Downregulation of hepatic cytochrome P450 in chronic renal failure

Chronic renal failure (CRF) is associated with a decrease in drug metabolism. The mechanism remains poorly understood. The present study investigated the repercussions of CRF on liver cytochrome P450 (CYP450). Three groups of rats were defined: control, control paired-fed, and CRF. Total CYP450 activity, protein expression of several CYP450 isoforms as well as their mRNA, and the in vitro N-demethylation of erythromycin were assessed in liver microsomes. The regulation of liver CYP450 by dexamethasone and phenobarbital was assessed in CRF rats. Compared with control and control paired-fed rats, creatinine clearance was reduced by 60% (P: < 0.01) in CRF rats. Weight was reduced by 30% (P: < 0.01) in control paired-fed and CRF rats, compared with control animals. There was no difference in the CYP450 parameters between control and control paired-fed. Compared with control paired-fed rats, total CYP450 was reduced by 47% (P: < 0.001) in CRF rats. Protein expression of CYP2C11, CYP3A1, and CYP3A2 were considerably reduced (>40%, P: < 0.001) in rats with CRF. The levels of CYP1A2, CYP2C6, CYP2D, and CYP2E1 were the same in the three groups. Northern blot analysis revealed a marked downregulation in gene expression of CYP2C11, 3A1, and 3A2 in CRF rats. Although liver CYP450 was reduced in CRF, its induction by dexamethasone and phenobarbital was present. N-demethylation of erythromycin was decreased by 50% in CRF rats compared with control (P: < 0.001). In conclusion, CRF in rats is associated with a decrease in liver cytochrome P450 activity (mainly in CYP2C11, CYP3A1, and 3A2), secondary to reduced gene expression.     

Effect of low dose cyclosporine and sirolimus on hepatic drug metabolism in the rat1.

BACKGROUND: We examined the effect cyclosporine (CsA) and sirolimus (SRL) alone and in combination on hepatic cytochrome P450-mediated metabolism in rats. METHODS: Rats were given 1 mg/kg of CsA or 0.4 mg/kg of SRL alone or in combination via constant intravenous infusion. Renal function was evaluated at the end of treatment. Blood samples were obtained to estimate CsA and SRL concentrations. Hepatic microsomes were prepared for immunoblotting and catalytic assays. RESULTS: CsA alone did not alter serum creatinine levels. SRL given alone or in combination with CsA produced a significant increase in urine output without changes in fluid balance. Although CsA and SRL administered alone caused damage to renal proximal tubules, the two-drug combination dramatically increased the renal structural damage. CsA alone suppressed cytochrome P450 (CYP) 3A2 protein levels by 39% (P=0.012) and catalytic activity by 30% (P=0.042). SRL alone reduced catalytic activity by 38% (P=0.012). Combination therapy reduced both CYP3A2 levels by 55% (P<0.001) and catalytic activity by 55% (P=0.001). CYP2C11 protein expression or catalytic activity were not changed in any group. CYP2A1 protein expression and catalytic activity were both significantly reduced in rats given CsA or/and SRL. Steady-state CsA levels were increased during concurrent SRL dosing, however, SRL concentrations were not changed by CsA coadministration. CONCLUSIONS: Concurrent SRL dosing increases CsA concentrations due to inhibition of hepatic CYP3A2 protein expression. Nephrotoxicity caused by combination therapy is due to CsA elevating levels of SRL or by SRL itself. Concurrent administration of CsA and SRL in transplant patients should be performed with caution.     

The effect of erythromycin and fluvoxamine on the pharmacokinetics of intravenous lidocaine

Inhibitors of CYP3A4 (cytochrome P450 3A4) have a minor effect on lidocaine pharmacokinetics. We studied the effect of coadministration of the antidepressant fluvoxamine (CYP1A2 inhibitor) and antimicrobial drug erythromycin (CYP3A4 inhibitor) on lidocaine pharmacokinetics in a double-blind, randomized, three-way crossover study. Nine volunteers ingested daily 100 mg fluvoxamine and placebo, 100 mg fluvoxamine and 1500 mg erythromycin, or their corresponding placebos for 5 days. On day 6, 1.5 mg/kg lidocaine was administered IV over 60 min. Concentrations of lidocaine and its major metabolite monoethylglycinexylidide were measured for 10 h. Fluvoxamine alone decreased the clearance of lidocaine by 41% (P < 0.001) and prolonged its elimination half-life from 2.6 to 3.5 h (P < 0.01). During the combination of fluvoxamine and erythromycin, lidocaine clearance was 53% smaller than during placebo (P < 0.001) and 21% smaller than during fluvoxamine alone (P < 0.05). During the combination phase the half-life of lidocaine (4.3 h) was longer than during the placebo (2.6 h; P < 0.001) or fluvoxamine (3.5 h; P < 0.01). We conclude that inhibition of CYP1A2 by fluvoxamine considerably reduces elimination of lidocaine and may increase the risk of lidocaine toxicity. Concomitant use of both fluvoxamine and a CYP3A4 inhibitor such as erythromycin can further increase plasma lidocaine concentrations by decreasing its clearance.     

CYP3A4 and P-Glycoprotein Activity in Healthy Controls and Transplant Patients on Cyclosporin vs. Tacrolimus vs. Sirolimus

This study aimed to determine the impact of maintenance immunosuppressive therapy with cyclosporin A (CsA), tacrolimus (FK506) and sirolimus (Rapa) on the in vivo activity of both intestinal and hepatic cytochrome P450 3A4 (CYP3A4) and P-glycoprotein (PGP) in renal transplant patients. The activity of these four elimination pathways was measured by the recently validated intravenous (iv) and per oral (po)14C erythromycin breath and urine test. In addition, overall hepatic P450 activity was measured by the (13)C aminopyrin breath test. Three groups of stable renal transplant patients on maintenance therapy with corticosteroids (CS) and mycophenolate mofetil (MMF) plus either CsA or FK506 or Rapa were examined. A significant increase in intestinal CYP3A4 activity and a significant decrease in hepatic and intestinal PGP activity was seen in patients on CsA in comparison with those on FK506 or Rapa (p < 0.01). A similar analysis in six healthy volunteers at baseline and after intake of CsA, FK506 and Rapa confirmed the results seen in the patients. There was no difference in CYP3A4 and PGP activity in the patients taking either FK506 or Rapa and healthy controls. These data suggest that a different pattern of drug interactions might be expected in patients treated with CsA vs. FK506/Rapa.     

Cryopreserved Porcine Hepatocytes: Expression and Induction of Cytochrome P450, Isoform CYP2E1

Cryopreservation of porcine hepatocytes for their use in bioartificial liver devices may result in reduced cytochrome P450 (CYP) enzyme activity. The aim of this study was to assess the effects of several CYP inducers on the isoform CYP2E1 protein expression in cryopreserved porcine hepatocytes. Isolated porcine hepatocytes were cryopreserved for 1 month, thawed, and cultured for 3 days. During medium culture, the hepatocytes were exposed to the following CYP inducers: dimethyl sulfoxide, rifampicin, phenobarbital, 3-methylcholanthrene, and dexamethasone. CYP2E1 protein expression was determined by immunoblotting. CYP2E1 protein levels were constantly detected in cryopreserved porcine hepatocytes. CYP inducers did not modify CYP2E1 protein levels. Long-term cryopreserved porcine hepatocytes preserved their capacity for CYP2E1 protein expression, although exposure of these hepatocytes to CYP inducers did not modify the CYP2E1 protein expression.     

Metabolic interaction between cyclosporine and sirolimus

BACKGROUND: When the immunosuppressants cyclosporine (CsA) and sirolimus (SRL) are co-administered to transplant patients, lower doses are used than when either drug is given alone. Since both drugs share similar transport and metabolic pathways, there is the potential for an interaction leading to unpredictable effects. Furthermore, both drugs affect the activity of cytochrome P450 3A1/2 (CYP3A1/2), the rat parallel to human CYP3A4, and the multidrug transporter P-glycoprotein (Pgp). METHODS: To clarify the role of metabolic enzymes and membrane transporters involved in the disposition of both drugs, we examined hepatic CYP3A1/2, Pgp, and multidrug resistance gene (mdr) mRNA during chronic therapy with CsA and SRL in salt-depleted rats. Specifically, rats were given intravenous doses of CsA 2.5 mg/kg and SRL 1 mg/kg, alone or in combination, for two weeks via constant rate intravenous infusion. RESULTS: CsA treatment inhibited hepatic CYP3A1/2 protein expression, catalytic activity, and mRNA levels. SRL dosing suppressed CYP3A1/2 protein expression and catalytic activity, without affecting mRNA. With combined dosing, however, there was a much greater reduction. Hepatic Pgp protein levels were elevated after treatment with either drug alone, as well as with combined dosing. Compared to controls, there were significant increases in mdr1a and mdr1b mRNA levels in all treatment groups, with the combined drugs causing the greatest increase. CONCLUSIONS: Both CYP3A1/2 and Pgp participate in the disposition of CsA and SRL in rats. Changes in the individual activities of CYP3A1/2 and Pgp may contribute to an interaction between CsA and SRL resulting in unanticipated effects during chronic therapy.     

Alterations of CYP3A4 and P-glycoprotein activity in vivo with time in renal graft recipients

BACKGROUND: Catabolism by intestinal and hepatic cytochrome P450 3A4 (CYP3A4), and excretion by P-glycoprotein (PGP), has a major influence on oral bioavailability of calcineurin inhibitors. In this study, the activity of intestinal and hepatic CYP3A4 and PGP in vivo was assessed in renal transplant recipients during the first year after transplantation (Tx). METHODS: Stable Caucasian renal transplant patients were tested at 1 week, 3 months, and 1 year after Tx, and compared with the results obtained in drug-free healthy volunteers. Intestinal and hepatic CYP3A4 and PGP activity were determined by measurement of (14)C-excretion dynamics in breath and urine after oral and intravenous administration of [N-methyl-(14)C]-erythromycin. RESULTS: Compared with 1 week after Tx, intestinal and hepatic CYP3A4 activity significantly decreased at 3 months and 1 year after Tx (-33% and -45%; -7% and -33%, respectively). Compared with the healthy volunteers, intestinal and hepatic CYP3A4 activity of the patients was significantly increased at 1 week after Tx, but normalized at 1 year after Tx. A similar pattern, though not significant, was seen for intestinal PGP activity. CONCLUSION: Phenotypic expression of hepatic and intestinal CYP3A4 was increased immediately after Tx, but gradually decreased to basal levels toward the end of the first year after Tx. The most plausible explanation for this evolution was the tapering of corticosteroid (CS) doses. These findings may also explain the increasing bioavailability of tacrolimus with time after Tx.     

细胞色素P450芳香化酶在大鼠多囊卵巢中的表达及意义

目的检测细胞色素P450芳香化酶(P450 arom)在大鼠多囊卵巢颗粒细胞中的表达水平,探讨P450 arom与多囊卵巢综合征(PCOS)的关系。方法应用半定量逆转录-聚合酶链反应(RT-PCR)检测PCOS实验组及正常对照组大鼠卵巢组织中P450 arom基因(CYP19)的表达;采用链霉素抗生物素蛋白-过氧化物酶(SP)法对大鼠卵巢组织中P450 arom进行检测。结果实验组和对照组卵巢组织中CYP19基因,相对表达强度分别为(0.138±0.072)和(0.759±0.236),差别有显著性意义(P<0.01);实验组中原始卵泡、初级卵泡和成熟卵泡比较,P450 arom蛋白表达下降(P<0.05)。对照组间初级卵泡、次级卵泡和成熟卵泡比较,arom蛋白表达有显著性差异(P<0.05)。结论P450 arom mRNA在PCOS大鼠卵巢组织中呈低表达;P450 arom蛋白在PCOS大鼠卵巢初级卵泡和成熟卵泡中表达下降;PCOS的发生可能与P450 arom在mRNA和蛋白水平的表达下调有关。     

Role of parathyroid hormone in the downregulation of liver cytochrome P450 in chronic renal failure

Chronic renal failure (CRF) is associated with a decrease in drug metabolism secondary to a decrease in liver cytochrome P450 (P450). The predominant theory to explain this decrease is the presence of factors in the blood of uremic patients. This study tested the hypothesis that parathyroid hormone (PTH) could be this factor. The objectives of this study were to determine (1) the role of PTH in the downregulation of hepatocyte P450 induced by rat uremic serum, (2) the role of PTH in the downregulation of liver P450 in rats with CRF, and (3) the effects of PTH on P450 in hepatocytes. For this purpose, (1) hepatocytes were incubated with serum from rat with CRF that was depleted with anti-PTH antibodies or with serum from parathyroidectomized (CRF-PTX) rat with CRF, (2) the effect of PTX on liver P450 was evaluated in rats with CRF, and (3) the effects of PTH on P450 in hepatocytes were determined. The depletion of PTH from CRF serum completely reversed the downregulating effect of CRF serum on P450 in hepatocytes. Addition of PTH (10(-9) M) to depleted CRF serum induced a decrease in P450 similar to nondepleted CRF serum. The serum of CRF-PTX rats had no effect on P450 in hepatocytes compared with CRF serum. Adding PTH to CRF-PTX serum induced a similar decrease in P450 as obtained with CRF serum. Finally, PTX prevented the decrease of liver P450 in rats with CRF. In summary, PTH is the major mediator implicated in the downregulation of liver P450 in rats with CRF.     

Cytochrome P450 2E1 inhibition prevents hepatic carcinogenesis induced by diethylnitrosamine in alcohol-fed rats

Chronic alcohol ingestion increases hepatic cytochrome P450 2E1 (CYP2E1), which is associated with hepatocarcinogenesis. We investigated whether treatment with chlormethiazole (CMZ), a CYP2E1 inhibitor, protects against alcohol-associated hepatic carcinogenesis in rats. Rats were fed either an ethanol liquid diet or a non-ethanol liquid diet, with or without CMZ for one and ten months. A single intraperitoneal injection of diethylnitrosamine (DEN, 20 mg/kg) was given to initiate hepatic carcinogenesis. CYP2E1 expression, inflammatory proteins, cell proliferation, protein-bound 4-HNE, etheno-DNA adducts, 8-hydroxy-2''-deoxyguanosine (8-OHdG), retinoid concentrations, and hepatic carcinogenesis were examined. Ethanol feeding for 1 month with DEN resulted in significantly increased hepatic CYP2E1 levels and increased nuclear accumulation of NF-κB protein and TNF-α expression, which were associated with increased cyclin D1 expression and p-GST positive altered hepatic foci. All of these changes induced by ethanol feeding were significantly inhibited by the one month CMZ treatment. At 10-months of treatment, hepatocellular adenomas were detected in ethanol-fed rats only, but neither in control rats nor in animals receiving ethanol and CMZ. The 8-OHdG formation was found to be significantly increased in ethanol fed animals and normalized with CMZ treatment. In addition, alcohol-reduced hepatic retinol and retinoic acid concentrations were restored by CMZ treatment to normal levels in the rats at 10 months of treatment. These data demonstrate that the inhibition of ethanol-induced CYP2E1 as a key pathogenic factor can counteract the tumor-promoting action of ethanol by decreasing TNF-α expression, NF-κB activation, and oxidative DNA damage as well as restoring normal hepatic levels of retinoic acid in DEN-treated rats.Key Words: Ethanol, hepatic carcinogenesis, cytochrome P450 2E1, NF-κB, cell proliferation     

Differential effects of traumatic brain injury on the cytochrome p450 system: a perspective into hepatic and renal drug metabolism

Traumatic brain injury is known to cause several secondary effects, one of which is altered drug clearance. Given the fact that patients who sustain TBI are subsequently treated with a variety of pharmacological agents for the purpose of either neuroprotection or physiological support, it is imperative to clarify changes in expression and/or activities of enzymes involved in clearing drugs. The mixed function oxidase system, which consists of cytochrome P450 and cytochrome P450 reductase, plays a vital role in phase I drug metabolism. This paper addresses the issue as to what extent TBI affects the levels and activity of various rat CYP450 subfamilies. Our results show that TBI induces tissue-specific and time-dependent alterations. Total hepatic CYP450 content showed a biphasic response with a decrease seen at 24 h followed by an increase at 2 weeks. CYP450 reductase, in contrast, showed an opposite temporal profile. Immunoblot analyses and marker substrate metabolism demonstrated a clear decrease in hepatic CYP1A levels while a significant increase in kidney was seen at both 24 h and 2 weeks. A dramatic induction of CYP3A was evident at 2 weeks in liver, while no changes were noticed in CYP2B or CYP2D subfamilies. CYP4F subfamily showed induction in kidney only. Collectively, the data reveal the differential effects of TBI on hepatic and renal drug metabolism.     

Protective effect of 20-hydroxyeicosatetraenoic acid (20-HETE) on glomerular protein permeability barrier

BACKGROUND: Proteinuria is a significant problem in medicine today, although glomerular events underlying it are unknown. Products of cytochrome P450 (CYP450) pathway of arachidonic acid metabolism are increasingly recognized as playing major roles in renal function. We used in vitro albumin permeability (P(alb)) as a measure of injury and puromycin aminonucleoside (PAN) as an injurious agent to test the hypothesis that 20-hydroxyeicosatetraenoic acid (20-HETE) protects the glomerular filtration barrier from increased P(alb). METHODS: We determined P(alb) in the following experimental groups: (1) isolated rat glomeruli incubated with PAN (5 microg/mL) for 5, 15, 30 or 60 minutes; (2) isolated glomeruli preincubated with 20-HETE (1.0 nmol/L to 100 nmol/L) for 15 minutes followed by additional incubation with PAN (5 microg/mL) for 15 minutes; (3) isolated glomeruli from rats treated with the CYP450 4A inducer clofibrate, and incubated with PAN (5 microg/mL) for 15 minutes; and (4) appropriate controls for each group. CYP450 4A levels were measured in glomeruli isolated from rats treated with clofibrate or vehicle. RESULTS: PAN increased P(alb) of isolated glomeruli as early as 5 minutes (P(alb) 0.33 +/- 0.21, P < 0.05 vs. control). Maximal effect occurred at 30 minutes (P(alb) 0.75 +/- 0.16, P < 0.001 vs. control). Inclusion of 20-HETE (100 nmol/L) blocked the increased P(alb) caused by PAN (P(alb) 0.05 +/- 0.13). Likewise, glomeruli isolated from rats treated with clofibrate were protected from PAN-induced increase in P(alb) (P(alb) 0.19 +/- 0.03). Treatment with clofibrate significantly increased glomerular CYP450 4A expression. CONCLUSION: PAN directly and immediately affects the glomerular permeability barrier. Furthermore, exogenous 20-HETE or clofibrate treatment protects glomeruli from increased P(alb) caused by PAN. Relative lack of 20-HETE may be a general characteristic of proteinuric states. Conversely, measures used to treat and/or prevent proteinuria may act to restore or increase glomerular 20-HETE levels.     

大鼠无肝状态下七氟醚代谢相关药酶细胞色素P450 2E1的表达

目的 探讨七氟醚代谢相关药酶细胞色素P450 2E1(CYP2E1)在肝移植无肝期肝外组织的表达变化.方法 24只SD大鼠随机均分为无肝期30 min组(A组)和60 min组(B组)和对照组(C组),各组均吸入七氟醚麻醉.A和B组通过阻断肝门模拟肝移植无肝期,相应时间终点截取各组大鼠肝外组织(小肠、肺脏和肾脏),C组麻醉后直接处死,截取大鼠肝外组织(小肠、肺脏和肾脏)应用RT-PCR、Western blot和免疫组化检测七氟醚代谢相关药酶CYP2E1在其中的表达.结果 A和B组肝外各组织中CYP2E1的表达显著强于C组(P<0.05);CYP2E1在小肠和肾脏的单一细胞中表达,而在肺脏的多种细胞中表达.结论 大鼠无肝状态下,CYP2E1在肝外器官组织中表达增多,可能促进七氟醚的肝外代谢;肺脏可能是最主要的肝外代谢器官.     

The effect of selective inhibition of inducible nitric oxide synthase on cytochrome P450 after liver transplantation in a rat model

BACKGROUND: Activity levels of cytochrome P450 (CYP) provide markers for liver function and graft rejection episodes after orthotopic liver transplantation (OLT). Some in vitro studies have shown decreased CYP activation of inducible nitric oxide synthase (iNOS) in rejecting liver grafts. The aim of this study was to evaluate CYP isoenzyme activity changes in vivo and to examine histopathologic aspects during inhibition of iNOS after treatment with aminoguanidine (AG) using OLT in the rat. MATERIALS AND METHODS: Thirty DA-(RT1av1) rats that served as donors and LEWIS-(RT(1)) rats as recipients were divided into three groups: group I (controls, syngeneic rats; n = 6), group II (allogeneic rats without immunosupression; n = 11), and group III (allogeneic rats with AG treatment; n = 13). On postoperative days 5, 8, and 10 we performed laboratory investigations and liver biopsies for histopathologic investigations. RESULTS: On postoperative day 5, activities of CYP-1A1 and -3A4 were significantly lower (P = .022) in group III and the activity of CYP-1A2 higher (P < .05) compared with group II. At postoperative days 8 and 10, the activities of all CYP isoenzymes were significant higher in AG-treated rats (group III) in contrast with group II after allogeneic OLT without immunosuppression. Histopathologic findings revealed less distinct rejection signs in group III specimens after AG treatment compared with group II. CONCLUSION: Summarizing our results, we concluded that AG treatment led to increased CYP activity and less distinction of graft rejection after OLT in rats.     

Multi-drug resistance gene-1 (MDR-1) haplotypes and the CYP3A5*1 genotype have no influence on ciclosporin dose requirements as assessed by C0 or C2 measurements

The intestinal efflux pump P-glycoprotein (P-gp), the product of the multi-drug resistance-1 (MDR-1) gene, significantly influences the pharmacokinetics of several drugs. Ciclosporin is a substrate for P-gp and is metabolized by cytochrome P450 (CYP) 3A enzymes. P-gp activity is affected by several known single nucleotide polymorphisms (SNPs) and haplotypes. MDR-1 genotypes of SNPs C1236T, G2677T/A and C3435T, as well as haplotypes C-G-C and T-T-T and CYP3A5*1 genotype (predictive of CYP3A5 expression), were related to ciclosporin blood concentrations measured at both 0 and 2 h after drug dosing in 197 stable renal transplant patients. Significant differences (of a magnitude unlikely to be relevant clinically) in dose-normalized blood ciclosporin concentrations were found only between MDR-1 genotypes of the C1236T SNP and between haplotype groups C-G-C and T-T-T in patients that were expressers of CYP3A5. MDR-1 SNPs and haplotypes and also CYP3A5*1 genotype, do not appear to have a major influence on ciclosporin pharmacokinetics.     

Acidosis, not azotemia, stimulates branched-chain, amino acid catabolism in uremic rats

To investigate branched-chain, amino acid metabolism (BCAA) in muscle in chronic renal failure (CRF), we studied rats with moderately severe uremia (PUN 110 approximately mg/dl) and spontaneous metabolic acidosis (bicarbonate, 19 +/- 1 mEq/liter). Plasma BCAA levels in CRF compared to pair-fed control rats were approximately 15% lower and muscle valine was 93 microM lower (P less than 0.05). BCAA metabolism was measured in incubated epitrochlearis muscles using L-[1-14C]valine or L-[1-14C]leucine in the presence and absence of insulin. BCAA decarboxylation was increased (P less than 0.05) and insulin-stimulated BCAA incorporation into protein was blunted (P less than 0.05) by CRF. Since we have found that metabolic acidosis, by itself, stimulates muscle branched-chain, ketoacid dehydrogenase activity, another group of CRF and control rats was given NaHCO3 which corrected the acidosis, but not the azotemia. BCAA decarboxylation in muscle was reduced in CRF rats given NaHCO3, and this was reflected in increased plasma and muscle BCAA concentrations. We conclude that in CRF, chronic metabolic acidosis stimulates BCAA decarboxylation in skeletal muscle and this could contribute to the reduced intra- and extracellular concentrations of BCAA. Correction of acidosis should be a goal of therapy in CRF, especially when dietary regimens restrict intake of BCAA.     

Propofol inhibits renal cytochrone P450 activity and enflurance defluorinationin vitro in hamsters

PURPOSE: To determine the effect of propofol on renal cytochrome P450 activity and defluorination of enflurane. METHODS: Renal microsomes were prepared by homogenization and differential centrifugation from pooled hamster kidneys. Defluorination of enflurane was assessed by measuring free fluoride metabolites after reacting enflurane with renal microsomes incubated with various concentrations, 0.05 - 1.0 mmol x L(-1) propofol in the NADPH-generating system. Drug metabolizing activities of renal cytochrome P450 mono-oxygenase enzymes were evaluated within microsomes preincubated with propofol and reacted with the specific marker substrates, aniline, benzo(a)pyrene, erythromycin and pentoxyresorufin, for cytochrome P450 2E1, 1A1, 3A4 and 2B1, respectively. RESULTS: Renal defluorination of enflurane was inhibited by clinical concentrations, 0.05 mmol x L(-1) of propofol (P < 0.05). Dose-dependent inhibition of defluorination, aniline and benzo(a)pyrene hydroxylase within kidney microsomes was related to propofol concentration. Propofol demonstrated a profound inhibition of renal pentoxyresorufin dealkylase activity even at low concentrations, 0.05 mmol x L(-1) (P < 0.01). Propofol did not exhibit inhibition of erythromycin N-demethylation of kidney microsomes except at high concentration, 1.0 mmol x L(-1). Spectral analyses of key coenzymes of renal cytochrome P450 monooxygenase, cytochrome b5 and cytochrome c reductase, demonstrated an inhibition when incubated with high concentrations of propofol (P < 0.05). CONCLUSION: In an in vitro study in an NADPH-generating system of hamster kidney microsomes, propofol, in clinical concentrations, exhibited a broad-spectrum of inhibition to renal monooxygenase activities and enflurane defluorination.     

胰岛素样生长因子1对慢性肾功能衰竭大鼠骨骼肌蛋白质代谢的影响

目的 探讨胰岛素样生长因子1（IGF-1）在慢性肾功能衰竭（CRF）大鼠骨骼肌蛋白质代谢上的作用。方法 用放射免疫分析法测定CRF大鼠和配对喂养的假手术（SO）对照大鼠血清及骨骼肌中IGF-1的含量：计算骨骼肌蛋白质的基础合成率及分解率；评估重组人胰岛素样生长因子-1（rhIGF-1）对骨骼肌蛋白质合成与分解的影响。结果（1）CRF组血清和骨骼肌中IGF-1含量明显低于SO组；（2）CRF组骨骼肌基础蛋白合成率显著低于SO组，基础蛋白分解率显著高于SO组；（3）rhIGF-1对CRF大鼠骨骼肌蛋白质合成的促进作用仅为SO大鼠的25%～44%，对其分解的抑制作用仅为SO大鼠的15%～42%。结论CRF时，血清及骨骼肌的IGF-1含量的减少和骨骼肌对IGF-1促进蛋白质合成代谢作用的抵抗可能是白质合成减少、分解增强，导致营养不良、肌肉萎缩的主要原因。     

Increased epoxyeicosatrienoic acid formation in the rat kidney during liver cirrhosis

Vascular complications during liver cirrhosis are often severe, particularly in the kidney. These complications are the result of complex and poorly understood interactions between the injured liver and other organs such as the lungs, heart, and kidney. The purpose of this study was to investigate the alterations to renal hemodynamics during cirrhosis, focusing on the actions of epoxyeicosatrienoic acids (EET), known to be potent regulators of renal hemodynamics. Cirrhosis was induced in rats by common bile duct ligation (CBDL), and they were compared with sham rats. Experiments were conducted 4 wk after either the sham or CBDL surgery. Vasoreactivity was assessed in isolated perfused kidneys. cPLA(2) expression and cytochrome P450 (CYP450) expression were measured using Western blot. cPLA(2) enzymatic activity was measured by radioenzymatic assay. EET production was measured using rpHPLC analysis. The major findings were that kidneys from CBDL rats had significantly greater acetylcholine-induced vasodilation that was partially blocked by nitric oxide (NO) and prostaglandin inhibition and fully blocked by the combined inhibition of NO, prostaglandins, and CYP450 metabolites. Expression and activity of cPLA(2) in CBDL kidneys was increased, providing arachidonic acid substrate to the CYP450 enzymes. Finally, expression and activity of CYP450 enzymes was elevated in CBDL kidneys, resulting in significantly greater production of the vasodilating 11,12-EET and 14,15-EET. While it is well documented that renal vasoconstriction leading to impaired renal function occurs during cirrhosis, our data clearly demonstrate that endogenous production of EET is increased in cirrhotic kidneys. This may be a homeostatic response to preserve renal perfusion.     

CYP3A4 is a human microsomal vitamin D 25-hydroxylase.

The human hepatic microsomal vitamin D 25-hydroxylase protein and gene have not been identified with certainty. Sixteen hepatic recombinant microsomal enzymes were screened for 25-hydroxylase activity; 11 had some 25-hydroxylase activity, but CYP3A4 had the highest activity. In characterized liver microsomes, 25-hydroxylase activity correlated significantly with CYP3A4 testosterone 6beta-hydroxylase activity. Activity in pooled liver microsomes was inhibited by known inhibitors of CYP3A4 and by an antibody to CYP3A2. Thus, CYP3A4 is a hepatic microsomal vitamin D 25-hydroxylase. INTRODUCTION: Studies were performed to identify human microsomal vitamin D-25 hydroxylase. MATERIALS AND METHODS: Sixteen major hepatic microsomal recombinant enzymes derived from cytochrome P450 cDNAs expressed in baculovirus-infected insect cells were screened for 25-hydroxylase activity with 1alpha-hydroxyvitamin D2 [1alpha(OH)D2], 1alpha-hydroxyvitamin D3 [1alpha(OH)D3], vitamin D2, and vitamin D3 as substrates. Activity was correlated with known biological activities of enzymes in a panel of 12 characterized human liver microsomes. The effects of known inhibitors and specific antibodies on activity also were determined. RESULTS: CYP3A4, the most abundant cytochrome P450 enzyme in human liver and intestine, had 7-fold greater activity than that of any of the other enzymes with 1alpha(OH)D2 as substrate. CYP3A4 25-hydroxylase activity was four times higher with 1alpha(OH)D2 than with 1alpha(OH)D3 as substrate, was much less with vitamin D2, and was not detected with vitamin D3. 1alpha(OH)D2 was the substrate in subsequent experiments. In a panel of characterized human liver microsomes, 25-hydroxylase activity correlated with CYP3A4 testosterone 6beta-hydroxylase activity (r = 0.93, p < 0.001) and CYP2C9*1 diclofenac 4'-hydroxylase activity (r = 0.65, p < 0.05), but not with activity of any of the other enzymes. Activity in recombinant CYP3A4 and pooled liver microsomes was dose-dependently inhibited by ketoconazole, troleandomycin, isoniazid, and alpha-naphthoflavone, known inhibitors of CYP3A4. Activity in pooled liver microsomes was inhibited by antibodies to CYP3A2 that are known to inhibit CYP3A4 activity. CONCLUSION: CYP3A4 is a vitamin D 25-hydroxylase for vitamin D2 in human hepatic microsomes and hydroxylates both 1alpha(OH)D2 and 1alpha(OH)D3.