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Inhibitory effects of Delta(9)-tetrahydrocannabinol (Delta(9)-THC), cannabidiol (CBD), and cannabinol (CBN), the three major constituents in marijuana, on catalytic activities of human cytochrome P450 (CYP) 1 enzymes were investigated. These cannabinoids inhibited 7-ethoxyresorufin O-deethylase activity of recombinant CYP1A1, CYP1A2, and CYP1B1 in a competitive manner. CBD most potently inhibited the CYP1A1 activity; the apparent K(i) value (0.155microM) was at least one-seventeenth of the values for other CYP1 isoforms. On the other hand, CBN more effectively decreased the activity of CYP1A2 and CYP1B1 (K(i)=0.0790 and 0.148microM, respectively) compared with CYP1A1 (K(i)=0.541microM). Delta(9)-THC less potently inhibited the CYP1 activity than CBD and CBN, and showed low selectivity against the CYP1 inhibition (K(i)=2.47-7.54microM). The preincubation of CBD resulted in a time- and concentration-dependent decrease in catalytic activity of all the recombinant CYP1 enzymes and human liver microsomes. Similarly, the preincubation of Delta(9)-THC or CBN caused a time- and concentration-dependent inhibition of recombinant CYP1A1. The inactivation of CYP1A1 by CBD indicated the highest k(inact)/K(I) value (540l/mmol/min) among the CYP1 enzyme sources tested. The inactivation of recombinant CYP1A1 and human liver microsomes by CBD required NADPH, was not influenced by dialysis and by glutathione, N-acetylcysteine, and superoxide dismutase as trapping agents. These results indicated that CBD and CBN showed CYP1 isoform-selective direct inhibition and that CBD was characterized as a potent mechanism-based inhibitor of human CYP1 enzymes, especially CYP1A1., (2010 Elsevier Inc. All rights reserved.)

Staurosporine is one of the most potent and well known inhibitors of protein kinases, and it is often used to study the involvement of protein kinases in signal transduction pathways. We now report that staurosporine can induce the production of hepatocyte growth factor (HGF) independently of protein kinase inhibition. Staurosporine markedly stimulated the production of HGF in various cell types, including human skin fibroblasts. Its effect was accompanied by up-regulation of HGF gene expression. The inhibition of protein kinases appears not to be involved in staurosporine-induced HGF production, because other protein kinase inhibitors, K-252a, H-7, GF 109203X and genistein, had no HGF-inducing activity. UCN-01, 7-hydroxystaurosporine, which differs from staurosporine only in its aglycone moiety, also showed HGF-inducing activity, and inactive K-252a differs from staurosporine only in its sugar moiety. These results indicate that the sugar moiety, a six-atom ring structure, is important in the HGF-inducing activity of staurosporine. Experiments were then carried out to determine whether the characteristics of staurosporine-induced HGF production have similarities to those of HGF production stimulated by other HGF inducers. The effect of staurosporine like that of 8-bromo-cAMP and that of cholera toxin was marked in human skin fibroblasts from all four different sources, whereas the effects of epidermal growth factor and phorbol 12-myristate 13-acetate were variable depending on cells. The net increase in HGF production induced by staurosporine was not reduced in protein kinase C-depleted human skin fibroblasts. Moreover, synergistic induction of HGF was detected between staurosporine and interferon-gamma as well as between 8-bromo-cAMP and interferon-gamma. Staurosporine, however, did not increase intracellular cAMP levels in human skin fibroblasts. These results indicate that staurosporine induced HGF in different cell types via a signaling pathway similar to the cAMP-mediated pathway without increasing cAMP levels.

In this study, we examined whether the production of hepatocyte growth factor (HGF) in fibroblasts is regulated by protein phosphatase(s). Inhibitors of the enzymes okadaic acid and calyculin A were used for this purpose. Both inhibitors markedly stimulated HGF production in human skin fibroblasts in a dose-dependent manner. The effects of okadaic acid and calyculin A were maximal at 25-37.5 and 1.25 nM, respectively. Highly active HGF production in MRC-5 human embryonic lung fibroblasts was also promoted by both inhibitors. The effect of okadaic acid was accompanied by an up-regulation of HGF gene expression. The stimulating effect of okadaic acid on HGF production was synergistic with that of phorbol 12-myristate 13-acetate (PMA) and epidermal growth factor (EGF), whereas it was additive to the effect of cholera toxin. The protein kinase C (PKC) inhibitor GF 109203X inhibited the effect of PMA, but not of okadaic acid and EGF. The effect of okadaic acid as well as EGF was not inhibited, but rather enhanced in human skin fibroblasts pretreated for 24 hr with a high dose of PMA to deplete PKC, as compared with its effect in untreated cells. PD 98059, an inhibitor of mitogen-activated protein (MAP) kinase kinase, suppressed the effects of okadaic acid and EGF, but not those of cholera toxin and 8-bromo-adenosine 3',5'-cyclic monophosphate (cAMP). These results suggest that HGF production in human skin fibroblasts is down-regulated by protein phosphatase(s) and that HGF production stimulated by okadaic acid is, at least in part, dependent on the activation of the MAP kinase cascade.

By employing the specific histone deacetylase inhibitor trichostatin A (TSA), we investigated whether histone acetylation modulates the production of antigen-specific antibodies in murine splenocytes in vitro. TSA caused a marked increase in both anti-sheep red blood cell (SRBC) and anti-trinitrophenyl (TNP) plaque-forming cell (PFC) responses in splenocytes at much lower concentrations than sodium butyrate. It also dose dependently augmented the production of anti-trinitrophenyl antibodies in splenic B cells with a concomitant, moderate increase in the level of histone H4 acetylation. Its optimal concentration for promoting the production of these antibodies was 10 nM. However, to gain such an effect on antibody production, TSA had to be added to cells before Day 2 in culture. Trichostatin C, an analog of TSA and a less potent inducer of Friend leukemia cell differentiation, also increased both the anti-trinitrophenyl PFC response and histone H4 acetylation in B cells, but at higher concentrations than TSA. TSA did not stimulate the production of lipopolysaccharide-induced polyclonal immunoglobulin M in B cells. These results suggest that a moderate increase in histone acetylation may play a significant role in promoting antigen-specific antibody production in B cells.

A cytochrome P450 (designated P450 MUT-2) which catalyses the oxidation of 11-oxo-delta 8-tetrahydrocannabinol (11-oxo-delta 8-THC) to delta 8-THC-11-oic acid has been purified from hepatic microsomes of untreated male mice. Analysis of NH2-terminal sequence suggests that the isozyme is a member of the P450 2C gene subfamily. P450 MUT-2 exhibited aldehyde oxygenase activity for 11-oxo-delta 8-TH, 11-oxo-delta 9-THC, 11-oxo-cannabinol (11-oxo-CBN) and 9-anthraldehyde together with high activity for the hydroxylation of cannabinoids at the 11-position. Antibody against P450 MUT-2 significantly inhibited the microsomal formation of delta 8-THC-11-oic acid from 11-oxo-delta 8-THC, but not that of 9-anthracene carboxylic acid from 9-anthraldehyde. Major metabolic reactions of delta 8-THC, delta 9-THC and CBN with mouse hepatic microsomes were the 11-hydroxylation (all cannabinoids), 7 alpha-(delta 8-THC) or 8 alpha-hydroxylation (delta 9-THC) and epoxide formation (delta 8- and delta 9-THC). All these reactions except for 7 alpha-hydroxylation of delta 8-THC and alpha-epoxide formation from delta 9-THC were also markedly inhibited by the antibody. These results indicate that P450 MUT-2 is a major enzyme for metabolizing cannabinoids by mouse hepatic microsomes.

Ascorbic acid 2-O-alpha-glucoside (AA-2G) is a stable ascorbate derivative which has vitamin C activity in vivo and in vitro. We studied whether AA-2G exerts a prooxidant action in cultured fibroblasts from chick embryo and human skin, as does ascorbic acid. At concentrations of 0.1-1.0 mM, ascorbic acid markedly reduced the viable cell number of low density cultures within 24 hr, whereas AA-2G had no such effect. The ascorbate cytotoxicity was dependent on the cell density at the time of its addition and it was characteristic of low density cultures. This cytotoxicity was completely prevented by catalase and partially by an Fe3+ ion chelator, desferrioxamine. In the early culture stage at which a morphological change in the fibroblasts began to occur, intracellular ascorbate concentrations in low density cultures after addition of ascorbic acid were much higher than in high density cultures. However, at the same concentrations, AA-2G did not cause an elevation even in low density cultures and it was also effective on collagen synthesis at high and medium densities. These results suggest that the abnormally accumulated ascorbic acid in the cells cultured at low density possibly amplifies the generation of oxygen radicals through the reduction of Fe3+ ions and subsequent oxidative reactions, leading to cell death. Therefore, it is concluded that AA-2G which supplies an adequate amount of ascorbic acid during culture period is a bioavailable ascorbate source without cytotoxicity.

A protein (designated as protein-B) was purified from liver microsomes of adult male guinea pigs by an affinity chromatography with omega-aminooctyl Sepharose 4B, followed by HPLC using DEAE-5PW and hydroxyapatite columns which had been used to purify a cytochrome P450 (P450) isozyme (P450-A) from the same subcellular fraction (Narimatsu et al., Biochem Biophys Res Commun 172: 607-613, 1990). Protein-B had a molecular mass of 49 kDa in SDS-PAGE, but did not show absorbance at 417 nm for heme. Further, it did not show any oxidative activities towards aniline (AN), d-benzphetamine (d-BP), p-nitroanisole (p-NA) or delta 9-tetrahydrocannabinol (delta 9-THC) in a reconstituted system including dilauroylphosphatidylcholine, NADPH-P450 reductase, and cytochrome b5. However, antiserum against protein-B raised in rabbits suppressed liver microsomal oxidative activities towards d-BP and p-NA dose-dependently. The antibody decreased delta 9-THC oxidative activity most effectively, but did not decrease AN hydroxylation activity. Antiserum against P450-A suppressed all the activities towards these four substrates, especially towards delta 9-THC, in liver microsomes of male guinea pigs. Moreover, reconstitution with hemin made it possible for protein-B to produce some oxidative activity toward delta 9-THC. These results suggest that protein-B is also a cytochrome P450 isozyme which has lost a heme moiety during purification steps. Both P450-A and protein-B could have a role as cytochrome P450 isozymes in the oxidative metabolism of drugs, especially that of delta 9-THC by the liver microsomes of adult male guinea pigs.

Cytochrome P450 isozymes purified from rat hepatic microsomes were able to catalyse the oxidation of 11-oxo-delta 8-tetrahydrocannabinol (11-oxo-delta 8-THC) to delta 8-THC-11-oic acid in the presence of NADPH, cytochrome P450 reductase and dilauroylphosphatidylcholine. The catalytic activities (nmol/min/nmol P450) of cytochrome P450s, UT-2 (IIC11), UT-4 (IIA2), UT-5 (IIC13), PB-1, PB-2 (IIC6), PB-4 (IIB1), MC-1 (IA2), MC-5 (IA1) and IF-3 (IIA1), were 0.69, 0.08, 0.07, 0.23, 0.46, 0.02, 0.06, 0.07 and 0.34, respectively, whereas the activities of cytochrome P450s, PB-5 (IIB2) and DM (IIE1), were less than 0.02 nmol/min/nmol P450. Cytochrome P450 IIC11 showed the highest catalytic activity of the cytochromes examined. The mechanism for the oxidation of 11-oxo-delta 8-THC to delta 8-THC-11-oic acid by cytochrome P450 IIC11 was established as being an oxygenation since one atom of oxygen-18 was exclusively incorporated into the carboxylic acid formed under 18O2. The antibody raised to cytochrome P450 IIC11 inhibited by 60% the hepatic microsomal oxidation of 11-oxo-delta 8-THC to delta 8-THC-11-oic acid in male rats. These results indicate that cytochrome P450 IIC11 is a major form of the cytochrome to catalyse the oxidation of 11-oxo-delta 8-THC to delta 8-THC-11-oic acid in the hepatic microsomes of male rats and that the oxidation of aldehyde to carboxylic acid is a catalytic activity common to most isozymes of P450.

In vivo formation of ascorbic acid 2-O-alpha-glucoside (AA-2G) in guinea pigs and rats given ascorbic acid (AA) orally in combination with maltose was examined. A metabolite of AA which has the same HPLC retention characteristics as authentic AA-2G was detected in the blood, urine and liver of guinea pigs 1-2 hr after their administration. The metabolite was isolated from the urine by chromatographic procedures and identified as AA-2G by its UV spectrum, non-reducibility, susceptibility to alpha-glucosidase hydrolysis, HPLC profile and elementary analysis. The same glucoside was also synthesized by rats and found in the urine, although it could not be determined qualitatively in the blood. AA-2G-forming activities of tissue homogenates from both animals were apparently correlated with their alpha-glucosidase activities and, moreover, both activities were completely inhibited by a specific neutral alpha-glucosidase inhibitor. Thus, we conclude that AA-2G is a possible metabolite produced by enzymatic alpha-glucosidation after a combined administration of AA and maltose to guinea pigs and rats.

Oxidative metabolism of delta 9-tetrahydrocannabinol (THC), one of the major components of marihuana, was studied using liver microsomes of adult male and female rats. There was no significant difference in the rates of the cannabinoid oxidation in terms of nmol per min per nmol of liver microsomal cytochrome P450 or of nmol per min per mg of microsomal protein between male and female rats. delta 9-THC was biotransformed to various metabolites including 11-hydroxy-delta 9-THC (11-OH-delta 9-THC), 8 alpha-OH-delta 9-THC, 8 alpha,11-diOH-delta 9-THC, 3'-OH-delta 9-THC by liver microsomes of male rats, while it was oxidized selectively to 11-OH-delta 9-THC by liver microsomes of female rats. After intraperitoneal administration of delta 9-THC, various metabolites were again found in the liver of the male rat, while in the female rat oxidation of the methyl group at the 9-position was a major metabolic pathway. These results demonstrate that an apparent sex-related difference exists in the oxidative metabolism of delta 9-THC in the rat.

Cannabidiol induced a carbon monoxide-like complex with mouse hepatic microsomal cytochrome P-450 during NADPH-dependent metabolism in vitro on a spectral basis. The reduction by dithionite was required for the maximal development of a spectrum. The complex showed a peak at 450 nm which shifted to 419 or 423 nm, respectively, by further addition of hemoglobin or myoglobin. Cannabidiol-induced complex formation required molecular oxygen, and was decreased by the addition of inhibitors of cytochrome P-450-dependent monoxygenase. Pretreatment of mice with phenobarbital (80 or 100 mg/kg, i.p. for 3 days) but not 3-methylcholanthrene (80 mg/kg, i.p.) increased the complex formation. In contrast, pretreatment with cobaltous chloride (40 mg/kg, i.p. for 3 days) decreased the complex formation. 8,9-Dihydro- and 1,2,8,9-tetrahydrocannabidiols also induced the same spectrum as that of above complex, whereas cannabidiol monomethyl- and dimethylethers reduced this ability. In addition, both cannabidivarin and cannabigerol induced the complex formation, although delta 9-tetrahydrocannabinol, cannabinol and cannabielsoin did not. Olivetol but not d-limonene induced the spectrum of the complex to some extent. These results indicate that cannabidiol induces a carbon monoxide-like complex with cytochrome P-450 during hepatic microsomal metabolism, and suggest that phenobarbital-inducible cytochrome P-450s mediate at least one of the metabolic steps of CBD to form the complex, as well as the importance of the resorcinol moiety of CBD for the complex formation.

When cannabidiol (CBD) was incubated with hepatic microsomes of mice in the presence of an NADPH-generating system, a significant decrease of cytochrome P-450 content was observed by measuring its carbon monoxide difference spectra. The decrease of cytochrome P-450 by CBD required NADPH and molecular oxygen. The effect was partially inhibited by SKF 525-A but not by various scavengers of active oxygen species, superoxide anion, hydroxyl radical and singlet oxygen. The incubation of CBD with hepatic microsomes did not affect total heme but decreased significantly free sulfhydryl contents in the microsomes. The derivatives of CBD modified in the resorcinol moiety, CBD-monomethyl- and dimethylethers, almost lost the effect on cytochrome P-450, whereas those modified in the terpene moiety, 8,9-dihydro- and 1,2,8,9-tetrahydro-CBDs exhibited some potency to inactivate cytochrome P-450. The inactivation of cytochrome P-450 by CBD and related compounds led to the inhibition of hepatic microsomal p-nitroanisole O-demethylase and aniline hydroxylase activities. These results suggest that the resorcinol moiety of CBD plays some role in the inactivation of cytochrome P-450 by the cannabinoid.

Male ddY mice were given one injection of (1) mercury (mercuric chloride) simultaneously with various doses of selenium (sodium selenite), (2) mercury alone, or (3) various doses of selenium alone. The interaction between mercury and selenium in the liver and kidneys at 1, 5, 24, 120, and 240 hr after administration was investigated. The concentrations of mercury in the liver of mice receiving mercury and selenium simultaneously were higher than those after administration of mercury alone, while the concentrations of mercury in the kidney decreased markedly over a 1-120 hr period after administration, depending on the dose of selenium administered simultaneously with mercury. Clearly, selenium had a different effect on the accumulation of mercury in the liver and kidneys. Subcellular distribution studies revealed that mercury and selenium which were administered simultaneously were incorporated into the crude nuclear and mitochondrial fractions as stable complexes. The transport of these complexes to the kidneys seems to be limited. In addition, gel filtration of supernatant fractions of liver and kidney through a Sephadex G-75 column indicated that the proportion of mercury bound to metallothionein fraction decreased depending on the dose of selenium administered simultaneously with the mercury. This reduction was attributed to the decreased synthesis of mercury-thionein due to a reduction in the activity of Hg2+ which results from binding between mercury and selenium in the cells.

Incubation of rat peritoneal mast cells with hydrogen peroxide results in a marked release of histamine. Maximal release is observed with 0.05-0.1 mM H(2)O(2), but higher concentrations of H(2)O(2) instead suppresses the release. Histamine release starts after about 2 min of lag time and reaches a plateau in about 10 min. Hydrogen peroxide-induced release does not exceed 50-60 per cent of total histamine if the incubations are prolonged or additional H(2)O(2) is given at 10 min. This would be explained by the fact that H(2)O(2) causes impairment of the histamine releasing system of mast cells simultaneously with the release of histamine. Hydrogen peroxide-induced release is not due to nonspecific lysis of the cells because lactate dehydrogenase, a cytoplasmic enzyme, is not liberated during the reaction. The reaction requires the presence of Ca(2+), is enhanced by D(2)O and suppressed by colchicine. It is not, however, affected by dibutyryl cAMP or dibutyryl cGMP. No significant alteration of intracellular levels of cyclic AMP and cyclic GMP is observed during the incubation of mast cells with 0.1 mM H(2)O(2). These results indicate that microtubular functions would be involved in the releasing reaction although they are not under the control of cyclic nucleotides. Microscopic observation shows that H(2)O(2)-induced release is accompanied by degranulation.

The effects of delta 8-tetrahydrocannabinol (delta 8-THC) and its major and active metabolite, 11-hydroxy-delta 8-tetrahydrocannabinol (11-OH-delta 8-THC), on the hepatic microsomal drug-metabolizing enzyme system were studied in mice. The repeated administration of 11-OH-delta 8-THC (5 mg/kg/day, i.v.) for 3 or 7 days increased significantly the activities of aniline hydroxylase and p-nitroanisole O-demethylase. By the same treatment, cytochrome P-450 content (3 days) or NADPH-cytochrome c reductase activity (7 days) was also increased significantly. The treatment with delta 8-THC for 7 days (5 mg/kg/day, i.v.) significantly increased aniline hydroxylase only. 11-OH-delta 8-THC increased the Vmax, but not the Km, values for both drug-metabolizing enzymes, whereas delta 8-THC decreases significantly the Km value (270 microM) for p-nitroanisole O-demethylase as compared with the control (398 microM). Repeated administration of these cannabinoids for 7 days also increased the metabolism of delta 8-THC by hepatic microsomes; this was attributed to an enhanced formation of 11-OH-delta 8-THC. In contrast, microsomal formation of 7 alpha-OH-delta 8-THC was decreased significantly by treatment with delta 8-THC. 11-OH-delta 8-THC, but not delta 8-THC, treatment increased the metabolism of 11-OH-delta 8-THC by hepatic microsomes. These findings indicate that delta 8-THC and 11-OH-delta 8-THC treatment can induce hepatic microsomal drug-metabolizing enzymes and affect differently the catalytic properties of the enzymes.