Coumarin 7-hydroxylase Activity Research Articles

Comparison of Mongolian gerbil and rat hepatic microsomal monooxygenase activities: High coumarin 7-hydroxylase activity in the gerbil

Comparison of Mongolian gerbil and rat hepatic microsomal monooxygenase activities: High coumarin 7-hydroxylase activity in the gerbil

Identification of the human liver cytochrome P-450 responsible for coumarin 7-hydroxylase activity

1. We have constructed a full-length human liver cytochrome P450IIA cDNA from a partial-length clone by oligonucleotide-directed mutagenesis, and subcloned it into the monkey kidney (COS-7) cell expression vector, pSVL. 2. The cDNA encodes a 49 kDa protein with coumarin 7-hydroxylase (COH) activity which cross-reacts with antisera to the mouse cytochrome P-450 isoenzyme responsible for COH activity and comigrates with a human liver microsomal protein. 3. Western blot analysis of a panel of human livers indicates that the level of the 49 kDa protein, detected using antisera to either the mouse COH P-450 or rat P450IIA1 protein, correlates very highly with COH activity. 4. Antisera to the rat P450IIA1 protein can inhibit COH activity in human liver microsomes. Taken together, these data indicate that a member of the P450IIA subfamily is responsible for most, if not all, of the COH activity in human liver.

The CYP2A3 gene product catalyzes coumarin 7-hydroxylation in human liver microsomes

Three cDNAs, designated IIA3, IIA3v, and IIA4, coding for P450s in the CYP2A gene subfamily were isolated from a lambda gt11 library prepared from human hepatic mRNA. Only three nucleotide differences and a single amino acid difference, Leu160----His, were found between IIA3 and IIA3v, indicating that they are probably allelic variants. IIA4 displayed 94% amino acid similarity with IIA3 and IIA3v. The three cDNAs were inserted into vaccinia virus, and recombinant viruses were used to infect human hepatoma Hep G2 cells. Only IIA3 was able to produce an enzyme that had a reduced CO-bound spectrum with a lambda max at 450 nm. This expressed enzyme was able to carry out coumarin 7-hydroxylation (turnover number of 15 min-1) and ethoxycoumarin O-deethylation. cDNA-expressed IIA3v and IIA4 failed to incorporate heme and were enzymatically inactive. Analysis of IIA proteins in human liver microsomes, using antibody against rat IIA2, revealed two proteins of 49 and 50 kDa, the former of which appeared to correlate with human microsomal coumarin 7-hydroxylase activity. A more striking correlation was found between IIA mRNA and enzyme activity. The rat antibody was able to completely abolish coumarin 7-hydroxylase activity in 12 liver samples. In addition, kinetics of coumarin metabolism in two livers were monophasic over the substrate concentration tested. Km values obtained from human liver (2.3 microM) were similar to those obtained from lysates of hepatoma cells expressing IIA3 (3.6-7.1 microM).(ABSTRACT TRUNCATED AT 250 WORDS)

BROMOBENZENE-INDUCED CLARA CELL DAMAGE: THE CONTRIBUTION OF CYTOCHROME P-450 SYSTEM LOCALIZED IN THE CLARA CELL

The relationship between the alterations of different pulmonary drug metabolizing systems and the selective damages of Clara cells in ddY mice caused by bromobenzene treatment was investigated in order to elucidate the mechanism of bromobenzene-induced pneumotoxicity. Pulmonary drug metabolizing systems showed different responses to oral administration of bromobenzene (5 mmole/kg body weight) in mice at 8 and 24 hours. The time-dependent decrease of coumarin hydroxylase activity was the severest compared to other drug metabolizing enzymes. Bromobenzene treatment caused selective damages of the Clara cells without influencing the ciliated bronchiolar cells and alveolar type II cells, and the degrees of Clara cell damage became severer with time. These results suggest that the degradation of pulmonary coumarin hydroxylase is closely related to the selective damage of Clara cells induced by bromobenzene, and this enzyme contributes to the occurrence of bromobenzene-induced Clara cell damages.

Human cytochrome P450IIA3: cDNA sequence role of the enzyme in the metabolic of promutagens comparison to nitrosamine activation by human cytochrome P450IIE1

We report that, in a human cell line, human cytochrome P450IIA3 is capable of metabolizing aflatoxin B1, benzo[a]-pyrene, N-nitrosodimethylamine (NDMA) and N-nitrosodiethylamine (NDEA) to cytotoxic and mutagenic species. Cytochrome P450IIA3-mediated activation of NDMA and NDEA was compared with human cytochrome P450IIE1-mediated activation in the same cell system. P450IIE1 was more effective at activating NDMA than P450IIA3, while P450IIA3 was more effective at activating NDEA than P450IIE1. Whole cells and microsomal fractions obtained from control cells and from cells expressing the P450IIA3 cDNA were characterized for expression of P450IIA3. Microsomal coumarin 7-hydroxylase activity was some 40 times greater in the transfected cells than in the control cells and was catalyzed by a protein that was immunochemically related to the rat liver cytochrome P450IIA gene family. Immunoblot analysis demonstrated that this protein was readily detectable in transfected cells but barely detectable in control cells. We also report the DNA and deduced amino acid sequence of the P450IIA3 cDNA isolate used in this study. Our isolate encodes a protein 489 amino acids that is five amino acids shorter at the N terminus but otherwise identical to a previously reported human P450IIA3 cDNA sequence.

Effects of nitrogen dioxide and ozone in combination on xenobiotic metabolizing activities of rat lungs

Male Jcl: Wistar rats were exposed continuously to 0.2 ppm ozone (O 3) and 4 ppm nitrogen dioxide (NO 2), alone and in combination, for 1 and 2 months to examine the effects of combined gas on the xenobiotic metabolizing systems of lung microsomes. The cytochrome P-450 content increased to 200% and 253% of the control values during 2-month exposures to O 3, while it was not increased by NO 2 exposures. Addition of NO 2 to O 3 reduced the increased level of cytochrome P-450 to 179% and 178% of the control values. The activities of cytochrome P-450-dependent monooxygenase, benzo[ a]pyrene hydroxylase and 7-ethoxy-coumarin O-deethylase were changed in the same fashion by exposures to NO 2 and O 3. The 7-ethoxycoumarin O-deethylase activity was increased to 147% and 142% of the control values by O 3 exposure, whereas it was decreased to 71% and 75% of the control values by NO 2 exposures. This activity was decreased to 124% and 97% of the control values by combination of O 3 with NO 2 after 1 and 2 months, respectively. Similarly, the benzo[ a]pyrene hydroxylase activity was increased to 157% and 153% of the control values during O 3 exposures, while it was not changed by NO 2 exposures. Addition of NO 2 to O 3 reduced the activity to 140% and 115% of the control values after 1 and 2 months, respectively. The alteration of coumarin hydroxylase activity was different from those of others. This activity decreased to 44% and 29% of the control values after 1- and 2-month exposures to NO 2, respectively, and was also decreased by O 3 exposures. However, the magnitude of decrease was not reinforced by combination of NO 2 with O 3. These results indicate that an increased level of xenobiotic metabolizing activity produced by O 3 exposures is lowered by combination with NO 2. These phenomena may be antagonistic effects of these gases on the xenobiotic metabolizing systems of lung microsomes.

Mouse steroid 15 alpha-hydroxylase gene family: identification of type II P-450(15)alpha as coumarin 7-hydroxylase.

We identified type II P-450(15)alpha as mouse coumarin 7-hydroxylase (P-450coh). Unlike type I P-450(15)alpha, the other member within the mouse steroid 15 alpha-hydroxylase gene family, type II catalyzed little steroid 15 alpha-hydroxylase activity, yet structurally there were only 11 substitutions between type I and type II P-450(15)alphaS within their 494 amino acid residues (Lindberg et al., 1989), and the N-terminal sequence (21 residues) of P-450coh was identical with that of both P-450(15)alphaS. Induction by pyrazole of coumarin 7-hydroxylase activity correlated well with the increase of type II P-450(15)alpha mRNA in 129/J male and female mice. Pyrazole, on the other hand, was less in males or not effective in females in inducing the 15 alpha-hydroxylase activity and type I P-450(15)alpha mRNA. Expression of type I and II in COS-1 cells revealed that the latter catalyzed coumarin 7-hydroxylase activity at 10 to approximately 14 pmol min-1 (mg of cellular protein)-1. The former, on the other hand, had a high testosterone 15 alpha-hydroxylase but little coumarin 7-hydroxylase activity. It was concluded, therefore, that type II P-450(15)alpha is the mouse coumarin 7-hydroxylase. Identification of type II as the P-450 specific to coumarin 7-hydroxylase activity and characterization of its cDNA and gene, therefore, were significant advances toward understanding the basis of genetic regulation of this activity in mice (known as Coh locus).

Mouse liver P450Coh: Genetic regulation of the pyrazole-inducible enzyme and comparison with other P450 isoenzymes

Genetic experiments with two inbred strains of mice, AKR/J and DBA/2N, show a single major gene inheritance of additive mode for pyrazole-inducible coumarin 7-hydroxylase. Intragroup variation in the enzyme activity further suggests the contribution of minor modifying genes to the final enzyme activity. Western blot analysis with a polyclonal antibody raised against the purified isozyme P450Coh (highly active in the 7-hydroxylation of coumarin) showed that a difference in the amounts of P450Coh protein between the D2 and AKR mice is the reason for the differences in the enzyme activity between the two mouse strains. Accordingly, changes at the regulatory level rather than at the structural gene would explain the genetic difference in the activity of coumarin 7-hydroxylase. This hypothesis is further supported by the identical K m values of the basal and induced enzyme. The inducibility of coumarin 7-hydroxylase by phenobarbital (PB) and its genetic regulation have been previously studied by A. W. Wood and colleagues ((1974) Science 185, 612–614; (1979); J. Biol. Chem. 254, 5641–5646 and 5647–5651). Our present experiments show that the regulation is the same for the pyrazole-inducible enzyme. Furthermore the experiments with anti-P450Coh antibody show that the PB-and pyrazole-inducible proteins have the same molecular weight and are immunologically indistinguishable. This suggests that PB and pyrazole may induce the same enzyme. Immunoinhibition of microsomal coumarin 7-hydroxylase is practically 100% for control animals and after pretreatment with pyrazole or PB. This suggests that in each case the same or immunologically closely related proteins are metabolizing coumarin and that the P450Coh may be the only P450 isoenzyme in mouse liver microsomes catalyzing the 7-hydroxylation of coumarin. The N-terminal amino acid sequence of P450Coh was found to be identical with those from Type I and Type II genes of the mouse P45015α family for the first 21 amino acids. With rat PB-inducible P450b the homology is only 33%. Also the immunological properties of P450Coh are different from those of P450b. This may suggest that P450Coh has a closer association to the steroid 15α-hydroxylase gene family than to the P450IIB subfamily of phenobarbital-inducible isoenzymes.

A selective decrease in the xenobiotic metabolizing activity of rat lungs by nitrogen dioxide exposures

Groups of male Wistar rats were continuously exposed to nitrogen dioxide (NO 2) ranging from 1.2 to 15 ppm for 1 or 2 weeks to examine the dose-effect relationship between NO 2 and the xenobiotic metabolizing activity of lung microsomes. The lung cytochrome P-450 decrease significantly after 1-week exposures to 10 and 15 ppm NO 2 and showed a decreasing tendency after 2-week exposures to 6–10 ppm NO 2. On the other hand, the cytochrome b 5, NADPH-cytochrome P-450 reductase and NADH-cytochrome b 5 reductase of lung microsomes were increased concomitant with increase in microsomal proteins during 2-week exposures to 6–10 ppm NO 2. These results show that the lung cytochrome P-450 decreases preferentially upon exposure to NO 2 at higher concentrations. The coumarin hydroxylase activity was the most sensitive to NO 2 exposures among activities metabolizing 4 kinds of xenobiotics examined. The coumarin hydroxylase activity was decreased in a dose-dependent fashion to 67-10% of the control level by 2-week exposures to 1.2–6 ppm NO 2 and became negligible at 10 ppm NO 2. The 7-ethoxycoumarin O-deethylase activity was also decreased to 82-56% of the control level by 2-week exposures to 1.2–6 ppm NO 2 and became a constantly reduced level at 10 ppm NO 2. The benzo[ a]pyrene hydroxylase activity was decreased by exposures to NO 2 above 10 ppm, and the benzphetamine N-demethylase activity also decreased during 2-werk exposures to 6–10 ppm NO 2. These results indicate that exposures to NO 2 above 1.2 ppm cause a consistent and preferential reduction in the activities of coumarin hydroxylase and 7-ethoxycoumarin O-deethylase of lung microsomes in a dose-dependent manner.

Comparison of the metabolism and disposition of [3- 14C]coumarin in the rat and marmoset ( Callithrix jacchus)

Male Sprague-Dawley rats and marmosets were given a single oral 25 mg/kg dose of [3- 14C]coumarin and the excretion of radioactivity in the expired air, urine and faeces monitored up to 96 h. Excretion profiles were similar in both species with the bulk of the dose being excreted in the urine and faeces within 24 h. Chromatographie analysis of 0–48 h urine samples revealed similar metabolic profiles with only small amounts of unchanged coumarin and very little 7-hydroxycoumarin. Coumarin 7-hydroxylase activity was not detectable in hepatic microsomes from either species. These results demonstrate that the disposition of [3- 14C]coumarin was similar in the rat and marmoset, a New World primate, and that both species, unlike man, are poor 7-hydroxylators of coumarin.

Close linkage of the human cytochrome P450IIA and P450IIB gene subfamilies: implications for the assignment of substrate specificity

We have isolated from human liver libraries two cytochrome P450 cDNA clones (lambda MP14 and lambda MP3) which are highly similar (83% over the coding region) to mouse testosterone 15 alpha hydroxylase and are therefore part of the cytochrome P450IIA gene subfamily. The P450IIA (CYP2A) gene subfamily was found to be closely linked to the P450IIB (CYP2B) subfamily and their chromosomal location could not be distinguished using somatic cell hybrids containing fragments of chromosome 19 between 19q12 and 19q13.2. Pulsed field gel electrophoresis indicates that both gene subfamilies are contained within 350-kb genomic DNA fragments, but were separated using various restriction enzymes. Northern blot analysis identified three P450IIA mRNAs each showing a wide inter-individual variation in their levels in the liver. High levels of P450IIA transcript were associated with high levels of P450IIB transcript suggesting that common factors may influence the expression of genes within these subfamilies. Genetic analysis has suggested previously that a member of the P450IIB subfamily is responsible for coumarin hydroxylase activity in the mouse. We discuss the possibility, based on our findings of tight linkage of the human P450IIA and IIB subfamilies, that a member of the IIA subfamily is a better candidate for this enzyme activity.

Pyrazole is different from acetone and ethanol as an inducer of the polysubstrate monooxygenase system in mice: Evidence that pyrazole-inducible P450Coh is distinct from acetone-lnducible P450ac

The induction of liver microsomal monooxygenase activities elicited by pyrazole, ethanol, and acetone, all shown to be inducers of rat P450j and rabbit P450LM3a, has been compared in inbred strains of DBA 2 N , AKR J , and Balb c mouse. Pyrazole strongly increases coumarin 7-hydroxylase (COH) activity in DBA 2 N but much less in other strains. The effect of pyrazole on aniline p-hydroxylase and ethanol oxidase activities is also strain dependent: an increase was seen only in the DBA 2 N strain. Ethanol and acetone were unable to induce COH, whereas aniline p-hydroxylase and ethanol oxidase were elevated about 1.4- to 3.3-fold in all strains. No strain difference could be detected in aniline p-hydroxylase or ethanol oxidase inducibility. There was a strong correlation between aniline p-hydroxylase and ethanol oxidase activities in every strain, whereas no positive correlation could be found between COH and aniline p-hydroxylase activities. Immunoinhibition experiments showed that a polyclonal antibody against purified pyrazole-inducible COH (P450Coh) blocked about 90% of COH activity, but only about 10% of aniline p-hydroxylase or ethanol oxidase in mouse liver microsomes. Monoclonal antibody 1-91-3 (raised against rat acetone-inducible P450ac) did not inhibit COH, whereas aniline p-hydroxylase was blocked 46–76% and ethanol oxidase 25–70%, depending on the source of microsomes. In immunoblots, anti-P450Coh recognized only its own antigen but not the P450ac, whereas monoclonal antibody 1-98-1 against P450ac detected P450ac and a corresponding form in the D2 mouse liver, but not the P450Coh. The purified P450ac and P450Coh had molecular masses of 52 and 50 kDa, respectively, on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These antigens were expressed differentially in response to pyrazole, ethanol, and acetone: P450Coh was increased only after pyrazole treatment, but 1-98-1-detectable protein was elevated in D2 mouse liver microsomes by ethanol and acetone, but not by pyrazole. We conclude that mouse P450Coh and rat P450ac are not corresponding forms of the same isozyme, and that a P450ac-like protein, responsible for most of aniline p-hydroxylation and ethanol oxidation, is present in the the mouse liver. These two P450 isozymes are also dissimilarly expressed in the mouse liver in response to inducer administration.

Hepatic mitochondrial coumarin 7-hydroxylase: Comparison with the microsomal enzyme

The specific activity of cytochrome P450-linked coumarin 7-hydroxylase (COH) of hepatic mitoplasts from DBA 2 N mice is up to 55% as great as the microsomal activity. According to Western blot and immunodiffusion analysis and inhibition studies with anti-P450Coh and metyrapone, the mitoplastic P450Coh had the same molecular weight and immunochemical and catalytic properties as the corresponding microsomal enzyme. The inducibility of the two proteins by pyrazole and their genetic regulation, as studied with DBA 2 N and AKR J mice, appears to be similar. However, the mitochondrial electron transfer system was not able to support the COH activity of reconstituted microsomal P450Coh although the enzyme was fully active with the microsomal NADPH-cytochrome P450 reductase. This indicates some differences between the two proteins with respect to their interaction with the electron transfer system. This was confirmed by the ability of anti-adrenodoxin reductase antibody to effectively inhibit the mitoplastic COH but not the COH reconstituted with purified microsomal P450Coh and NADPH-P450 reductase. We have previously found that P450Coh does not react with anti-P450b or anti-P450c antibodies, which recognize respective isoforms in rat liver mitoplasts. While P450Coh from microsomes and mitoplasts possess a number of properties in common, the mitoplast P450Coh represents a new subspecies of mitochondrial P450. Some characteristics of mitoplast P450Coh may be the result of post-translational modifications necessary for processing and translocation into the mitochondria.

Immunochemical and catalytical studies on hepatic coumarin 7-hydroxylase in man, rat, and mouse

The cytochrome P-450-mediated coumarin 7-hydroxylase (COH) was studied in microsomal preparations from Wistar rat, DBA/2N mouse, and human liver. Human liver contained the highest constitutive COH activity of up to about 500 pmol mg microsomal protein min . The rat liver contained low levels of COH (about 3–5 pmol mg protein min) which could be demonstrated only with high substrate concentrations. Rabbit polyclonal antibody generated against P-450 Coh (a P-450 isozyme purified from pyrazole-treated DBA/2N mouse liver showing high activity for coumarin 7-hydroxylation) inhibited COH activity by almost 100% in human liver microsomes and 86–99% in mouse liver microsomes. Also the deethylation of 7-ethoxycoumarin was inhibited somewhat by the antibody, whereas no inhibition was obtained in ethoxyresorufin O-deethylase and aryl hydrocarbon hydroxylase activities. None of these enzyme activities was affected by the antibody in the rat liver microsomes. In Ouchterlony immunodiffusion analysis precipitin lines were obtained with human, mouse and rat liver microsomes. Complex coalescence patterns were obtained suggesting full identity between human and pyrazole-treated mouse antigens, partial identity between mouse and rat antigens, and no identity between human and rat antigens. Western blot analysis with the anti-P-450 Coh antibody revealed a distinct 48-kDa protein in all four human samples tested. A 50-kDa protein comigrating exactly with P-450 Coh was observed in microsomes from PB and pyrazole-treated mouse liver microsomes. No distinct protein bands appeared in rat liver samples. These data suggest that despite slightly differing molecular masses, the human and mouse P-450s supporting COH are structurally conserved at their active centers. The corresponding rat P-450 appears to differ from that of mouse and man.

Purification and characterization of a liver microsomal cytochrome P-450 isoenzyme with a high affinity and metabolic capacity for coumarin from pyrazole-treated D2 mice.

Microsomal coumarin 7-hydroxylase activity is regulated differently from several other monooxygenase enzymes, at least in mice [Wood, A. W. and Conney, A. H. (1974) Science (Wash. DC) 612-614]. Recently we found that in D2 mice this activity is strongly and selectivity induced by pyrazole [Juvonen, R. O., Kaipainen, P. K. and Lang, M. A. (1985) Eur. J. Biochem. 152-3-8]. This paper describes the purification of the pyrazole-inducible cytochrome P-450 isoenzyme. Because of the lability of the protein, a special procedure for the purification was developed. The procedure is based on a combination of hydrophobic and ion-exchange chromatography and the presence of 100 microM coumarin in the preparations throughout the whole purification. Coumarin effectively protected the P-450 from degradation and also converted the pyrazole-inducible P-450 to its high-spin state. This enabled us to choose only those fractions for further purification where the P-450(s) was in its high-spin state (rather than measuring the content of the total P-450). As a result the purified protein had an apparent molecular mass of 49.7 kDa, a specific content of 19.9 nmol/mg protein and a very high affinity and metabolic capacity for coumarin.

Pyrazole as a modifier of liver microsomal monooxygenase in DBA/2N and AKR/J mice

Effects of pyrazole on liver microsomal monooxygenase was studied in two inbred strains of mice, DBA/2N (D2) and AKR/J (AKR). A selective effect on microsomal monooxygenase was found. In the D2 mouse pyrazole strongly increases the coumarin 7-hydroxylase (CoH) and 7-ethoxycoumarin O-deethylase (ECDE) activities while on the total cytochrome P-450 (P-450) content and ethylmorphine N-demethylase (EMDM) and benzo( a)pyrene hydroxylase (AHH) activities the effect is biphasic (increased with lower doses and decreased with higher). For AKR the effect of pyrazole is different from the D2. The increase of CoH and ECDE is weaker and no biphasic effect for the other three parameters can be seen. Instead only a decrease takes place. The optimal dose of pyrazole for the induction of CoH in the D2 mice is 200 mg/kg once a day during three days. The effect of pyrazole is strongest in animals (D2) of 4–10 weeks old. For young animals (2 weeks old) no effect except of a weak decrease in AHH can be seen. Also for old animals the effect is weak. Recovery of the monooxygenase after pyrazole induction takes place in about 120 hr except for the total P-450 content which is still below normal. No sex dependence in the effect of pyrazole on CoH was found.

The effect of cigarette smoking on 7-ethoxyresorufin O-deethylase and other monooxygenase activities in human liver: analyses with monoclonal antibodies.

Four cytochrome P-450 enzyme activities, 7-ethoxyresorufin O-deethylase (ERDE), coumarin 7-hydroxylase (CH), 7-ethoxycoumarin O-deethylase (ECDE) and aryl hydrocarbon hydroxylase (AHH) were measured in human liver needle biopsy samples from smokers and non-smokers. Cigarette smoking was verified and quantitated by measuring plasma cotinine levels. Enzyme inhibitory monoclonal antibodies (MAb) to a 3-methylcholanthrene-induced (MAb 1-7-1) and phenobarbitone-induced (MAb 2-66-3) rat hepatic cytochrome P-450 were used to measure the contribution of MAb-defined, epitope-specific cytochromes P-450 to the total reaction measured for each of the above activities. ERDE activity was significantly elevated in the livers of cigarette smokers, whereas AHH, CH or ECDE activities were not affected by cigarette smoking. No correlation was observed between plasma cotinine concentration and ERDE activity. MAb 1-7-1 inhibited hepatic ERDE activity to a variable extent (from 0 to 65%), but had very little or no effect on AHH, CH or ECDE activities. The inhibitory effect of MAb 1-7-1 on ERDE activity was greater than 50% in the non-smokers. MAb 2-66-3 had no inhibitory effect on any of the enzyme activities studied. In contrast to liver both ERDE and AHH on human placental microsomes from cigarette smokers were inhibited by MAb 1-7-1. The MAb 2-66-3 was without effect. Cigarette smoking induces a form of P-450 in human liver, responsible for ERDE activity, that contains an epitope recognized by MAb 1-7-1. This form of cytochrome P-450 is insensitive to MAb 2-66-3 and is not contributing to AHH, CH or ECDE activities of human liver.

3-Methylcholanthrene-induced ethoxyresorufin O-deethylase activity in alveolar type II cells of rabbit lung.

Lungs of 3-methylcholanthrene-treated rabbits and those of untreated rabbits were digested with protease, and the cells were separated into an alveolar type II cell-rich fraction and a Clara cell-rich fraction by the use of counter-flow centrifugal elutriation. Drug oxidation activities of the cell homogenate of each fraction were examined. Marked elevation of ethoxyresorufin O-deethylase activity was found mainly in the cell homogenate of the alveolar type II cell-rich fraction after 3-methylcholanthrene treatment. The activity increased 6-fold with the drug treatment. Coumarin hydroxylase activity was found mainly in the cell homogenates of the Clara cell-rich fraction of the 3-methylcholanthrene-treated and untreated rabbits. The activity increased 3.5-fold with the drug treatment.

Inhibition by methylglyoxal bis(guanylhydrazone) of drug oxidation reactions catalyzed by mouse liver microsomes in vivo and in vitro.

The activity of coumarin 7-hydroxylase (coumarin 7-hydroxylation) was inhibited in B6 mouse liver after a single injection of methylglyoxal bis(guanylhydrazone (MGBG). The decrease in the activity in vivo was greatest (70%) one day after the drug injection and the hydroxylase activity in microsomal fraction prepared from livers of MGBG-treated B6 mice was still 25% decreased 5 days after the drug. The amount of cytochrome P-450 also was decreased in MGBG-treated livers with the same time-dependency as the inhibition of coumarin 7-hydroxylation. MGBG and its close derivative 1,1'-[methylethanediylidene)dinitrilo)bis(3-aminoguanidine) (MBAG) inhibited the activity in vitro of coumarin 7-hydroxylase, benzo(a)pyrene hydroxylase and 7-ethoxy 0-de-ethylase when microsomes were prepared from livers of untreated B6 mice. In every case MBAG was a better inhibitor than MGBG in vitro. The in vitro inhibition of MGBG of several drug metabolizing enzymes was not reversed when microsomes were preincubated with 1 mM putrescine, spermidine or spermine. These results suggest that the anti-cancer drug, MGBG, has a severe effect(s) on the drug metabolizing system at concentrations reached during the treatment of patients with MGBG.

1,4-Bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP) and related compounds as inducers of hepatic monooxygenases: Structure-activity effects

1,4-Bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP) resembles phenobarbital (PB) in its mode of induction of the hepatic drug-metabolizing enzymes in mice. The structural features of this molecule include: a linear tricyclic aromatic ether ring system, an internal 1,4-disubstituted benzene ring and two 3,5-dichloropyridyloxy substituents. Ten analogs of TCPOBOP have been synthesized and their activities as microsomal enzyme inducers evaluated. Dose-response induction of mice hepatic microsomal cytochrome P-450, aldrin epoxidase and dimethylaminoantipyrine N-demethylase gave ed 50 values for TCPOBOP and five homologs. The results illustrate that changes in the structure of the pyridyloxy ring markedly affect enzyme induction activity. The order of activity for the substituents was 3,5-dibromopyridyloxy ≈ 3,5-dichloropyridyloxy > 5-bromopyridyloxy ≈ 5-chloropyridyloxy > 3-chloropyridyloxy > pyridyloxy. In addition, the effects of altered substitution pattern of the benzene ring and structural alterations of the internal ring moiety were evaluated by measuring hepatic microsomal coumarin hydroxylase activity. The results confirm the microsomal monooxygenase enzyme induction activity of TCPOBOP, and the observed structure-dependent potencies of several related homologs support a receptor-mediated mechanism of action for the process.