Halothane-dependent lipid peroxidation in human liver microsomes is catalyzed by cytochrome P4502A6 (CYP2A6)

BACKGROUND: Halothane is extensively (approximately 50%) metabolized in humans and undergoes both oxidative and reductive cytochrome P450-catalyzed hepatic biotransformation. Halothane is reduced under low oxygen tensions by CYP2A6 and CYP3A4 in human liver microsome to an unstable free radical, and then to the volatile metabolites chlorodifluoroethene (CDE) and chlorotrifluoroethane (CTE). The free radical is also thought to initiate lipid peroxidation. Halothane-dependent lipid peroxidation has been shown in animals in vitro and in vivo but has not been evaluated in humans. This investigation tested the hypothesis that halothane causes lipid peroxidation in human liver microsomes, identified P450 isoforms responsible for halothane-dependent lipid peroxidation, and tested the hypothesis that lipid peroxidation is prevented by inhibiting halothane reduction. METHODS: Halothane metabolism was determined using human liver microsomes or cDNA-expressed P450. Lipid peroxidation was quantified by malondialdehyde (MDA) formation using high-pressure liquid chromatography-ultraviolet analysis of the thiobarbituric acid-MDA adduct. CTE and CDE were determined by gas chromatography-mass spectrometry. RESULTS: Halothane caused MDA formation in human liver microsomes at rates much lower than in rat liver microsomes. Human liver microsomal MDA production exhibited biphasic enzyme kinetics, similar to CDE and CTE production. MDA production was inhibited by the CYP2A6 inhibitor methoxsalen but not by the CYP3A4 inhibitor troleandomycin. Halothane-dependent MDA production was catalyzed by cDNA-expressed CYP2A6 but not CYP3A4 or P450 reductase alone. CYP2A6-catalyzed MDA production was inhibited by methoxsalen or anti-CYP2A6 antibody. CONCLUSIONS: Halothane causes lipid peroxidation in human liver microsomes, which is catalyzed by CYP2A6, and inhibition of halothane reduction prevents halothane-dependent lipid peroxidation in vitro.     

Halothane-induced lipid peroxidation and glucose-6-phosphatase inactivation in microsomes under hypoxic conditions

Halothane-induced lipid peroxidation was studied in microsomes from phenobarbital-pretreated male rats at defined steady state oxygen partial pressures (PO2). At PO2 less than 10 mmHg on addition of halothane to NADPH-reduced microsomes, significant increases in malondialdehyde (MDA) formation, oxygen uptake, and conjugated dienes were measured. At the maximum, near a PO2 of 1 mmHg, halothane induced the formation of about 0.75 nmol MDA X mg microsomal protein-1 X min-1; it also stimulated microsomal oxygen uptake twofold to threefold, and caused an almost threefold increase in conjugated diene absorption. Moreover, at this PO2 microsomal glucose-6-phosphatase lost about 70% of its activity. At PO2 greater than 10 mmHg, no significant effects of halothane on MDA formation, oxygen uptake, conjugated diene absorption, and glucose-6-phosphatase activity were observed; likewise under anaerobic conditions there was only a slight increase in conjugated dienes. The findings demonstrate that halothane induces microsomal lipid peroxidation at low PO2 and in the presence of particular cytochrome P-450 isoenzymes, and that the halothane-induced lipid peroxidation leads to severe microsomal lesions, as indicated by the loss of glucose-6-phosphatase activity.     

Demonstration of Halothane-induced Hepatic Lipid Peroxidation in Rats by Quantification of Flourine sub 2 -Isoprostanes

Background: Halothane can be reductively metabolized to free radical intermediates that may initiate lipid peroxidation. Hypoxia and phenobarbital pretreatment in Sprague-Dawley rats increases reductive metabolism of halothane. Flourine2 -isoprostanes, a novel measure of lipid peroxidation in vivo, were used to quantify halothane-induced lipid peroxidation in rats.

Methods: Rats were exposed to 1% halothane at 21% or 14% Oxygen2 for 2 h. Pretreatments included phenobarbital, isoniazid, or vehicle. Rats also were exposed to halothane, enflurane, and desflurane at 21% Oxygen2. Lipid peroxidation was assessed by mass spectrometric quantification of Flourine2 -isoprostanes.

Results: Exposure of phenobarbital-pretreated rats to 1% halothane at 21% Oxygen2 for 2 h caused liver and plasma Flourine2 -isoprostane concentrations to increase fivefold compared to nonhalothane control rats. This halothane-induced increase was enhanced by 14% Oxygen sub 2, but hypoxia alone had no significant effect. Alanine aminotransferase activity at 24 h was significantly increased only in the 1% halothane/14% Oxygen2 group. The effect of cytochrome P450 enzyme induction on halothane-induced Flourine2 -isoprostane production and liver injury was determined by comparing the effects of isoniazid and phenobarbital pretreatment with no pretreatment under hypoxic conditions. Halothane caused 4- and 11-fold increases in plasma and liver Flourine2 -isoprostanes, respectively, in non-pretreated rats, whereas isoniazid pretreatment had no effect. Phenobarbital pretreatment potentiated halothane-induced lipid peroxidation with 9- and 20-fold increases in plasma and liver Flourine2 -isoprostanes, respectively. Alanine aminotransferase activity was increased only in this group. At ambient oxygen concentrations, halothane but not enflurane or desflurane, caused Flourine2 -isoprostanes to increase.     

Cytochrome P-450 2B6 is responsible for interindividual variability of propofol hydroxylation by human liver microsomes

BACKGROUND: Oxidation of propofol to 4-hydroxypropofol represents a significant pathway in the metabolism of this anesthetic agent in humans. The aim of this study was to identify the principal cytochrome P-450 (CYP) isoforms mediating this biotransformation. METHODS: Propofol hydroxylation activities and enzyme kinetics were determined using human liver microsomes and cDNA-expressed CYPs. CYP-specific marker activities and CYP2B6 protein content were also quantified in hepatic microsomes for correlational analyses. Finally, inhibitory antibodies were used to ascertain the relative contribution of CYPs to propofol hydroxylation by hepatic microsomes. RESULTS: Propofol hydroxylation by hepatic microsomes showed more than 19-fold variability and was most closely correlated to CYP2B6 protein content (r = 0.904), and the CYP2B6 marker activities, S-mephenytoin N-demethylation (r = 0.919) and bupropion hydroxylation (r = 0.854). High- and intermediate-activity livers demonstrated high-affinity enzyme kinetics (K(m) < 8 microm), whereas low-activity livers displayed low-affinity kinetics (K(m) > 80 microm). All of the CYPs evaluated were capable of hydroxylating propofol; however, CYP2B6 and CYP2C9 were most active. Kinetic analysis indicated that CYP2B6 is a high-affinity (K(m) = 10 +/- 2 microm; mean +/- SE of the estimate), high-capacity enzyme, whereas CYP2C9 is a low-affinity (K(m) = 41 +/- 8 microm), high-capacity enzyme. Furthermore, immunoinhibition showed a greater contribution of CYP2B6 (56 +/- 22% inhibition; mean +/- SD) compared with CYP2C isoforms (16 +/- 7% inhibition) to hepatic microsomal activity. CONCLUSIONS: Cytochrome P-450 2B6, and to a lesser extent CYP2C9, contribute to the oxidative metabolism of propofol. However, CYP2B6 is the principal determinant of interindividual variability in the hydroxylation of this drug by human liver microsomes.     

Metabolism of a New Local Anesthetic, Ropivacaine, by Human Hepatic Cytochrome P450

Background: Ropivacaine is a local anesthetic with a long duration of action. Although it is less toxic than bupivacaine, local anesthetic toxicity is possible when the plasma concentration is increased. Because ropivacaine is an amide-type local anesthetic, it is metabolized by cytochrome P450 (P450) in the liver, and its elimination and plasma concentration can be dependent on the level of P450. The purpose of this investigation was to elucidate the metabolism of ropivacaine by human hepatic P450.

Methods: The metabolism of ropivacaine was compared using recombinant human and purified rat hepatic P450 isozymes. An inhibition study using antibodies against rat P450 was performed using hepatic microsomes from human and rat to identify which P450s are involved in ropivacaine metabolism.

Results: Ropivacaine was metabolized to 2',6'-pipecoloxylidide (PPX), 3'-hydroxyropivacaine (3'-OH Rop), and 4'-hydroxyropivacaine (4'-OH Rop) by hepatic microsomes from human and rat. PPX was a major metabolite of both human and rat hepatic microsomes. In a reconstituted system with rat P450, PPX was produced by CYP2C11 and 3A2, 4'-OH Rop by CYP1A2, and 3'-OH Rop by CYP1A2 and 2D1. Formation of PPX in rat hepatic microsomes was inhibited by anti CYP3A2, but not by CYP2C11 antibody, and formation of 3'-OH Rop was inhibited by CYP1A2 and 2D1 antibodies. Anti CYP3A2 and 1A2 antibodies inhibited the formation of PPX and 3'-OH Rop in human hepatic microsomes, respectively. Recombinant human P450s expressed in lymphoblast cells were used for further study. CYP3A4 and 1A2 formed the most PPX and 3'-OH Rop, respectively. Ropivacaine N-dealkylation and 3'-hydroxylation activities correlated well with the level of CYP3A4 and 1A2 in human hepatic microsomes, respectively.     

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.     

Cytochrome P-450 2B6 Is Responsible for Interindividual Variability of Propofol Hydroxylation by Human Liver Microsomes

Background: Oxidation of propofol to 4-hydroxypropofol represents a significant pathway in the metabolism of this anesthetic agent in humans. The aim of this study was to identify the principal cytochrome P-450 (CYP) isoforms mediating this biotransformation.

Methods: Propofol hydroxylation activities and enzyme kinetics were determined using human liver microsomes and cDNA-expressed CYPs. CYP-specific marker activities and CYP2B6 protein content were also quantified in hepatic microsomes for correlational analyses. Finally, inhibitory antibodies were used to ascertain the relative contribution of CYPs to propofol hydroxylation by hepatic microsomes.

Results: Propofol hydroxylation by hepatic microsomes showed more than 19-fold variability and was most closely correlated to CYP2B6 protein content (r = 0.904), and the CYP2B6 marker activities, S-mephenytoin N-demethylation (r = 0.919) and bupropion hydroxylation (r = 0.854). High- and intermediate-activity livers demonstrated high-affinity enzyme kinetics (Km < 8 [mu]m), whereas low-activity livers displayed low-affinity kinetics (Km > 80 [mu]m). All of the CYPs evaluated were capable of hydroxylating propofol; however, CYP2B6 and CYP2C9 were most active. Kinetic analysis indicated that CYP2B6 is a high-affinity (Km = 10 +/- 2 [mu]m; mean +/- SE of the estimate), high-capacity enzyme, whereas CYP2C9 is a low-affinity (Km = 41 +/- 8 [mu]m), high-capacity enzyme. Furthermore, immunoinhibition showed a greater contribution of CYP2B6 (56 +/- 22% inhibition; mean +/- SD) compared with CYP2C isoforms (16 +/- 7% inhibition) to hepatic microsomal activity.     

Stimulatory effects of halothane and isoflurane on fluoride release and cytochrome P-450 loss caused by metabolism of 2-chloro-1,1-difluoroethene, a halothane metabolite

The structural similarity of the halothane metabolite, 2-chloro-1,1-difluoroethene (CDE), to haloethenes that are metabolized by and inactivate cytochrome P-450, suggests that CDE may undergo secondary metabolism and degrade these isozymes. This possibility was examined in hepatic microsomes by determining fluoride release and cytochrome P-450 loss due to CDE metabolism in the presence of several anesthetics. CDE alone decreased cytochrome P-450 from phenobarbital-treated rats by as much as 37%, but the addition of isoflurane or halothane to incubations containing CDE increased the loss of cytochrome P-450 nearly twofold. Fluoride release was enhanced approximately 2.5 to 3 times by halothane or isoflurane; however, fluroxene inhibited fluoride release and did not enhance the loss of cytochrome P-450. Extrapolation of these results to the clinical situation suggests that the metabolism of CDE produced during halothane anesthesia and the accompanying cytochrome P-450 loss may contribute to the inhibition of drug metabolism produced by halothane.     

Effects of halothane, isoflurane, and sevoflurane on lipid peroxidation following experimental closed head trauma in rats

Background: In a rat closed head trauma model we examined both the time course of lipid peroxidation and the effects of halothane, isoflurane, and sevoflurane on it by analysis of malondialdehyde (MDA) formation.
Methods: Animals were divided randomly into five groups: sham-operated (SO), n =18; control-closed head trauma to left frontal pole, n =18; closed head trauma model+halothane, n =18; closed head trauma model+isoflurane, n =18; and closed head trauma model+sevoflurane, n =18. Halothane, isoflurane, or sevoflurane were applied 15 min after trauma for 30 min. Rats were euthanized 1,3, and 5 h after the inhalation agents. Brain tissue samples were taken 5 mm from the left and right frontal poles. MDA was considered to reflect the degree of lipid peroxidation.
Results: MDA concentrations were greater in the control, halothane, sevoflurane, and isoflurane groups than in SO animals ( P <0.001). No statistical difference between the hemispheres was found between the halothane, isoflurane, or sevoflurane groups, but MDA levels were lower with isoflurane than in the halothane, sevoflurane, and control groups at 1, 3, and 5 h ( P <0.001). MDA levels were higher as compared with the halothane and sevoflurane groups at 1 h but not at 3 or 5 h ( P <0.001).
Conclusion: MDA levels with the isoflurane group were lower than in the other trauma groups, which suggest that isoflurane, given after closed head trauma, might be protective against lipid peroxidation of cerebral injury.     

Fentanyl inhibits metabolism of midazolam: competitive inhibition of CYP3A4 in vitro

Fentanyl decreases clearance of midazolam administered i.v., but the mechanism remains unclear. To elucidate this mechanism, we have investigated the effect of fentanyl on metabolism of midazolam using human hepatic microsomes and recombinant cytochrome P450 isoforms (n = 6). Midazolam was metabolized to l'-hydroxymidazolam (l'-OH MDZ) by human hepatic microsomes, with a Michaelis-Menten constant (K(m)) of 5.0 (SD 2.7) microgramsmol litre-1. Fentanyl competitively inhibited metabolism of midazolam in human hepatic microsomes, with an inhibition constant (Ki) of 26.8 (12.4) microgramsmol litre-1. Of the seven representative human hepatic P450 isoforms, CYP1A2, 2A6, 2C9, 2C19, 2D6, 2E1 and 3A4, only CYP3A4 catalysed hydroxylation of midazolam, with a K(m) of 3.6 (0.8) microgramsmol liter-1. Fentanyl competitively inhibited metabolism of midazolam to l'-OH MDZ by CYP3A4, with a Ki of 24.2 (6.8) microgramsmol litre-1, comparable with the Ki obtained in human hepatic microsomes. These findings indicate that fentanyl competitively inhibits metabolism of midazolam by CYP3A4.      

Cytochrome P450 3A and 2B6 in the developing kidney: implications for ifosfamide nephrotoxicity

Repeated administration of agents (e.g., cancer chemotherapy) that can cause drug-induced nephrotoxicity may lead to acute or chronic renal damage. This will adversely affect the health and well-being of children, especially when the developing kidney is exposed to toxic agents that may lead to acute glomerular, tubular or combined toxicity. We have previously shown that the cancer chemotherapeutic ifosfamide (IF) causes serious renal damage substantially more in younger children (less than 3 years of age) than among older children. The mechanism of the age-related IF-induced renal damage is not known. Our major hypothesis is that renal CYP P450 expression and activity are responsible for IF metabolism to the nephrotoxic chloroacetaldehyde. Presently, the ontogeny of these catalytic enzymes in the kidney is sparsely known. The presence of CYP3A4, 3A5 and 2B6 was investigated in human fetal, pediatric and adult kidney as was the metabolism of IF (both R-IF and S-IF enantiomers) by renal microsomes to 2-dechloroethylifosfamide (2-DCEIF) and 3-dechloroethylifosfamide (3-DCEIF). Our analysis shows that CYP 3A4 and 3A5 are present as early as 8 weeks of gestation. IF is metabolized in the kidney to its two enantiomers. This metabolism can be inhibited with CYP 3A4/5 and 2B6 specific monoclonal inhibitory antibodies, whereby the CYP3A4/5 inhibitory antibody decreased the production of R-3-DCEIF by 51%, while the inhibitory CYP2B6 antibody decreased the production of S-2-DCEIF and S-3-DCEIF by 44 and 43%, respectively, in patient samples. Total renal CYP content is approximately six-fold lower than in the liver.     

甘草对豚鼠肝微粒体氟烷还原代谢的抑制作用

观察了甘草提取液对豚鼠肝微粒体氟烷还原代谢的影响，目的在寻找预防和治疗氟烷性肝炎的方法。随着氟烷的剂量增加，对照组及实验组ＣＤＥ和ＣＴＥ的生成量均增加，与对照组相比，１％组、２％组、５％组的氟烷还原代谢产物 ＣＤＥ和 ＣＴＥ生成量明显减少。甘草提取液对ＣＤＥ和 ＣＴＥ的生成呈非竟争性抑制和剂量依赖性抑制，甘草对ＣＤＥ和ＣＴＥ的抑制系数分别为０．３８％和０．７５％。结果表明甘草提取液可明显抑制氟烷的还原代谢，使其中间代谢产物亦减少。由于甘草还具有抗脂质过氧化作用．因此对预防氟烷性肝炎是有益的。     

Effect of propofol on ropivacaine metabolism in human liver microsomes

A combination of the general anesthestic propofol and epidural anesthesia with a local anesthetic is widely used. The metabolism of ropivacaine and that of lidocaine are mediated by similar P450 isoforms. Previously, propofol was found to inhibit the metabolism of lidocaine in vitro. Here we investigated whether propofol inhibits the metabolism of ropivacaine using human liver microsomes in vitro. Ropivacaine (6.0 μmol·l⁻¹) as the substrate and propofol (1–100 μmol·l⁻¹) were reacted together using human microsomes. The concentrations of ropivacaine and its major metabolite 2′,6′-pipecoloxylidide (PPX) were measured using high-performance liquid chromatography. The metabolic activity of ropivacaine was reflected in the production of PPX. The inhibitory effects of propofol on ropivacaine metabolism were observed to be dose-dependent. The IC₅₀ of propofol was 34.9 μmol·l⁻¹. Propofol shows a competitive inhibitory effect on the metabolism of ropivacaine (i.e., PPX production mediated by CYP3A4) in human CYP systems in vitro.     

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.     

Cytochrome P4502B6 and 2C9 do not metabolize midazolam: kinetic analysis and inhibition study with monoclonal antibodies

We determined the contribution of cytochrome P450 (CYP) isoformsto the metabolism of midazolam by kinetic analysis of humanliver microsomes and CYP isoforms and by examining the effectof chemical inhibitors and monoclonal antibodies against CYPisoforms in vitro. Midazolam was metabolized to 1'-hydroxymidazolam(1'-OH MDZ) by human liver microsomes with a Michaelis–Mentenconstant (K_m) of 4.1 (1.0) (mean (SD)) µmol litre^–1and a maximum rate of metabolism (V_max) of 5.5 (1.1) nmol min^–1mg protein^–1 (n=6). Of the nine representative human liverCYP isoforms, CYP1A2, 2A6, 2B6, 2C9, 2C19, 2D6, 2E1, 3A4 and3A5, three (CYP2B6, 3A4 and 3A5) showed midazolam 1'-hydroxylationactivity, with K_ms of 40.7, 1.7 and 3.0 µmol litre^–1,respectively, and V_max values of 12.0, 3.3 and 13.2 nmol min^–1nmol P450^–1, respectively (n=4). Midazolam 1'-hydroxylationactivity of human liver microsomes correlated significantlywith testosterone 6ß-hydroxylation activity, a markerof CYP3A activity (r²=0.77, P=0.0001), but not with S-mephenytoinN-demethylation activity, a marker of CYP2B6 activity (r²<0.01,P=0.84) (n=11). Troleandomycin and orphenadrine, chemical inhibitorsof CYP isoforms, inhibited the formation of 1'-OH MDZ by humanliver microsomes. Monoclonal antibody against CYP3A4 inhibitedthe formation of 1'-OH MDZ by 79%, whereas monoclonal antibodyagainst CYP2B6 had no effect on midazolam 1'-hydroxylation byhuman liver microsomes (n=5). These results indicate that onlyCYP3A4, but not CYP2B6 or CYP2C, is involved in the metabolismof midazolam in vitro. Br J Anaesth 2001; 86: 540–4     

DRUG METABOLIZING ENZYMES IN THE RAT AFTER INHALATION OF HALOTHANE AND ENFLURANE: Different pattern of response in liver, kidney and lung and possible implications for toxicity

Male Sprague-Dawley rats were exposed in inhalation chambersto halothane and enflurane in concentrations from 50 to 1000p.p.m. (0.0025 MAC-0.05MAC) 6 h a day for 3–11 days. Nosigns of general toxicity were found. There was a normal increasein weight, and normal food consumption, organ to body weightratios and normal histological findings in liver, kidney andlung. Exposure to 500 p.p.m. (0.05 MAC) of halothane inducedthe activity of NADPH-cytochrome-c-reductase in the liver, decreasedthe concentration of cytochrome P-450 in the kidney and decreasedall the enzyme concentrations measured in lung microsomes. Exposureto halothane 50 p.p.m. (0.005 MAC) and enflurane produced onlyminor changes. It is concluded that the inhalation of halothane,in contrast to enflurane, may affect drug metabolism and therebydrug kinetics and toxicity. Halothane may increase its own toxicityby increasing the activity of NADPH-cytochrome-c-reductase inliver. An organ differentiation in enzymatic response was observed.     

Human Kidney Methoxyflurane and Sevoflurane Metabolism: Intrarenal Fluoride Production as a Possible Mechanism of Methoxyflurane Nephrotoxicity

Background: Methoxyflurane nephrotoxicity is mediated by cytochrome P450-catalyzed metabolism to toxic metabolites. It is historically accepted that one of the metabolites, fluoride, is the nephrotoxin, and that methoxyflurane nephrotoxicity is caused by plasma fluoride concentrations in excess of 50 micro Meter. Sevoflurane also is metabolized to fluoride ion, and plasma concentrations may exceed 50 micro Meter, yet sevoflurane nephrotoxicity has not been observed. It is possible that in situ renal metabolism of methoxyflurane, rather than hepatic metabolism, is a critical event leading to nephrotoxicity. We tested whether there was a metabolic basis for this hypothesis by examining the relative rates of methoxyflurane and sevoflurane defluorination by human kidney microsomes.

Methods: Microsomes and cytosol were prepared from kidneys of organ donors. Methoxyflurane and sevoflurane metabolism were measured with a fluoride-selective electrode. Human cytochrome P450 isoforms contributing to renal anesthetic metabolism were identified by using isoform-selective inhibitors and by Western blot analysis of renal P450s in conjunction with metabolism by individual P450s expressed from a human hepatic complementary deoxyribonucleic acid library.

Results: Sevoflurane and methoxyflurane did undergo defluorination by human kidney microsomes. Fluoride production was dependent on time, reduced nicotinamide adenine dinucleotide phosphate, protein concentration, and anesthetic concentration. In seven human kidneys studied, enzymatic sevoflurane defluorination was minimal, whereas methoxyflurane defluorination rates were substantially greater and exhibited large interindividual variability. Kidney cytosol did not catalyze anesthetic defluorination. Chemical inhibitors of the P450 isoforms 2E1, 2A6, and 3A diminished methoxyflurane and sevoflurane defluorination. Complementary deoxyribonucleic acid-expressed P450s 2E1, 2A6, and 3A4 catalyzed methoxyflurane and sevoflurane metabolism, in diminishing order of activity. These three P450s catalyzed the defluorination of methoxyflurane three to ten times faster than they did that of sevoflurane. Expressed P450 2B6 also catalyzed methoxyflurane defluorination, but 2B6 appeared not to contribute to renal microsomal methoxyflurane defluorination because the P450 2B6-selective inhibitor had no effect.     

Interleukin-6 (IL-6) in seminal plasma of infertile men, and lipid peroxidation of their sperm

Concentrations of tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) in seminal fluid, as well as levels of sperm lipid membrane peroxidation, were investigated in fertile and infertile men. Semen samples, obtained by masturbation from 37 infertile and 14 fertile men, were examined for the presence of TNF-alpha and IL-6. The level of lipid peroxidation of the sperm membrane was measured by determining malondialdehyde (MDA) formation. The correlation between the IL-6 and the TNF-alpha concentrations in seminal plasma with the levels of lipid peroxidation of the sperm membranes was statistically evaluated. The IL-6 concentration in seminal plasma of infertile men was significantly higher than that of fertile men (p < .05). Similarly, the level of membrane lipid peroxidation was higher for the semen of infertile men than that of fertile men (p < .001). A significant positive correlation was found between IL-6 levels in seminal plasma and membrane sperm lipid peroxidation (p < .002), but not between this parameter and TNF-alpha levels in seminal plasma. These findings suggest a possible association between IL-6 seminal plasma levels and lipid peroxidation of sperm membrane. Stimulation of reactive species production by human sperm and leucocytes, induced by the high levels of IL-6, could explain these results.     

Cytochrome P450‐2D6*4 polymorphism seminal relationship in infertile men

This study aimed to assess cytochrome (CY) P450‐2D6*4 polymorphism relationship with semen variables in infertile men. In all, 308 men were included; fertile normozoospermia (N) (n = 77), asthenozoospermia (A) (n = 70), asthenoteratozoospermia (AT) (n = 75) and oligoasthenoteratozoospermia (OAT) (n = 86). They were subjected to history taking, clinical examination, semen analysis, sperm acrosin activity, seminal malondialdehyde (MDA) and CYP450‐2D6*4 genotyping. CYP450‐2D6*4 wild‐type allele was represented in 76.5% of N, 70% of A, 66.7% of AT and 57.7% of OAT men where homozygous gene mutation was present in 5.9% of N, 20% of A, 26.6% of AT and 26.9% of OAT men, respectively. Sperm acrosin activity, sperm concentration, sperm motility, linear sperm velocity and sperm normal forms were significantly higher, and seminal MDA level was significantly lower in men with CYP450‐2D6*4 wild‐type allele compared with men with homozygous mutation. It is concluded that CYP450‐2D6*4 wild‐type allele has higher frequency where homozygous‐type allele has lower frequency in N men compared with A, AT and OAT men. Sperm acrosin activity index, sperm concentration, sperm motility, linear sperm velocity and sperm normal forms were significantly higher, and seminal MDA level was significantly lower in men with CYP450‐2D6*4 wild‐type allele compared with men with homozygous mutation.     

Downregulation of intestinal cytochrome p450 in chronic renal failure

Chronic renal failure (CRF) is associated with a decrease in intestinal drug metabolism. The mechanisms remain poorly understood, but one hypothesis involves a reduction in cytochrome P450 levels. This study aimed to investigate the effects of CRF on intestinal cytochrome P450. Two groups of rats were defined, i.e., rats with CRF (induced by 5/6 nephrectomy) and control pair-fed rats. Total cytochrome P450 levels and protein and mRNA expression of cytochrome P450 isoforms, as well as in vitro N-demethylation of erythromycin (a probe for CYP3A activity) and 7-ethoxyresorufin o-deethylase activity (a probe for CYP1A), were assessed in intestinal microsomes. Body weights were similar in the two groups. Creatinine clearance was reduced by 77% (P < 0.001) in CRF rats, compared with control pair-fed animals. Total intestinal cytochrome P450 activity was reduced by 32% (P < 0.001) in CRF rats. CYP1A1 and CYP3A2 protein expression was considerably reduced (>40%, P < 0.001) in rats with CRF. CYP2B1, CYP2C6, and CYP2C11 levels were the same in the two groups. RT-PCR assays revealed marked downregulation of CYP1A1 and CYP3A2 gene expression in CRF rats (P < 0.001). Although intestinal cytochrome P450 levels were reduced in CRF, induction by dexamethasone was present. N-Demethylation of erythromycin and 7-ethoxyresorufin o-deethylase activity were decreased by 25% (P < 0.05) in CRF rats, compared with control rats. In conclusion, CRF in rats is associated with decreases in intestinal cytochrome P450 activity (mainly CYP1A1 and CYP3A2) secondary to reduced gene expression.