Segment-specific proximal tubule injury in tripterygium glycosides intoxicated rats.

Tripterygium glycosides (TG), extracted from the Chinese herb Tripterygium wilfordii Hook. f, is a widely used anti-inflammatory and immunosuppressive agent with definite nephrotoxicity. However, its toxic mechanism remained undiscovered. The aim of this study was to characterize the potential toxicity of TG on segments of proximal tubule in rats. Six hundred and 1200 mg/kg of TG was administered by daily intragastric instillation for 16 days. A significant reduction of p-aminohippurate accumulation by renal cortical slices indicated that TG damaged organic anion transporter (OAT) system that localized at the proximal tubule, especially S(2) segment. A dramatic loss of kidney glutamine synthetase (GS) activity induced by TG reflected S(3) segment damage. Because mRNA expression of OAT1, OAT3, and GS was decreased substantially, we ascribe the fall of p-aminohippurate accumulation and GS activity to alterations at the transcriptional level. A dose-related diminution of kidney glutathione S-transferase activity was noted simultaneously, suggesting oxidative stress involvement. Histopathological lesions were observed in the TG intoxicated rat kidney even though there were no obvious changes of serum urea and creatinine at this dose level. In summary, we provided evidences supporting that TG caused segment-specific dysfunction in the kidney proximal tubule.

Effects of 18alpha-glycyrrhizin on the pharmacodynamics and pharmacokinetics of glibenclamide in alloxan-induced diabetic rats.

This paper investigated the effects of 18alpha-glycyrrhizin (18alpha-GL) on the pharmacodynamics and pharmacokinetics of glibenclamide in experimental diabetic rats. 18alpha-GL (25 mg/kg) and/or glibenclamide (1 mg/kg) were given to alloxan-induced diabetic rats for consecutive 5 days. When the rats were co-treated with 18alpha-GL and glibenclamide, fasting plasma glucose concentration was further reduced, plasma insulin content and liver glycogen level were increased markedly as compared with glibenclamide-treated animals. Meanwhile, in co-treated group, elimination rate constant (Ke) of glibenclamide was reduced while peak plasma concentration (C(max)), area under the plasma concentration vs time curve (AUC(0-14 h)) and elimination half-life (T(1/2Ke)) were increased significantly vs glibenclamide alone administered rats. The activities of hepatic CYP3A and the markers of liver injury, plasma alanine aminotransferase (ALT) and aspartate aminotransferase (AST), were significantly decreased in rats treated with 18alpha-GL alone and in combination with glibenclamide. Results of immunohistochemistry showed that 18alpha-GL improved the effects of glibenclamide on the pathological morphology of pancreatic islet beta cells and the intensities of positive immunostaining for insulin. Our results revealed that 18alpha-GL led to the enhancement of the hypoglycemic effect of glibenclamide by inhibiting the activity of CYP3A; on the other hand, 18alpha-GL protected the pancreatic islet beta cells and liver from damage in diabetes which suggested that 18alpha-GL might be beneficial as an adjuvant drug of glibenclamide in a proper dose, especially to the diabetic patients associated with liver dysfunction.

Changes of multiple biotransformation phase I and phase II enzyme activities in human fetal adrenals during fetal development.

AIM: Fetal adrenal, which synthesizes steroid hormones, is critical to fetal growth and development. Our recent research showed that some xenobiotics could interfere with steroidogenesis and induce intrauterine growth retardation in rats. The study on the characteristics of biotransformation enzymes in fetal adrenals still seems to be important with respect to possible significance in xenobiotic-induced fetal development toxicity. In this study, the activities of several important xenobiotic-related phase I and phase II enzymes in human fetal adrenals were examined and compared with those in fetal livers. METHODS: The activity and mRNA expression were determined by enzymatic analysis and RT-PCR. RESULTS: The levels of cytochrome (CYP)2A6, CYP2E1, and CYP3A7 isozymes in fetal adrenals were 82%, 92%, and 33% of those in fetal livers, respectively. There was a good positive correlation between adrenal CYP2A6 activity and gestational time. The values of alpha glutathione S-transferase (GST), pi-GST, and microGST in adrenals were 0.5, 4.4, and 8.3-fold of those in the livers, respectively, and the activity of adrenal pi-GST was negatively correlated with gestational time. The uridine diphosphoglucuronyl transferase activities, which were measured using p-hydroxy-biphenyl and 7-hydroxy-4-methylcoumarin as substrates, were 9% and 3%, respectively, of those in the fetal livers. CONCLUSION: Our investigation suggested that adrenal could be an important xenobiotic-metabolizing organ in fetal development and may play a potential role in xenobiotic-induced fetal development toxicity.

Expression of epoxygenases belonging to CYP2 family in rat myocardial ischemia/reperfusion injury in vivo.

OBJECTIVE: To elucidate the expression of epoxygenases belonging to cytochrome P-450 mono-oxygenases (CYP2) family in rat ischemic myocardium at varying reperfusion periods, and the effect of epoxygenase inhibition on the post-ischemic heart. METHODS: The current study was conducted in the Department of Pharmacology, Medical College of Wuhan University, China, between September 2004 and June 2005. Rats were subjected to 40 minutes of myocardial ischemia, followed by 0, 15, 60, and 180 minutes of reperfusion. Superoxide generation was assayed by confocal microscopy, CYP2B1/2, 2C6, 2E1, 2J3 gene expressions were determined by reverse transcriptase polymerase chain reaction. Fourteen, 15-dihydroxyeicosatrienoic acid (DHET) concentration was measured by enzyme-linked immunosorbent assay. The effects of the CYP epoxygenase inhibitor N-methylsulphonyl-6-(2-propargyloxyphenyl) hexanamide (MS-PPOH) on myocardial damage and superoxide generation caused by 60 minutes of reperfusion were also evaluated. RESULTS: During myocardial ischemia/reperfusion, CYP2C6 and 2J3 mRNA expression were up-regulated with the peak level at 15 minutes of reperfusion; CYP2E1 gene expression decreased in a time dependent manner and reached the minimum level at 180 minutes of post-ischemia. Meanwhile, no obvious variations of CYP2B1/2 gene expression were detected during different reperfusion periods. Fourteen, 15-DHET significantly increased during reperfusion in ischemic hearts. The MS-PPOH pretreatment (15 mg/kg) effectively reduced myocardial damage and superoxide production. CONCLUSION: There are changes in gene expression of individual isozymes and an elevation of CYP epoxygenase activity involved in myocardial reperfusion injury in vivo. Epoxygenase inhibition plays a protective role in cardiac post-ischemic damage.

Fast evaluation of oxidative DNA damage by liquid chromatography-electrospray tandem mass spectrometry coupled with precision-cut rat liver slices.

OBJECTIVE: To establish a fast and sensitive method for the detection of 8-hydroxy-2'-deoxyguanosine (8-OHdG) in precision-cut rat liver slices by HPLC-MS/MS and to investigate isoniazid (INH) -induced oxidative DNA damage. METHODS: Precision-cut liver slices (300 microm) were prepared from male rats, and incubated with INH (0.018 mol/L) for 2 h after 1 h preincubation. DNA in the slices was extracted and digested into free nucleosides at 37 degrees C. The samples were injected into HPLC-MS/MS after the proteins were removed. The level of oxidative DNA damage was estimated using the ratio of 8-OHdG to deoxyguanosine (dG). RESULTS: The limit of detection of 8-OHdG was 1 ng/mL (S/N=3) and the intra-assay relative standard variation was 3.38% when one transition 284.3/168.4 was used as a quantifier and another two transitions 284.3/140.2, 306.1/190.2 as qualifiers. 8-OHdG and dG were well separated, as indicated by elution at 10.02 and 7.37 min, respectively. INH significantly increased the ratio of 8-OHdG to dG in rat liver slices (P<0.05). CONCLUSION: 8-OHdG in precision-cut liver slices could be sensitively determined by HPLC-MS/MS. HPLC-MS/MS coupled with precision-cut tissue slices is a fast and reliable analytical technique to evaluate oxidative DNA damage of target tissues caused by procarcinogens and cytotoxins.

Influences of 3-methylcholanthrene, phenobarbital and dexamethasone on xenobiotic metabolizing-related cytochrome P450 enzymes and steroidogenesis in human fetal adrenal cortical cells.

AIM: To explore the influence and possible mechanism of xenobiotics on adrenal steroidogenesis during fetal development. METHODS: Primary human fetal adrenal cortical cells were prepared, cultured and treated with 3-methylcholanthrene, phenobarbital and dexamethasone. The activities of 7-ethoxyresorufin O-dealkylase, benzphetamine, aminopyrine and erythromycin N-demethylases were measured by enzyme assays. At the same time, quantitative analysis of steroid hormones cortisol, aldosterone, testosterone and progesterone were carried out in cultural medium by radioimmunoassays. RESULTS: The activities of benzphetamine and aminopyrine N-demethylase were increased in the cultural fetal adrenal cells treated with phenobarbital (0.25-1 mmol/L) for 24 h. Dexamethasone (25-100 micromol/L) also increased the activity of erythromycin N-demethylase. The activity of 7-ethoxyresorufin O-dealkylase was undetected in the cells treated without and with 3-methylcholanthrene (0.5-2 micromol/L). Meanwhile, the contents of medium cortisol, aldosterone and progesterone were decreased after treatment with 3-methylcholanthrene. Cortisol, aldosterone and progesterone concentrations were also slightly decreased with phenobarbital. Dexamethasone enhanced the productions of cortisol and progesterone remarkably. The trend of testosterone concentration was uncertain after 3-methylcholanthrene, phenobarbital or dexamethasone treatment. CONCLUSION: 3-Methylcholanthrene, phenobarbital or dexamethasone could interfere with the synthesis of cortisol, aldosterone and progesterone in primary human fetal adrenal cortical cells, which likely act through xenobiotic metabolizing-related cytochrome P450 isoform activation.

[Activation of the immunologic function of rat Kupffer cells by the polysaccharides of Angelica sinensis].

AIM: To observe the influence of polysaccharides of Angelica sinensis (ASP) on the immunologic function of rat Kupffer cells. METHODS: Normal rat Kupffer cells were treated with ASP in vitro. Absorbance at 540 nm ( A540) of neutral red absorption and supernatant NO, TNF-alpha in the cells were measured to evaluate the immunologic function of Kupffer cells; LDH leakage was measured to estimate the severity of cellular damage; Rats were given ASP 0.025, 0.1, 0.25 and 1.0 g x kg(-1) ig (qd x 7 d) in vivo. The above indices and ACP of Kupffer cells were measured, sGST and sALT activity were detected as indices of hepatotoxicity. RESULTS: ASP markedly enhanced the phagocytic activity, ACP and supernatant NO, TNF-alpha of Kupffer cells both in vitro and in vivo . The increase of sGST was observed after administration of ASP 1.0 g x kg(-1), but the LDH leakage of the hepatocytes was not increased in vitro. CONCLUSION: ASP with suitable dose could activate the function of Kupffer cells. Slight liver injury was caused by ASP 1.0 g x kg(-1) in vivo, which was likely caused by factors, such as NO, TNF-alpha, indirectly.

CYP2E1 mediated isoniazid-induced hepatotoxicity in rats.

AIM: To investigate the role of CYP2E1 in isoniazid (INH)-induced hepatotoxicity and the influence of rifampicin (RFP) on INH-induced liver injury. METHODS: Rats were treated with INH alone (100 mg/kg, ip) or co-administered with RFP (100 mg/kg, ig) for 10 d and 21 d. Hepatotoxicity was assayed by plasma enzymes (sALT, sAST) and histopathological examinations. Hepatic CYP2E1 activity was measured by aniline hydroxylase (ANH), and CYP2E1 mRNA expression was determined by RT-PCR. Plasma hydrazine concentration was determined by RP-HPLC. RESULTS: For a 10 d INH-treatment, hepatic CYP2E1 level was increased to 3.7-fold over the control; liver impairment appeared after 21 d treatment, while CYP2E1 and plasma hydrazine were, respectively, increased to 4.6-fold and 1.7-fold. However, in INH-RFP group for 10 d, CYP2E1 and plasma hydrazine were, respectively, decreased by 13 % and 18 % over INH group; similarly, hepatic injury is equal to INH group appeared after 21 d, and CYP2E1 was further decreased by 26 %. Correlation analysis showed that sALT had a positive correlation with plasma hydrazine and with CYP2E1 activity; CYP2E1 activity was also markedly correlated with plasma hydrazine. And compared with control, there is no difference in changes of CYP2E1 mRNA expression in INH and INH-RFP treatment for 21 d. CONCLUSION: The metabolite of INH, hydrazine, plays an important role in INH-induced hepatotoxicity in rats. The induction of CYP2E1 by hydrazine is involved in the hepatotoxicity of INH. RFP does not exacerbate INH-induced hepatotoxicity in short term, which relates to down-regulation of CYP2E1.

[Effects of Angelica sinensis polysaccharides on hepatic drug metabolism enzymes activities in mice].

OBJECTIVE: To study the effects of Angelica sinensis Polysaccharides (ASP) on the hepatic drug metabolism enzymes activities in normal mice and those prednisolone (PSL)-induced liver injury. METHOD: The activities of phase II enzymes (GSH-related enzymes) and cytochrome P450 enzymes were measured by biochemical method. RESULT: ASP increased the activities of glutathione S-transferase in liver microsomes and mitochondria. The cytochrome P450 content, NADPH-cytochrome c reductase, aminopyrine N-demethylase, and aniline hydroxylase activities in liver microsomes were also increased. PSL significantly increased serum ALT levels, and decreased the liver mitochondrial glutathione content. At the same time, other enzymes activities were all increased. When mice were treated with ASP 2.0 g.kg-1, the PSL-induced changes on cytochrome P450 enzymes, glutathione S-transferase, and GSH content were restored. CONCLUSION: ASP can modulate the activities of drug metabolism enzymes.

[The effects of silymarin on hepatic microsomal and mitochondrial membrane fluidity in mice].

OBJECTIVE: To observe the effects of silymarin on hepatic microsomal and mitochondrial membrane fluidity in mice. METHOD: Liver microsomal and mitochondrial membranes were labled by ANS and DPH. Membrane fluorensent intensity (F), fluorensent polarization(P) and microviscosily(eta) of liver microsome and mitochondria were determined. RESULT: Sil increased the external membrane fluidities of liver microsome and mitochondria, and decreased the internal membrane fluidities of liver microsome and mitochondria. Pretreatment with CCl4, the external membrane fluidity of liver microsome and mitochondria were increased, and the internal membrane fluidities of liver microsome and mitochondria were decreased. After given sil 140,280 mg.kg-1, the increased external membrane fluidities of liver microsome and mitochondria were lowered, and the decreased internal membrane fluidities of liver microsome and mitochondria were enhanced in a dose-dependent manner. CONCLUSION: The protective effects of sil on liver injury may be related to the recovery of the membrane fluidities of liver microsome and mitochondria.

[Antagonistic effect of sodium ferulate on glycerol-induced renal oxidative injury in mice].

AIM: To investigate the effect of sodium ferulate (SF) on glycerol-induced renal injury. METHODS: Glycerol solution 50% was injected intramuscularly to establish a model of acute tubular necrosis in mice. SF was administered intraperitoneally at the dose of 100-200 mg.kg-1 at the beginning of establishing the model and its effect was observed by monitoring renal function, antioxidative functions and renal pathologic histology. RESULTS: At 6 and 72 h after glycerol injection, SF treatment (100-200 mg.kg-1) showed significant and dose-dependent antagonistic actions on the increment of blood urea nitrogen (BUN), creatinine (Cr), and N-acetyl-beta-glucosaminidase (NAG) induced by glycerol. The increase of renal malondialdehyde (MDA) content and the decrease of glutathione content, glutathione peroxidase (GSH-Px), glutathione S-transferase (GST), catalase (Cat) and superoxide dismutase (SOD) activities resulting from glycerol injection were remarkably inversed by SF at the dose of 200 mg.kg-1. Meanwhile, improvement of the renal histology was observed as well. CONCLUSION: SF showed beneficial effect on glycerol-induced acute tubular necrosis due to its antioxidative action.

In vitro metabolic characteristics of cytochrome P-450 2A6 in Chinese liver microsomes.

AIM: To investigate the metabolic characteristics of cytochrome P-450 C YP2A6 in human liver microsomes. METHODS: Cytochrome P-450 enzyme activities were measured by biochemical assays. Xenobiotics were employed to observe their effects on CYP2A6 in vitro. The kinetics of coumarin 7-hydroxylase was determined, and the correlation between CYP2A6 and UDP-glucuronosyltransferase (UGT) was analyzed. RESULTS: CYP2A6 activities of human liver microsomes were from 0.47 to 4.14 micromol . min-1 . g-1, with a 8.8-fold variation. The Km and Vmax of CYP2A6 ranged from 0.25 to 1.56 micromol/L and 1.41 to 8.70 micromol . min-1 . g-1, respectively. CYP2A6 activity was markedly inhibited (> 50 %) by pilocarpine, diethyldithio carbamic (DDC), and rifampicin, the IC50 was 5.31 micromol/L, 156.35 micromol/L, and 38.81 micromol/L, respectively. alpha-Naphthoflavone, sulfaphenazole, troleandomycin (TAO), ketoconazole, phenobarbital, prednisolone, and azithromycin had little or no effects on coumarin 7-hydroxylation. A significant correlation was observed between CYP2A6 and UGT2 (r = 0.9453, P < 0.05). CONCLUSION: CYP2A6 activity and kinetics exhibited a considerable variation in human liver microsomes in vitro, and a significant correlation was existed between CYP2A6 and phase II enzyme UGT2. Not only pilocarpine, CYP2A6 specific inhibitor, but also rifampicin and DDC inhibited CYP2A6 activity selectively.