The effects of age on the pharmacokinetics and biotransformation of theophylline in vivo and in vitro in the Mongolian gerbil (Meriones unguiculatus).

The effect of post maturational aging on the in vivo disposition of theophylline was examined in the Mongolian gerbils (Meriones unguiculatus) aged 30-39 (old), 12-18 (middle-aged) and 3 (young) months following a 20 mg/kg i.p. dose. Biotransformation of theophylline was also examined in liver microsomes from non-induced and 3-methylcholanthrene induced gerbils. Analysis of theophylline plasma kinetics showed decreased clearance, increased half-life and increased volume of distribution in old vs. young animals. Clearance to the 1,3-dimethyluric acid metabolite was similar for all age groups, while clearance to the 1-methyluric acid metabolite was significantly lower in the middle-aged group compared to that of young and old gerbils. Urinary recovery of 1-methylurate was increased in old vs. young and middle-aged animals while recovery of theophylline was decreased. 3-Methylcholanthrene induction resulted in decreased recovery of theophylline and increased recovery of 1,3-dimethylurate and 1-methylurate in young and middle-aged gerbils compared to non-induced controls. Decreased microsomal protein content was observed in old vs. young and middle-aged gerbils and an age-related decrease in cytochrome P-450 content (nmol P-450/g liver) was also observed. The rate of dimethylurate formation was decreased 37% in microsomes from old vs. young and middle-aged gerbils. 3-Methylcholanthrene administration resulted in a 2- and 1.5-fold increase in the rate of 1,3-dimethylurate formation in young and middle-aged gerbils, respectively. The results of these experiments indicate that the Mongolian gerbil may be useful for the study of the biochemical mechanisms underlying age-related changes in the biotransformation and kinetics of theophylline.     

Effects of inhibition and induction of cytochrome P-450 isozymes on hyperoxic lung injury in rats.

Pulmonary oxygen toxicity most likely results from excessive production of reactive oxygen species. The role of the cytochromes P-450 in this process is controversial because these enzymes have been reported both to enhance hyperoxic lung injury and to protect from the damaging effects of 100% oxygen. We sought to further determine the role of the cytochromes P-450 in hyperoxic lung injury by inhibiting and inducing pulmonary cytochrome P-450 isozymes in rats. Treatment with the cytochrome P-450 inhibitor cimetidine or 8-methoxypsoralen did not improve survival or reduce lung edema in rats exposed to 100% oxygen. The activity of cytochrome P-450IIB1, the major pulmonary cytochrome P-450 isozyme in rats, was clearly inhibited by 8-methoxypsoralen. beta-Naphthoflavone (beta NF), a selective inducer of cytochrome P-450IA1, was administered in two-dose and five-dose regimens. The two-dose regimen produced significant and sustained induction of cytochrome P-450IA1 activity, but survival in these rats was not improved when exposed to 100% oxygen. In rats treated with five doses of beta NF, a small increase in survival time was found from 71.1 +/- 8.7 to 88.0 +/- 20.2 h; however, there was no difference in the induction of cytochrome P-450IA1 activity between this five-dose regimen and the two-dose regimen. The small improvement in survival after five doses of beta NF is thus unrelated to cytochrome P-450IA1 induction. We conclude that neither inhibition of cytochrome P-450IIB1 activity nor induction of cytochrome P-450IA1 activity protects adult rats against hyperoxic lung injury.     

Induction of tolerance in mice and rats to the effect of endotoxin to decrease the hepatic microsomal mixed-function oxidase system. Evidence for a possible macrophage-derived factor in the endotoxin effect

Daily administration of low, non-lethal doses of bacterial endotoxin to mice and rats has been shown to induce tolerance to the effect of an acute challenge dose of endotoxin to decrease the hepatic microsomal drug metabolizing activity, the level of cytochrome P-450, and to increase heme oxygenase activity. The serum collected at various times after injection of endotoxin into control animals when injected into untreated animals markedly depressed aniline hydroxylase activity, ethylmorphine N-demethylase activity, and the level of cytochrome P-450. Tolerant animals were not affected by the post-endotoxin serum injection, suggesting the decreased activity caused by the serum in untreated animals was probably due to endotoxin contained in the serum. Injection of tolerant mice and rats with supernatant medium obtained from cultures of peritoneal macrophages incubated with 100 micrograms/ml of endotoxin caused a loss of hepatic microsomal drug-metabolizing activity, and a decrease in the level of cytochrome P-450. These results suggest that peritoneal macrophages release a factor in response to endotoxin that mediates the decreased hepatic mixed-function oxidase activity.     

Effect of PSK, a protein-bound polysaccharide from Coriolus versicolor, on drug-metabolizing enzymes in sarcoma-180 bearing and normal mice

The effects of PSK and Propionibacterium acnes (anaerobic Corynebacterium) on hepatic drug-metabolizing enzymes were studied using sarcoma-180 bearing and non-tumor bearing mice. PSK had no influence on aminopyrine N-demethylase and aniline hydroxylase activities, cytochrome P-450 concentration in hepatic microsomes, and the reductase activity of cytochrome c in normal mice. The content of cytochrome P-450 was not significantly reduced in S-180 bearing mice. On the other hand, P. acnes administration significantly decreased the amount of cytochromes P-450 and b5 and aminopyrine N-demethylase activity. When FT-207 (Tegafur) was administered orally to S-180 bearing mice combined with the immunoadjuvants, only P. acnes significantly reduced the 5-FU levels in the serum and some organs.     

Immunohistochemical localization of cytochrome P-450 in rat brain

The presence of cytochrome P-450 in rat brain was studied by immunohistochemistry, using antibodies to cytochrome P-450 purified from livers of phenobarbital- or 3-methylcholanthrene-treated rats. Immunoreactive nerves were observed only in brain sections incubated with immunoglobulin-G to 3-methylcholanthrene-induced cytochrome P-450. This immunoreactivity was abolished by preabsorption of the antibody with highly purified rat liver cytochrome P-450c, the major cytochrome P-450 isozyme induced by 3-methylcholanthrene, but was not affected by other cytochrome P-450 isozymes induced by phenobarbital, isosafrole or pregnenolone-16-carbonitrile.

The most abundant concentration of nerve fibers with cytochrome P-450 immunoreactivity was observed in the globus pallidus. Immunoreactive fibers were also observed in the caudate putamen, amygdala, septum, ventromedial nucleus of the hypothalamus, medial forebrain bundle, ansa lenticularis, and ventromedial portion of the internal capsule and crus cerebri. Cell bodies with cytochrome P-450 immunoreactivity were observed in the caudate putamen and in the perifornical area of the hypothalamus. The cytochrome P-450 immunoreactive fibers in the globus pallidus and caudate putamen do not appear to emanate from cell bodies in the substantia nigra, since there was no reduction in the density of these fibers after unilateral stereotaxic electrolytic destruction of the substantia nigra (zona compacta and reticulata). Our data suggest that these striatal nerve processes are derived from cell bodies within the caudate putamen itself.

The present results indicate that rat brain contains a form of cytochrome P-450 with antigenic relatedness to the hepatic 3-methylcholanthrene-inducible cytochrome P-450c. This cytochrome P-450 isozyme was detected in brain areas which metabolize morphine and convert estradiol and estrone into catecholestrogens, which suggests an important role for this enzyme in the metabolism of both ex´ogenous and endogenous compounds in brain.     

Drug metabolism in rats with arthritis induced by 6-sulfanilamidoindazole (6-SAI)

Hexobarbital sleeping time was prolonged and ethylmorphine N-demethylation was inhibited after a single dosage or seven administrations of 6-SAI to old rats. These effects were independent of the development of arthritis. Changes in cytochrome P-450 concentration after 6-SAI treatment were insignificant and thus not responsible for the decrease in drug metabolism.In vitro 6-SAI inhibited ethylmorphine N-demethylation; the inhibition was of a mixed type. 6-SAI bound to cytochrome P-450 and induced a type II spectrum. The magnitude of hexobarbital-induced type I spectral changes was diminished by 6-SAI.It is concluded that 6-SAI inhibits cytochrome P-450-dependent drug metabolism by binding to cytochrome P-450.     

Interactions between cyclosporin A, indomethacin and 16,16-dimethyl prostaglandin E2: effects on renal, hepatic and gastrointestinal toxicity in the rat

Rats were treated for 3 or 14 days with cyclosporin A (CsA, 50 mg/kg) or indomethacin (2 or 5 mg/kg) either alone or in combination, or with CsA plus 16,16-dimethylprostaglandin E2 (DMPGE2, 0.25 mg/kg). Hepatic and renal function were unaffected by treatment with indomethacin at either dose and only at the higher dose was severe intestinal ulceration observed. CsA caused renal and hepatic toxicity, evidenced by increased urine N-acetyl-beta-D-glucosaminidase activity, serum urea, creatinine and bilirubin and decreased serum albumin and total protein. In rats cotreated with CsA and either dose of indomethacin the increases in serum urea and creatinine and decreases in serum albumin and total protein were accentuated, but serum bilirubin was not further increased. Intestinal lesions were present in rats treated for 14 days with CsA plus the lower dose of indomethacin, but not in rats treated with either drug alone. In rats treated with DMPGE2 plus CsA, serum urea and creatinine were normal and urine N-acetyl-beta-D-glucosaminidase activity was reduced compared to rats treated with CsA alone, but DMPGE2 cotreatment had no effect on the CsA induced hyperbilirubinaemia. Hepatic microsomal cytochrome P-450 concentration and aminopyrine N-demethylase activity were lower in rats treated with CsA plus indomethacin than in untreated rats or those treated with either drug alone. Coadministration of indomethacin or DMPGE2 had no effect on serum trough CsA levels. The results are interpreted as showing an exacerbation by CsA of the intestinal toxicity of indomethacin, an increase by indomethacin in the renal toxicity of CsA and a protection by DMPGE2 against CsA renal toxicity. Possible mechanisms involving drug interactions and either hepatic cytochrome P-450, renal cyclooxygenase or other renal sites are discussed.     

Enzym-Induktion in der Hefe Lodderomyces in Gegenwart von n-Alkanen

The utilization of n-alkanes is connected with extensive modifications of the yeast cell, especially of the cytochrome P-450-containing membrane. Beside the cytochrome P-450 the NADPH cytochrome P-450 reductase, the cytochrome b₅, long-chain alcohol and long-chain aldehyde dehydrogenases are induced. The activity of the alkane-hydroxylating enzyme system grows more than the concentration of its terminal oxidase. The induction of the cytochrome P-450 is inhibited by cycloheximide. A low concentration of oxygen in the culture medium amplifies the induction both of the alkane-hydroxylating enzyme system and of catalase and cytochrome oxidase, which are localized in the peroxisomes and mitochondria, respectively.     

照射和电击对大鼠性激素,肝功能和脂质过氧化物的影响

本文观察了长期低剂量ν射线照射和照射加电击对中年大鼠血浆性激素水平、肝微粒体细胞色素P-450浓度和混合功能氧化酶(MFO)活力的影响。照射组与照射加电击组雄性大鼠血浆睾酮水平明显降低(分别为对照组的67％和58％,P<0.05)。照射加电击还使雄鼠肝微粒体细胞色素p-450浓度和MFO潘力较对照组、照射组明显下降(P<0.01),但雌性大鼠血浆性激素水平与肝功能均无明显变化。照射使睾丸线粒体、肝微粒体脂质过氧化物明显升高,而照射加电击使有关组织脂质过氧化物明显降低。这些结果说明照射与照射加电击对中年大鼠具有不同的作用特点。     

Biochemical studies of promoters of carcinogenesis in rat liver

Adult female rats were orally dosed with 1/5 to 3/5 the published LD50 of either promoters or putative promoters of carcinogenesis [hexachlorobenzene (HCB), alpha-hexachlorocyclohexane (alpha-HCH), kepone and toxaphene] or noncarcinogens [coumaphos, EDTA, caprolactam, 8-hydroxyquinoline, titanium (IV) oxide, sodium diethyldithiocarbamate (DEDTC), and sucrose] at 21 and 4 h before sacrifice. The promoters selected in this study were all of the halogenated hydrocarbon class. At doses of 1/5 to 3/5 the LD50, all four promoters or putative promoters induced rat hepatic ODC activity. The seven noncarcinogens produced several biochemical effects at doses of 1/5 the LD50: increased serum alanine aminotransferase activity (SGPT) (caprolactam and DEDTC), decreased hepatic cytochrome P-450 content (DEDTC), and increased hepatic ODC activity (8-hydroxyquinoline and DEDTC). None of the seven noncarcinogens caused hepatic DNA damage or coordinate induction of hepatic ODC and cytochrome P-450. The results support the interpretation that several of these biochemical parameters are useful in distinguishing potential tumor promoters and noncarcinogens.     

The effect of age on antipyrine metabolism by liver microsomes isolated from phenobarbital-treated rats

A study was performed to test the hypothesis that ageing influences the activities of diverse forms or populations of cytochrome P-450 isoenzymes in different ways. The formation of antipyrine metabolites in induced rats is mediated by such different forms or populations of cytochrome P-450 isoenzymes. To test the hypothesis, the formation rates of antipyrine metabolites by liver microsomes isolated from phenobarbital-treated rats of different ages was determined. After phenobarbital induction in vitro, the maximal velocity for norantipyrine formation decreased from 12 to 24 months and then showed a tendency to increase with age. Hydroxymethylantipyrine formation did not change with age. 4-Hydroxyantipyrine increased between 3 and 12 months and remained constant afterwards. This is in agreement with data obtained in vivo in uninduced male BN/BiRij rats. It can be concluded that age does indeed influence the activities of different forms or populations of the cytochrome P-450 isoenzymes in different ways. Consequently, determining the overall clearance of antipyrine, which is metabolized by several isoenzymes, especially in the induced situation, is not to be recommended for measuring the activity of cytochrome P-450 isoenzymes as a function of age.     

The antitoxic function of the liver in D-galactosamine poisoning

Two intragastric administrations of 500 mg/kg of D-galactosamine reduce the RNA and the cytochrome P-45, and b5 content in the hepatic microsomes of rats; inhibit the activity of aminopyrine-N-demethylase, hexobarbital hydroxylase, aniline-p-hydroxylase, and glutathione-S-transferase; reduce the rate of NADP.H and NAD.H oxidation; accelerate inactivation of cytochrome P-450 to cytochrome P-420; reduce the number of points of hexobarbital binding with N-octilamine, though increase the hemoprotein affinity to these substrates. Destruction of the nucleus, endoplasmic reticulum, and mitochondria occurs in the hepatocytes of D-galactosamine poisoned rats.     

Hepatotoxicity of vinyl chloride and 1,1-dichloroethylene. Role of mixed function oxidase system.

Vinyl chloride, an occupational carcinogen, produces acute liver injury in rats pretreated with phenobarbital or Aroclor 1254. Injury appears related to morphologic changes in the endoplasmic reticulum. The degree of injury, as indicated by elevation of serum enzymes derived from the liver, correlates with the magnitude of induction of cytochrome P-450 and its reduction by NADPH. Hepatic injury following 1,1-dichloroethylene exposure differs strikingly from that caused by vinyl chloride and appears to involve plasma membranes, mitochondria, and chromatin and spares endoplasmic reticulum. Induction of cytochrome P-450 appears to protect against 1,1-dichloroethylene but not vinyl chloride.     

Substrate specificity of age-related changes in the inducibility of hepatic microsomal monooxygenases in middle-aged rats

Hepatic microsomal monooxygenase induction was investigated in young-adult and middle-aged male Fischer 344 rats. Monooxygenase components and drug metabolism activities were determined in liver microsomes prepared from rats treated with phenobarbital (PB), β-naphthoflavone (BNF) or methyltestosterone (MT) and compared with values from untreated rats. PB and BNF effects on cytochrome P-450 concentration and cytochrome c reductase activity were similar in young-adult and middle-aged animals. However, the extent of cytochrome P-450 induction by MT was less in the older animals. The age-related changes in induction of drug metabolism activities differed with different substrates for the monooxygenase system. In contrast to the inducibility of benzphetamine N-demethylation and aniline hydroxylation, which was dimished in the older rats, the inducibility of nitroanisole O-demethylation was enhanced. The results imply that qualitative changes in the microsomal enzyme system occurred as the animals progresses from young to middle adulthood.     

Halonal is a phenobarbital inductor of the monooxygenase system

Experiments on rats demonstrated induction of the hepatic monooxygenase system with halonal. The effects of halonal and phenobarbital on the contents of cytochrome P-450 and its isoform P-450b+e and on the rate of substrate metabolism were similar. This suggests that enzyme-inducing activity of halonal is determined by the effect of its major metabolite. It cannot be excluded that halonal molecules possess intrinsic enzyme-inducing activity.     

Effect of endotoxin to differentially affect cytochrome P-450 monooxygenase activities of untreated rats and animals induced with phenobarbital or 3-methylcholanthrene

The effect of endotoxin in decreasing the cytochrome P-450-dependent metabolism of aniline, aminopyrine and ethoxycoumarin was examined in untreated rats, and in rats pretreated with either phenobarbital or 3-methylcholanthrene. Ethoxycoumarin metabolism was determined at two substrate concentrations (5 microM and 500 microM) to determine the effect of endotoxin on the high and low affinity enzyme activities. In untreated animals, endotoxin depressed both aniline and ethoxycoumarin metabolism by the high and low affinity enzymes by approximately 70%, but aminopyrine was decreased by only 47%. In phenobarbital pretreated rats, endotoxin decreased enzyme activities less than in untreated animals. Aniline metabolism and low affinity ethoxycoumarin metabolism were decreased by only 24%, and aminopyrine metabolism was decreased by 35%. The high affinity ethoxycoumarin metabolism was least affected, being decreased by only 12%. In 3-methycholanthrene pretreated rats, aniline and ethoxycoumarin (500 microM) metabolism were decreased by approximately 45%, but aminopyrine metabolism was only decreased by 20%. In these animals, endotoxin did not significantly affect the activity of ethoxycoumarin metabolism assayed with the low substrate concentration. Endotoxin decreased total cytochrome P-450 level of untreated rats by 32%, of phenobarbital pretreated rats by 39%, and in 3-methylcholanthrene pretreated animals the decrease was only 21%. Heme oxygenase activity of untreated animals was induced most by endotoxin administration and least in phenobarbital treated rats. The data suggest that endotoxin may differentially affect the various isozymes of cytochrome P-450 associated with the metabolism of aniline, aminopyrine and ethoxycoumarin. The results also suggest that the isozymes associated with these activities in untreated, phenobarbital or 3-methylcholanthrene pretreated rats may differ in their sensitivity to the effect of endotoxin.     

Turnover of hepatic cytochrome P-450 in experimental cholestasis

In mechanical experimental chllestasis, hypertrophy of smooth microsomal membranes was observed. In contrast to typical induction, the membranes were deficient in cytochrome P-450. The total cytochrome P-450 content of the liver, however, as determined in the liver homogenate remained unchanged. To clarify the mechanism of the development of cytochrome P-450 deficient membranes in cholestasis, the half life of the heme portion of cytochrome P-450, and the initial rate of synthesis of cytochrome P-450 and b₅ hemes were compared in bile duct ligated rats and in control animals after labeling the heme by injection of the precursor δ-[4-¹⁴C]aminolevulinic acid. The half lives were not significantly different, which eliminates the possibility that selective destruction of cytochrome P-450 has occurred. Depression of cytochromal heme synthesis was not observed. During mechanical cholestasis, the relative cytochrome P-450 deficiency is probably caused by proliferation of components of the endoplasmic reticulum other than the hemoprotein.     

Efficiency of enzyme-inducing agents in rats with intrahepatic cholestasis

Inductors of the monooxygenase system benzonal, halonal, and halodif prevented the development of intrahepatic cholestasis induced by a-naphthylisothiocyanate and stimulated detoxifying function of the liver in rats. These agents increased the content of microsomal protein and cytochrome P-450 and accelerated metabolism of types I and II substrates. This was accompanied by a decrease of serum concentrations of total and free bilirubin and activity of liver-specific enzymes. Phenobarbital did not prevent the development of hepatocyte cytolysis.     

Phenotyping cytochromes P450 with monoclonal antibodies

Monoclonal antibodies (MAbs) to cytochrome P-450 isozymes can be used to phenotype tissues for epitope-specific cytochrome P-450 content. MAbs that inhibit specific cytochrome P-450 dependent drug or carcinogen reactions are useful tools for quantitative measurement of the individual or classes of cytochromes P-450 that catalyze these reactions. This method has been applied successfully to animal as well as human tissues. Radioimmunoassays based on MAbs have been developed and provide a rapid and efficient means for detecting cytochromes P-450 independent of functional enzyme activity. In addition, MAbs coupled to a Sepharose support can be used to immunopurify cytochromes P-450 in a procedure that is more rapid and efficient than conventional purification schemes. MAbs add a new dimension to analyses of cytochrome P-450 multiplicity and will find numerous applications in elucidation of the relationship between cytochrome P-450 phenotype and carcinogen or drug metabolism.     

Aldehydic products of lipid peroxidation inactivate cytochrome P-450.

The effects of aldehydic products of lipid peroxidation, malondialdehyde (MDA) and 4-hydroxynonenal (HNE), on the structure of rat liver microsomal membrane and cytochrome P-450 was studied. MDA (15-30 microM) similarly to p-chlormercuribenzoate decreased the cytochrome P-450 content by 50 % and lowered microviscosity of lipid surrounding of the spin label OTMB bound to SH-groups of membrane proteins. OTMB was effectively reduced by K3Fe(CN)6 in microsomes preincubated with MDA (20 (M), but not in native microsomes. HNE (10 microM) decreased the cytochrome P-450 content by 90 %. Reduced glutathione and cysteine (5 mM) prevented the decrease of cytochrome P-450 under influence of both MDA or HNE, whereas cytochrome P-420 formation remains unchanged. MDA and HNE decreased activities of NADPH oxidase and NADPH cytochrome c reductase. HNE increased microviscosity of the OTMB lipid environment. The further increase of HNE concentration did not affect this parameter. Both MDA and HNE increased the absorbance at 420 nm, which indicated inactivation of cytochrome P-450 by changes in hydrophobicity of lipid surrounding. We suggest that HNE and aliphatic aldehydes at low concentrations can enter into hydrophobic environment of cytochrome P-450 binding to its SH-groups, which led to inactivation of cytochrome P-450. At the same time, the modification of membrane surface layer and subsequent decrease of hydrophobicity of cytochrome P-450 environment preceded the binding of MDA to SH-groups of cytochrome P-450 to develop its inactivating effect.