Distinct responses of mouse hepatic CYP enzymes to corn oil and peroxisome proliferators.

We studied the response of male DBA/2N mouse liver monooxygenases to acute (one-day) and subacute (7-day) exposure to clofibrate, gemfibrozil, and corn oil. The day following a single treatment with clofibrate (200 mg/kg), coumarin 7-hydroxylase (COH) activity decreased significantly (by 70%) with a concomitant decrease in the CYP2A4/5 protein and mRNA levels. The 7-day treatment schedule also decreased COH activity by only by 30%, though the levels of CYP2A4/5 protein and mRNA were still low. Treatment 1 and 7-day with clofibrate decreased 7-pentoxyresorufin O-dealkylase (PROD) activity by 40%. No changes were seen in testosterone 15 alpha-hydroxylase (T15 alpha OH) activity after 1 day of treatment with clofibrate but, after 7 days, it was decreased by 50%. Clofibrate treatment had no significant effects on testosterone 7 alpha-hydroxylase (T7 alpha OH), 7-ethoxyresorufin O-deethylase (EROD), or benzphetamine N-demethylase (BZDM) activities. Gemfibrozil (200 mg/kg) did not alter COH activity or CYP2A4/5 protein content after a single treatment, but a slight decrease was seen in the mRNA level. Treatment for 7 days significantly increased (2.5-fold) the activity and mRNA content but the amount of protein remained unchanged. Gemfibrozil enhanced (2-2.7-fold PROD and EROD (2-2.5-fold) activities by both treatments, whereas T15 alpha OH, T7 alpha OH, or BZDM activities were not significantly affected. Treatment with corn oil for 7 days significantly decreased (65%) COH activity and CYP2A4/5 protein and mRNA levels. PROD (55%) and T15 alpha OH (65%) activities were significantly decreased even after a single dose although injection for 7 days had no effect. Neither of the corn oil schedules had any marked effect on T7 alpha OH, EROD, or BZDM activities. These results demonstrate: 1. a decrease in the expression of CYP2A4/5 gene by clofibrate and corn oil; 2. substantial differences within the CYP2A subfamily in their responses to corn oil, clofibrate, and gemfibrozil; and 3. distinct responses of other xenobiotic metabolizing CYP subfamily enzymes to clofibrate and gemfibrozil.

Modification of hepatic cytochrome P450 profile by cocaine-induced hepatotoxicity in DBA/2 mouse.

Previous studies in our laboratory have shown that a hepatotoxic dose of cocaine increases coumarin 7-hydroxylase activity in male DBA/2 mouse liver. In the present study, the dose- and time-dependent responses of the hepatic CYP2A4/5 complex to cocaine-induced liver damage were studied. Cocaine increased CYP2A4/5 levels in a dose-dependent manner. The maximal increases in coumarin 7-hydroxylase activity (4-fold), microsomal CYP2A4/5 content (3-fold) and steady-state mRNA levels (10-fold) were observed at 24 h after administration of a single dose of 60 mg/kg cocaine coinciding with morphologically detectable diffuse liver damage, while the total P450 content was not changed. 3 and 5 days after the daily administration of cocaine severe, mainly pericentral (zone III of Rappaport), liver damage was apparent in parallel with a clear decline in CYP2A4/5 mRNA, protein content and coumarin 7-hydroxylase activity. After 5 days of treatment, CYP2A5 still remained at a very low level but an induction in CYP2B10 protein and related pentoxyresorufin O-dealkylase activity was observed. No marked changes in microsomal CYP2Cx and CYP1A1/2 contents or associated activities were observed. Dimethylnitrosamine N-demethylase activity, a marker for CYP2E1, decreased in parallel with increased cocaine dose and time and the severity of liver damage.(ABSTRACT TRUNCATED AT 250 WORDS)

Cocaine N-demethylation and the metabolism-related hepatotoxicity can be prevented by cytochrome P450 3A inhibitors.

Cocaine is eliminated and detoxified principally through the metabolism of nonspecific plasma and tissue esterases. Microsomal oxidative metabolism is of importance in cocaine N-demethylation, this being a principal pathway of cocaine bioactivation and hepatotoxicity. The contribution of different cytochrome P450 (CYP) enzymes to cocaine N-demethylase activity was studied in vitro with DBA/2 mouse and human liver microsomes, and cocaine hepatotoxicity was examined in vivo in DBA/2 male mice. Species dependent enzyme kinetics was observed. Cocaine N-demethylase displayed two Km values in murine liver (40-60 microM and 2-3 mM), whereas only one Km value was observed in human liver microsomes (2.3-2.7 mM). We suggest that CYP3A plays a prominent role in the N-demethylation of cocaine for the following reasons: (i) pregnenolone-16 alpha-carbonitrile, an inducer of CYP3As increases cocaine N-demethylase in parallel with testosterone 6 beta-hydroxylase activity and immunoreactive 3A protein in mouse liver; (ii) human and mouse cocaine N-demethylase and testosterone 6 beta-hydroxylase activities can be inhibited by triacetyloleandomycin, cannabidiol, or gestodene, all selective inhibitors of CYP3A P450s; (iii) antibodies directed against P450s within subfamilies 1A, 2A, 2B, 2C, or 2E inhibited cocaine N-demethylase activity only marginally, and finally, (iv) treatment of mice with triacetyloleandomycin or cannabidiol in vivo significantly attenuated the cocaine-elicited hepatotoxicity as assessed by the serum alanine aminotransferase activity and liver histology in parallel with decreased cocaine N-demethylase activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Response of mouse liver coumarin 7-hydroxylase activity to hepatotoxins: dependence on strain and agent and comparison to other monooxygenases.

Acute effects of a single intraperitoneal dose of allyl alcohol (AA, 64 mg/kg), dimethylnitrosamine (DMNA, 30 mg/kg), hexachlorobutadiene (HCBD, 50 mg/kg), carbon tetrachloride (CCl4, 24 mg/kg), cocaine (60 mg/kg) and pyrazole (300 mg/kg) on the hepatic histology and monooxygenases in DBA/2 and C57Bl/6 strains of mice were investigated. All substances caused histologically verified injury to the liver, which varied in appearance and severity depending on the compound and the mouse strain. Responses of P450-catalyzed reactions were highly dependent on the toxin and varied between different monooxygenase (MO) reactions and two mouse strains. In DBA/2 strain, coumarin 7-hydroxylase (COH) activity was increased from 3- to 5-fold by pyrazole, cocaine, HCBD and CCl4. With respect to P450 content and other MO activities, no changes or even decreases were generally observed. Some exceptions to this rule were found: HCBD significantly increased T15 alpha OH, PROD and EROD activities in C57Bl/6 mice, whereas cocaine caused a significant stimulation of T15 alpha OH and PROD in DBA/2 mice, It is concluded that i) different hepatoxins cause different types of liver injury and responses of the monooxygenase complex ("hepatotoxinspecific finger prints"), ii) although DBA/2 and C57Bl/6 mice responded rather similarly to hepatotoxins, also with respect to P450 content and most MO activities, they displayed a profound difference in the behaviour of COH activity, and iii) within the P450 superfamily, the regulation of COH activity seems to be rather unique, also when compared to its structurally close enzyme, testosterone 15 alpha-hydroxylase.

Posterior crus stapedectomy.

The results of stapedectomy were compared between 162 otosclerotic ears operated on using the posterior crus technique and 182 otosclerotic ears undergoing Teflon piston stapedectomy. The large fenestra technique with fascia seal to the oval window was used in all cases. Mean follow-up period was 9.6 years. Neither short-nor long-term hearing results showed any significant differences between the two surgical technique groups. Complications of surgery were more common in patients undergoing Teflon piston stapedectomy. On the other hand, a re-operation for recurring conductive deafness was performed significantly more often in patients undergoing posterior crus stapedectomy.

Stress fractures of the sesamoid bones of the first metatarsophalangeal joint in athletes.

Over a period of 11 years 15 cases of stress fractures of the sesamoid bones of the first metatarsophalangeal (MTP) joint were treated in athletes. The mean age of the patients was 22.3 years, and there were 9 males and 6 females in the series. All patients were athletes, who began to suffer from the symptoms during training without any trauma. Eight fractures were located in the medial, six in the lateral sesamoid bone, and in one case both sesamoids were affected. The diagnosis was performed on the basis of the history, symptoms, clinical examination, and radiological, or isotope scanning findings. Ten of the patients were treated conservatively by prescribing an avoidance of excessive physical activity and better training shoes. In five cases surgical excision of the fragmented painful sesamoid bone was performed. There were no complications in the series and the athletes could start gradually training 6-8 weeks after the operation. The histology showed fibrotic non-union at the fracture site and supported the diagnosis of stress fracture. Three of the conservatively treated athletes had mild symptoms in intensive training, others had a good end result.

Kinetic characteristics of norcocaine N-hydroxylation in mouse and human liver microsomes: involvement of CYP enzymes.

The first step in the oxidative metabolism of cocaine is N-demethylation to norcocaine, which is further N-hydroxylated to more toxic N-hydroxynorcocaine. In this study we examined the kinetics of norcocaine N-hydroxylation mediated by cytochrome P450 (CYP) in mouse and human liver microsomes. N-hydroxynorcocaine was identified by analytical HPLC-MS after incubation of norcocaine with mouse liver microsomes in the presence of NADPH. In mouse liver microsomes, there was no apparent difference in Km values for norcocaine N-hydroxylation between male and female microsomes, while the Vmax rate was approximately two times higher in female than in male microsomes (34+/-10 v. 16+/-4 pmol/min per mg protein). The Km value for norcocaine N-hydroxylation in human liver microsomes was approximately three times higher than that observed in comparable incubations using mouse liver microsomes, whereas the Vmax rate was ten times lower. Both cocaine and norcocaine induced type I difference spectra upon interaction with CYP in mouse liver microsomes. In contrast, in human microsomes both type I and type II spectra were recorded. In the 0.01 to 1 mM concentration range, cocaine and norcocaine inhibited mouse microsomal testosterone 6alpha-, 7alpha- and 16alpha-hydroxylation reactions by 20% to 30%. Testosterone 6beta- and 15alpha-hydroxylations were blocked by 60% and 50%, respectively, by 1 mM norcocaine, while only 40% inhibition was obtained with 1 mM cocaine. Coumarin 7-hydroxylation and pentoxyresorufin O-deethylation were inhibited by 50% by 1 and 0.4 mM norcocaine, respectively. In contrast, 10 and 2 mM cocaine, respectively, were needed to obtain the same degrees of inhibition. In human liver microsomes, 1 mM norcocaine and cocaine blocked testosterone 6beta-hydroxylase by 60% and 40%, respectively. Coumarin 7-hydroxylation was inhibited by only 30% by norcocaine (5.4 mM) and cocaine (10 mM). Norcocaine N-hydroxylation in mouse and human liver microsomes was blocked by 30% and 60%, respectively, by alpha-naphthoflavone (0.1 mM). The reaction was inhibited by 30-40% by metyrapone, cimetidine and gestodene at a concentration of 1 mM in mouse microsomes, while in human microsomes, 70% inhibition was obtained with 1 mM metyrapone and cimetidine. Taken together, these results indicate that (1) norcocaine N-hydroxylation is at least partly a CYP-mediated reaction, (2) the rate of reaction is considerably lower in human liver microsomes than in mouse liver microsomes and (3) several CYP subfamilies including 1A, 2A, 3A and possibly 2B may contribute to the formation of N-hydroxynorcocaine.

Regenerative changes in hepatic morphology and enhanced expression of CYP2B10 and CYP3A during daily administration of cocaine.

The effects of daily cocaine administration for up to 14 days were studied in terms of hepatic morphology and the expression of cytochrome P450 (CYP) enzymes in the mouse liver. Daily intraperitoneal doses of 60 mg/kg of cocaine for 3 days induced severe hepatocellular necrosis in the pericentral zone and decreased activities of CYP1A2, CYP2A4/5, and CYP2Cx. The microsomal CYP2B10 protein content was increased by about 2.5-fold, but 2B10-dependent 7-pentoxyresorufin O-dealkylase (PROD) activity was barely detectable. Five or seven daily cocaine doses caused prominent pericentral inflammation and a significant (up to 14-fold) increase in the microsomal protein content and PROD activity. An increase in microsomal CYP3A was also evident, but CYP2A5 and CYP1A2 still remained at a low level. Immunohistochemical examination showed that the relative induction of CYP2B10 and CYP3A after treatment with cocaine was strongest in perivenous hepatocytes. Immunoinhibition experiments showed that CYP2B10 accounted for catalysis of only 15% to 20% of the enhanced microsomal cocaine N-demethylase (CNDM) activity, which correlated well with immunoreactive 3A protein, and could be blocked 70% to 90% by triacetyloleandomycin. After 10 or 14 daily doses of cocaine, regenerative changes with hepatocyte ballooning were observed, coinciding with increases in CYP1A2, CYP2A4/5, and CYP3A. These results suggest the following: (1) cocaine enhances its own cytochrome P450-dependent metabolism; (2) increased production of norcocaine in microsomes is catalyzed mainly by CYP3A enzyme(s), whereas 2B10, although markedly increased by cocaine treatment, has only a minor role in cocaine hepatotoxicity; and (3) despite increased microsomal CNDM activity, cocaine-induced liver injury is reversible in mice.

The role of CYP enzymes in cocaine-induced liver damage.

Cocaine is hepatotoxic in several species, including man. A high dose of cocaine produces metabolism-dependent, mainly pericentral, liver damage. At 24 h after a single dose of cocaine, mouse hepatic P450 content decreases but CYP2A activities; coumarin 7-hydroxylase and testosterone 15 alpha-hydroxylase increase concomitant with prominent diffuse cell necrosis. Repeated administration of cocaine for up to 5 days decreases CYP1A1/2, 2A4/5, 2Cx, and 2E1 related enzymatic activities. However, after five doses of cocaine, CYP2B10 increases in conjunction with the healing process. In the acute phase, the increased CYP2A activities do not participate in cocaine bioactivation. CYP3A enzymes are principally responsible for the cocaine N-demethylation in human and mouse liver microsomes. The hepatic metabolic CYP enzyme profile will change during prolonged cocaine intake, this being accompanied by altered cell morphology. Possible connections to cocaine toxicity in man are discussed.

Effect of drinking water fluoridation on hearing of patients with otosclerosis in a low fluoride area: a follow-up study.

Fluorine content in bone samples taken from the middle ears of otosclerotic patients was determined. Otosclerotic stapes footplate was found to have a significantly higher content of fluorine than skeletal bone from the meatus. Fluorine contents in footplate and meatal wall samples of otosclerotic patients drinking fluoridated water were slightly higher than those of patients drinking low-fluoride water. In the clinical part of the study, hearing levels of 280 patients with otosclerosis living in an area with low-fluoride water were assessed. In 344 operated ears, the preoperative and long-term postoperative air conduction and bone conduction thresholds of patients drinking fluoridated water did not differ significantly from those of patients drinking low-fluoride water. After a mean follow-up period of 9.6 years, air conduction thresholds of non-operated ears in patients drinking fluoride-poor water were found to be significantly worse than those of patients drinking fluoridated tap water, likewise there were significant differences in bone conduction thresholds at 2 and 4 kHz. Thus, fluoridation of drinking water has a beneficial effect on non-operated otosclerotic ears but has no significant effect on hearing levels of operated ears.