The atherogen 3-methylcholanthrene induces multiple DNA adducts in mouse aortic smooth muscle cells: role of cytochrome P4501B1.

OBJECTIVE: 3-Methylcholanthrene (MC), a polycylic aromatic hydrocarbon, induces atherogenesis in mice fed an atherogenic diet. In this study, we tested the hypothesis that MC would induce DNA adducts in mouse aortic smooth muscle cells (SMCs) and that cytochrome P4501B1 (CYP1B1) plays an important role in the activation of MC to genotoxic intermediates. METHODS: Cultured SMCs were treated with MC or the vehicle dimethyl sulfoxide (DMSO), and DNA was isolated after 24 h. In some experiments, the cells were pre-treated with the CYP1B1 inhibitor 1-ethynylpyrene (EP) prior to exposure to MC. DNA adducts were determined by the 32P-postlabeling assay. Aryl hydrocarbon hydroxylase assay was measured by fluorimetry. RESULTS: MC induced formation of 12 DNA adducts that were not observed in DMSO-treated cells. DNA adduct formation was dose-dependent, with maximum response observed at 3 microM. Pre-treatment of cells with EP dramatically suppressed DNA adduct formation by MC. MC treatment caused induction of CYP1B1, but not CYP1A1. CONCLUSION: The induction of high levels of multiple DNA adducts in SMCs by MC suggests that SMCs have a functional enzymatic machinery capable of metabolically activating MC to genotoxic metabolites. The significant inhibition by EP of MC-induced DNA adduct formation indicated that CYP1B1 was the primary CYP enzyme responsible for formation of genotoxic metabolites that may play a role in the induction of atherosclerosis by MC.

In vitro and in vivo effects of naringin on cytochrome P450-dependent monooxygenase in mouse liver.

In vitro and in vivo effects of naringin on microsomal monooxygenase were studied to evaluate the drug interaction of this flavonoid. In vitro addition of naringin up to 500 microM had no effects on benzo(a)pyrene hydroxylase (AHH) activity of mouse liver microsomes. In contrast, the aglycone naringenin at 300 to 500 microM decreased AHH activity by 50% to 60%. Analysis of Lineweaver-Burk and Dixon plots indicated that naringenin competitively inhibited AHH activity with an estimated Ki of 39 microM. Naringenin at 100 microM also reduced metabolic activation of benzo(a)pyrene to genotoxic products as monitored by umuC gene expression response in Salmonella typhimurium TA1535/pSK1002. In the presence of equimolar naringenin and benzo(a)pyrene, umuC gene expression presented as beta-galactosidase activity was reduced to a level similar to the control value. Administration of a liquid diet containing 10 mg/ml naringin for 7 days caused 38% and 49% decreases of AHH and 7-methoxyresorufin O-demethylase activities, respectively. In contrast, the administration had no effects on cytochrome P450 (P450)-catalyzed oxidations of 7-ethoxyresorufin, 7-ethoxycoumarin, N-nitrosodimethylamine, nifedipine, erythromycin and testosterone. Microsomal P450 and cytochrome b5 contents and NADPH-P450 reductase activity were not affected. Immunoblot analysis using MAb 1-7-1, which immunoreacted with both P450 1A1 and 1A2, revealed that the level of P450 1A2 protein was decreased by 38%. These results demonstrate that naringenin is a potent inhibitor of AHH activity in vitro and naringin reduces the P450 1A2 protein level in vivo. These effects may indicate a chemopreventive role of naringin against protoxicants activated by P450 1A2.

Tellurite specifically affects squalene epoxidase: investigations examining the mechanism of tellurium-induced neuropathy.

A peripheral neuropathy characterized by a transient demyelinating/remyelinating sequence results when young rats are fed a tellurium-containing diet. The neuropathy occurs secondary to a systemic block in cholesterol synthesis. Squalene accumulation suggested the lesion was at the level of squalene expoxidase, a microsomal monooxygenase that uses NADPH cytochrome P450 reductase to receive its necessary reducing equivalents from NADPH. We have now demonstrated directly specificity for squalene epoxidase; our in vitro studies show that squalene epoxidase is inhibited 50% in the presence of 5 microM tellurite, the presumptive in vivo active metabolite. Under these conditions, the activities of other monooxygenases, aniline hydroxylase and benzo(a)pyrene hydroxylase, were inhibited less than 5%. We also present data suggesting that tellurite inhibits squalene epoxidation by interacting with highly susceptible -SH groups present on this monooxygenase. In vivo studies of specificity were based on the compensatory response to feeding of tellurium. Following tellurium intoxication, there was up-regulation of squalene epoxidase activity both in liver (11-fold) and sciatic nerve (fivefold). This induction was a specific response, as demonstrated in liver by the lack of up-regulation following exposure to the nonspecific microsomal enzyme inducer, phenobarbital. As a control, we also measured the microsomal monooxygenase activities of aniline hydroxylase and benzo(a)pyrene hydroxylase. Although they were induced following phenobarbital exposure, activities of these monooxygenases were not affected following tellurium intoxication, providing further evidence of specificity of tellurium intoxication for squalene epoxidase.

Inhibition of microsomal lipid peroxidation and monooxygenase activities by eugenol.

Previously we reported that eugenol (4-allyl-2-methoxyphenol) inhibits non-enzymatic peroxidation in liver mitochondria (E. Nagababu and N. Lakshmaiah, 1992, Biochemical Pharmacology. 43, 2393-2400). In the present study, we examined the effect of eugenol on microsomal mixed function oxidase mediated peroxidation using Fe+3-ADP-NADPH, carbon tetrachloride (CCL4)-NADPH and cumene hydroperoxide (CumOOH) systems. In the presence of eugenol the formation of thiobarbituric acid reactive substances (TBARS) was decreased in all the systems (IC50 values: 14 microM for Fe+3-ADP-NADPH, 4.0 microM for CCl4-NADPH and 15 microM for CumOOH). Oxygen uptake was also inhibited to a similar extent with Fe+3-ADP-NADPH and CumOOH systems. A comparative evaluation with other antioxidants showed that in Fe+3-ADP-NADPH and CumOOH systems, the antioxidant efficacy was in the order: butylated hydroxytoluene (BHT) > eugenol > alpha-tocopherol, while in CCl4-NADPH system the order was alpha-tocopherol > BHT > eugenol. Time course of inhibition by eugenol indicated interference in initiation as well as propagation of peroxidation. Eugenol did not inhibit cytochrome P-450 reductase activity but it inhibited P-450 - linked monooxygenase activities such as aminopyrine-N-demethylase, N-nitrosodimethylamine demethylase, benzo(a)pyrene hydroxylase and ethoxyresorufin-O-deethylase to different extents. However, CumOOH supported monooxygenases (aminopyrine-N-demethylase and benzo(a)pyrene hydroxylase) required much higher concentrations of eugenol for inhibition. The concentration of eugenol required to inhibit monooxygenase activities was more than that required to inhibit peroxidation in all the systems. Eugenol elicited type 1 changes in the spectrum of microsomal cytochrome P-450. These results suggest that the inhibitory effect of eugenol on lipid peroxidation is predominantly due to its free radical quenching ability. Eugenol significantly protected against the degradation of cytochrome P-450 during lipid peroxidation with all the systems tested. These findings suggest that eugenol has the potential to be used as a therapeutic antioxidant. Further evaluation may throw more light on this aspect.

Determinants of protein modification versus heme alkylation: inactivation of cytochrome P450 1A1 by 1-ethynylpyrene and phenylacetylene.

Reaction of cytochrome P450 enzymes with arylacetylenes results in heme N-alkylation [e.g., Komives, E. A., and Ortiz de Montellano, P. R., (1985) J. Biol. Chem. 260, 3330-3336] and/or protein modification [e.g., Gan, L.-S. L., Acebo, A. L. and Alworth, W. L. (1984) Biochemistry 23, 3827-3836]. To clarify the factors that determine whether heme or protein alkylation occurs, we have investigated the cytochrome P450 1A1-catalyzed oxidation of 1-ethynylpyrene (1-EP) and phenylacetylene (PA). Cytochrome P450 1A1 in microsomes from beta-naphthoflavone-induced rats is inactivated in a time- and NADPH-dependent manner by 1-EP and PA. Parallel loss of the heme chromophore is observed with PA but not with 1-EP, although partial heme chromophore loss is observed when the purified, reconstituted enzyme is inactivated by either agent. Product analysis shows that 1-EP and PA are oxidized to, respectively, (1'-pyrenyl)-acetic and phenylacetic acids. In contrast to the inactivation of cytochrome P450 2B1 by PA, no isotope effect is observed on enzyme inactivation or metabolite formation when the acetylenic hydrogen is replaced by deuterium in either 1-EP or PA. Inactivation of cytochrome P450 1A1 by 1-EP results in covalent binding of 0.8-0.9 equiv (relative to total cytochrome P450 content) of the inhibitor to the microsomal protein. The results demonstrate that a single isozyme can be inactivated, depending on the structure of the arylacetylene, by heme or protein alkylation. Spectroscopic binding constants (Ks) show that 1-EP binds to the enzyme with > 2000 times greater affinity that PA.(ABSTRACT TRUNCATED AT 250 WORDS)

[Effect of the monooxygenase activity inhibitor ketoconazole on cholesterol esterification in mouse peritoneal macrophages]. / Vliianie ingibitora monooksigenaznoi aktivnosti ketokonazola na ésterifizkatsiiu kholesterina v peritoneal'nykh makrofagakh myshi.

In order to determine the feasible role of monooxygenases in regulation of the macrophage acyl-CoA: cholesterol acyltransferase (ACAT) activity, the effects of ketoconazole on the activities of benz(a)pyrene hydroxylase and ACAT as well as on the [14C]oleate incorporation into cholesterol esters in cultured mouse peritoneal macrophages (MPM) were studied. Ketoconazole (0.5-50 M) inhibited the benz(a)pyrene hydroxylase activity but increased the free cholesterol (FC) level in MPM cultured with an acetylated low density lipoprotein (acetyl-LDL). An addition of ketoconazole (1-50 M) eliminated the increase in the rate of FC esterification after incubation of MPM with acetyl-LDL (but not with 25-hydroxycholesterol). In contrast, progesterone, an ACAT activity inhibitor, used at 5-30 M diminished the rate of FC esterification, when MPM were incubated with acetyl-LDL of 25-hydroxycholesterol. Ketoconazole provoked a dose-dependent decrease of the [3H]FC incorporation into macrophage polar oxysteroids. The data obtained suggest that the ketoconazole (1-30 M) effect on FC esterification in MPM cultured with acetyl-LDL is determined by its inhibiting monooxygenases, which produce oxidized forms of FC that are potential activators of ACAT.

[The role of secondary metabolism in formation of benz(a)pyrene dihydrodiol profile in microsome membranes]. / Rol' vtorichnogo metabolizma v formirovanii profilia digidrodiolov benz(a)pirena v membranakh mikrosom.

4,5-, 7,8- and 9,10-dihydrodiols of benz(a)pyrene (BP) were separated by thin-layer chromatography and their influence on BP-hydroxylase activity was studied in liver microsomes isolated from rats treated with phenobarbital (PB-microsomes) and 3-methylcholanthrene (MC-microsomes). All diols studied inhibited hydroxylation of BP by the competitive type. Accumulation of BP-diols in the incubation media correlated with their affinity to cytochrome P-450 isoenzymes which catalyzed the secondary metabolism of these diols. This correspondence allowed to formulate the kinetic and temperature dependence of BP oxidation suggesting that two main groups of hemoprotein isoforms were contained which were dissimilar in the active site orientation. Treatment with 3-methylcholanthrene induced specifically those hemoproteins which had the active site directed inside the membrane lipids; treatment with phenobarbital involved induction of two groups of hemoproteins active site of which was directed both to lipid and to water. The primary metabolism of the hydrophobic BP involved cytochrome P-450 isoenzymes which had the active site directed inside the lipids; the secondary metabolism of more polar diols was realized using both groups of hemoprotein isoenzymes with active sites oriented into lipids and water.

Monoclonal antibody characterization of NADH- and NADPH-dependent hydroxylation of benzo(a)pyrene in liver microsomes from 5,6-benzoflavone-induced C57Bl/6 mice.

Monoclonal antibodies (MAb 1-7-1) directed against the isoenzymes of rat liver cytochrome P-450 induced by methylcholanthrene, inhibited benzo(a)pyrene hydroxylase less strongly at low (0.04 mM) than at high (2 mM) NADH concentration. Inhibition of NADPH dependent hydroxylation was the same irrespective of NADPH concentrations and corresponded to that observed at high NADH concentration. The same was also the h.p.l.c. pattern of the reaction products of benzo(a)pyrene hydroxylation supported by high concentration of both coenzymes. It is postulated that different cytochrome P-450 isoenzymes participate in benzo(a)pyrene hydroxylation, whereas the second one acts at high concentration of both NADH and NADPH.

[The cytotoxic and antimutagenic effect of dithranol]. / Zur cytotoxischen und antimutagenen Potenz von Dithranol.

The anti-psoriatic compound anthralin (dithranol, cignolin) was determined to exhibit a strong cytostatic activity on HeLa-Köln cells; an ED50 concentration of 1.2 microM was determined for the cells. These growth-inhibition data were confirmed by thymidine-uptake experiments. The drug anthralin was determined to be neither direct a mutagen nor a premutagen in the Ames test using Salmonella typhimurium strain TA 100 (anthralin-concentration = 5 microM). Moreover, this compound was a strong inhibitor of benzo(a)pyrene monooxygenase, an enzyme which causes the metabolic conversion of premutagens to mutagens. These data demonstrate anthralin to be an antipsoriatic compound devoid of mutagenic property in vitro with regard to base-pair substitutions and provided at least with some antimutagenic potential.

Inhibition of hepatic microsomal drug metabolism by the steroid hydroxylase inhibitor SU-10'603.

SU-10'603 is a pyridine derivative that has been widely used as a steroid 17-hydroxylase inhibitor. Studies were done to compare the effects of SU-10'603 with those of the structurally related compound, metyrapone, on hepatic microsomal drug metabolism in vitro in rats and guinea pigs. In rat liver microsomes, SU-10'603 produced a concentration-dependent (0.01 to 1.0 mM) inhibition of ethylmorphine demethylation, aniline hydroxylation, and benzo[a]pyrene hydroxylation. A concentration of 0.1 to 0.2 mM decreased the metabolism of all three substrates by approximately 50%. SU-10'603 was a more potent inhibitor of ethylmorphine metabolism than metyrapone, and its relative potency was even greater with respect to aniline and benzo[a]pyrene metabolism. Similar results were obtained with guinea pig liver microsomes. SU-10'603 and metyrapone produced type II spectral changes in hepatic microsomes, but the apparent affinity of SU-10'603 for cytochrome(s) P-450 was greater than that of metyrapone. Both compounds inhibited the binding of type I substrates to microsomal cytochromes P-450; SU-10'603 was the more potent inhibitor. The results indicate that SU-10'603 is a potent inhibitor of hepatic microsomal monooxygenases whose mechanism of action is similar to that of metyrapone.

Inhibition of cytochrome P-450c-mediated benzo[a]pyrene hydroxylase and ethoxyresorufin O-deethylase by dihydrosafrole.

1. Inhibitory activity of dihydrosafrole towards benzo[a]pyrene (BP) hydroxylase activity in hepatic microsomes from beta-naphthoflavone (BNF)-induced rats, and in reconstituted systems containing cytochrome P-450c, increased dramatically on preincubation of the inhibitor with NADPH; no inhibition occurred without preincubation. The level of BP hydroxylase inhibition was associated with the progressive formation of the 456 nm dihydrosafrole metabolite-cytochrome P-450c spectral complex during preincubation. 2. Inhibition of BP hydroxylase by dihydrosafrole in control microsomes, and inhibition of ethoxyresorufin O-deethylase (EROD) in microsomes (control or BNF-induced) and in reconstituted systems with cytochrome P-450c, did not require preincubation and apparently was not dependent on prior formation of the dihydrosafrole metabolite-cytochrome P-450 complex. 3. Kinetic studies established that, following preincubation with NADPH, dihydrosafrole was a noncompetitive inhibitor of both BP hydroxylase and EROD activities. In the absence of preincubation, dihydrosafrole was an effective competitive inhibitor of EROD in BNF-induced microsomes and in reconstituted systems with cytochrome P-450c. 4. Both ethoxyresorufin and benzo[a]pyrene inhibited the development of the type I optical difference spectrum of dihydrosafrole in reconstituted systems containing cytochrome P-450c. Inhibition by ethoxyresorufin was competitive while that caused by benzo[a]pyrene was noncompetitive in nature. 5. The type II ligand phenylimidazole was an effective noncompetitive inhibitor of EROD activity but failed to exert any inhibitory effect on cytochrome P-450c-mediated BP hydroxylase activity. Phenylimidazole inhibited formation of the dihydrosafrole type I optical difference spectrum non-competitively. 6. The results indicate that ethoxyresorufin and benzo[a]pyrene may occupy different binding sites on cytochrome P-450c and that dihydrosafrole binds primarily to the site utilized by ethoxyresorufin.

Inactivation of human liver cytochrome P-450 by the drug methoxsalen and other psoralen derivatives.

The effects of psoralen derivatives on cytochrome P-450 have been studied in human liver microsomes. CO-binding cytochrome P-450 was decreased by 33% after 10 min of incubation with 1.5 mM EDTA, an NADPH-regenerating system and 20 microM methoxsalen (8-methoxypsoralen). No destruction of cytochrome P-450 was observed when either NADPH or methoxsalen was omitted. A similar (27%) decrease in CO-binding required a 100-times higher concentration of allylisopropylacetamide (2 mM). The activities of 7-ethoxycoumarin deethylase and benzo(a)pyrene hydroxylase were decreased by about 50% in the presence of 12.5 microM methoxsalen. At this low concentration, neither cimetidine nor SKF 525-A or piperonyl butoxide had any significant inhibitory effect. Monooxygenase activities were also decreased in the presence of 12.5 microM bergapten (5-methoxypsoralen) or 12.5 microM psoralen, but not with 12.5 microM trioxsalen (trimethylpsoralen). CO-binding cytochrome P-450 was not decreased after 10 min of incubation with 1.5 mM EDTA, an NADPH-regenerating system and 20 microM trioxsalen. We conclude that methoxsalen is an extremely potent suicide inhibitor of cytochrome P-450 in human liver microsomes. Bergapten and psoralen are also inhibitory whereas trioxsalen has little effects. In the latter derivative, a methyl group is attached on the furan ring and may hinder its metabolic activation and the inactivation of cytochrome P-450.

Metabolism of some carcinogenic aromatic amines in four species of marine sponges.

Postmitochondrial fractions from marine sponges Geodia cydonium, Tethya aurantium, Verongia aerophoba and Pellina semitubulosa activate precarcinogenic aromatic amine 2-aminoanthracene, but not precarcinogenic polycyclic aromatic hydrocarbon benzo(a)pyrene, to Salmonella typhimurium TA 98 mutagens. All four sponge species lack a benzo(a)pyrene monooxygenase activity, but possesses the enzyme activity whose characteristics (selective activation of aromatic amines, NADPH-dependency, pH optimum at 8.4) are similar to FAD-containing monooxygenase. Tethya postmitochondrial fraction possesses an UDP-glucuronyl transferase activity which catalyzes the conjugation of a considerable part of metabolized 2-acetylamino [9-14C]fluorene to water soluble glucuronides. The possible ecological significance of exuded aromatic amine metabolites as well as the significance of the presence of the selective potential for the activation of aromatic amines to mutagens among sponges for our understanding of the fate and effects of carcinogens in the marine environment are discussed.

Suppressed mutagenicity of benzo[a]pyrene by the liver S9 fraction and microsomes from eugenol-treated rats.

The mutagenicity of benzo[a]pyrene (B[a]P) in the Ames test using liver S9 fraction prepared from rats pretreated with eugenol (4-allyl-2-methoxyphenol) was suppressed to a lower level than that obtained using liver S9 from untreated rats. There was a reverse correlation between the mutagenicity of B[a]P and the dose of eugenol administered to the animals. Similarly suppressed mutagenicity was observed when liver microsomes, instead of the S9 fraction, were used in the Ames test. The mutagenic activity of B[a]P and arylhydrocarbon hydroxylase (AHH) could not be inhibited by the direct addition of eugenol into the assay mixtures. In eugenol-treated microsomes, cytochrome P-450 content, AHH activity and total B[a]P hydroxylase activity were decreased to 81, 29 and 48% of the control values, respectively. The mutagenicity of B[a]P catalyzed by microsomes from rats fed ad libitum on a diet containing 5% eugenol in the Ames test was significantly decreased. AHH activity and total B[a]P hydroxylase activity were also decreased in these liver microsomes. These results indicate that the activation of B[a]P in rat liver by cytochrome P-450, which metabolizes B[a]P to ultimate mutagens or carcinogens, is reduced by the administration of eugenol.

Inhibition of hepatic microsomal drug metabolism by the immunosuppressive agent cyclosporin A.

Cyclosporin A (CsA), an orally active immunosuppressive agent, was shown to inhibit cytochrome P-450 dependent biotransformation of drugs in the mouse. It competitively inhibited the hydroxylation of benzo[a]pyrene and the N-demethylation of aminopyrine in hepatic microsomes with Ki values of 93 and 1540 microM respectively. This selective inhibition for benzo[a]pyrene hydroxylase by CsA was substantiated in vivo by selective inhibition of total body clearance of theophylline, but not of antipyrine. CsA was itself N-demethylated by hepatic microsomes with a Km of 808 microM. CsA interacted directly with cytochrome P-450, causing a reverse type I spectral change in hepatic microsomes. No metabolic intermediate complexes could be demonstrated. These results suggest that CsA has the potential to cause drug interactions involving inhibition of drug biotransformation, particularly of drugs that are metabolised by the same types of cytochrome P-450 which oxidise benzo[a]pyrene and theophylline.

[Catalytic properties of monooxygenases from isolated immunocompetent cells]. / Kataliticheskie svoistva monooksigenaz izolirovannykh immunokompetentnykh kletok.

The kinetic parameters of two monoxygenase (N-demethylase and benzo(a)pyrene hydroxylase) reactions occurring in murine immunocompetent cells were determined. It was shown that the pH optimum for macrophage enzymes lies at 7.4, whereas that for thymocytes and spleen cells at 8.5. The effects of cytochrome P-450 inhibitors (methyrapone, SKF-525A, tilorone) on monooxygenase systems of immunocytes were investigated. Methyrapone and SKF-525A were shown to be effective inhibitors of N-demethylase. The mode of inhibition changes from non-competitive to competitive, depending on the inhibitor concentration. Tilorone, an immunostimulator and inhibitor of liver cytochrome P-450-dependent enzymes, competitively inhibits N-demethylase and benzo(a)pyrene hydroxylase of immunocytes.

In vitro effects of straight-chain alkanes (n-hexane through n-dodecane) on rat liver and lung cytochrome P-450.

To evaluate the effect of straight-chain alkanes on normal detoxication reactions, we studied the in vitro effect of the homologous series n-hexane through n-dodecane on two cytochrome P-450 (EC 1.14.14.1) enzyme activities. Benzo[a]pyrene hydroxylase (BaPOHase) and 7-ethoxycoumarin deethylase activities were measured in liver and lung microsomes of control and beta-naphthoflavone-treated rats. In the presence of 2 mM n-hexane through n-dodecane, liver BaPOHase activity decreased from 67% of control with n-dodecane to 21% of control with octane. Lung benzo[a]pyrene hydroxylase was insensitive to all tested alkanes at 2 mM. In the presence of 2 mM alkanes, liver 7-ethoxycoumarin deethylase activity decreased from 73% of control with n-octane to 28% with n-octane. Lung 7-ethoxycoumarin deethylase was also sensitive to the alkane series. In the presence of 2 mM alkane the greatest effect was obtained with n-octane and represented a 56% loss in activity. Alkane concentration-dependence measurements showed 0.02-0.20 mM as the sensitive region of the curve for n-octane with maximal loss of activity achieved at 0.20 mM. Liver ethoxycoumarin deethylase activity from beta-naphthoflavone-treated rats was less sensitive towards the reactive alkane, n-octane, than the activity from control rats. Double-reciprocal-plot analysis revealed the maximal velocity (Vmax) was decreased in the presence of 0.2 mM n-octane. Hence this hydrocarbon did not exert its effect solely as an alternate substrate. The data show the n-alkanes, n-hexane through n-dodecane, interfered with a normal detoxication pathway in a manner that was chainlength-dependent, tissue-specific, and dependent on the preexposure history of the animal.

Benzo(a)pyrene hydroxylase activity in human bronchial mucus.

Sputum collected from patients with respiratory diseases were examined for presence of benzo(a)pyrene hydroxylase (BPH) activity. The human bronchial mucus used in these studies had significant capability to metabolize benzo(a)pyrene. Clarification of the sputum by agents such as N-acetylcysteine or pancreatin in presence of antibiotics was found to be essential for the detection of BPH activity. In vitro incubation of the clarified human bronchial mucus with benzoflavone caused inhibition, while 7,8-dimethyl-benzanthracene induced BPH enzyme activity.

Antimutagenic activity of the novel antileukemic agents, avarone and avarol.

The two antileukemic agents, avarone and avarol, were determined to be neither direct nor indirect mutagenic agents in the Ames microsomal test. Moreover, the two sesquiterpenoid compounds drastically reduced the mutagenic effect of benzo[a]pyrene in the same system. Subsequent enzymic studies demonstrated that avarone and avarol are powerful inhibitors of benzo[a]pyrene monooxygenase.