptin may be an important factor in the development of liver fibrosis.     

Effects of pregnant mare serum gonadotrophin and oestradiol benzoate on enzymes associated with xenobiotic metabolism in immature and hypophysectomized female rats

Treatment of immature (22-day-old) female Sprague-Dawley rats with pregnant mare serum gonadotrophin (PMSG) resulted in a significant increase in hepatic microsomal cytochrome P-450 concentrations when compared with animals treated with 0.9% (w/v) NaCl solution. Cytochrome P-450 concentrations in microsomes from control and PMSG-treated animals were 0.49 +/- 0.07 and 0.78 +/- 0.18 nmol/mg microsomal protein respectively (mean +/- S.D.). When hypophysectomized rats were treated with PMSG no significant difference was observed between saline-treated and PMSG-treated animals (0.84 +/- 0.06 vs 0.88 +/- 0.08 nmol/mg microsomal protein). Cytochrome b5 concentration and NADPH-cytochrome c reductase and ferricyanide reductase activities were not significantly affected by PMSG treatment. Similar results were obtained when immature and hypophysectomized rats were treated with oestradiol benzoate. Hepatic glutathione (GSH) S-transferase activity was significantly stimulated over control values when hypophysectomized or immature animals were treated with PMSG, oestradiol benzoate or 3-methylcholanthrene. Treatment of animals simultaneously with PMSG or oestradiol benzoate and 3-methylcholanthrene resulted in higher transferase activity than did either treatment alone. Oestradiol benzoate or PMSG treatment of immature female rats resulted in an approximate twofold increase in hepatic aryl hydrocarbon hydroxylase (AHH) activity. However, AHH activity in hypophysectomized animals was not significantly increased by PMSG or oestradiol benzoate treatment. It is concluded that PMSG or oestradiol benzoate treatment of immature female rats resulted in increased total cytochrome P-450 concentration and AHH activity in hepatic microsomes. Glutathione S-transferase activity was also stimulated. Neither PMSG nor oestradiol benzoate stimulated cytochrome P-450 levels or AHH activity in hypophysectomized rats.(ABSTRACT TRUNCATED AT 250 WORDS)     

Manipulation of cytochrome P-450 dependent renal thromboxane synthase activity in spontaneously hypertensive rats

Thromboxane synthase is a cytochrome P-450-like enzyme requiring an iron-centered oxygen attack of the prostaglandin endoperoxide substrate (PGH2) for subsequent thromboxane A2 (TxA2) formation. The activity and levels of P-450 enzymes can be manipulated by decreasing heme availability. Stannous chloride (SnCl2) selectively induces renal heme oxygenase activity, depleting heme and decreasing hemoprotein synthesis. We therefore manipulated the renal cytochrome P-450 system to influence thromboxane synthase activity, as measured by the conversion of 14C-PGH2 to thromboxane B2 (TxB2) in renal cortical microsomes from spontaneously hypertensive rats (SHR). Seven-week-old SHR were treated subcutaneously with SnCl2 (1, 10 and 15 mg/100 g body weight) for 4 consecutive days, and cortical microsomal heme oxygenase activity, heme content, P-450 content, thromboxane synthase activity and systolic blood pressure were measured. Heme oxygenase activity was significantly increased from 1058 +/- 62 nmol/mg protein in controls to 3125 +/- 918, 5057 +/- 690--and 4236 +/- 581 nmol/mg protein in SHR treated with 1, 10 and 15 mg/100 g body weight SnCl2, respectively. The increase in heme oxygenase activity was associated with corresponding decreases in heme content (0.29 mumol/mg protein, for control to 0.12 mumol/mg protein for SHR treated with SnCl2, 10 mg/100 g body weight) and cytochrome P-450 content (0.18 +/- 0.1 nmol/mg protein for control to 0.06 +/- 0.01 nmol/mg protein for SHR treated with SnCl2 10 mg/100 g body weight). The reduction in heme and P-450 content was associated with a reduction in thromboxane synthase activity, i.e., decreases of 38, 35 and 47% from control levels at doses of 1, 10 and 15 mg/100 g body weight.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of short-term leptin exposure on triglyceride deposition in rat liver

Leptin has recently been suggested to play a role in the pathogenesis of hepatic steatosis. Consequently, this study was designed to examine the direct effects of portal leptin on the intrahepatic lipid contents in the postabsorptive state. Rat livers (n = 6 per group) were perfused in a recirculating system and portally infused with leptin (0.5 nmol/L, 5 nmol/L, and 25 nmol/L), insulin (10 nmol/L), leptin (5 nmol/L) plus insulin (10 nmol/L), glucagon (1 nmol/L), or vehicle (control). Intrahepatic contents of triglycerides, free cholesterol, cholesteryl esters, and free fatty acids were determined from the lipid extract of frozen livers by capillary gas chromatography. Short-term leptin infusion increased total triglycerides in a concentration-dependent (0.5 nmol/L: 2.8 +/- 0.4 mg/g, 5 nmol/L: 7.0 +/- 0.5 mg/g, 25 nmol/L: 8.3 +/- 1.0 mg/g) and time-dependent manner. Total triglycerides also rose during exposure to insulin plus leptin (7.2 +/- 0.6 mg/g) but fell during glucagon infusion (2.6 +/- 0.2 mg/g; control: 4.3 +/- 0.3 mg/g; P <.05). Leptin, insulin, and glucagon increased intrahepatic free cholesterol (P <.05). Free fatty acids were also higher during leptin exposure (0.5 nmol/L: 1.28 +/- 0.08 mg/g, 5 nmol/L: 0.47 +/- 0.01 mg/g, 25 nmol/L: 0.48 +/- 0.04 mg/g, control: 0.38 +/- 0.03 mg/g; P <.05). In conclusion, hyperleptinemia increases hepatic triglyceride content and may therefore contribute to hepatic steatosis in hyperleptinemic obese patients.     

Biochemical effect of methyl chloride in relation to its tumorigenicity

Summary The biochemical effects of methyl chloride were investigated in tissues of F-344 rats and B₆C₃F₁ mice (both sexes). Activities of GST were 2–3 times higher in livers of male B₆C₃F₁ mice, compared with those of female mice, and with rats of both sexes. In kidneys GST activities of (male) mice were about 7 times lower than those found in livers. The activity of FDH was higher in livers of mice (both sexes) than in those of rats. No obvious sex difference was found in livers of rats and mice with respect to FDH. In kidneys, however, (minor) differences in FDH activities occurred between male and female B₆C₃F₁ mice (4.7 vs. 3.1 nmol/min per mg). Sex differences of FDH activity in kidneys were not observed in F-344 rats. The microsomal transformation (by cytochrome P-450) of methyl chloride and S-methyl-L-cysteine to formaldehyde in tissues of B₆C₃F₁ mice occurred preferentially in the liver. More formaldehyde was produced in liver microsomes of male, compared to those of female mice. Kidney microsomes metabolized methyl chloride to formaldehyde much less than liver microsomes. After a single exposure of mice of both sexes to 1000 ppm methyl chloride no elevation in formaldehyde concentrations was observed in livers and kidneys ex vivo. The determination of DNA lesions, using the alkaline elution technique, revealed no DNA-protein crosslinks in kidneys of male B₆C₃F₁ mice after exposure to methyl chloride (1000 ppm, 6 h day^-1, 4 days) and gave only minor evidence of singlestrand breaks. Lipid peroxidation (production of TBA reactive material), induced by single exposure to methyl chloride (1000 ppm, 6 h), was very pronounced in livers of male and female mice. Smaller increases in peroxidation were observed in the kidneys of exposed mice. The theory that renal tumors observed in male mice after chronic exposure of the test animals to high (1000 ppm) concentrations of methyl chloride, are evoked by intermediates and in situ produced formaldehyde is proven unlikely by our results.Abbreviations used GST glutathione-S-transferase - FDH formaldehyde dehydrogenase - GSH glutathione - CDNB 1-chloro-2,4-dinitrobenzene (substrate for GST's) - TBA Thiobarbituric acid (indicative of lipid peroxidation) - TCA trichloroactetic acid Dedicated to Professor Werner Kunz on occasion of his 65th birthday     

Identification and functional characterization of leukotriene B4 20-hydroxylase of human polymorphonuclear leukocytes.

A single reaction product was formed during the incubation of 1.5 microM (5S,12R)-dihydroxy-6,14-cis-8,10-trans-[3H]icosatetraenoic acid (leukotriene B4, LTB4) for 30 min at 37 degrees C in 10 mM potassium phosphate buffer (pH 7.5) with 100 microM NADPH and the 150,000 X g supernatant of sonicated human polymorphonuclear leukocytes (PMN). The reaction product exhibited the same mobility on reversed-phase HPLC (RP-HPLC) and TLC as standard 20-hydroxy-LTB4 (20-OH-LTB4). When the omega-oxidation product of [3H]LTB4 was eluted from a Sep-Pak, resolved by RP-HPLC, and analyzed by GC/MS, its structure was determined to be solely 20-OH-LTB4. The Km of the 20-hydroxylase for [3H]LTB4 at its optimal pH of 7.5 was 0.22 +/- 0.08 microM (mean +/- SD, n = 4) and the Vmax was 48 +/- 11 pmol/min X mg of protein (mean +/- SD, n = 4). When the concentration of [3H]LTB4 was fixed at 1.5 microM, the Km for NADPH was 1.01 +/- 0.59 microM (mean +/- SD, n = 3). The location in the 150,000 X g supernatant of the LTB4 20-hydroxylase distinguishes it from the cytochrome P-450 system of liver, lung, and kidney microsomes and from the NADPH oxidase-cytochrome b-245 system of the human PMN. The LTB4 20-hydroxylase is either a unique cytochrome P-450 or other monooxygenase.     

Possible contributions of reactive oxygen species and mitogen-activated protein kinase to renal injury in aldosterone/salt-induced hypertensive rats

Studies were performed to test the hypothesis that reactive oxygen species (ROS) and mitogen-activated protein kinase (MAPK) contribute to the pathogenesis of aldosterone/salt-induced renal injury. Rats were given 1% NaCl to drink and were treated with one of the following combinations for 6 weeks: vehicle (0.5% ethanol, SC, n=6); aldosterone (0.75 microg/H, SC, n=8); aldosterone plus a selective mineralocorticoid receptor antagonist; eplerenone (0.125% in chow, n=8); aldosterone plus an antioxidant; and tempol (3 mmol/L in drinking solution, n=8). The activities of MAPKs, including extracellular signal-regulated kinases (ERK)1/2, c-Jun-NH2-terminal kinases (JNK), p38MAPK, and big-MAPK-1 (BMK1) in renal cortical tissues were measured by Western blot analysis. Aldosterone-infused rats showed higher systolic blood pressure (165+/-5 mm Hg) and urinary excretion of protein (106+/-24 mg/d) than vehicle-infused rats (118+/-3 mm Hg and 10+/-3 mg/d). Renal cortical mRNA expression of p22phox, Nox-4, and gp91phox, measured by real-time polymerase chain reaction, was increased in aldosterone-infused rats by 2.3, 4.3, and 3.0-fold, respectively. Thiobarbituric acid-reactive substances (TBARS) content in renal cortex was also higher in aldosterone (0.23+/-0.02) than vehicle-infused rats (0.09+/-0.01 nmol/mg protein). ERK1/2, JNK, and BMK1 activities were significantly elevated in aldosterone-infused rats by 3.3, 2.3, and 3.0-fold, respectively, whereas p38MAPK activity was not changed. Concurrent administration of eplerenone or tempol to aldosterone-infused rats prevented the development of hypertension (127+/-2 and 125+/-5 mm Hg), and the elevations of urinary excretion of protein (10+/-2 and 9+/-2 mg/day) or TBARS contents (0.08+/-0.01 and 0.11+/-0.01 nmol/mg protein). Furthermore, eplerenone and tempol treatments normalized the activities of ERK1/2, JNK, and BMK1. These data suggest that ROS and MAPK play a role in the progression of renal injury induced by chronic elevations in aldosterone.     

Glutathione protects the rat liver against reperfusion injury after hypothermic preservation.

BACKGROUND & AIMS: The extracellular generation of reactive oxygen species (ROS) by Kupffer cells contributes to reperfusion injury of the liver allograft. The endogenous antioxidant glutathione (GSH) can detoxify these ROS; however, this effect might be limited by the low extracellular concentration of GSH. We therefore investigated whether an increase of extracellular GSH protects the liver against reperfusion injury after cold preservation. METHODS: Livers of male Sprague-Dawley rats subjected to 24 hours of cold ischemia in University of Wisconsin solution (4 degrees C) were reperfused for 2 hours in the absence (controls) or presence of 0.5, 1, 2, or 4 mmol/L GSH (n = 4-6 each). RESULTS: Two hours after starting reperfusion of control livers, the sinusoidal release of lactate dehydrogenase and purine nucleoside phosphorylase increased to 247 +/- 96 and 27 +/- 13 mU. min(-1). g liver(-1), respectively, but only to 76 +/- 43 and 10 +/- 4 mU. min(-1). g liver(-1) in the presence of 4 mmol/L GSH. This cytoprotective effect was confirmed histologically by a marked reduction of trypan blue staining of hepatocytes. Compared with control livers, postischemic bile flow was significantly enhanced by GSH (0.15 +/- 0.02 vs. 0.41 +/- 0.11 microL. min(-1). g liver(-1)), indicating improved liver function. During reperfusion of control livers, intracellular GSH content declined from 4.5 +/- 0.3 to 2.3 +/- 0.1 micromol/g liver, but only to 3.8 +/- 0.4 micromol/g liver in the presence of 4 mmol/L GSH. Reperfusion of untreated livers was accompanied by a prolonged increase of portal pressure to maximally 12.5 +/- 1.9 cm H2O, which was significantly attenuated by 4 mmol/L GSH (7.2 +/- 1.4 cm H2O). Similar cytoprotective and hemodynamic effects were observed with 2 mmol/L GSH, but not with 0.5 and 1 mmol/L GSH. CONCLUSIONS: Treatment of cold-preserved livers with GSH upon reperfusion prevents damage of hepatocytes, deterioration of the hepatic circulation, and loss of intracellular GSH. In view of these protective effects and its low toxicity in humans, GSH should be considered a candidate drug for prevention of ROS-related reperfusion injury of the liver allograft.     

Coproporphyrinogen oxidase, protoporphyrinogen oxidase and ferrochelatase activities in human liver biopsies with special reference to alcoholic liver disease

The activities of coproporphyrinogen oxidase, protoporphyrinogen oxidase and ferrochelatase have been assayed in human liver biopsies using recently developed highly sensitive specific enzyme assays. The specific activities (nmol/min/mg protein) in controls were 0.010 +/- 0.003 (mean +/- S.D., n = 11), 0.18 +/- 0.07 (n = 9) and 0.062 +/- 0.022 (n = 8), respectively. The total activities (mumol/min/liver) were determined, using ultrasound to determine liver volumes, and were 2.6 +/- 0.6 (n = 5), 36.6 +/- 13.9 (n = 6) and 14.2 +/- 5.4 (n = 3), respectively. Both specific and total enzyme activities in alcoholics with fatty liver were not significantly different from normal controls. Decreased protoporphyrinogen oxidase activity (0.08 nmol/min/mg protein or 20.2 mumol/min/liver) was found in two patients with variegate porphyria. In a patient with erythrohepatic protoporphyria a reduction of the ferrochelatase activity (0.01 nmol/min/mg protein) was demonstrated.     

Effect of amphotericin B on hepatic cytochrome P-450 and glucose-6-phosphatase in the rat.

The effect of amphotericin B on hepatic microsomal cytochrome P-450 (P-450) concentration was measured in vitro, in vivo and ex vivo in the rat. In vitro, both amphotericin B (0-500 micrograms/ml) and its vehicle, sodium deoxycholate (0-410 micrograms/ml), caused similar dose-dependent decreases of P-450 concentrations and glucose-6-phosphatase activity. Intravenous amphotericin B (3 mg/kg) given daily for 3 days decreased antipyrine clearance from control values of 1.24 +/- 0.24 ml/min to 0.67 +/- 0.12 ml/min (p less than 0.001); whereas antipyrine clearance was unchanged by sodium deoxycholate. The P-450 concentration on the third day was reduced from 0.74 +/- 0.14 nmol/mg protein in control rats to 0.33 +/- 0.09 nmol/mg protein in rats treated with amphotericin B (p less than 0.001). Sodium deoxycholate had no effect on P-450 concentration. In contrast, amphotericin B had no effect on either antipyrine clearance or P-450 concentration following enzyme induction by phenobarbital. Amphotericin B had no effect on microsomal glucose-6-phosphatase activity in vivo. Neither amphotericin B nor sodium deoxycholate induced lipid peroxidation, measured as malondialdehyde production. These results show that amphotericin B decreases hepatic cytochrome P-450 content and function in the rat. These effects can not be observed in the enzyme induced state. Amphotericin B has no effect on glucose-6-phosphatase in vivo, the key enzyme of the gluconeogenesis, indicating selective effects on hepatic microsomal function.     

The influence of age on steroidogenic enzyme activities of the rat adrenal gland: enhanced expression of cholesterol side-chain cleavage activity

The ability of isolated adrenocortical cells to secrete corticosterone in response to ACTH challenge declines as rats age, but the site or mechanism(s) of this impairment is still unknown. To test the functionality of steroidogenic capacity per se, we measured the key enzyme activities involved in corticosterone biosynthesis. We also measured the mitochondrial cytochrome P-450 content and nonsteroidogenic enzymes specific for subcellular fractions. Mitochondria and microsomal fractions were isolated from the adrenals of 2-, 12-, and 18-month-old animals and used for various enzyme measurements. Mitochondrial side-chain cleavage enzyme activity (nanomoles per min mg protein-1) increased from a mean of 0.43 +/- 0.06 in 2-month-old rats to 1.26 +/- 0.11 and 1.51 +/- 0.06 in 12- and 18-month old rats, respectively. After incubation with 5-cholesten-3 beta,25-diol (25-hydroxycholesterol; 25 micrograms/ml) side-chain cleave activity rose to 5.0 +/- 0.6, 12.4 +/- 1.2, and 16 +/- 1.4 nmol min-1 mg protein-1 in adrenal mitochondrial fractions from 2-, 12-, and 18-month-old rats, respectively. In contrast, mitochondrial cytochrome P-450 content did not vary with advancing age. Microsomal delta 5-3 beta-hydroxysteroid dehydrogenase-delta 5-delta 4-isomerase activities were similar in 2- and 12-month-old rats, but 21-hydroxylase (nanomoles per min mg protein-1) activity was significantly increased in 12-month-old rats (2-month-old, 5.2 +/- 0.2; 12-month-old, 7.7 +/- 0.5). Finally, mitochondrial 11 beta-hydroxylase was comparable in both age groups. In addition, activities of mitochondrial nonsteroidogenic enzymes, such as monoamine oxidase, amytal insensitive NADH cytochrome c reductase, cytochrome c oxidase, succinate dehydrogenase, and malate dehydrogenase, did not change with age. It appears from the evidence presented that the activities of the steroidogenic enzymes are not responsible for the diminished capacity in corticosterone production seen with aging in the rat.     

Differential inhibition of individual human liver cytochromes P-450 by cimetidine

Cimetidine binds to cytochrome P-450 and inhibits hepatic metabolism of various drugs in humans. However, cytochrome P-450 is a family of enzymes rather than a single protein, and effects of cimetidine on individual human liver cytochromes P-450 have not been previously characterized. Metabolism of selected substrates and cimetidine-binding assays have been performed using human liver microsomes, purified human liver cytochromes P-450, and cytochrome P-450 complementary DNA-expressed yeast proteins to probe interaction of cimetidine with these individual enzymes. Cimetidine (3.0 mmol/L) in incubations reduced bufuralol hydroxylase activity by 80% and strongly inhibited microsomal nifedipine oxidation (23% +/- 13% of control activity). The same concentration of cimetidine produced intermediate inhibition of cytochrome enzymes responsible for ethoxyresorufin deethylation and aniline hydroxylation (77% +/- 6% and 68% +/- 17% of activity in control microsomal incubations, respectively), but little effect on tolbutamide hydroxylation was observed. Concordantly, the calculated binding constant for the binding of cimetidine to a purified cytochrome P-450 with high tolbutamide hydroxylase activity was 4.4 mmol/L, whereas the calculated binding concentration constant for a purified cytochrome P-450-metabolizing nifedipine was 0.7 mmol/L. These studies show a high variability in the effect of cimetidine on drug metabolism by individual human liver cytochromes P-450. In vitro studies using human liver microsomes and genetically engineered human cytochromes P-450 can be very useful in exploring important clinical questions of hepatic drug metabolism.     

Quantitation of intrinsic drug-metabolizing capacity in human liver biopsy specimens: support for the intact-hepatocyte theory.

Hepatic drug metabolism is decreased in patients with severe liver disease, but it is unclear to what extent this is due to altered hepatic blood flow or reduced intrinsic metabolic capacity. In this study we quantitated in needle-biopsy specimens the intrinsic capacity of liver tissue from 67 patients with mild liver disease (n = 36), chronic active hepatitis (n = 16) and cirrhosis (n = 15) to metabolize two model compounds in vitro. Hydroxylation of the low-extraction drug bufuralol resulted in the formation of 251 +/- 25 nmol 1'OH-bufuralol/gm wet wt/hr in mildly diseased liver tissue and was significantly (p less than 0.01) reduced in liver tissue exhibiting chronic active hepatitis (166 +/- 23 nmol/gm wet wt/hr) and cirrhosis (124 +/- 21 nmol/gm wet wt/hr). The formation rates of monoethylglycinexylidide, the main metabolite of the high-extraction drug lidocaine, varied widely and were not significantly different among the three groups. To relate the drug-metabolizing capacity to the hepatocyte content of liver tissue, morphometrical study was performed in the biopsy pieces originally submitted. The metabolic activity of each biopsy piece was then related to the fractional volume of hepatocytes it was calculated to contain. In mildly diseased liver tissue 355 +/- 35 nmol 1'OH-bufuralol/ml hepatocytes x hr or 12.4 +/- 1.0 mumol monoethylglycinexylidide/ml hepatocytes x hr- and in cirrhotic liver tissue 306 +/- 49 nmol 1'OH-bufuralol/ml hepatocytes x hr or 15.3 +/- 3.0 mumol monoethylglycinexylidide/ml hepatocytes x hr--were formed, respectively, and these differences were not significant.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ethanol-Inducible Cytochrome P-450: Assessment of Substrates'' Specific Chemical Probes in Rat Liver Microsomes

The capacity of liver microsomes to oxidize various substrates known to be specific of alcohol-inducible cytochrome P-450 was studied in rats treated with different xenobiotics such as 3-methylcholanthrene, phenobarbital, acetone, and ethanol. Analysis of results showed a significantly marked increase following ethanol and acetone treatments of the p-nitrophenol hydroxylation (283 +/- 19% and 304 +/- 21%), N-nitrosodimethylamine (NDMA) demethylation (280 +/- 105% and 228 +/- 95%), benzene hydroxylation (258 +/- 60% and 236 +/- 61%), butanol oxidation (173 +/- 34% and 154 +/- 32%), aniline hydroxylation (147 +/- 22% and 95 +/- 8%), and ether de-ethylation (95 +/- 17% and 83 +/- 17%) and a not significant increase of N-nitrosodiethylamine (NDEA) de-ethylation (34 +/- 11% and 9 +/- 8%) in rat microsomes, respectively, versus control animals (mean +/- SD, values expressed as nmol/min/nmole P-450). All of these activities significantly decreased after 3-MC treatment, except for the p-nitrophenol hydroxylation. PB treatment markedly enhanced NDEA de-ethylation, p-nitrophenol, and benzene hydroxylations (106 +/- 38%, 109 +/- 14%, and 153 +/- 62%, respectively) versus controls. These results suggest that NDMA and especially 1-butanol are the most specific and useful probes of alcohol-inducible cytochrome P-450 in crude liver microsomes.     

四氯化碳诱导大鼠肝纤维化脂质过氧化相关蛋白表达的动态变化及一贯煎的干预效应

目的 探讨四氯化碳(CCl4)诱导大鼠肝纤维化脂质过氧化相关蛋白表达的动态变化及一贯煎的干预效应.方法 Wistar雄性大鼠57只,其中模型组39只,正常组18只.模型大鼠腹腔注射50％的CCl4橄榄油溶液(1ml/lg),每周2次,共9周.造模3、6周后,随机抽取正常及模型大鼠各6只,处死作动态观察.其余模型大鼠随机分为模型组15只及干预组12只,模型组大鼠在8周时处死4只观察成模情况.第7周开始,继续造模的同时,干预组用一贯煎(2.682 g/kg)蒸馏水稀释灌胃,1次/d,共计3周.用药3周结束后,处死大鼠,检测肝功能、肝组织羟脯氨酸(Hyp)和丙二醛(MDA)含量、超氧化物歧化酶(SOD)与谷胱甘肽(GSH)活性,以及热休克蛋白70 (HSP70)、血红素加氧酶-1(HO-1)、转铁蛋白(Transferrin)、过氧化还原酶(Prxd)6、肝脏型脂肪酸结合蛋白(L-FABP)等的表达.计量资料采用单因素方差分析,计数资料采用Ridit分析. 结果 (1)与对照组比较,模型组大鼠6、9周时肝组织MDA含量显著升高[(4.23±0.45) nmol/mg比(2.22±0.59)nmol/mg; (6.29±1.23) nmol/mg比(2.22±0.59) nmol/mg,F值分别为60.13、66.99,P值均＜ 0.05];SOD活性显著降低[(196.94±39.20) U/mg比(264.50±30.44)U/mg,F=11.12,P＜ 0.05; (152.21±51.65) U/mg比(264.50±30.44) U/mg,F=23.11,P＜0.01];GSH含量显著降低[(48.47±7.27) nmol/mg比(60.74±9.04) nmol/mg,F=6.71,P＜0.05;(37.89±9.01) nmol/mg比(60.74±9.04)nmol/mg,F=24.06,P＜0.01];与9周模型组比较,干预组MDA显著降低[(4.25±0.86) nmol/mg比(6.29±1.23) nmol/mg,F=19.52,P＜ 0.01],SOD显著升高[(198.35±46.48) U/mg比(152.21±51.65) U/mg,F=4.65,P＜0.05],GSH显著升高[(53.73±7.54) nmol/mg比(37.89±9.01) nmol/mg,F=19.23,P＜0.01];(2)与正常组比较,9周时模型组大鼠HSP70蛋白表达量升高(1.21±0.06比0.58±0.07,F=166.87,P＜0.0l),HO-1蛋白表达量也升高(1.11±0.06比0.58±0.06,F=123.96,P＜ 0.01),Prdx6蛋白表达量降低(0.04±0.05比1.49±0.05,F=1215.85,P＜0.01),L-FABP表达量降低(0.24±0.02比1.44±0.14,F=219.05,P＜0.01),Transferrin蛋白表达量降低(0.67±0.03比1.67±0.04,F=301.35,P＜0.01).9周时,干预组HSP70和HO-1蛋白表达量分别为0.82±0.04、0.90±0.04,与9周时模型组比较,F值分别为92.31、26.89,P值均＜0.01,差异有统计学意义;9周时,干预组Prdx6、L-FABP和Transferrin蛋白表达分别为0.88±0.11、1.36±0.13、1.04±0.12,与9周时模型组比较,F值分别为150.17、237.19、27.53,P值均＜0.01,差异有统计学意义. 结论 一贯煎具有促进机体抗氧化物质生成、减轻脂质过氧化损伤的作用.     

Decreased hepatic glycogen content and accelerated response to starvation in rats with carbon tetrachloride-induced cirrhosis

Glucose homeostasis and fatty acid metabolism are abnormal in patients with cirrhosis. To assess the metabolic response to starvation in an animal model of cirrhosis, glycogen and fuel metabolism were characterized in rats with CCl4-induced cirrhosis studied 2 wk after 10 weekly doses of CCl4. Plasma concentrations of glucose and beta-hydroxybutyrate were not different between fed CCl4-treated and control rats, but plasma nonesterified fatty acid concentrations were higher in cirrhotic animals (0.25 +/- 0.01 vs. 0.39 +/- 0.04 mmol/L; p less than 0.05). After 12 hr of starvation, the plasma nonesterified fatty acid concentration had reached 0.58 +/- 0.04 mmol/L in CCl4-treated rats, compared with 0.38 +/- 0.04 mmol/L in control rats (p less than 0.05). The redistribution of the hepatic carnitine pool toward acylcarnitines, which is characteristic of starvation, was complete after fasting for 12 hr in the CCl4-treated rats, compared with the 24 hr required in control rats. In fed cirrhotic rats, liver glycogen content per gram liver was decreased by 64% compared with control rats (30.0 +/- 5.1 vs. 10.8 +/- 1.1 mg/gm liver wet wt; p less than 0.05). After 12-hr fasting, hepatic glycogen content had fallen to 14.3 +/- 3.9 and 4.8 +/- 0.4 mg/gm liver wet wt (p less than 0.05) in control and cirrhotic animals, respectively. To further characterize the status of glycogen metabolism in cirrhotic livers, activities of glycogen synthase and glycogen phosphorylase were determined. Hepatic active and total glycogen phosphorylase activities normalized to hepatocellular content were unaffected by CCl4 treatment, whereas total glycogen synthase activity was increased by 45%.(ABSTRACT TRUNCATED AT 250 WORDS)     

Role of angiotensin II and reactive oxygen species in cyclosporine A-dependent hypertension

Treatment with cyclosporine A (CysA), a potent immunosuppressive agent, is associated with systemic and renal vasoconstriction, leading to hypertension. The present study was conducted to elucidate the contribution of angiotensin II (Ang II) to CysA-induced hypertension and reactive oxygen species (ROS) generation. CysA (30 mg/kg per day SC), given for 3 weeks in rats, increased systolic blood pressure (SBP) from 119+/-2 to 145+/-3 mm Hg (n=7). Plasma and kidney Ang II levels were significantly higher in CysA-treated rats (136+/-10 fmol/mL and 516+/-70 fmol/g) than in vehicle-treated (1 mL olive oil) rats (76+/-10 fmol/mL and 222+/-21 fmol/g, n=7). CysA treatment increased AT1 receptor protein expression in the aorta (by 251+/-35%), whereas it was reduced in the kidney (by -32+/-4%). Superoxide anion production in aortic segments and kidney thiobarbituric acid-reactive substance (TBARS) contents were higher in CysA-treated rats (26+/-2 counts/min per milligram and 37+/-3 nmol/g) than in vehicle-treated rats (17+/-1 counts/min per milligram and 24+/-3 nmol/g). Concurrent administration of an AT1 receptor antagonist, valsartan (30 mg/kg per day, in drinking water), to CysA-treated rats (n=7) significantly decreased SBP (113+/-4 mm Hg) and prevented increases in vascular superoxide (16+/-2 counts/min per milligram) and kidney TBARS contents (21+/-3 nmol/g). Similarly, treatment with a superoxide dismutase mimetic, 4-hydroxy-2,2,6,6,-tetramethylpiperidine-N-oxyl (Tempol; 3 mmol/L in drinking water, n=7), prevented CysA-induced increases in SBP (115+/-3 mm Hg), vascular superoxide (16+/-1 counts/min per milligram), and kidney TBARS contents (19+/-2 nmol/g). These data suggest that ROS generation induced by augmented Ang II levels contributes to the development of CysA-induced hypertension.     

Mitochondrial redox components of human adrenocortical steroid hydroxylases under physiological conditions and in focal hyperplasia of the zone fasciculata.

Cytochrome P450, aa3 and adrenodoxin were characterized in human adrenocortical mitochondria. Human adrenodoxin demonstrated a distinctive electron paramagnetic spectrum exhibiting anisotropic g values indicative of axial symmetry. The observed g values were (formula: see text). Adrenodoxin content of crude human adrenal mitochondria was 0.65 +/- 0.01 nmol/mg of mitochondrial protein and was in a stoichiometric 1:1 molar ration with cytochrome P450. Mitochondrial cytochrome P450 (0.62 nmol/mg mitochondrial protein) and aa3 (0.19 nmol/mg mitochondrial protein) levels were determined in a normal human adrenal A patient receiving ACTH (3d) demonstrated a doubling of P450 content, while cytochrome aa3 levels remained unchanged. ACTH plus hydrocortisone therapy (3d) increased both P450 and aa3 levels, however 24 h hydrocortisone therapy alone was without effect. An area of focal hyperplasia of the zona fasciculata also demonstrated P450 and aa3 levels elevated above the contralateral and normal human adrenal levels. The possibility of hormonal control of human adrenal mitochondrial cytochromes P450 and aa3 is suggested.     

Expression of TIMP-1 and TIMP-2 in rats with hepatic fibrosis

AIM: To investigate the location and expression of TIMP-1 and TIMP-2 in the liver of normal and experimental hepatic fibrosis in rats. METHODS: The rat models of experimental immunity hepatic fibrosis (n=20) were prepared by the means of immunologic attacking with human serum albumin (HSA),and normal rats (n=10) served as control group. Both immunohistochemistry and in situ hybridization methods were respectively used to detect the TIMP-1 and TIMP-2 mRNA and related antigens in liver. The liver tissue was detected to find out the gene expression of TIMP-1 and TIMP-2 with RT-PCR. RESULTS: The TIMP-1 and TIMP-2 related antigens in livers of experimental group were expressed in myofibroblasts and fibroblasts (TIMP-1: 482&#177;65 vs 60&#177;20; TIMP-2:336&#177;48 vs 50&#177;19, P&lt;0.001). This was the most obvious in portal area and fibrous septum. The positive signals were located in cytoplasm, not in nucleus. Such distribution and location were confirmed bysitu hybridization (TIMP-1/β-actin: 1.86&#177;0.47 vs 0.36&#177;0.08; TIMP-2/β-actin: 1.06&#177;0.22 vs 0.36&#177;0.08,P&lt;0.001). The expression of TIMP-1 and TIMP-2 was seen in the liver of normal rats, but the expression level was very low. However, the expression of TIMP-1 and TIMP-2 in the liver of experimental group was obviously high. CONCLUSION: In the process of hepatic fibrosis, fibroblasts and myofibroblasts are the major cells that express TIMPs.The more serious the hepatic fibrosis is in the injured liver,the higher the level of TIMP-1 and TIMP-2 gene expression.