Synthesis and in vitro activity of some epimeric 20 alpha-hydroxy, 20-oxime and aziridine pregnene derivatives as inhibitors of human 17 alpha-hydroxylase/C17,20-lyase and 5 alpha-reductase

Some epimeric 20-hydroxy, 20-oxime, 16 alpha, 17 alpha-, 17,20- and 20,21-aziridine derivatives of progesterone were synthesized and evaluated as inhibitors of human 17 alpha-hydroxylase/C17,20-lyase (P450(17) alpha) and 5 alpha-reductase (5 alpha-R). The reduction of 16-dehydropregenolone acetate (3a) was reinvestigated. NaBH4 in the presence of CeCl3 gave better stereo-selectivity for 20 beta-ol [20 alpha/20 beta-OH (4 alpha/4 beta) = 1/2.7] than LTBAH or the Meerwein-Pondroff method reported; reduction with Zn in HOAc formed exclusively 20 alpha-ol (4 alpha b). The 20 alpha- and 20 beta-hydroxy-4,16-pregnadien-3-one (9 alpha) and (9 beta) were synthesized from the alcohols 4 alpha b and 4 beta b. Several 20-oxime pregnadienes and 16 alpha, 17 alpha-, 17,20- and 20,21-aziridinyl-5-pregnene derivatives were also synthesized. LiAlH4 reduction of the 16-en-20-oxime (12b) yielded 20 (R)-(13a) and 20(S)-17 alpha,20-aziridine (13b) and 20(R)-17 beta,20-aziridine (14a). Several compounds inhibited the human P450(17) alpha with greater potency than ketoconzole. The 5 alpha-R enzyme assay showed that while (9 alpha) did not have any activity, (9 beta) and (3b) were potent 5 alpha-reductase (IC50 = 21 and 31 nM) inhibitors with activities similar to finasteride. The 20-oximes (17a) and (17b) were potent dual inhibitors for both 5 alpha-R (IC50 = 63 and 115 nM, compared to 33 nM for finasteride) and P450(17) alpha (IC50 = 43 and 25 nM, compared to 78 nM for ketoconazole).     

Time-dependent effects of Klebsiella pneumoniae endotoxin on hepatic drug-metabolizing enzyme activity in rats

The time-dependent effects of Klebsiella pneumoniae endotoxin on hepatic cytochrome P450-dependent drug-metabolizing capacity (cytochrome P450 and b5 content, activity of aminopyrine N-demethylase, p-nitroanisole O-demethylase, aniline hydroxylase and benzphetamine N-demethylase) and on the pharmacokinetics of antipyrine have been determined in rats. Measurement of enzyme activity and antipyrine (after intravenous injection of 20 mg kg(-1)) were performed 2, 24 and 96 h after a single intraperitoneal injection of endotoxin (1 mg kg(-1)) and after repeated doses (once daily for 4 days). The contribution of tumour necrosis factor alpha (TNFalpha) to the endotoxin-induced changes was also examined in rats pretreated with granulocyte colony-stimulating factor (G-CSF). The systemic clearance of antipyrine and the activity of hepatic cytochrome P450-dependent drug-metabolizing enzymes were dramatically reduced 24 h after a single injection of endotoxin, but had returned to control levels by 96h. The magnitudes of these decreases in these measurements after repeated doses of endotoxin were similar to those seen 24h after the single dose. The systemic clearance of antipyrine correlated significantly with cytochrome P450 content and aminopyrine N-demethylase activity. In histopathological experiments, moderate hypertrophy of Kupffer cells was observed, with no evidence of severe liver-tissue damage. G-CSF pretreatment suppressed the increased plasma concentrations of TNFalpha produced 2 h after single endotoxin injection, but did not eliminate the endotoxin-induced decrease in the systemic clearance of antipyrine, suggesting that TNFalpha is not the sole component responsible for the reduction of cytochrome P450-mediated drug-metabolizing enzyme activity. These results provide evidence that a single intraperitoneal injection of 1.0 mgkg(-1)K. pneumoniae endotoxin in rats reduces hepatic P450 and b5 levels, and reduces the activity of various cytochrome P450-mediated drug-metabolizing enzymes without causing severe liver-tissue damage. This suggests that the effect of endotoxin on hepatic cytochrome P450-mediated drug-metabolizing isozymes is non-selective.     

Tetrahydronaphthalenes: influence of heterocyclic substituents on inhibition of steroid enzymes P450 arom and P450 17

In search of new leads for selective inhibition of estrogen and androgen biosynthesis, respectively, heterocyclic substituted 2-(arylmethylene)-1-tetralones (1-4, 9-17), 2-(aryl-hydroxymethyl)-1-tetralones (5-8), exo-1a,2,3,7b-tetrahydro-1H-cyclopropa[alpha] naphthalenes (18-24), and 3-alkyl substituted 4,5-dihydronaphtho[1,2-c]pyrazoles (25-27) were synthesized and tested for inhibitory activity toward four steroidogenic enzymes (P450 arom, P450 17, P450 18, and P450 scc, as well as another P450 enzyme, thromboxane A(2) (TXA(2)) synthase. The test compounds inhibited human placental P450 arom, showing a wide range of inhibitory potencies. (Z)-4-Imidazolyl compound 17 was the most potent inhibitor, with a relative potency (rp) of 110 [rp of aminoglutethimide (AG) = 1), rp of fadrozole = 359]. A competitive type of inhibition was shown by the (E)-4-imidazolyl compound 16(rp = 71). On the other hand some of these compounds inhibited rat testicular P450 17. Maximum activity was shown by the 3-pyridyl compound 20 (rp = 10, ro of ketoconazole = 1). 20 was the only compound which exhibited a marked inhibition of TXA(2) synthase (IC(50) = 14.5 microM; IC(50) of dazoxiben = 1.1 microM). Regarding selectivity toward the steroidogenic enzymes, compound 16 was relatively selective toward P450 arom, whereas compound 20 was relatively selective toward P450 17. (P450 arom: K(m) testosterone = 42 nM, K(i)16 = 33 nM, K(i)20 = 3 microM. P450 17: K(m)progesterone = 7 microM, K(i)16 = 9 microM, K(i)20 = 80 nM). 17 and 24 were not selective since they showed strong inhibition of P450 arom (K(i)17 = 26 nM, K(i)24 = 0.12 microM) and P450 17 (K(i) 17 = 0.7 microM, K(i)24 = 0.11 microM).     

Effects of cannabinoids on preimplantation mouse embryo development and implantation are mediated by brain-type cannabinoid receptors

We examined the relative importance of G (Gi) protein-coupled brain-type (CB1-R) and spleen-type (CB2-R) cannabinoid receptors in preimplantation embryo development using agonists and antagonists specific to CB1-R and CB2-R. The results establish that endogenous cannabinoid ligands, anandamide and sn-2 arachidonoylglycerol, arrest embryo development in vitro, and this effect is reversed by CB1-R antagonists SR141716A or AM 251, but not by SR144528, a CB2-R antagonist. A CB2-R selective agonist AM 663 failed to affect embryo development. These results suggest that cannabinoid effects on embryo development are mediated by CB1-R. We also observed that delta9-tetrahydrocannabinol ([-]THC) infused in the presence of cytochrome P450 inhibitors interfered with blastocyst implantation. This adverse effect was reversed by coinfusion of SR141716A. The less active stereoisomer (+)THC plus the inhibitors failed to affect implantation. Analysis of tissue levels demonstrated that uterine accumulation of (-)THC occurred when it was infused in the presence of the P450 inhibitors. These results demonstrate that the uterus and perhaps the embryo have the cytochrome P450 enzymes to metabolize (-)THC and neutralize its adverse effects on implantation. Collectively, the present study demonstrates that cannabinoid effects on embryo development and implantation are mediated by embryonic and/or uterine CB1-R, but not CB2-R.     

Effect of SKF-525A on liver metabolism and hepatotoxicity of tri- and dibutyltin compounds in mice

The effect of pretreatment with SKF-525A, which inhibits hepatic cytochrome P450 enzymes, on metabolism and hepatotoxicity was examined in mice orally administered tributyltin chloride (TBTC) or dibutyltin dichloride (DBTC) at a dose of 180 mumol/kg. Analysis of butyltin compounds showed that the main metabolites in liver of mice treated with TBTC alone were DBTC (40%) and dibutyl(3-carboxylpropyl)tin chloride (TCOOH; 12-26%), with the levels of other butyltin compounds including TBTC comprising < 12% of total butyltin compounds at 3-24 h following treatment. The pretreatment with SKF-525A resulted in four- to tenfold increased TBTC levels and a significant decrease of debutylated metabolites, particularly DBTC (60 and 37% decrease) at both 3 and 6 h in liver of mice treated with TBTC, leading to complete inhibition of hepatotoxicity at 24 h. At 24 h after TBTC treatment, hepatic levels of TBTC and most of the debutylated metabolites in mice pretreated with SKF-525A did not differ significantly when compared to those in unpretreated mice, resulting in the induction of hepatotoxicity at 48 h, although levels of TCOOH decreased even at 24 h. In the case of DBTC treatment, > 95% of the butyltin compounds were detected as DBTC in liver, and the levels of DBTC inside cells as well as the induction of DBTC hepatotoxicity were unaffected by pretreatment with SKF-525A. These results suggest that debutylated metabolites, in particular DBTC, are the main metabolites of butyltin compounds responsible for the induction of hepatotoxicity following in vivo administration of TBTC. The results also indicate that cytochrome P450 enzymes may play a greater role in the metabolism of TBTC to form DBTC or butyltin trichloride (MBTC) than that of DBTC to form MBTC in liver of mice.     

The 16,17-double bond is needed for irreversible inhibition of human cytochrome p45017alpha by abiraterone (17-(3-pyridyl)androsta-5, 16-dien-3beta-ol) and related steroidal inhibitors

Abiraterone (17-(3-pyridyl)androsta-5,16-dien-3beta-ol, 1) is a potent inhibitor (IC50 4 nM for hydroxylase) of human cytochrome P45017alpha. To assist in studies of the role of the 16,17-double bond in its mechanism of action, the novel 17alpha-(4-pyridyl)androst-5-en-3beta-ol (5) and 17beta-(3-pyridyl)-16,17alpha-epoxy-5alpha-androst-3beta-ol (6) were synthesized. 3beta-Acetoxyetienic acid was converted in three steps into 5 via photolysis of the thiohydroxamic ester 8. Oxidation of an appropriate 16,17-unsaturated precursor (21) with CrO3-pyridine afforded the acetate (23) of 6. Inhibition of the enzyme by 1, the similarly potent 5,6-reduced analogue 19 (IC50 5 nM), and the 4, 16-dien-3-one 26 (IC50 3 nM) and by the less potent (IC50 13 nM) 3,5, 16-triene 25 is slow to occur but is enhanced by preincubation of the inhibitor with the enzyme. Inhibition following preincubation with these compounds is not lessened by dialysis for 24 h, implying irreversible binding to the enzyme. In contrast under these conditions the still potent (IC50 27 nM) 17alpha-(4-pyridyl)androst-5-en-3beta-ol (5) showed partial reversal after 5 h of dialysis and complete reversal of inhibition after 24 h. This behavior was also shown by the less potent 16,17-reduced 3-pyridyl compounds 3 and 24. Further, in contrast to the compounds (1, 19, 25, 26) with the 16,17-double bond, the inhibition of the enzymic reaction was not enhanced by preincubation either with 5 or with the 17beta-pyridyl analogues 3, 4, and 24 which also lack this structural feature. The results show that the 16,17-double bond is necessary for irreversible binding of these pyridyl steroids to cytochrome P45017alpha. However oxidation to an epoxide is probably not involved since epoxide 6 was only a moderately potent inhibitor (IC50 260 nM).     

Purification and characterization of constituent androstenedione 15 alpha-hydroxylase (cytochrome P450(15 alpha AD)) from mouse liver. Sex- and tissue-dependent expression

Hepatic microsomal androstenedione 15 alpha-hydroxylase (i.e.cytochrome P450(15)alpha AD was purified from female CD-1 mice. Protein purification was monitored in eluates from Fractogel, DEAE-Sephacel, and hydroxylapatite columns at heme absorbing 417 nm, by cytochrome P450 content, reactivity to monoclonal antibody against female-specific rat cytochrome P450 2C12, and androstenedione 15 alpha-hydroxylase activity. The catalytic activity for androgens of the purified cytochrome P450(15)alpha AD, exhibiting a high degree of regioselectivity and stereospecificity, was restricted to the 7 alpha- and 15 alpha-hydroxylation of androstenedione, representing, respectively, > 5% and > 93% of the total metabolites. Polyclonal antibodies against cytochrome P450(15)alpha AD exhibited a concentration-dependent and very selective inhibition of hepatic microsomal androstenedione 7 alpha- and 15 alpha-hydroxylation and a 60% inhibition of benzphetamine demethylation, the latter drug appearing to be a much more effective substrate than androgens. Cytochrome P450(15)alpha AD accounted for about 3% of the total P450 in female mouse liver microsomes. The apparent subunit molecular weight of P450(15)alpha AD was 53,000, and the protein appeared as a single band or sodium dodecyl sulfate-polyacrylamide gels. The isoform was intensely expressed in both liver and lung of CD-1 female mice and was female-predominant in the livers of five or eight strains examined; it was sex-independent in the remaining three strains. Amino-terminal sequence analysis indicates that cytochrome P450(15)alpha AD is a member of the murine cytochrome P450 2c subfamily.     

Enzymology of mitomycin C metabolic activation in tumour tissue. Characterization of a novel mitochondrial reductase

In this study, the enzymology of mitomycin C (MMC) bioactivation in two murine colon adenocarcinomas, MAC 16 and MAC 26, was examined. Subcellular quinone reductase assessment via cytochrome c reduction confirmed a number of active enzymes. MAC 16 exhibited 22-fold greater levels of cytosolic DT-diaphorase than MAC 26, while microsomal NADPH:cytochrome P-450 reductase levels were similar in both tumour types. Metabolism of MMC by subcellular fractions isolated from both MAC 16 and MAC 26 was quantitated by monitoring the formation of the principle metabolite 2,7-diaminomitosene (2,7-DM) via high-performance liquid chromatography (HPLC). In MAC 16 only, activity displaying the properties of cytosolic DT-diaphorase and microsomal NADPH:cytochrome P-450 reductase was detected and confirmed, using the enzyme inhibitors dicoumarol and cytochrome P-450 reductase antiserum, respectively. The highest level of MMC metabolism was associated with the mitochondrial fraction from both tumours and was the sole enzyme activity detected in MAC 26. The greatest mitochondrial drug metabolism was achieved in the presence of NADPH as cofactor and hypoxia (MAC 16-specific activity, 3.67 +/- 0.58 nmol/30 min/mg; MAC 26 specific-activity, 3.87 +/- 0.71 nmol/30 min/mg) and was unaffected by the addition of the inhibitors dicoumarol and cytochrome P-450 reductase antiserum. NADH-dependent mitochondrial activity was only observed in MAC 16 at approximately 4-fold less than that seen with NADPH. MAC 26 homogenate incubations displayed enhanced metabolism under hypoxia, presumably due to the presence of the identified mitochondrial enzyme. MAC 16 homogenates showed no increase in metabolism under hypoxia, suggesting that other enzyme(s) may be predominant. These data indicate the presence of a novel mitochondrial one-electron reductase capable of metabolising MMC in MAC 16 and MAC 26.     

Purification and characterization of constituent testosterone 2 alpha-hydroxylase (cytochrome P450(2)alpha) from mouse liver

Hepatic microsomal testosterone/androstenedione 2 alpha-hydroxylase (i.e., cytochrome P450(2)alpha) was purified from female CD-1 mice. Protein purification was monitored in eluates from Fractogel, DEAE-sephacel, and hydroxylapatite columns at heme absorbing 417 nm and by cytochrome P450 content, reactivity to a monoclonal antibody against female-specific rat cytochrome P450 2C12, and testosterone 2 alpha-hydroxylase activity. The catalytic activity of the purified cytochrome P450(2)alpha, exhibiting a high degree of regioselectivity and stereospecificity, was basically restricted to the 2 alpha-hydroxylation of testosterone and androstenedione; representing > 96% and > 92% of these respective metabolites. Polyclonal antibodies against cytochrome P450(2)alpha exhibited a dose-dependent and very selective inhibition of testosterone 2 alpha-hydroxylation. The specific cytochrome P450 content of the purified cytochrome P450(2)alpha fraction was 12.06 nmol/mg protein. The specific testosterone 2 alpha-hydroxylase activity of the purified protein was 14 nmol/min/nmol cytochrome P450, which was about 60-fold higher than the respective microsomes. The apparent subunit molecular weight of cytochrome P450(2)alpha was 51,000 and the protein appeared as a single band on sodium dodecyl sulfate polyacrylamide gels. The amino-terminal sequence analysis indicates that cytochrome P450(2)alpha is a member of the murine cytochrome P450 2d family.     

Rational drug design approach for overcoming drug resistance: application to pyrimethamine resistance in malaria

Pyrimethamine acts by selectively inhibiting malarial dihydrofolate reductase-thymidylate synthase (DHFR-TS). Resistance in the most important human parasite, Plasmodium falciparum, initially results from an S108N mutation in the DHFR domain, with additional mutation (most commonly C59R or N51I or both) imparting much greater resistance. From a homology model of the 3-D structure of DHFR-TS, rational drug design techniques have been used to design and subsequently synthesize inhibitors able to overcome malarial pyrimethamine resistance. Compared to pyrimethamine (Ki 1.5 nM) with purified recombinant DHFR fromP. falciparum, the Ki value of the m-methoxy analogue of pyrimethamine was 1.07 nM, but against the DHFR bearing the double mutation (C59R + S108N), the Ki values for pyrimethamine and the m-methoxy analogue were 71.7 and 14.0 nM, respectively. The m-chloro analogue of pyrimethamine was a stronger inhibitor of both wild-type DHFR (with Ki 0.30 nM) and the doubly mutant (C59R +S108N) purified enzyme (with Ki 2.40 nM). Growth of parasite cultures of P. falciparum in vitro was also strongly inhibited by these compounds with 50% inhibition of growth occurring at 3.7 microM for the m-methoxy and 0.6 microM for the m-chloro compounds with the K1 parasite line bearing the double mutation (S108N + C59R), compared to 10.2 microM for pyrimethamine. These inhibitors were also found in preliminary studies to retain antimalarial activity in vivo in P. berghei-infected mice.     

Modulation of constitutive and inducible hepatic cytochrome(s) P-450 by interferon beta in mice

AIMS/METHODS: Interferon beta is used as a therapeutic agent, but its effects on the hepatic cytochrome P-450-dependent drug metabolizing system have not yet been characterized. We investigated the effect of interferon beta on cytochrome P-450 in mice. RESULTS: Interferon beta (2 x 10(5) units/mouse) significantly reduced total hepatic cytochrome P-450 (20%) and the activity of NADPH cytochrome C reductase (12%) 24 h after administration; lower doses had no such effect. Various monooxygenase activities were slightly reduced, the one most affected being 7-ethoxycoumarin O-deethylase (29%). In phenobarbital-treated mice, interferon beta reduced the induction of total cytochrome P-450 (22%), the activities of pentoxyresorufin O-dealkylase (38%), benzyloxyresorufin O-dealkylase (30%), erythromycin N-demethylase (30%), 7-ethoxycoumarin O-deethylase (16%) and cytochrome P-450 2B1 (33%) and 3A (45%) proteins. In beta-naphthoflavone-treated mice, interferon beta lowered the induction of total cytochrome P-450 (18%), the activities of ethoxyresorufin O-deethylase (31%) and of 7-ethoxycoumarin O-deethylase (25%) and of cytochrome P-450 1A1 protein (31%). CONCLUSIONS: Thus it appears that induced cytochrome(s) P-450 were susceptible to interferon beta, this effect not being influenced by the type of inducer. Since various members of the same cytochrome P-450 subfamilies catalyze oxidation of drugs in humans, our findings have potential significance as regards the fate of drugs or exogenous compounds given to patients receiving interferon beta.     

Inhibition of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone mouse lung tumorigenesis by arylalkynes, mechanism-based inactivators of cytochrome P450

Arylalkynes such as 4-phenyl-1-butyne (PBY), 5-phenyl-1-pentyne (PPY) and 2-ethynylnaphthalene (2-EN) are suicide inhibitors of cytochrome P450 enzymes. Arylalkyl isothiocyanates such as 6-phenylhexyl isothiocyanate (PHITC) are structurally related to arylalkynes and are known to inhibit the cytochrome P450 mediated metabolic activation and tumorigenicity of a tobacco-specific lung carcinogen, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). In this study, we compared the ability of PBY, PPY, 2-EN and PHITC to inhibit A/J mouse lung tumorigenesis by NNK. Groups of 20 female mice were gavaged with 5 mumol of arylalkyne or PHITC in corn oil. Two hours later they were given a single i.p. injection of 10 mumol NNK. The mice were killed 16 weeks later. PPY and PHITC were both potent inhibitors of tumorigenesis by NNK, reducing lung tumor multiplicity from 8.35 tumors per mouse to 0.40 and 0.35 respectively. PBY and 2-EN also significantly inhibited tumor multiplicity. The results of this study demonstrate that arylalkynes and PHITC are potent inhibitors of NNK induced lung tumorigenesis in A/J mice, consistent with the hypothesis that inhibition of specific cytochrome P450 enzymes is involved in inhibition of tumorigenesis.     

Heterogeneous expression of neurofilament proteins in forebrain and cerebellum during development: clinical implications for spinocerebellar ataxia

Previously it was shown that methylenedioxybenzenes (MDBs), particularly isosafrole, were highly effective at preventing CCl4-induced liver necrosis in vivo (Z.S. Zhao, P.J. O'Brien, The prevention of CCl4-induced liver necrosis in mice by naturally occurring methylenedioxybenzenes, Toxicol. Appl. Pharmacol., 140 (1996) 411-421), probably as a result of forming metabolic intermediate complexes with cytochrome P450. In the following it was shown that pretreatment of mice with isosafrole also completely prevented ferric nitrilotriacetate (FeNTA)-induced renal necrosis and lipid peroxidation, even though metabolic activation by cytochrome P450 is not involved. The naturally occurring or synthetic MDBs that prevented CCl4 hepatotoxicity also prevented hepatocyte lipid peroxidation. induced by FeNTA, but other cytochrome P450 inhibitors were ineffective. These compounds, in decreasing order of antioxidant effectiveness, were sesamol, 4-t-butyl-methylenedioxybenzene, isosafrole, piperonyl butoxide and 4-bromo-methylenedioxybenzene and safrole, whereas, benzodioxole, 3,4-(methylenedioxy)-toluene and 1,2-(methylenedioxy)-4-nitrobenzene were ineffective. Pre-incubating the hepatocytes with P450 inhibitors decreased the protective effects of isosafrole, suggesting that the catecholic metabolites of MDBs were responsible for the antioxidant activity. A greater inhibition of FeNTA-induced lipid peroxidation by catecholic metabolites was observed. Since cytochrome P450 did not participate in FeNTA-induced hepatocyte or microsomal lipid peroxidation, it is likely that the antioxidant properties of MDBs or their catecholic metabolites also contribute to their in vivo protection against CCl4 or FeNTA-induced hepato- or nephrotoxicity.     

17 alpha-alkyl- or 17 alpha-substituted benzyl-17 beta-estradiols: a new family of estrone-sulfatase inhibitors

A series of 17 alpha-derivatives of 17 beta-estradiol was synthesized and tested for their ability to inhibit the estrone-sulfatase activity transforming estrone sulfate to estrone. A strong inhibitory activity was obtained when an alkyl side chain or a substituted benzyl was introduced at position 17 alpha of estradiol. The 17 alpha-(3'-bromobenzyl)-estradiol (26) and 17 alpha-(4'-t-butylbenzyl)-estradiol (30) were the most potent estrone-sulfatase inhibitors obtained in our study with IC50 values of 24 and 28 nM, respectively. They also represent a new family of estrone-sulfatase inhibitors. These compounds are about 300-fold more effective in interacting with the enzyme than the substrate estrone sulfate itself.     

Characterization of endothelium-dependent relaxation independent of NO and prostaglandins in guinea pig coronary artery

In the presence of N omega-nitro-L-arginine and indomethacin, acetylcholine (ACh) induced endothelium-dependent relaxation in guinea pig coronary artery preconstricted with 9,11-dideoxy-9 alpha, 11 alpha-epoxymethano prostaglandin F2 alpha. Dexamethasone and arachidonyltrifluoromethyl ketone, inhibitors of phospholipase A2, and 17-octadecynoic acid, an inhibitor of cytochrome P450 epoxygenase, had no effect on the response to ACh. Although proadifen, which is used widely as an inhibitor of cytochrome P450-dependent enzymes, suppressed the ACh-induced relaxation, the drug also inhibited the relaxation induced by cromakalim, a K+ channel opener. In isolated smooth muscle cells of guinea pig coronary artery, proadifen, but not 17-octadecynoic acid, almost abolished delayed rectifier K+ current. Epoxyeicosatrienoic acids failed to relax the artery. Apamin and iberiotoxin, inhibitors of small- and large-conductance Ca(++)-activated K+ channels, respectively, did not affect the relaxation induced by ACh. A combination of charybdotoxin plus apamin, but not iberiotoxin plus apamin, abolished the response. However, the combination of charybdotoxin plus apamin had no effect on ACh-induced increase in intracellular free Ca++ concentration in endothelial cells. These results suggest that epoxyeicosatrienoic acids do not contribute to N omega-nitro-L-arginine/indomethacin-resistant relaxation induced by ACh in the guinea pig coronary artery. The present study also proposes that K+ channels on vascular smooth muscle cells, which both charybdotoxin and apamin must affect for inhibition to occur, are the target for endothelium-derived hyperpolarizing factor.     

Inhibition and inactivation of murine hepatic ethoxy- and pentoxyresorufin O-dealkylase by naturally occurring coumarins

The present study was designed to evaluate the effects of a series of natural coumarins on ethoxyresorufin O-dealkylase (EROD) and pentoxyresorufin O-dealkylase (PROD) activities in vitro using hepatic tissues from SENCAR mice. Fifteen different coumarins were examined for potential modulating activities. Several naturally occurring coumarins, found in the human diet, were effective inhibitors of hepatic EROD activity in vitro, including coriandrin, bergamottin, isoimperatorin, and ostruthin. Notably, coriandrin and bergamottin were approximately as potent as 7,8-benzoflavone, a relatively selective inhibitor of cytochrome P450 1A1. Several naturally occurring coumarins were also potent inhibitors of hepatic PROD activity, including imperatorin, bergamottin, isopimpinellin, and angelicin. Kinetic studies of the type of inhibition revealed that these compounds inhibited hepatic EROD and PROD activity by a variety of modes rather than by a uniform one. Furthermore, experiments using a two-stage incubation assay revealed that coriandrin, imperatorin, ostruthin, and several other natural coumarins inactivated hepatic EROD activity (i.e., predominantly cytochrome P450 1A1-mediated) and that isopimpinellin inactivated hepatic PROD activity (i.e., predominantly cytochrome P450 2B1-mediated). Finally, the results indicate that some coumarins had selective inhibitory effects for EROD vs PROD and preliminary analyses suggested a possible structural basis for the observed differences. The current data suggest that certain naturally occurring coumarins, to which humans are exposed in the diet, are potent modulators of cytochrome P450. Furthermore, these compounds may be capable of influencing the metabolic activation of other xenobiotics, including chemical carcinogens.     

PMP-22 expression in the central nervous system of the embryonic mouse defines potential transverse segments and longitudinal columns

Treatment of rats with p-chlorotoluene (PCT, 1 g/kg, ip) resulted in peak PCT blood and lung concentrations at 4 h, which declined to very low levels at 12 h. The concentration of PCT in the liver attained its highest value at 1 h and also declined to low levels at 12 h. In the dose-response study, PCT significantly decreased hepatic and pulmonary AHH activities at 0.5 g/kg, 1 h. Maximum inhibition was attained at 1 g/kg, 1 h, and further increase in the dose did not enhance the enzyme inhibition in the time-course investigation, PCT (1 g/kg) maximally inhibited hepatic and pulmonary aryl hydrocarbon hydroxylase (AHH) activities at 1 h and the decrease in enzyme activity was sustained through 12 h. Administration of PCT (1 g/kg, 1 h) also markedly decreased pulmonary cytochrome P-450 content, while hepatic cytochrome P-450 content was only slightly reduced. The partial decrease in cytochrome P-450 content indicated altered levels of the P-450 isozymes, which may have profound effects on the metabolic disposition of benzo[a]pyrene [BaP]. BaP is regioselectively metabolized by two major isoforms of P-450 to toxic dihydrodiols and nontoxic phenol derivatives and there is a balance between these two metabolite groups. PCT (1 g/kg, 1 h) significantly inhibited the phenolic 3-OH BaP formation in both lung (52%) and liver (56%). The formations of BaP 7,8-dihydrodiol (146%) and 9,10-dihydrodiol (90%) were significantly elevated in the lung. The toxication to detoxication ratios were significantly elevated in both organs. Total quinone formation was markedly enhanced in the liver. Since PCT inhibited phenolic metabolite formation and increased dihydrodiol production, the activities of the isozymes that are responsible for their formations were determined. PCT (1 g/kg, 1 h) significantly inhibited cytochrome P-4502B1 in the lung (50%) and 2B1/2B2 in the liver (40%), while cytochrome P-4501A activity was not altered in either lung or liver. PCT increased phospholipid (PL) levels (45%) and conjugated diene (CD) formation (58%) in lung but not in liver, while membrane fluidity was increased [phospholipid/cholesterol (PL/CL) ratio; diphenylhexatriene (DPH) and trimethylammonium DPH (TMA-DPH) fluorescence polarization] in both organs. There was no apparent relationship between these membrane changes and alterations in MFO activity. Taken together the results suggest that PCT is capable of nonselectively inactivating the hepatic and pulmonary isozymes of P-450 that are responsible for the detoxication of BaP. The observed shift in the metabolism of BaP toward potentially more toxic metabolites suggests that concurrent exposure to BaP and PCT may result in greater toxicity, compared to exposure to BaP alone.     

Major cytochrome P450 enzyme responsible for oxidation of secondary alcohols to the corresponding ketones in mouse hepatic microsomes. Oxidation of 7-hydroxy-delta8-tetrahydrocannabinol to 7-oxo-delta8-tetrahydrocannabinol

The oxidative activities of 7alpha- and 7beta-hydroxy-Delta8-tetrahydrocannabinol (7alpha- and 7beta-hydroxy-Delta8-THC) to 7-oxo-Delta8-THC in hepatic microsomes of mice were significantly increased by the treatment of mice with dexamethasone or phenobarbital. A cytochrome P450 enzyme, named P450MDX-B, was purified from hepatic microsomes of dexamethasone-treated mice, and its apparent molecular mass was estimated to be 51,000. The NH2-terminal amino acid sequence of P450MDX-B was the same as that of CYP3A11. The oxidative activities of 7alpha- and 7beta-hydroxy-Delta8-THC were 2.55 and 4.92 nmol/min/nmol P450, respectively. The antibody against P450MDX-B almost completely inhibited the oxidative activities of 7alpha- and 7beta-hydroxy-Delta8-THC in mice. These results indicate that P450MDX-B (CYP3A11) is a major enzyme responsible for the oxidation of 7alpha- and 7beta-hydroxy-Delta8-THC to 7-oxo-Delta8-THC in mouse liver.     

Messenger RNA synthesis, transport, and storage in silkmoth ovarian follicles

The effect on liver microsomal enzyme activity of three steroid contraceptive drug (SCD) combinations was compared in rats, mice and guinea-pigs. Lynestrenol plus mestranol, norethisterone plus mestranol and norethynodrel plus mestranol were given orally for 4 consecutive days (acute treatment) or 30 days (chronic treatment) at various doses eliciting an experimentally controlled antifertility activity which varied in its extent. In rats and mice all the combined treatments (with the exception of norethynodrel plus mestranol in mice) were active as inducers of liver microsomal enzymes. This induction seems to be mediated mainly by the progestogenic compounds. Oestrogens showed a very poor effect bordering on significance only in a few cases. No effect on liver microsomal protein or cytochrome P 450 concentration was obtained after treatment with doses capable of increasing the microsomal enzyme activity. The activity of the liver microsomal enzymes did not appear to be reduced immediately (2 h) after the last administration of the SCD given during 4 or 30 days. Contraceptive treatments at doses capable of eliciting complete antifertility activity were inactive on liver microsomal enzyme activity in guinea-pigs.     

Leptin down-regulates the steroid producing system in the adrenal

OB protein leptin inhibits the secretion of cortisol in primary cultures of bovine adrenocortical cells and down-regulates 17alpha-hydroxylase cytochrome P450 mRNA expression. To analyze if leptin regulates other major enzymes involved in adrenal steroidogenesis we tested its effect on mRNA expression for two further key enzymes, C21-hydroxylase (P450C21) and side-chain cleavage enzyme (P450SCC). Cultured bovine cortical cells were stimulated for 24 hours with 10 nM ACTH, with 10 nM ACTH plus 100 ng/ml leptin or left unstimulated as controls. Stimulation with ACTH led to a 1.75-fold increase of P450C21 mRNA and a 3.31-fold increase of P450SCC mRNA compared to unstimulated controls. Addition of leptin led to a reduction of ACTH-stimulated mRNA accumulation of 73% for P450C21 and of 45% for P450SCC. We therefore suggest that leptin reduces cortisol synthesis in the adrenal by down-regulating the steroid producing enzyme cascade in the cortical cell.