Induction and inhibition of cytochrome P450 and drug-metabolizing enzymes by climbazole

To determine the effect of climbazole on hepatic microsomal cytochrome P450 (P450) and drug-metabolizing enzymes, four different P450 isoforms (CYP2B1, 3A2, 2E1, and 2C12) were examined in female Long-Evans rats. Treatment of rats with climbazole resulted in the induction of P450 content. Climbazole both induced and inhibited aminopyrine N-demethylase activity, but not erythromycin N-demethylase activity. Uridine 5'-phosphate (UDP)-glucuronosyl transferase and glutathione S-transferase activities were also increased with climbazole treatment. Immunoblot analyses revealed that climbazole induces CYP2B1 and CYP3A2 at the lower dose examined, but it failed to increase CYP2B1 at the higher dose. Northern blot analysis revealed that climbazole markedly increases P450 2B1 mRNA. These results indicate that climbazole induces and inhibits P450-dependent drug-metabolizing enzymes in vivo and may have the dose-differential effect on CYP2B1 in rat liver.     

Down-regulation of hepatic cytochrome p450 in chronic renal failure: role of uremic mediators

1. Chronic renal failure (CRF) is associated with a decrease in liver cytochrome p450 (p450). The mechanism remains poorly understood. The present study aimed to investigate the effects of the serum of rats with CRF on liver p450. 2. Normal rat hepatocytes were incubated for 24 h with serum (concentration of 10%) from rats with CRF and from control animals in order to measure (1). total p450 level, (2). protein expression and mRNA levels of major p450 isoforms, and (3). some of their specific metabolic activities (N-demethylation of erythromycin). Time-course experiments (incubation time from 12 to 48 h) and dose-response curves (concentration of serum ranging from 1 to 30%) have been conducted. 3. In normal hepatocytes incubated for 24 h with serum (concentration of 10%) from rats with CRF, total p450 level, protein expression and mRNA levels of several p450 isoforms (CYP2C6, 2C11, 3A1 and 3A2) were decreased by more than 35% (P<0.001) compared to serum from control animals. The protein expression as well as the mRNA levels of CYP2D were similar in hepatocytes incubated with serum from either control or CRF rats. The N-demethylation of erythromycin was decreased by more than 35% (P<0.001) in hepatocytes incubated with serum from rats with CRF. The inhibitory effect of serum from rats with CRF tended to peak at 48 h of incubation and was maximum at a concentration of 20%. 4. In conclusion, uremic serum contains mediator(s) that down-regulate the cytochrome p450 of normal hepatocytes secondary to reduced gene expression.     

Contribution of human hepatic cytochrome P450s and steroidogenic CYP17 to the N-demethylation of aminopyrine

1. Aminopyrine N-demethylase activity was determined for 11 forms of human hepatic cytochrome P450s (P450s) expressed in yeast Saccharomyces cerevisiae and for human steroidogenic CYP17 expressed in Escherichia coli. 2. Among the hepatic P450s, the N-demethylation of aminopyrine was catalysed most efficiently by CYP2C19, followed by CYP2C8, 2D6, 2C18 and 1A2, whereas the activity with CYP2E1 was negligible. The kinetics of the N-demethylation process by CYP1A2, 2C8, 2C19 and 2D6 were studied by fitting to Michaelis-Menten kinetics by Lineweaver-Burk plots. CYP2C19 exhibited the highest affinity and a high capacity for the aminopyrine N-demethylation. CYP2C8 showed the highest Vmax, followed by CYP2C19, 2D6 and 1A2, whereas the Km for CYP2C8, 2D6 and 1A2 were 10-17 times higher than that for CYP2C19. Accordingly, the Vmax/Km for CYP2C19 was more than nine times higher than that of other P450s. 3. Human steroidogenic CYP17 also catalysed aminopyrine N-demethylation and the activity was comparable with that for CYP3A4 which is a dominant P450 in human liver. The activity was increased 1.5-fold by the addition of cytochrome b5, whereas the activity was not affected by the addition of Mg2+. 4. These results suggest that several human hepatic P450s, especially CYP2C19, and steroidogenic CYP17 exhibit aminopyrine N-demethylase activity.     

Microsomal Function in Hepatitis B Surface Antigen Healthy Carriers: Assessment of Cytochrome P450 1A2 Activity by the 14C-Caffeine Breath Test

Abstract: The hepatitis B surface antigen (HBsAg) carrier state is associated with changes in hepatocellular function involving the cytochrome P450 (CYP) system. Among this system, CYP1A2 enzyme plays an important role in chemical carcinogenesis and in the metabolism of several drugs. We have thus investigated CYP1A2 function using two ¹⁴C-caffeine breath tests (3-methyl-¹⁴C; C3BT and 7-methyl-¹⁴C caffeine; C7BT) in 12 HBsAg healthy carriers and 8 healthy volunteers matched for ¹⁴C-aminopyrine breath test values. HBsAg carriers exhibited lower C3- and C7BT values than normal controls. This difference, however, did not reach statistical significance except for C7BT values normalised for aminopyrine breath test values. Our data thus do not support the association between viral presence and CYP1A2 dysfunction.     

Contribution of human hepatic cytochrome P450s and steroidogenic CYP17 to the N-demethylation of aminopyrine

1. Aminopyrine N -demethylase activity was determined for 11 forms of human hepatic cytochrome P450s (P450s) expressed in yeast Saccharomyces cerevisiae and for human steroidogenic CYP17 expressed in Escherichia coli. 2. Among the hepatic P450s, the N-demethylation of aminopyrine was catalysed most efficiently by CYP2C19, followed by CYP2C8, 2D6, 2C18 and 1A2, whereas the activity with CYP2E1 was negligible. The kinetics of the N-demethylation process by CYP1A2, 2C8, 2C19 and 2D6 were studied by fitting to Michaelis-Menten kinetics by LineweaverBurkplots. CYP2C19 exhibited the highest affinityanda high capacity forthe aminopyrine N-demethylation. CYP2C8 showed the highest Vmax, followed by CYP2C19, 2D6 and 1A2, whereas the K m for CYP2C8, 2D6 and 1A2 were 10-17 times higher than that for CYP2C19. Accordingly, the Vmax/Km for CYP2C19 was more than nine times higher than that of other P450s. 3. Human steroidogenic CYP17 also catalysed aminopyrine N-demethylation and the activity was comparable with that for CYP3A4 which is a dominant P450 in human liver. The activity was increased 1.5-fold by the addition of cytochrome b, whereas the activity was not affected by the addition of Mg. 4. These results suggest that several human hepatic P450s, especially CYP2C19, and steroidogenic CYP17 exhibit aminopyrine N-demethylase activity.     

Effects of serum from patients with chronic renal failure on rat hepatic cytochrome P450

1. In humans, chronic renal failure (CRF) is associated with decreased hepatic drug metabolism, particularly that mediated by the cytochrome P450 (P450). The mechanisms remain poorly understood. The present study aimed to investigate the effects of the serum of patients with CRF on liver P450, and to evaluate whether renal replacement therapies (dialysis or transplantation) impede the inhibition of CRF serum on P450. 2. Rat hepatocytes were incubated for 24 h with serum from patients with severe CRF and from controls to measure (1) P450 level, (2) protein expression and mRNA levels of P450 isoforms and (3) metabolic activities of CYP3A and CYP1A. Similar experiments were performed with serum of patients once on chronic hemodialysis and after kidney transplantation. 3. In rat hepatocytes incubated for 24 h with serum from patients with CRF, P450 level and protein expression, as well as mRNA levels of P450 isoforms (CYP1A2, 2C6, 2C11, 2D1/2D2, 3A2 and 4A1/4A3), were decreased by more than 45% (P<0.001) compared to control serum, while the levels of CYP2E1 were not modified. CYP3A and CYP1A activities were decreased by 51 and 59% (P<0.001), respectively. The inhibitory effect of serum obtained from patients before first dialysis was similar after 1 or 6 months on chronic hemodialysis but was lost after successful kidney transplantation. In CRF serum, the fraction containing proteins between 10 and 15 kDa decreases P450. 4. Human uremic serum contains mediator(s) that decreases rat hepatic P450 activity and expression secondary to reduced gene expression. The inhibitory effect of serum persists even after initiation of dialysis, but disappears after normalization of renal function following kidney transplantation.     

大黄素对大鼠肝脏CYP450酶的诱导研究

目的 探讨大黄素对大鼠肝脏细胞色素P450酶(CYP450)及其主要亚型的影响。方法 20只雄性SD大鼠, 随机分成4组, 每组5只, 分别为溶剂对照组, 170、500和1 500 mg/kg大黄素染毒组, 大黄素蒸馏水混悬后连续经口给药16 d, 结束后次日取大鼠肝脏组织制作微粒体, 分别采用CO还原差示光谱法、分光光度法及化学发光法检测大鼠肝脏微粒体总CYP450水平, 红霉素脱甲基酶(CYP3A)、氨基比啉-N-脱甲基酶, CYP1A、CYP2B和CYP2E1酶活性变化。结果 大黄素连续经口给药16 d, 能够引起大鼠肝脏微粒体总CYP450显著升高、可轻度诱导CYP3A、CYP1A、CYP2E1和CYP2B酶, 500 mg/kg剂量组最明显。结论 大黄素对大鼠肝脏中CYP3A、CYP1A、CYP2B和CYP2E1酶均有诱导作用。     

Mdr1 limits CYP3A metabolism in vivo

We determined whether the drug efflux protein P-glycoprotein (Pgp) could influence the extent of CYP3A-mediated metabolism of erythromycin, a widely used model substrate for CYP3A. We compared CYP3A metabolism of erythromycin (a Pgp substrate) using the erythromycin breath test in mice proficient and deficient of mdr1 drug transporters. We first injected mdr1(+/+) mice with [(14)C]N-methyl erythromycin and measured the rate of appearance of (14)CO(2) in the breath as a measure of hepatic CYP3A activity. Animals treated with CYP3A inducers or inhibitor showed accelerated or diminished (14)CO(2) in the breath, respectively. The erythromycin breath test was next administered to mdr1a(-/-) and mdr1a/1b(+/+) and (-/-) mice. These animals had equivalent levels of immunoreactive CYP3A and CYP3A activity as measured by erythromycin N-demethylase activity in liver microsomes. Nevertheless, the rate of (14)CO(2) appearance in the breath showed no relationship with these measurements of CYP3A, but changed proportionally to expression of mdr1. The average breath test (14)CO(2) area under the curves were 1.9- and 1.5-fold greater in mdr1a/1b(-/-) and mdr1a(-/-) mice, respectively, compared with (+/+) mice, and CER(max) was 2-fold greater in mdr1a/1b(-/-) compared with (+/+) mice. We conclude that Pgp, by limiting intracellular substrate availability can be an important determinant of CYP3A metabolism of numerous medications that are substrates for CYP3A and Pgp.     

Suppression of male-specific cytochrome P450 isoforms by bisphenol A in rat liver

We examined the effect of bisphenol A (BPA) on microsomal cytochrome P450 (P450) enzymes in rats. Rats were treated intraperitoneally with BPA daily for 4 days, at doses of 10, 20, and 40 mg/kg. Among the P450-dependent monooxygenase activities, testosterone 2α-hydroxylase (T2AH) and testosterone 6β-hydroxylase (T6BH) activities, which are associated with CYP2C11 and CYP3A2 respectively, were remarkably decreased by 40 mg/kg BPA. The levels of the control activities were 13 and 50%, respectively. Furthermore, immunoblotting showed that BPA (20 or 40 mg/kg) significantly reduced CYP2C11/6 and CYP3A2/1 protein levels in rat liver microsomes. In addition, estradiol 2-hydroxylase (ED2H) and benzphetamine N-demethylase (BZND) activities were significantly decreased by BPA at 20 and 40 mg/kg (by 19–73%). The K _m values for T2AH and T6BH in 20 and 40 mg/kg BPA-treated rats were significantly high compared with that in control rats. The V _max for T2AH was dose-dependently decreased by BPA treatment, whereas that of T6BH was only decreased by BPA at 40 mg/kg. On the other hand, lauric acid ω-hydroxylase (LAOH) activity was significantly increased by BPA at 20 and 40 mg/kg (1.5- and 1.7-fold, respectively). Immunoblot analysis showed that 20 and 40 mg/kg BPA induced CYP4A1/2 protein expression. However, the activities 7-ethoxyresorufin O-deethylase (EROD), 7-methoxyresorufin O-demethylase (MROD), 7-ethoxycoumarin O-deethylase (ECOD), 7-benzyloxyresorufin O-debenzylase (BROD), aminopyrine N-demethylase (APND), chlorzoxazone 6-hydroxylase (CZ6H), erythromycin N-demethylase (EMND), and testosterone 7α-hydroxylase (T7AH) were not affected by BPA at any dose. These results suggest that BPA affects male-specific P450 isoforms in rat liver, and that these changes closely relate to the toxicity of BPA. Received: 26 January 1998 / Accepted: 26 February 1998     

Climbazole is a new potent inducer of rat hepatic cytochrome P450

We examined the effect of climbazole on the induction of rat hepatic microsomal cytochrome P450 (P450), and compared the induction potency with other N-substituted azole drugs such as clorimazole. We found that climbazole is found to be a potent inducer of rat hepatic microsomal P450 as clorimazole. Induced level of P450 by climbazole was almost similar in extent to clorimazole when compared with other imidazole drugs in a dose- and time-dependent manner. Parallel to the increase in P450, climbazole increased aminopyrine and erythromycin N-demethylase, ethoxycoumarin O-deethylase, and androstenedione 16 beta- and 15 alpha/6 beta hydroxylase activities; however, clorimazole did not induce aminopyrine N-demethylase activity irrespective of its marked increase in P450 content. Immunoblot analyses revealed that climbazole induced CYP2B1, 3A2 and 4A1. The present findings indicate that climbazole is a new potent inducer of hepatic microsomal P450 and drug-metabolizing enzymes like clorimazole, but it may have some differential mechanism(s) for these enzymes' induction in rat liver.     

Down-regulation of hepatic cytochrome P450 enzymes associated with cisplatin-induced acute renal failure in male rats

Hepatic drug metabolism is impaired in experimental animals and humans with renal diseases. An anticancer drug, cisplatin induces acute renal failure (ARF) in rats. Under the same experimental conditions, cisplatin causes down-regulation of hepatic cytochrome P450 (P450) enzymes in an isozyme selective manner. The present study examined the pathological role of ARF in the down-regulation of hepatic P450 enzymes in the cisplatin-treated rats. Male rats with single dose of intraperitoneally cisplatin (5 mg/kg) caused marked changes in renal parameters, BUN and serum creatinine but not hepatic parameters, serum alanine aminotransferase or aspartate aminotransferase. The rats also suffered from down-regulation of hepatic microsomal CYP2C11 and CYP3A2, male specific P450 isozymes, but not CYP1A2, CYP2E1, or CYP2D2. The decrease in serum testosterone level was also observed in injured rats, which was consistent with the selective effects on male specific P450 enzymes. Protection of rats against cisplatin-induced ARF by dimethylthiourea, a hydroxyl radical scavenger, also protected rats against the decrease in serum testosterone levels and the down-regulation of CYP2C11 and CYP3A2. Carboplatin, an analogue to cisplatin but no ARF inducer, did not cause decrease in serum testosterone levels and down-regulation of hepatic male specific P450 enzymes. These results suggest that down-regulation of hepatic P450 enzymes in male rats given cisplatin is closely related to the cisplatin-induced ARF and the resultant impairment of testis function.     

Inhibition of drug-metabolizing enzyme activity in human hepatic cytochrome P450s by bisphenol A

Effect of bisphenol A on drug-metabolizing enzyme activities by human hepatic cytochrome P450s (CYP) was investigated. We measured aminopyrine N-demethylation by eleven kinds of cDNA-expressed CYPs. CYP2C19 and CYP2B6 catalyzed most efficiently the aminopyrine N-demethylation, followed by CYP2C8 and CYP2D6. Bisphenol A (1 mM) most efficiently inhibited aminopyrine N-demethylation by CYP2C8 and CYP2C19 by 82% and 85%, respectively, whereas inhibition of the activities by CYP 2B6 and 2D6 was less than 40%. Bisphenol A exhibited a noncompetitive-type inhibition of aminopyrine N-demethylase activity by CYP2C8 with Ki value of 97 microM. Additionally, we investigated the inhibitory effect of bisphenol A on CYP2C19-mediated S-mephenytoin 4-hydroxylation. Bisphenol A exhibited a mixed-type inhibition with Ki value of 113 microM. These results suggest that bisphenol A inhibits human hepatic CYP activities, especially CYP2C8 and CYP2C19.     

Comparative study on nobiletin metabolism with liver microsomes from rats, Guinea pigs and hamsters and rat cytochrome p450

In vitro metabolism of nobiletin, a polymethoxy-flavonoid abundantly present in citrus peels, was studied using liver microsomes of rats, hamsters and guinea pigs and ten cDNA-expressed rat cytochrome P450 (P450). The effects of P450 inducers on nobiletin metabolism were also investigated. Aerobical incubation with NADPH and animal liver microsomes transformed nobiletin to five metabolites, M-1, M-2, M-3, M-4 and M-5. From LC-MS and (1)H-NMR data and a time-course study, these were assumed to be 4'-hydroxy (OH)-, 7-OH-, 6-OH-, 3',4'-diOH- and 6,7-diOH-metabolites, respectively. Pretreatment of animals with phenobarbital increased M-2 and M-3 to about 2-fold that in untreated animals. Pretreatment with 3-methylcholanthrene (MC) resulted in remarkable increases of both M-1 and M-4 (3 to 9-fold that of untreated). Males had 2-3 times higher M-2 and M-3 formation activities in rats, and for M-2 in hamsters than did females. Immunoinhibition study using antiserum against P450 revealed the involvement of hamster CYP1A2 in the formation of M-1 and M-4 in hamster liver. Of ten rat P450s, CYP2C11, CYP3A1, CYP3A2 and CYP2D1 had high activities for the formation of M-1, M-2 and M-3. Another P450s (CYP1A1, CYP2C12 and CYP1A2) also showed activity for the formation of M-1. Only CYP1A1 produced 3',4'-diOH-metabolites (M-4). However, CYP2A1, CYP2B1 and CYP2E1 had no activity for nobiletin. These results suggested that constitutive P450s such as CYP2C11, CYP2D1, CYP3A1, CYP3A2 and CYP2C12 are responsible for the demethylation at the 6-, 7-, 3'- and 4'-positions; whereas, MC-inducible P450s, CYP1A1 and CYP1A2, preferentially catalyzed demethylation at the 3'-and 4'-positions.     

Changes in cytochrome P450 enzymes by 1,1-dichloroethylene in rat liver and kidney

We examined the effect of 1,1-dichloroethylene (1,1-DCE) on microsomal cytochrome P450 (P450) enzymes in rat liver and kidney. Rats were treated intraperitoneally with 1,1-DCE daily for 4 days, at doses of 200, 400, and 800 mg/kg. Among the P450-dependent monooxygenase activities in liver microsomes, testosterone 2α-hydroxylase (T2AH), which is associated with CYP2C11 activity, was remarkably decreased by 800 mg/kg 1,1-DCE. The level relative to control activity was <10%. Furthermore, immunoblotting showed that 1,1-DCE (≥400 mg/kg) significantly decreased CYP2C11/6 protein levels in liver microsomes. In addition, 7-methoxyresorufin O-demethylase (MROD), 7-ethoxycoumarin O-deethylase (ECOD), benzphetamine N-demethylase (BZND), chlorzoxazone 6-hydroxylase (CZ6H), and testosterone 6β-hydroxylase (T6BH) activities were significantly decreased by the highest dose of 1,1-DCE (by 40–70%). However, the activities of other P450-dependent monooxygenases, namely 7-ethoxyresorufin O-deethylase (EROD), 7-benzyloxyresorufin O-debenzylase (BROD), aminopyrine N-demethylase (APND), erythromycin N-demethylase (EMND), lauric acid ω-hydroxylase (LAOH), and testosterone 7α-hydroxylase (T7AH) were not affected by 1,1-DCE at any dose. Immunoblotting showed CYP1A1/2, CYP2B1/2, CYP2E1, and CYP3A2/1 protein levels were significantly decreased by 60–66% by 1,1-DCE (800 mg/kg), whereas that of CYP4A1/2 was not affected by any dose of 1,1-DCE. By contrast, among the P450-dependent monooxygenase activities in kidney microsomes, only CZ6H activity was increased by 1,1-DCE (1.6-fold at 800 mg/kg). Also, it was␣observed that 1,1-DCE (800 mg/kg) significantly increased CYP2E1 protein levels by immunoblotting (∼1.5-fold). These results suggest that 1,1-DCE changes the constitutive P450 isoforms in the rat liver and kidney, and that these changes closely relate to the toxicity of 1,1-DCE. Received: 28 January 1997 / Accepted: 18 August 1997     

Preliminary evaluation of progestins as inducers of cytochrome P450 3A4 activity in postmenopausal women

The effects of intramuscularly and orally administered medroxyprogesterone acetate on cytochrome P450 3A4 (CYP3A4) activity were investigated in twelve postmenopausal women in a randomized, crossover study. Unbound prednisolone clearance and the erythromycin breath test were used as markers of CYP3A4 activity. After 2 months of intramuscular progestin therapy, unbound prednisolone clearance increased by 25% in five of six subjects. Similarly, after intramuscular progestin therapy, results from the erythromycin breath test showed a 23% mean increase in CYP3A4 activity. In contrast, 2 months of oral progestin therapy had no effect on prednisolone pharmacokinetics or erythromycin metabolism. These results suggest that parenterally but not orally administered progestins may induce or activate the CYP3A4 enzyme system, leading to an increased metabolism of many CYP3A4 substrates.     

Rat cytochrome p450 1A and 3A enzymes involved in bioactivation of tegafur to 5-fluorouracil and autoinduced by tegafur in liver microsomes.

Tegafur, an anticancer prodrug, is reported to be bioactivated to 5-fluorouracil (5-FU) by cytochrome P450 (P450) enzymes. Liver microsomal P450 enzymes involved in the biotransformation of tegafur into 5-FU in rats and the effect of tegafur in vivo on P450 levels in rats were investigated. Of 12 cDNA-expressed rat P450 enzymes tested, CYP1A2, CYP3A1, and CYP2C11 had high 5-FU formation rates from 100 microM and 1.0 mM tegafur concentrations. The contributions of CYP1A, CYP2C, and CYP3A enzymes to 5-FU formation in male rat liver microsomes were supported by immunoinhibition studies. 5-FU formation from tegafur, at substrate concentrations of 100 microM and 1.0 mM, was increased by intraperitoneal treatment of tegafur (50 mg/kg for 5 days) as well as by beta-naphthoflavone, phenobarbital, and dexamethasone. Orally administered tegafur (100 mg/kg daily for 20 days) caused the induction of CYP2B (5-fold) and of CYP1A and CYP3A (approximately 2-fold) and of 5-FU formation (approximately 2-fold) in rat liver microsomes. These results suggest that CYP1A and CYP3A enzymes, autoinduced by tegafur, have important roles in 5-FU formation from tegafur in rat liver microsomes. Coadministration of tegafur and P450-inducing drugs could markedly enhance the biotransformation of tegafur into 5-FU via P450 induction.     

Cytochrome p450-dependent metabolism of trichloroethylene in rat kidney.

The metabolism of trichloroethylene (Tri) by cytochrome P450 (P450) was studied in microsomes from liver and kidney homogenates and from isolated renal proximal tubular (PT) and distal tubular (DT) cells from male Fischer 344 rats. Chloral hydrate (CH) was the only metabolite consistently detected and was used as a measurement of P450-dependent metabolism of Tri. Pretreatment of rats with pyridine increased CH formation in both liver and kidney microsomes, whereas pretreatment of rats with clofibrate increased CH formation only in kidney microsomes. Pyridine increased CYP2E1 expression in both liver and kidney microsomes, whereas clofibrate had no effect on hepatic but increased renal CYP2E1 and CYP2C11 protein levels. These results suggest a role for CYP2E1 in both the hepatic and renal metabolism of Tri and a role for CYP2C11 in the renal metabolism of Tri. Studies with the general P450 inhibitor SKF-525A and the CYP2E1 competitive substrate chlorzoxazone provided additional support for the role of CYP2E1 in both tissues. CH formation was higher in PT cells than in DT cells and was time and reduced nicotinamide adenine dinucleotide phosphate (NADPH) dependent. However, pretreatment of rats with either pyridine or clofibrate had no effect on CYP2E1 or CYP2C11 protein levels or on CH formation in isolated cells. These data show for the first time that Tri can be metabolized to at least one of its P450 metabolites in the kidneys and quantitate the effect of P450 induction on Tri metabolism in the rat kidney.     

Differential selectivity in carbamazepine-induced inactivation of cytochrome P450 enzymes in rat and human liver

Oxidative metabolism of carbamazepine results in covalent binding of its reactive metabolite to liver microsomal proteins, which has been proposed as an important event in pathogenesis of the hypersensitivity reactions to this drug. Although the proposed reactive metabolites are produced by cytochrome P450 enzymes (P450 or CYP), the impact of the formation of unstable metabolites on the enzyme itself has not been elucidated. The present study examines the alteration of P450 enzyme activities during the metabolism of carbamazepine. Liver microsomes from rats and humans were preincubated with carbamazepine in the presence of NADPH, and subsequently assayed for monooxygenase activities representing several P450s. No evidence was obtained for inactivation of CYP2C11, CYP3A, CYP1A1/2 or CYP2B1/2 in rat liver microsomes during the carbamazepine metabolism, whereas the CYP2D enzyme was inactivated in a manner related to the preincubation time. Interestingly, under the same protocol human liver microsomes did not exhibit inactivation of CYP2D6, as well as there being no CYP2C8, CYP2C9 or CYP3A4 inactivation, whereas CYP1A2 was inactivated. Reduced glutathione could not protect against the observed inactivation of the P450s. These results suggest that CYP2D enzyme(s) in rats and CYP1A2 in humans biotransform carbamazepine into reactive metabolites, resulting in inactivation of the enzyme themselves, and raise the possibility that the P450 isoforms participate in toxicity induced by the drug in both animal species.     

Inducibility of male-specific isoforms of cytochrome p450 by sex-dependent growth hormone profiles in hepatocyte cultures from male but not female rats.

Although in vivo expression levels of the male-specific hepatic isoforms of cytochrome P450 (P450) (CYP2C11, CYP2C13, CYP2A2, and CYP3A2) are determined by the episodic growth hormone profile secreted by male rats, these isoforms have been completely refractory to growth hormone regulation in hepatocyte culture. By using species-specific rat growth hormone, at subphysiologic in vivo concentrations administered in two daily episodic pulses, we successfully induced CYP2C11 and CYP2A2 to near normal concentrations. Whereas inductive levels of CYP2C13 were subnormal, CYP3A2 was unresponsive to all hormonal treatments, quickly declining to undetectable concentrations. In agreement with in vivo findings, we observed that induction levels of the isoforms were always greatest when the male hepatocytes were exposed to the masculine-like episodic growth hormone profile and least stimulated by the continuous feminine-like hormone profile. When administered alone, dexamethasone consistently increased isoform levels. However, when administered with growth hormone, the glucocorticoid was always antagonistic, suppressing growth hormone induction of CYP2C11, CYP2C13, and CYP2A2. Finally, the P450 isoforms were completely unresponsive to all treatments when the hepatocytes were derived from female rats, supporting earlier findings that expression levels of sexually dimorphic P450 isoforms are inherently irreversible between sexes.