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P E Reilly

Publications and source records attributed to P E Reilly.

At least 19 recordsLinked to original sources
PubMed2001

Relationships between non-occupational cadmium exposure and expression of nine cytochrome P450 forms in human liver and kidney cortex samples.

This study was undertaken to assess associations between age, gender, cigarette smoke and non-workplace cadmium exposure, and liver pathology and inter-individual variation in cytochrome P450 (CYP) expression in human tissues. Autopsy specimens of twenty-eight Queensland residents whose ages ranged from 3 to 89 years were analyzed for the presence of nine CYP protein isoforms by immunoblotting. All subjects were Caucasians and their liver cadmium contents ranged from 0.11 to 3.95 microg/g wet weight, while their kidney cadmium contents were in the range of 2 to 63 microg/g wet weight. CYP1A2, CYP2A6, CYP2D6, CYP3A4, and CYP3A5 were detected in liver but not in kidney, and CYP1A1 and CYP1B1 were not found in liver or kidney. Lowered liver CYP2C8/19 protein contents were found to be associated with liver pathology. Importantly, we show elevated levels of CYP2C9 protein to be associated with cadmium accumulation in liver. No mechanism that explains this association is apparent, but there are two possibilities that require further study. One is that variation in CYP2C9 protein levels may be, in part, attributed to an individual's non-workplace exposure to cadmium, or an individual's CYP2C9 genotype may be a risk factor for cadmium accumulation. A positive correlation was found between liver CYP3A4 protein and subject age. Levels of liver CYP1A2 protein, but not other CYP forms, were increased in people more exposed to cigarette smoke, but there was no association between CYP1A2 protein and cadmium. CYP2A6 protein was found in all liver samples and CYP2A6 gene typing indicated the absence of CYP2A6 null allele (CYP2A6(D)) in this sample group, confirming very low prevalence of homozygous CYP2A6(D) in Caucasians. CYP2A6 gene types W/W, W/C, and C/C were not associated with variations in liver microsomal CYP2A6 protein. CYP2D6 protein was absent in all twenty-five kidney samples tested but was detectable in liver samples of all but two subjects, indicating the prevalence of the CYP2D6 null allele (CYP2D6(D)) in this sample group to be about 7%, typical of Caucasian populations.

Adolescent↗
PubMed2001

Changes in zinc and copper homeostasis in human livers and kidneys associated with exposure to environmental cadmium.

This study was undertaken to assess changes in zinc and copper homeostasis in human tissues that could be attributed to human exposure to environmental cadmium, using samples of lung, liver and kidney cortex of 61 Queensland residents, aged 2 to 89 years, who had died of accidental causes. None of the subjects were exposed to cadmium in the workplace. Levels of zinc in liver and kidney cortex samples showed inverse associations with donor age whereas zinc in lung only showed inverse association with gender. Lung zinc levels in females were 14% lower than in males. Zinc in liver and kidney cortex samples were found to exist in at least two pools; one was associated with cadmium that bound to metallothionein (MT) and the other was associated with non-MT bound copper. In liver, the amounts of zinc in the MT pool were smaller compared to those in non-MT pool given that only 7% of zinc variations were explained by cadmium whereas 22% of the liver zinc variations were accounted for by non-MT bound copper. In sharp contrast, larger amounts of zinc in kidney cortex samples were in the MT pool, compared to those in the non-MT pool given that cadmium was found to explain 69% of total zinc variation whereas copper explained only 17% of kidney zinc variations. The levels of copper in liver were found to be increased by 45-50% in subjects with high cadmium exposure level, compared to subjects of similar ages with medium exposure level. The levels of zinc and copper in kidney cortex samples in the subjects with high cadmium exposure were both found to be significantly elevated compared to those found in the medium-exposure group whereas copper contents were about 19-23% greater than in medium- as well as low-exposure groups. Taken together these results indicate increased sequestration of zinc and copper in liver and kidney cortex samples. The increases in metal sequestrations were observed in liver samples having cadmium contents of greater than 1 microg/g wet weight and in kidney cortex having cadmium contents of greater than 26 microg/g wet weight. Zinc and copper contents in lung of this sample group, however, were not associated with cadmium due probably to lower exposure levels compared to those of liver and kidney.

Adolescent↗
PubMed2000

Evidence for a synergistic interaction between cadmium and endotoxin toxicity and for nitric oxide and cadmium displacement of metals in the kidney.

This study was undertaken to examine changes in Zn and Cu homeostasis in the liver and kidney of rats caused by cadmium (Cd) or lipopolysaccharide (LPS) administration. Twenty-five male, 7- to 8-week-old Wistar rats were divided into five groups: saline only treatment, saline treatment and food deprivation, exposure to a single dose of Cd, exposure to LPS alone, and exposure to Cd + LPS. Changes in plasma nitrate concentrations and hepatic and renal Zn and Cu contents were measured together with urinary excretion rates for the metals and nitrate on 3 consecutive days: 24 h before treatment and 24 and 48 h after treatments. Cd exposure alone for 48 h caused a nearly 2-fold increase in plasma nitrate levels with no changes in urinary nitrate excretion whereas LPS treatment caused plasma nitrate levels to increase by 10-fold and urinary nitrate excretion to increase by 4-fold. Administration of LPS 24 h after Cd exposure caused a 10-fold increase in plasma nitrate concentrations and a 100-fold increase in urinary nitrate excretion compared to the rates prior to LPS administration. These results indicate a synergistic interaction between Cd and LPS toxicity. Cd exposure also caused a marked increase in hepatic Zn levels, but LPS did not cause any changes in hepatic Zn or Cu content. In sharp contrast, both Zn and Cu contents were decreased in the kidneys by 16 and 36% in animals exposed to Cd or LPS. A correlation analysis of measured variables reveals that renal Cu contents were inversely associated with plasma nitrate concentrations while urinary Cu excretion on day 3 showed a strong positive correlation with both urinary nitrate and Cd excretions on the same day. A linear regression analysis shows 20% of the variation in urinary Cu excretion was associated with urinary Cd excretion on the same day. It is concluded that reductions in renal Cu contents caused by Cd or LPS administration may be a result of Cd and NO displacement of Cu previously bound to metallothionein.

Animals↗
PubMed1999

Pyrrolizidine alkaloids in human diet.

Pyrrolizidine alkaloids are the leading plant toxins associated with disease in humans and animals. Upon ingestion, metabolic activation in liver converts the parent compounds into highly reactive electrophiles capable of reacting with cellular macromolecules forming adducts which may initiate acute or chronic toxicity. The pyrrolizidine alkaloids present a serious health risk to human populations that may be exposed to them through contamination of foodstuffs or when plants containing them are consumed as medicinal herbs. Some pyrrolizidine alkaloids (PA) adducts are persistent in animal tissue and the metabolites may be re-released and cause damage long after the initial period of ingestion. PAs are also known to act as teratogens and abortifacients. Chronic ingestion of plants containing PAs has also led to cancer in experimental animals and metabolites of several PAs have been shown to be mutagenic in the Salmonella typhimurium/mammalian microsome system. However, no clinical association has yet been found between human cancer and exposure to PAs. Based on the extensive reports on the outcome of human exposure available in the literature, we conclude that while humans face the risk of veno-occlusive disease and childhood cirrhosis PAs are not carcinogenic to humans.

Animals↗
PubMed1999

Mechanism-based inhibition of rat liver microsomal diazepam C3-hydroxylase by mifepristone associated with loss of spectrally detectable cytochrome P450.

Since initial studies with the steroids norethindrone and ethynylestradiol, reported by White and Muller-Eberhard in 1977 (Biochem. J. 166, 57-64), there has been continuing interest in xenobiotics that bear terminal or sub-terminal acetylenic groups which can cause catalysis-dependent inhibition of CYP monooxygenases associated either with loss of prosthetic group heme or protein adduct formation. Mifepristone is a synthetic steroid bearing a propyne substitution on carbon 17 and this suggested to us that it may act as a mechanism-based inhibitor of the CYP isoforms responsible for its metabolism. In human and rat liver, CYP3A isoforms have been implicated in mifepristone clearance and mifepristone administration to rats has also been shown to induce CYP3A enzymes and the associated diazepam C3-hydroxylase activity (Cheesman, Mason and Reilly, J. Steroid Biochem. Mol. Biol., 58, 1996, 447-454). With microsomes prepared from the livers of untreated female rats and others in which diazepam C3-hydroxylase has been induced, we show here that mifepristone can cause catalysis-dependent inhibition of this monooxygenase. In addition, incubation of microsomes with mifepristone in the presence, but not in the absence, of NADPH caused loss of spectrally detectable cytochrome P450. These results suggest that heme adduct formation may result from mifepristone metabolism by CYP3A monooxygenases which undergo self-catalysed irreversible inactivation with this drug as substrate. Since mifepristone administration in vivo is able also to cause induction of the synthesis of hepatic CYP3A apoprotein, mifepristone may have the potential in human medicine for complex interactions with other co-administered drugs which are also substrates for CYP3A monooxygenases.

Animals↗
PubMed1999

Human, rat and crocodile liver microsomal monooxygenase activities measured using diazepam and nifedipine: effects of CYP3A inhibitors and relationship to immunochemically detected CYP3A apoprotein.

Nifedipine oxidase and diazepam C3-hydroxylase were tested as activities for selectively measuring CYP3A enzymes using liver microsomes from male and female human organ donors, male and female Wistar rats and male and female estuarine crocodiles. The association between CYP3A enzymes and these monooxygenations was confirmed for the human samples. Male rat samples had lower specific contents of CYP3A apoprotein than the human samples but had equivalent (nifedipine) or higher (diazepam) monooxygenase specific activities. CYP3A apoprotein was undetectable in female rat samples which had very low activities towards both substrates. Enzyme inhibition studies showed that diazepam C3-hydroxylase of male rat liver was attributable to CYP3A but corresponding results for female rats suggested a contribution from non-CYP3A enzyme. Western blotting with immunochemical detection using anti-CYP3A4 IgG suggested the presence of putative CYP3A apoprotein in male and female crocodile liver samples and inhibition studies with diazepam as substrate suggested the presence of CYP3A subfamily monooxygenase activity in these enzyme preparations. Results for nifedipine oxidase with male and female rat liver and male crocodile liver suggested major contributions to catalysis from non-CYP3A enzymes. Inhibition studies suggested that a higher proportion of nifedipine oxidase in female crocodile liver may be attributable to the putative CYP3A enzyme(s) than in male crocodile liver. These results show the need for care in the assessment of CYP3A activity of fractionated tissues when using these substrates in cross-species studies and where gender is a variable.

Alligators and Crocodiles↗
PubMed1998

Dehydromonocrotaline generates sequence-selective N-7 guanine alkylation and heat and alkali stable multiple fragment DNA crosslinks.

Monocrotaline is a pyrrolizidine alkaloid known to cause toxicity in humans and animals. Its mechanism of biological action is still unclear although DNA crosslinking has been suggested to a play a role in its activity. In this study we found that an active metabolite of monocrotaline, dehydromonocrotaline (DHM), alkylates guanines at the N7 position of DNA with a preference for 5'-GG and 5'-GA sequences. In addition, it generates piperidine- and heat-resistant multiple DNA crosslinks, as confirmed by electrophoresis and electron microscopy. On the basis of these findings, we propose that DHM undergoes rapid polymerization to a structure which is able to crosslink several fragments of DNA.

Alkylating Agents↗
PubMed1998

Differential inducibility of specific mRNA corresponding to five CYP3A isoforms in female rat liver by RU486 and food deprivation: comparison with protein abundance and enzymic activities.

The induction of cytochrome P450 3A (CYP3A) protein and mRNA by RU486 [17beta-hydroxy-11beta-(4-dimethylaminophenyl)-17alpha-1-pro pyl-estra-4,9-dien-3-one] treatment and food deprivation in female rat liver was studied using Western blotting and competitive reverse transcription-polymerase chain reaction (RT-PCR). CYP3A apoprotein levels increased in response to food deprivation and to RU486 treatment, and the combination of RU486 treatment plus food deprivation had an apparent additive effect. Food deprivation and RU486 treatment also caused increases in CYP3A1, CYP3A18, and CYP3A23 mRNA, and the combined effects of these treatments on each of these mRNA forms were synergistic. CYP3A2 mRNA was not detected in any of the treatment groups, and there was a lack of concordance between CYP3A9 mRNA levels and the specific messages corresponding to the other CYP3A isoforms. CYP3A9 mRNA levels were highest in food-deprived animals, whereas RU486 inhibited CYP3A9 mRNA expression and suppressed the induction effect of food deprivation. Food deprivation and RU486 treatment each separately caused increased microsomal diazepam C3-hydroxylase activity, and the combined effects of these treatments on this monooxygenase were additive. In contrast, the [N-methyl-14C]erythromycin demethylase activity of the fasted, RU486-treated group of rats did not differ from that of the untreated group, and kinetic analyses revealed that both groups of animals exhibited similar Km and Vmax values. These results suggest that CYP3A9 may be primarily responsible for erythromycin N-demethylation and that the isoforms induced by the combination of fasting and RU486 administration are CYP3A1, CYP3A23, and, to a lesser extent, CYP3A18.

Animals↗
PubMed1997

Expression of cytochrome P450 3A7 in Escherichia coli: effects of 5' modification and catalytic characterization of recombinant enzyme expressed in bicistronic format with NADPH-cytochrome P450 reductase.

Cytochrome P450 3A7 is the major P450 form present in fetal liver tissue and may be responsible for the detoxification of many drugs that reach the fetal circulation. We report the development of bacterial expression systems for P450 3A7. Maximal yields (up to 50 nmol P450/liter culture) were obtained with a construct in which the 5'-terminus of the 3A7 cDNA was modified to include the MALLLAVFL N-terminal sequence of recombinant bovine P450 17A (H. J. Barnes, M. P. Arlotto, and M. R. Waterman, Proc. Natl. Acad. Sci. USA 88, 5597-5601, 1991) and to incorporate several downstream amino acid substitutions derived from the P450 3A5 sequence. This sequence also appeared optimal for expression of P450 3A4 and 3A5. Recombinant P450 3A7 was partially purified using ion-exchange and hydroxylapatite chromatography and reconstituted with NADPH-cytochrome P450 reductase, cytochrome b5, and lipids. Activity comparable to that of P450 3A4 was demonstrated toward a number of procarcinogens. An alternative approach was used to further characterize recombinant 3A7 due to low yields of recombinant protein in the expression and poor recovery in the purification. P450 3A7 was subcloned into a bicistronic vector containing human NADPH-cytochrome P450 reductase and expressed in bacteria. Recombinant P450 3A7 coexpressed in bacterial membranes with NADPH-cytochrome P450 reductase showed similar levels of activity toward erythromycin (N-demethylation) and ethylmorphine (N-demethylation) to P450 3A4 and 3A5 expressed in the same system, whereas 3A7 was less active toward midazolam (1'- and 4-hydroxylation) and nifedipine (oxidation).

Amino Acid Sequence↗
PubMed1996

Effects of food deprivation and adrenalectomy on CYP3A induction by RU486 in female rats.

We have studied the effects of food deprivation and adrenalectomy on the induction by RU486 of female rat liver microsomal CYP3A apoprotein, erythromycin N-demethylase and diazepam C3-hydroxylase activities. RU486 was a potent inducer of CYP3A apoprotein in intact animals and food deprivation enhanced this response. Food deprivation alone caused only weak CYP3A apoprotein induction suggesting a synergistic interaction in the regulation of protein expression. These results were reflected in the measurements of diazepam C3-hydroxylase activity. This confirms diazepam C3-hydroxylase as a useful and easily measured index of CYP3A monooxygenase content in female rat liver microsomes. Erythromycin N-demethylase did not show concordance with this pattern; this monooxygenase was much more strongly induced by food deprivation alone than by RU486 administration and, in addition, adrenalectomy abolished the induction response to food deprivation. The lack of correspondence between the apoprotein and erythromycin N-demethylase results suggests that non-CYP3A or novel, hitherto uncharacterized CYP3A isoforms may contribute to erythromycin N-demethylation in female rats. The close agreement between the results for CYP3A apoprotein and diazepam C3-hydroxylase indicates that although RU486 possesses a terminal acetylenic moeity it does not, at the dosages used here, cause mechanism-based inactivation of the CYP3A monooxygenase protein it induces. Current studies are directed to characterizing the particular CYP3A isoform(s) whose production is stimulated by RU486.

Adrenalectomy↗
PubMed1995

Expression of cytochrome P450 3A5 in Escherichia coli: effects of 5' modification, purification, spectral characterization, reconstitution conditions, and catalytic activities.

Cytochrome P450 (P450) 3A5 is a human enzyme with 85% amino acid sequence identity to the more predominantly expressed P450 3A4 and has been reported to have overlapping catalytic specificity. The 5'-terminus of a P450 3A5 cDNA was modified for optimal expression in Escherichia coli using the vector pCW, by aligning the MALLLAVFL N-terminal sequence of recombinant bovine P450 17A (H. J. Barnes, M. P. Arlotto, and M. R. Waterman, (1991) Proc. Natl. Acad. Sci. USA 88, 5597-5601) to the 3A5 cDNA. Two constructs were made, differing by their identity with the modified 3A4 N-terminal sequence (E. M. J. Gillam, T. Baba, B-R. Kim, S. Ohmori, and F. P. Guengerich, (1993) Arch. Biochem. Biophys. 305, 123-131). The first modified sequence (3A5#1) was identical to recombinant P450 3A4 up to codon 15, the 3A5 sequence being introduced thereafter. In the other (3A5#2), the successful 3A4 N-terminal nucleotide sequence was attached to codon 30. The yield was greater than fourfold higher in the first construct [up to 260 nmol (liter culture)-1]. The recombinant P450 3A5 (construct 1) was purified to electrophoretic homogeneity using a variation of a three-step procedure developed previously for P450 3A4, with an overall yield of approximately 40%. Purified P450 3A5 was active in nifedipine oxidation, testosterone 6 beta-hydroxylation, aflatoxin 3 alpha-hydroxylation and 8,9-epoxidation, ethylmorphine N-demethylation, erythromycin N-demethylation, and d-benzphetamine N-demethylation. The reconstitution of nifedipine oxidation, testosterone 6 beta-hydroxylation, and the aflatoxin oxidation activities showed dependence upon the presence of cytochrome b5, divalent cations, phospholipid mixtures, glutathione, and cholate similar to that previously found for purified P450 3A4. However, rates of the N-demethylations of ethylmorphine, erythromycin, and d-benzphetamine were as high or higher for P450 3A5 than P450 3A4 and were not particularly dependent upon modifications of reconstitution systems [corrected].

Amino Acid Sequence↗
PubMed1992

Fluorimetric detection of serum corticosterone using high-performance liquid chromatography.

A simple, sensitive, specific and reproducible method for the determination of corticosterone concentrations in rat serum using high-performance liquid chromatography (HPLC) with fluorimetric detection is described. Corticosterone is detectable down to 0.1 ng injected onto the HPLC column. Cortisol is used as an internal standard. Ethyl acetate was used for both initial serum corticosteroid extraction and the subsequent fluorophore extraction after sulfuric acid hydrolysis; thus sulfuric acid does not enter the HPLC system. The resultant fluorophores for both corticosterone and cortisol are stable for at least two weeks at ambient temperature not requiring storage at -20 degrees C. The procedure is highly suitable for use with HPLC systems utilising automatic sample injectors. The method is specific for corticosterone; dexamethasone, cortisone and gonadal steroids are not detectable and do not interfere in this assay.

Animals↗
PubMed1992

Comparative metabolism of clinically important precursors of N-desmethyldiazepam using phenobarbitone-pretreated rat liver microsomes.

Phenobarbitone-pretreated male Sprague-Dawley rat liver microsomes were used to examine C3-hydroxylation and N-dealkylation of four clinically important benzodiazepines: diazepam (DZP), prazepam (PZP), pinazepam (PIN) and halazepam (HZP). These substrates differ only in the nature of the N-substituent of the B ring and N-desmethyldiazepam (DMD) is the N-dealkylation product in each case. C3-Hydroxylation was accordingly also studied with DMD as substrate. All monooxygenations were studied with substrates at a concentration of 10 microM, in the absence of solubilizing agents, and under conditions where the production of secondary metabolites was minimized. A 20-fold variation in the rate of C3-hydroxylation was recorded across the five substrates with HZP showing the highest rate and DMD showing the lowest rate. An almost equally large range of variation was shown for the N-dealkylation reaction, with PZP undergoing this biotransformation more than 17 times faster than DZP. Log P values (a measure of lipophilicity) for the five substrates were determined using an HPLC method and a remarkable lack of correspondence between this substrate parameter and either of the monooxygenations was noted. This suggests that multiple substrate determinants govern the relative rates of these monooxygenations. It was, however, notable that the additive rate of metabolism of these substrates by both monooxygenase routes did show an excellent correlation with substrate lipophilicity.

Animals↗
PubMed1992

Benzodiazepine metabolism in ethanol-treated male rats: use of pair-fed and age-matched controls.

The effects of chronic moderate (15%) ethanol consumption and ageing on rat hepatic cytochrome P450 monooxygenase activities were examined using diazepam, nordazepam, d-benzphetamine, erythromycin, ethylmorphine and nitrosodimethylamine (NDMA) as substrates. In addition, the effects of moderate ethanol alone on the oxidation of metoprolol, morphine and temazepam were examined. Cytochrome P450 specific content increased significantly only in the 6-week ethanol-treated rats, and no changes in percentage liver to body weights were apparent in any of the ethanol-treated animals compared with pair-fed controls. Only cytochrome P450IIIA enzyme activities displayed age-related decreases, these being identified in the pair-fed animals. C3-hydroxylation of diazepam and nordazepam (36% of controls) and N-demethylation of erythromycin and ethylmorphine (58% and 64% of controls) were decreased in 6-week ethanol-treated animals, these effects being less pronounced in the 12, 24 and 48-week ethanol-treated groups. The decrease seen for diazepam and d-benzphetamine N-demethylation caused by ethanol consumption was approximately 80% of control groups for the duration of the treatment. NDMA and morphine N-demethylations were increased to 120% of control activities and metoprolol alpha-hydroxylase was increased to 140% of control activities at 6 weeks, whilst metoprolol O-demethylase activity remained unaltered. NDMA N-demethylase activity showed a two-fold induction at 24 and 48 weeks of ethanol treatment, compared with corresponding pair-fed control groups. These results support previous findings from this laboratory showing that the same or similar P450IIIA family isozymes are involved in the C3-hydroxylation of diazepam and nordazepam.(ABSTRACT TRUNCATED AT 250 WORDS)

Age Factors↗
PubMed1991

Effects of ethinylestradiol and testosterone implants on hepatic microsomal cytochrome P450 monooxygenases of birth gonadectomized male and female Dark Agouti rats.

Monooxygenases in the cytochrome P450 IIIA subfamily are induced by a number of their xenobiotic substrates and by testosterone, an endobiotic substrate of importance in their regulation. 17 alpha-Ethinylestradiol (EE) is also metabolized by these enzymes and in this study Dark Agouti rats were used to examine the effects of subcutaneous implantation of controlled release silastic capsules containing EE to determine if this steroid also induces these enzymes. Data were compared with results obtained from equivalent groups of animals implanted with capsules containing testosterone propionate (TP). Liver microsomes prepared from male and female rats were used to identify intrinsic gender differences in the monooxygenases studied and gender differences in the responses to the implanted steroids were also determined. Effects due to imprinting of growth hormone secretion patterns were controlled by using male and female birth gonadectomized animals. Results obtained from groups with blank implants showed there were no effects due to the silastic implant material itself on the monooxygenases studied. The specific activities of erythromycin N-demethylation in liver microsomes of both EE and TP implanted male and female birth gonadectomized animals were enhanced relative to corresponding blank implanted controls consistent with both steroids having an effect to induce activity attributable to cytochrome P450 IIIA isoforms. Immunoinhibition studies using microsomes from EE treated female rats with erythromycin as substrate provided further evidence for this steroid having this induction effect. The specific activity of ethylmorphine N-demethylation was however not increased in microsomes prepared from the EE implanted female animals and was decreased in the corresponding male preparations. These findings distinguished the response to this steroid from that to TP and suggested induction by this estrogen of an isoform(s) having a more limited range of substrates than has characteristically been found in this subfamily. EE treatment also caused an increase in diazepam C3 hydroxylase consistent with an effect to induce P450 IIIA activity but this was found only in microsomes from birth gonadectomized female animals. This was in contrast to the effect of TP treatment which produced increases in this monooxygenase in both male and female animals. Another gender specific effect of EE was a striking decrease in morphine N-demethylase activity seen only in birth gonadectomized male rats. This again contrasted with the effect of TP which caused a marked increase in this activity in liver microsomes of both male and female birth gonadectomized animals consistent with the proposal that testosterone is important in the regulation of this activity.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1991

Cimetidine: an inhibitor and an inducer of rat liver microsomal cytochrome P-450.

1. Cimetidine pretreatment of male Sprague-Dawley rats caused a significant increase in the specific content of total hepatic cytochrome P-450, supporting the hypothesis that this H2-receptor antagonist has monooxygenase induction effects. 2. Quantitative ultrastructural studies of liver of cimetidine-pretreated animals also supported this hypothesis in showing a significant proliferation of smooth endoplasmic reticulum. These ultrastructural changes were qualitatively similar to those produced by treatment of rats with phenobarbital, a well-characterized monooxygenase-inducing agent whose effects were studied for comparative purposes. 3. Competitive inhibition of metoprolol alpha-hydroxylation by cimetidine in liver microsomes prepared from untreated animals (Ki = 18.8 microM) was also demonstrated. 4. These results allowed testing of the hypothesis (Burnet et al. 1986) that inhibition of a defined monooxygenase should lead to induction of the synthesis of the relevant cytochrome P-450 isozyme. 5. The finding that metoprolol alpha-hydroxylase activity of liver microsomes was lowered, not elevated, by pretreatment of animals with cimetidine argues against the concept of a causal link between monooxygenase inhibition and induction.

Animals↗
PubMed1990

Cytochrome P450IIIA enzymes in rat liver microsomes: involvement in C3-hydroxylation of diazepam and nordazepam but not N-dealkylation of diazepam and temazepam.

Microsomes prepared from livers of male and female rats of nine inbred and outbred strains and of male Sprague-Dawley rats pretreated with monooxygenase-inducing agents were used to study N-dealkylation of diazepam and temazepam and C3-hydroxylation of diazepam and nordazepam. Both C3-hydroxylation reactions were more rapid in male than in female liver preparations, but this gender-dependent pattern was not seen with the N-dealkylation reactions. These results indicate the lack of identity of the monooxygenases responsible for the two kinds of reaction and suggest that male-specific enzyme(s) are responsible for the C3-hydroxylations. Induction studies were undertaken to further define these enzymes. To do this, liver microsomes prepared from male Sprague-Dawley rats pretreated with a variety of agents known to have different monooxygenase induction effects were used. With triacetyloleandomycin, dexamethasone, and phenobarbital pretreatment, the specific activities of the C3-hydroxylation reactions were selectively elevated over corresponding control values. These particular xenobiotics are known to enhance the abundance of cytochrome P450IIIA family enzymes, and our results strongly suggest the involvement of these enzymes in the benzodiazepine B ring monooxygenations. Formation of temazepam was also shown to be inhibited by triacetyloleandomycin. This effect was demonstrated to be equal in both saline-treated and dexamethasone-treated male Sprague-Dawley rat liver microsomes, with the antibiotic present either with diazepam throughout the entire incubation period or initially with NADPH in a preincubation mix for 15 min, following which C3-hydroxylation was initiated by the addition of diazepam. These results confirm the uniformity of the involvement of cytochrome P450IIIA family enzymes in diazepam C3-hydroxylation in untreated and inducer-treated rat liver microsomes. Recent studies with human and rabbit liver microsomal preparations have shown that orthologues of these enzymes also catalyze an equivalent hydroxylation in the B ring of midazolam. These findings, considered with the present results showing that the adjacent methyl N-substituent (absent in nordazepam but present in diazepam) did not affect the selectivity of these enzymes for the C3-hydroxylation reaction, suggest that neither steric nor electronic factors markedly influence catalysis of this monooxygenation by these enzymes.

Animals↗