Coal fly ash induces hepatic and pulmonary cytochrome P-450 and sigma-aminolevulinic acid synthetase in rats

The effect of intratracheal administration of coal fly ash, its benzene-soluble and benzene-insoluble fractions has been studied on the levels of hepatic and pulmonary cytochrome P-450, cytochrome b5, and the activities of sigma-aminolevulinic acid synthetase and heme oxygenase. Fly ash and both its fractions significantly increased the levels of hepatic and pulmonary cytochrome P-450. Benzene-soluble and benzene-insoluble fractions of coal fly ash significantly increased the levels of cytochrome b5 also in both lung and liver. Fly ash and both its fractions increased the activity of sigma-aminolevulinic acid synthetase and reduced the activity of heme oxygenase in lung and liver. Glass bead particles of similar size did not show any effect on hepatic and pulmonary cytochrome P-450 and cytochrome b5.     

Reduction in pulmonary and hepatic respiratory cytochrome contents by fly ash inhalation in rats

Exposure of rats to fly ash for 15 days, 6 hours daily, inhibited pulmonary and hepatic NADH-oxidase activity. The content of cytochrome b and cytochromes a + a3 was significantly lower in the lungs of the fly-ash-exposed group. However, in liver, fly ash exposure reduced the cytochrome a + a3 level without affecting the cytochrome b content, indicating a tissue-specific effect. Mitochondrial protein content in both organs was the same in both groups.     

Induction of pulmonary drug metabolizing enzymes by coal fly ash in rats

The effect of intratracheal administration of fly ash, benzene extracted fly ash residue and benzene extract of fly ash has been studied on the activity of pulmonary mixed function oxidase. Fly ash, its benzene extract and benzene extracted residue significantly increased the levels of cytochrome P-450, cytochrome b5 and the activities of NADPH-cytochrome c reductase, NADH cytochrome b5 reductase, aminopyrine N-demethylase and glutathione S-transferase in a dose dependent manner. Phenobarbital or 3-methylcholanthrene treatment along with administration of fly ash, its benzene extracted residue and benzene extract of fly ash showed a synergistic effect on the activity of mixed function oxidase. The observed effects were due to chemical causes, i.e. organic and inorganic fractions of fly ash and not, due to its particulate nature. This was shown by the administration of glass beads which caused no alteration in the activity of pulmonary mixed function oxidase.     

Induction of the hepatic microsomal cytochrome P-450 system by trialkyl phosphorothioates in rats

Single i.p. doses of O,O,O-triethyl phosphorothioate [OOO-Et(S)], one of the suicide substrates for cytochrome P-450, caused a rapid increase of NADPH-cytochrome c reductase activity in rat liver microsomes. The increase was dose dependent but did not coincide with the recovery from the inhibition of drug-metabolizing activities. There was no change of Km value of the reductase in the induced state. The co-administration of cycloheximide repressed the stimulatory effect of OOO-Et(S), suggesting that a de novo synthesis of enzyme protein may be responsible for the increase in activity. Of four homologous tri-n-alkyl esters tested, the triethyl compound was the most effective at 24 and 48 hr after administration. Triethyl phosphate, the oxygen analog of OOO-Et(S), also caused an increase of the reductase activity, but carbon disulfide had no influence on this activity. Although O,O,S-triethyl phosphorodithioate [OOS-Et(S)] and its n-alkyl homologs also caused the inhibition of drug-metabolizing activities and the increase of the reductase activity, the recovery and the stimulation of enzyme activity were different from that of O,O,O-tri-n-aklyl phosphorothioates.     

Induction of hepatic microsomal cytochrome P-450 by mirex and kepone.

The chlorinated insecticides, mirex and Kepone, pose a threat to human health as a consequence of their pollution of the environment. We investigated their potential to affect synergistically the toxicity of other xenobiotics and the pharmacological function of drugs by induction of hepatic microsomal enzymes. Male rats were induced by ip injection of mirex (50 or 5 mg/kg/day for 5 days) or Kepone (10 or 1 mg/kg/day for 5 days). Metabolic activity was tested with warfarin and biphenyl using high-performance liquid chromatographic assays. The high doses of both compounds induced cytochrome P-450 with absorbance bands (reduced, CO complex) at 449 nm. Cytochrome concentrations were enhanced twofold relative to controls. Mirex resembled 3-methylcholanthrene and benzo[a]pyrene by inducing formation of 6-hydroxywarfarin but differed in not inducing 8-hydroxywarfarin. Kepone resembled phenobarbital in inducing 7-hydroxywarfarin but differed in its effects on the other metabolites. The low dose of mirex induced higher amounts of 4′-hydroxywarfarin than did the high dose. The metabolite profiles with high and low doses of Kepone also showed marked variations from one another. Mirex and Kepone are carcinogenic in rats and mice but, in contrast to the polycyclic aromatic carcinogens, do not markedly enhance the activity of microsomal biphenyl 2-hydroxylase relative to biphenyl 4-hydroxylase. We conclude that mirex and Kepone induce hepatic mixed-function oxidase profiles which differ from one another and from the classical inducers, phenobarbital and 3-methylcholanthrene. Mirex apparently only induces one of the enzymes induced by 3-methylcholanthrene. The enzyme profiles arising from the insecticides are dose dependent and will thus potentiate qualitatively differing effects depending on the level of ingestion.     

The destruction of pulmonary and hepatic cytochrome P-450 by phthaladehyde.

Addition of phthalaldehyde to pulmonary and hepatic microsomes resulted in destruction of cytochrome P-450 at all concentrations of phthalaldehyde tested. Phthalaldehyde destruction of pulmonary and hepatic cytochrome P-450 did not require NADPH. Addition of phthalaldehyde to pulmonary and hepatic microsomal suspension also resulted in inhibition of p-xylene hydroxylase and benzphetamine N-demethylase activities. The in vitro inhibition of p-xylene hydroxylase and benzphetamine N-demethylase as well as the destruction of P-450 could be prevented by supplementing microsomes with hepatic cytosol or purified aldehyde dehydrogenase. Lipid peroxidation did not occur when phthalaldehyde was added to either pulmonary or hepatic microsomes. It is suggested that phthalaldehyde can be used to study the in vitro drug metabolism reactions in these tissue preparations.     

Induction of rat hepatic cytochrome P-450 by ketamine and its toxicological implications

Ketamine is a common intravenous anesthetic and a frequent drug of abuse, alone or in combination with cocaine. However, the pharmacokinetic effects of ketamine have not been fully investigated. This study determined the effects of ketamine on cytochrome P-450 (P-450)-dependent catalytic activities, protein levels, and hepatotoxicity using male Wistar rats treated with 10, 20, 40, or 80 mg/kg ketamine intraperitoneally twice daily for 4 d. Treatment with ketamine produced a dose-dependent increase of pentoxyresorufin O-dealkylation activity of liver microsomes. Treatment with 80 mg/kg ketamine resulted in 14-, 3-, and 2-fold rise in O-dealkylation of pentoxyresorufin, ethoxyresorufin, and methoxyresorufin of rat liver microsomes, respectively. The treatment produced 31% and 86% increases in 7-ethoxycoumarin O-deethylation and erythromycin N-demethylation, respectively. In addition, aniline hydroxylation activity was elevated by 62%. Protein blot analysis of liver microsomal proteins revealed that 80 mg/kg ketamine induced P-450 1A, 2B, 2E1, and 3A proteins by 2-, 13-, 2-, and 2-fold, respectively. In reversibility study, ketamine-induced pentoxyresorufin O-dealkylation, 7-ethoxycoumarin O-deethylation, erythromycin N-demethylation, and methoxyresorufin O-demethylation activities of liver microsomes prepared from rats 4 d after ketamine treatment were 75%, 48%, 29%, and 38% lower than the respective activities of liver microsomes prepared from rats 1 d after treatment. Protein blot analysis showed that ketamine-induced P-450 2B1/2 proteins also decreased in a time-dependent manner in 4 d. In hepatotoxicity study, treatment of rats with 1 ml/kg CCl4 produced a 7-fold increase in serum alanine aminotransferase activity level and a 17-fold rise in rats pretreated with 80 mg/kg ketamine for 4 d. Treatment of ICR mice with 120 mg/kg cocaine produced a 17% mortality, whereas the same dose of cocaine produced a 50% mortality in mice pretreated with ketamine. Treatment of mice with 100 mg/kg cocaine produced a 76-fold increase in serum alanine aminotransferase activity level and a 260-fold rise in mice pretreated with 80 mg/kg ketamine for 4 d. The present study shows that ketamine induces the expression of multiple forms of P-450 in rat liver microsomes and increases CCl4-induced liver toxicity and cocaine-mediated acute toxicity. Other potential pharmacological or toxicological events related to ketamine use need to be further explored.     

Induction of hepatic cytochrome P-450c-dependent monooxygenase activities by dantrolene in rat

The effect of dantrolene sodium, a skeletal muscle relaxant, on drug metabolizing enzymes has been investigated after treatment of rats with a dose of 200 mg/kg for five days. We observed an induction of cytochrome P-450c and epoxide hydrolase in immunoassays and activities. An enhancement of the UDP-glucuronosyltransferase (GT1) activity was observed. We also reported a decrease of both liver cytochrome P-450 content and microsomal cytochrome P-450b dependent N-demethylation activities. On the other hand, the binding of dantrolene on microsomal cytochrome P-450 produced a type I difference spectrum, these data were obtained with liver microsomal cytochrome P-450c induced by 3-methylcholanthrene.     

Induction of hepatic and extrahepatic cytochrome P-450 and monooxygenase activities by N-substituted imidazoles

1. Seven N-substituted imidazoles, with abilities to induce rat hepatic cytochrome P-450 from 1.5- to 4-fold after 3 days of treatment (75 mg/kg daily), were investigated for their concurrent inductive effect in kidney, intestine and lung. 2. The ability of a compound to induce cytochrome P-450 in the liver did not correlate with the ability to induce in extrahepatic tissues, the highest magnitude hepatic inducer (clotrimazole) having little inductive effect in other organs. 3. Induction of cytochrome P-450 concentration was greater in kidney and intestine than in lung but, with the exception of the two imidazoles bearing either a benzyl or a 2-naphthylmethyl substituent, the degree of induction in the extrahepatic organs did not approach that seen in liver. 4. Different monooxygenase activities were preferentially induced by the individual N-substituted imidazoles in a single tissue, and activities induced by a compound in one tissue were not uniformly induced by that compound in other tissues. Induction of activities did not always correlate with an increase in cytochrome P-450 concentration.     

Elevation of rat pulmonary, hepatic and lung surfactant lipids by fly ash inhalation

Fly ash contains many polycyclic aromatic hydrocarbons and genotoxic trace elements. In rats, fly ash exposure profoundly affects lung and liver histology. In the present study, the effect of fly ash inhalation on lung and liver lipids of rats was examined. Male Wistar strain rats were exposed daily to fly ash (0.27 +/- 0.01 mg/L air) in an inhalation chamber, 6 hr daily over a period of 15 days, and were killed on various days, i.e. 16, 30, 60, and 120. Fly ash inhalation significantly (P less than 0.05) increased total phospholipids (PL), phosphatidylcholine (PC) and phosphatidylethanolamine (PE) in lungs. PC and dipalmitoylphosphatidylcholine (DPPC) contents in microsomes and lung surfactant also were significantly (P less than 0.05) higher in rats exposed to fly ash compared to control group animals. Radiolabeled precursor incorporation studies indicated that fly ash induced the synthesis of PC and DPPC by both CDP-choline pathway and N-methylation of PE in lung microsomes and enhanced their secretion into lung surfactant. In liver, PC and PE contents were elevated significantly (P less than 0.05) by fly ash exposure on days 16 and 30 respectively. A similar elevation of PC was observed in hepatic microsomes; this increase was due to its increased synthesis. However, the increased synthesis of PC in liver occurred to a greater extent by the N-methylation pathway than by the CDP-choline pathway.     

Induction of rat hepatic cytochrome P-450 I proteins by the antimutagen anthraflavic acid

Administration of the antimutagen anthraflavic acid to rats gave rise to significant increases in the hepatic microsomal O-deethylations of ethoxyresorufin and ethoxycoumarin, but not in the O-dealkylation of pentoxyresorufin nor in cytosolic glutathione S-transferase activity. Immunoblot studies of solubilized microsomes from anthraflavic acid-treated rats revealed that anthraflavic acid induced the apoproteins P-450 I A1 and A2 but not P-450 B1 and B2. Pretreatment with anthraflavic acid resulted in a marked increase in the in vitro bioactivation of 2-amino-6-methyldipyrido[1,2-a:3',2'-d]imidazole and 2-amino-3,2-amino-3-methylimidazomethylimidazo[4,5-f]-quinoline (IQ) to mutagenic intermediate(s); IQ is a carcinogen against which anthraflavic acid has displayed strong antimutagenic effect in the Ames test when incorporated into the metabolic activation system. The increase in mutagenicity of IQ was the result of enhancement of both the microsomal and cytosolic activation steps. It is concluded that anthraflavic acid is a specific inducer of P-450 I proteins in the rat and this compound is not only unlikely to exhibit any anticarcinogenic effect in vivo but may act as a co-carcinogen.     

酒精性肝损伤大鼠细胞色素P450 CYP2E1和细胞色素P450 CYP3A的代谢活性

目的观察酒精性肝损伤对大鼠细胞色素P450CYP3A(CYP3A)和细胞色素P450CYP2E1(CYP2E1)代谢活性的影响。方法采用ig给予白酒制备大鼠酒精性肝损伤模型,检测血清中谷丙转氨酶(GPT)和谷草转氨酶(GOT)活性,采用HE染色法光镜下观测酒精对肝脏损伤程度。大鼠ip给予CYP3A探针药物咪达唑仑10mg·kg-1或ig给予CYP2E1探针药物氯唑沙宗50mg·kg-1后,采用高效液相色谱法测定不同时间点大鼠血浆中咪达唑仑和氯唑沙宗的血药浓度,并应用3P87软件计算其药代动力学参数,以考察CYP2E1和CYP3A的代谢活性的变化。大鼠ig给予氯唑沙宗80mg·kg-1后,热板方法测定大鼠添足次数和添足反射潜伏期。结果酒精性肝损伤可致大鼠肝小叶结构不清,肝索排列紊乱,肝细胞体积增大,呈弥漫性中度水变性,肝窦受压,大部分肝细胞胞浆内见大小不等的脂肪空泡;与正常对照组相比,酒精性肝损伤组大鼠GPT和GOT活性分别增加了16.0%和20.0%(P<0.05,P<0.01)。酒精性肝损伤致大鼠CYP2E1对探针药物氯唑沙宗的代谢活性增强,AUC,t1/2和cmax分别降低了38.0%,30.5%和35.0%(P<0.05);酒精肝损伤组大鼠氯唑沙宗镇痛效果明显降低;酒精性肝损伤致大鼠CYP3A对探针药物咪达唑仑的代谢活性增强,AUC,t1/2和cmax分别降低了122.6%,54.9%和56.9%(P<0.01,P<0.05)。结论酒精性肝损伤可使大鼠CYP2E1和CYP3A代谢活性增强。     

Structural comparison of monoclonal antibody immunopurified pulmonary and hepatic cytochrome P-450 from 3-methylcholanthrene-treated rats

A pulmonary cytochrome P-450 was purified from lung microsomes of 3-methylcholanthrene (MC)-treated rats by immunoaffinity chromatography using a monoclonal antibody to MC-induced rat liver cytochrome P-450. The isolated pulmonary cytochrome P-450 was MC-inducible and had an apparent molecular weight of 57 kD on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weight, as well as the NH2-terminal sequence of the first nine amino acids of the pulmonary cytochrome P-450, was identical to that of an epitopically related MC-induced rat liver cytochrome P-450. In addition, partial proteolysis of both cytochromes P-450 yielded indistinguishable peptide patterns on SDS-Page. Treatment of rats with MC, therefore, induces a pulmonary cytochrome P-450 which is structurally identical to the MC-induced hepatic enzyme by several criteria.     

Imprinting of hepatic microsomal cytochrome P-450 enzyme activities and cytochrome P-450IIC11 by peripubertal administration of testosterone in female rats.

The influence of peripubertal exposure to physiological doses of testosterone on the adult androgen responsiveness of hepatic microsomal cytochrome P-450 was investigated. Male and female Sprague-Dawley rats were sham-operated or gonadectomized before puberty, at 25 days of age. They were injected subcutaneously with testosterone enanthate (5 mumol/kg/day) during the pubertal time period, on days 35-49. Responsiveness to this same dose of testosterone was tested by administering the compound during adulthood, on days 81-89. The females provided a model that had not been exposed to neonatal androgen imprinting, in contrast to the males. Testosterone 2 alpha-hydroxylase activity and cytochrome P-450IIC11, which are normally expressed only in adult males, were expressed in the gonadectomized females administered testosterone during puberty with no further exposure to the hormone for the next 40 days. The levels found were similar to those in the gonadectomized male group. When the combined pubertal and adult testosterone regimen was used, a synergistic effect was produced; the 2 alpha-hydroxylase activity reached control male levels in both gonadectomized and sham-operated females and, in addition, cytochrome P-450IIC11 attained control male levels in the gonadectomized females. Testosterone 6 beta-hydroxylase and erythromycin N-demethylase activities were used as indicators of the cytochrome P-450IIIA subfamily. These activities were significantly increased only in the females treated with testosterone during both the pubertal and adult periods, reaching control male levels of 6 beta-hydroxylation. A similar effect, but in the opposite direction, was found with testosterone 7 alpha-hydroxylase, an enzyme activity indicative of cytochrome P-450IIA1. A decrease in this enzyme was produced in the females administered testosterone during both time periods, resulting in levels equivalent to those found in control males. In general, a highly significant interaction was found between the pubertal and adult treatment periods for the females, indicating a chronic effect of the pubertal exposure. The experiments with castrated males did not result in synergistic interactions, although there was some evidence of an additive effect. The results of this study support the hypothesis that the peripubertal period is a time during which testosterone imprinting of both increased basal levels and adult androgen responsiveness of some hepatic cytochrome P-450 enzymes can occur in the female rat.     

Relation between hepatic microsomal metabolism of N-nitrosamines and cytochrome P-450 species

Effects of SKF 525A (0.1 mM), metyrapone (0.1 mM), alpha-naphthoflavone (ANF) (0.5 mM) and pyrazole (1.0 mM) on N-nitrosodimethylamine (NDMA), N-nitrosomethylbutylamine (NMBuA) and N-nitrosomethylbenzylamine (NMBeA) metabolism by hepatic microsomes from rats pretreated with inducers were investigated. NDMA demethylation was weakly increased by phenobarbital (PB) treatment. The demethylation was inhibited by SKF 525A and enhanced by metyrapone in non-treated and PB-treated microsomes, and weakly inhibited by ANF in 3-methylcholanthrene(MC)-treated microsomes. NMBuA demethylation was increased by PB treatment and inhibited by SKF 525A in all microsomes. Metyrapone inhibited the demethylation in PB-treated microsomes. NMBuA debutylation was increased by PB and MC treatments, and inhibited by metyrapone in all microsomes. The strongest inhibition by metyrapone was observed in PB-treated microsomes. The debutylation was inhibited by SKF 525A in non-treated and PB-treated microsomes and by ANF in MC-treated microsomes. NMBeA demethylation was decreased by MC treatment and weakly inhibited by SKF 525A in all microsomes. The effects of the inducers and inhibitors on NMBeA debenzylation were almost the same as those on NMBuA debutylation except that the increasing effect of MC was small. Pyrazole was a relatively selective inhibitor of NDMA demethylation. These results suggest the following: NDMA demethylation is catalyzed by PB-induced cytochrome P-450 species (P450-PB) and MC-induced cytochrome P-450 species (P448-MC). But their specific activity is low and the other cytochrome P-450 species demethylate NDMA. NMBuA demethylation is catalyzed by P450-PB. But the specific activity is not high and the other cytochrome P-450 species also demethylate NMBuA. NMBuA debutylation is catalyzed by P450-PB and P448-MC. Almost all of NMBeA demethylation is catalyzed by cytochrome P-450 species other than P450-PB and P448-MC. NMBeA debenzylation is catalyzed by P450-PB and P448-MC, but the specific activity of P448-MC is not high.     

Induction of hepatic cytochrome P-450 and xenobiotic metabolizing enzymes in rats gavaged with an Alberta crude oil

Changes in body weight gain and in biochemical parameters of blood and liver were assessed in Sprague-Dawley rats after multiple oral administration of three test doses of an Alberta crude oil (ACO). Rats treated with ACO (1.25-5 ml/kg) did not show statistically significant (p greater than .05) differences from control, corn-oil treated (5 ml/kg) rats, in body weight gains, liver weight, and blood biochemical indicators of liver (alanine aminotransferase, gamma glutamyltransferase), kidney (blood urea nitrogen, creatinine), and erythrocyte (adenosine 5'-triphosphate, 2,3-diphosphoglyceric acid, reduced glutathione) cytotoxicity. Treatment with ACO, however, caused statistically significant (p less than .05) and dose-related increases from control in (1) microsomal protein and cytochrome P-450 content, and NADPH-cytochrome c reductase, aryl hydrocarbon hydroxylase (AHH), and 7-ethoxycoumarin-O-deethylase (7-ECOD) activities, and (2) cytosolic glutathione transferase activity of liver. The induction of hepatic cytochrome P-450 and xenobiotic-metabolizing enzymes in microsomes of ACO-treated rats was probably associated with dose-related changes in isozymic forms of cytochrome P-450, as evidenced by (1) appearance of a 448-nm spectral peak in microsomes of ACO-treated rats and (2) differences in the inhibition pattern of AHH and 7-ECOD activities in microsomes of control and ACO-treated rats upon treatment with metyrapone and 7,8-benzoflavone.