Oral administration of naturally occurring coumarins leads to altered phase I and II enzyme activities and reduced DNA adduct formation by polycyclic aromatic hydrocarbons in various tissues of SENCAR mice

Several naturally occurring coumarins, to which humans are routinely exposed in the diet, were previously found to inhibit P450-mediated metabolism of benzo[a]pyrene (B[a]P) and 7,12-dimethylbenz[a]anthracene (DMBA) in vitro, block DNA adduct formation in mouse epidermis and inhibit skin tumor initiation by B[a]P and/or DMBA when applied topically to mice. The present study was designed to investigate the effects of two of these compounds, of the linear furanocoumarin type, when given orally (70 mg/kg per os, four successive daily doses), on P450 and glutathione S-transferase (GST) activities and DNA adduct formation by B[a]P and DMBA in various mouse tissues. Imperatorin and isopimpinellin significantly blocked ethoxyresorufin O-deethylase (EROD) and pentoxyresorufin O:-dealkylase (PROD) activities in epidermis at 1 and 24 h after oral dosing. Imperatorin and isopimpinellin modestly inhibited EROD activities in lung and forestomach at 1 h and significantly inhibited PROD activities in lung and forestomach at 1 h after the final oral dose. Twenty-four hours after the final oral dose of imperatorin or isopimpinellin EROD and PROD activities remained inhibited in epidermis and lung. However, forestomach P450 activity had returned to control levels. Interestingly, imperatorin and isopimpinellin treatment inhibited liver EROD activity at 1 h, had no effect on PROD activity at this time point, but elevated both these enzyme activities at 24 h. Elevated EROD and PROD activities coincided with elevated hepatic P450 content. Imperatorin and isopimpinellin treatment also increased liver cytosolic GST activity at both 1 and 24 h after the final oral dose by 1.6-fold compared with corn oil controls. Oral administration of imperatorin and isopimpinellin also had a protective effect against DNA adduct formation by B[a]P and DMBA. Imperatorin pretreatment decreased formation of DNA adducts by DMBA in forestomach. Pretreatment with isopimpinellin led to reduced DNA adduct levels in liver (B[a]P), lung (B[a]P) and mammary epithelial cells (DMBA). These results suggest that imperatorin and isopimpinellin may have potential chemopreventive effects when administered in the diet.     

Structure-activity relationships of arylalkyl isothiocyanates for the inhibition of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone metabolism and the modulation of xenobiotic-metabolizing enzymes in rats and mice

Many arylalkyl isothiocyanates are potent inhibitors of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone(NNK)-induced lung tumorigenesis in rats and mice. In the mouse,4-phenylbutyl isothiocyanate (PBITC) and 6-phenylhexyl isothiocyanate(PHTTC) exhibited greater inhibition than benzyl isothiocyanate(BITC) and phenethyl isothiocyanate (PEITC). The present studywas conducted to investigate the structure-activity relationshipsof these four arylalkyl isothiocyanates for their inhibitionof NNK oxidation and effects on xenobiotic-metabolizing enzymesin rats and mice. A single dose (0.25 or 1.00 mmol/kg) of eachisothiocyanate was given to F344 rats 6 or 24 h before death.The rates of NNK oxidation were decreased in microsomes fromthe liver, lung and nasal mucosa of rats. Generally, PEITC wasmore potent than BITC but less potent than PBITC and PHlTC.The rates in rat liver microsomes were decreased at 6 h butrecovered or increased at 24 h; and the rates in rat lung microsomeswere markedly decreased at both 6 and 24 h; and the rates inrat nasal mucosa microsomes were also significantly decreased.The same treatment decreased the rat liver N-nitrosodimethyl-aminedemethylase activity dramatically and ethoxyresorufin O-dealkylaseand erythromycin N-demethylase activities moderately. However,the rat liver microsomal pentoxy-resorufin O-dealkylase activitywas decreased at 6 h but increased at 24 h, with PEITC showingthe most marked induction. The rat liver NAD(P)H: quinone oxidoreductaseactivity was increased 1.4- to 3.3-fold, with PEITC being mosteffective; and the glutathione S-transferase activity was increasedslightly. Similarly, at a single dose of 0.25 mmol/kg (5 µmol/mouse)24 h before death, PEITC, PBITC, PHlTC but not BITC, decreasedNNK oxidation in mouse lung microsomes by 40–85%, withPBITC and PHlTC showing greater inhibition. Furthermore, allfour isothiocyanates extensively inhibited NNK oxidation inrat lung and nasal mucosa microsomes as well as mouse lung microsomesin vitro, with PEITC (IC₅₀ of 120–300 nM) being more potentthan BITC (IC₅₀ of 500–1400 nM) but less potent than PBITCand PHITC (IC50 of 15–180 nM). PHITC was a very potentcompetitive inhibitor of NNK oxidation in mouse lung microsomeswith apparent K₁ values of 11–16 nM. These results indicatethat PBITC and PHITC are more potent inhibitors of NNK bioactivationin rats and mice than PEITC. In addition, these arylalkyl isothiocyanatescould be effective in protecting against the actions of a broadspectrum of carcinogenic or toxic compounds.     

Inhibition of rat liver cytochrome P450 isozymes by isothiocyanates and their conjugates: a structure-activity relationship study

A series of arylalkyl and alkyl isothiocyanates, and their glutathione,cysteine, and N-acetylcysteine conjugates were used to studytheir inhibitory activity toward the dealkylation of ethoxyresorufin(EROD), pentoxyresorufin (PROD), and methoxyresorufin (MROD)in liver microsomes obtained from the 3-methylcholanthrene orphenobarbital-treated rats. These reactions are predominantlymediated by cytochrome P450 (P450) isozymes 1A1 and 1A2, 2B1and 1A2, respectively. All isothiocyanates inhibited PROD morereadily than EROD. Increases in the alkyl chain length of arylalkylisothiocyanates to C₆ resulted in an increased inhibitory potencyin these assays; at longer alkyl chain lengths (C₈-C₁₀) theinhibitory potency declined. The IC₅₀s for phenethyl isothiocyanate(PEITC) were 47, 46 and 1.8 µM for EROD, MROD and PROD,respectively. Substitution of an additional phenyl group onPEITC also increased the inhibitory potency; the IC₅₀s for 1,2-diphenylethyl isothiocyanate (1, 2-DPEITC) and 2, 2-diphenylethylisothiocyanate (2,2-DPEITC) were 0.9 and 0.26 µM for EROD,and 0.045 and 0.13 µM for PROD, respectively. The relativeinhibitory potency of PEITC and its conjugates was N-acetylcysteine-PEITC(PEITC-NAC) < glutathione-PEITC (PEITC-GSH) < cysteine-PEITC(PEITC-CYS) < PEITC. The observations that the parent isothiocyanateswere more potent inhibitors than the conjugates suggest thatdissociation of the conjugate is required for activity. Naturallyoccurring alkyl isothiocyanates, sulforaphane (SFO) and allylisothiocyanate (AITC), were very weak inhibitors in the assays.These results suggest the potential of isothiocyanates as structuralprobes for studying P450 isozymes. In addition, the inhibitoryactivity of isothiocyanates for PROD correlated with the previouslydemonstrated tumor inhibitory potency in (4-methylnitrosamino)-1-(3-pyridyl)-1-butanone(NNK) induced A/J mouse lung tumor bioassays, which supportsearlier findings that P450 2B1 is one of the major isozymesinvolved in NNK activation and that inhibition of this isozymeis an important mechanism for the chemopreventive activity ofisothiocyanates.     

Metabolic activation of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone as measured by DNA alkylation in vitro and its inhibition by isothiocyanates

The bioactivation of the tobacco-specific nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), by microsomes from target organs was studied with an in vitro microsome-mediated DNA alkylation system. Mouse lung, rat lung, and rat nasal microsomes catalyzed a time- and protein-dependent DNA methylation by [methyl-3H]NNK with activities of 4.11, 0.95, and 137.4 pmol/mg DNA/mg protein/h, respectively. The DNA methylation of NNK catalyzed by all three microsomal systems was inhibited by cytochrome P-450 inhibitors, such as carbon monoxide and metyrapone, but not by the cyclooxygenase inhibitor, aspirin, or by prolonged preincubation in the absence of NADPH. The possible involvement of specific P450 isozymes was assessed by specific inhibitory antibodies. An anti-P450IIB1&2 antibody significantly inhibited the DNA methylation by 45 and 32% in mouse lung and rat lung, respectively, whereas anti-P450IA1 and anti-P450IIE1 antibodies failed to show significant inhibition. All antibodies showed no inhibition in rat nasal microsomes. Glutathione inhibited the DNA methylation in a concentration-dependent manner in all three microsomal systems. Phenethyl isothiocyanate (PEITC), at doses of 0.25 and 1.00 mmol/kg body weight, was given intragastrically 2 h before sacrifice to mice and 24 h before sacrifice to rats, respectively; both mouse and rat lung microsomal activities were inhibited by about 40 and 90% by the low- and high-dose PEITC treatments, respectively. The rat nasal microsomes were only inhibited by the high-dose PEITC treatment by about 40%. PEITC, 4-phenylbutyl isothiocyanate, and 6-phenylhexyl isothiocyanate all inhibited the microsome-mediated DNA methylation of NNK in vitro, with 4-phenylbutyl isothiocyanate and 6-phenylhexyl isothiocyanate being more potent than PEITC and the mouse lung microsomes more sensitive than the rat lung and nasal microsomes. All three microsomal systems were shown to catalyze the in vitro DNA pyridyloxobutylation by [5-3H]NNK. On an equal protein basis, the rat nasal microsomes were much more active in catalyzing the DNA pyridyloxobutylation.     

Effect of diallyl sulfide on rat liver microsomal nitrosamine metabolism and other monooxygenase activities

It has been reported that p.o. administration of diallyl sulfide (DAS), a naturally occurring component of garlic (Allium sativum), inhibits 1,2-dimethylhydrazine-induced colon and liver cancer in rodents. A possible mechanism for this protective effect is inhibition of hepatic activation of the procarcinogen. The effect of DAS on P450IIE1, an isozyme of cytochrome P-450 which is active in the oxidative metabolism of dimethylhydrazine, was conveniently assayed in the present study by determination of N-dimethylnitrosamine demethylase (NDMAd) activity at 1 mM N-dimethylnitrosamine in Sprague-Dawley rat liver microsomal incubations. DAS was found to be a competitive inhibitor of NDMAd, in contrast to the irreversible inactivation of NDMAd produced by carbon tetrachloride incubated under similar conditions. The inhibition by DAS of the demethylation of several substrates was selective. The thioether was most potent against N-dimethylnitrosamine, less effective against N-nitrosomethylbenzylamine, and essentially ineffective against benzphetamine and ethylmorphine. Microsomes prepared at 3 h after DAS administration (200 mg/kg in corn oil intragastrically) showed moderate inhibition (less than 30% inhibition compared to control microsomes) of several demethylase activities; however, microsomes prepared 18 h posttreatment showed a marked decrease (about 80% inhibition compared to controls) in NDMAd activity, minor effects on other demethylase activities, and a 6-fold increase in pentoxyresorufin dealkylation. These trends at 18 h agreed with immunoblot analyses which showed suppression in the level of P450IIE1 and an elevation in P450IIB1. The selective inhibition of P450IIE1 activity and suppression of its level in microsomes may contribute to the reported chemoprotective effects of DAS.     

Metabolism and activation of N-nitrosodimethylamine by hamster and rat microsomes: comparative study with weanling and adult animals

It has been reported that hamster liver preparations are more effective for the metabolic activation of N-nitrosodimethylamine (NDMA) to a mutagen than rat liver preparations. The enzymatic basis for this phenomenon, however, has not been clearly elucidated. The present study was undertaken to examine the enzymology of NDMA metabolism by different hepatic subcellular fractions prepared from hamsters and rats of two different ages, and to investigate the correlation between the metabolism and the activation of NDMA to a mutagen for Chinese hamster V79 cells. The content of cytochrome P-450 was approximately 1.5-fold higher in hamster microsomes than in rat microsomes from both ages (1.19-1.38 versus 0.73-0.83 nmol P-450/mg protein). Weanling hamster microsomes exhibited multiple apparent Km values for NDMA metabolism as did weanling rat microsomes. The apparent Km I value of NDMA demethylase (NDMAd) in hamster microsomes was about one-half that in rat microsomes (36 versus 83 microM) with corresponding Vmax values of 2.09 and 2.57 nmol/min/nmol P-450. The Km I values for denitrosation did not differ from the corresponding values for NDMAd with Vmax values of 0.17 and 0.22 nmol/min/nmol P-450 for hamster and rat microsomes, respectively. These apparent Km values were affected neither by sonication nor by the presence of cytosolic proteins in S9 fractions. Adult rat liver microsomes showed less than one-half the NDMAd activity in weanling rat liver microsomes, whereas such age difference was not observed in hamster liver microsomes. This result was confirmed by Western blotting showing the levels of P-450ac (an acetone-inducible form of P-450) of these microsomes at comparable levels to their NDMAd activities. NDMAd was highly correlated to the metabolic activation of NDMA to a mutagen for V79 cells in an activation system mediated by microsomes prepared from hamsters and rats of different ages. The results from this study clearly demonstrate the enzymatic basis for the more effective metabolism of NDMA in adult hamsters than in adult rats.     

Effect of cigarette smoke on CYP1A1, CYP1A2 and CYP2B1/2 of nasal mucosae in F344 rats

Enzymes of the nasal tissue, one of the first tissues to contact inhaled toxicants, are relatively resistant to induction by traditional inducers. Because tobacco smoke has been shown to induce cytochrome P450 1A1 (CYP1A1) in rat and human lung tissue, we hypothesized that it would also alter levels of xenobiotic-metabolizing enzymes in nasal mucosae. In the present study, the effect of mainstream cigarette smoke (MCS) on nasal CYP1A1, CYP1A2 and CYP2B1/2 was explored. Four groups of 30 F344 rats were exposed to MCS (100 mg total particulate matter/m3) or filtered air for 2 or 8 weeks. Western analysis of microsomes from nasal tissue of MCS-exposed rats showed an induction of CYP1A1 in respiratory and olfactory mucosae, as well as liver, kidney and lung. Relative to controls, CYP1A2 levels increased slightly in the liver and olfactory mucosa. CYP2B1/2, which increased in the liver, appeared to decrease in upper and lower respiratory tissues. Little to no immunoreactivity with CYP1A1 antibody was observed in fixed nasal sections of control rats, yet intense immunoreactivity was seen in epithelia throughout the nasal cavity of MCS-exposed rats. Ethoxyresorufin O-deethylase activity (associated with CYP1A1/2) decreased approximately 2-fold in olfactory mucosa, but increased in non-nasal tissues of rats exposed to MCS. Methoxy- and pentoxyresorufin O-dealkylase activities (associated with CYP1A2 and CYP2B1/2, respectively) decreased in olfactory and respiratory mucosae, as well as lung (CYP2B1/2), yet increased in liver. These data suggest that xenobiotic-metabolizing enzymines of the nasal mucosae may be regulated differently than other tissues.      

Effects of benzyl isothiocyanate and phenethyl isothiocyanate on benzo[a]pyrene metabolism and DNA adduct formation in the A/J mouse

Benzyl isothiocyanate (BITC) inhibits lung tumorigenesis induced in A/J mice by benzo[a]pyrene (B[a]P). In contrast, phenethyl isothiocyanate (PEITC) does not. We tested the hypothesis that BITC inhibits B[a]P tumorigenicity in mouse lung by inhibiting DNA adduct formation, and compared the effects of BITC and PEITC. In mouse liver or lung microsomal incubations, BITC and PEITC inhibited formation of 7,8-dihydro-7,8-dihydroxybenzo[a]pyrene (B[a]P-7, 8-diol) and some other B[a]P metabolites. The metabolism of B[a]P was compared in mouse lung and liver microsomes, 6 or 24h after treatment with BITC or PEITC. In lung, 6 h after treatment, B[a]P-7, 8-diol and some other metabolites were inhibited by BITC and PEITC. However, 24 h after treatment, no inhibition of B[a]P-7,8-diol was observed in microsomes from BITC-treated mice, whereas it was substantially increased in mice treated with PEITC. Effects on B[a]P metabolism in liver microsomes were generally modest. Conversion of B[a]P-7,8-diol to mutagens by mouse liver microsomes was more strongly inhibited by BITC than PEITC. Effects on 7,8-dihydroxy-9, 10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE)-DNA adduct formation were evaluated in DNA from mice treated with isothiocyanates and B[a]P, and killed 2-120h later. The area under the curve (AUC) for BPDE-DNA adducts in lung was 29.5% less (P = 0. 001) in the BITC-B[a]P treated mice and 19.0% less (P = 0.02) in the PEITC-B[a]P mice than in the mice treated with B[a]P alone. Similar results were obtained in liver DNA. There were no significant differences between the reduction of BPDE-DNA AUC values by BITC versus PEITC. The results of this study support the hypothesis that BITC inhibits B[a]P-induced lung tumorigenesis in A/J mice by inhibiting the metabolic activation of B[a]P to BPDE-DNA adducts. However, differences in BPDE-DNA adduct formation do not appear to explain fully the contrasting effects of BITC and PEITC on B[a]P-induced lung tumorigenesis.     

The induction and competitive inhibition of a high affinity microsomal nitrosodimethylamine demethylase by ethanol

The effects of ethanol on the metabolism of nitrosamines byrat liver microsomes have been studied. Treatment of rats with10 or 15% ethanol in drinking water for 3 days causes a 4- to5-fold enhancement in microsomal N-nitrosodimethylamine demethylase(NDMAd) activity and a 40–60% increase in gross P-450content. The enhancement is mainly due to the induction of alow K_m form (K_m = 0.07 mM) of NDMAd. The treatment induces proteinspecies with molecular weights between 50 000 and 52 000, someof which are believed to be P-450 isozymes with high affinityto NDMA. In addition to NDMA, treatment with ethanol also enhancesthe metabolism of N-nitroso-N-methylethylamine, N-nitrosomethylamline,and N-nitroso-N-methylbenzylamine. When added to the incubationmixture, ethanol and its homologs inhibit the demethylationof these nitrosamines by microsomes. Ethanol is a competitiveinhibitor of the low K_m NDMAd with a K_i of 0.31 mM and is lesseffective in inhibiting the metabolism of more lipophilic nitrosamines.     

Chemoprevention of lung tumorigenesis induced by a mixture of benzo(a)pyrene and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone by the organoselenium compound 1,4-phenylenebis(methylene)selenocyanate

We evaluated the chemopreventive efficacy of the organoselenium compound 1,4-phenylenebis(methylene)selenocyanate (p-XSC) against the development of tumors of the lung and forestomach induced by a mixture of benzo(a)pyrene (B(a)P) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), two of the major lung carcinogens present in tobacco smoke. A/J mice (20 mice/group) were given intragastric doses of a mixture of B(a)P (3 micromol/mouse) and NNK (3 micromol/mouse) in cottonseed oil (0.1 ml) once a week for eight consecutive weeks. Mice were fed either AIN-76A control diet or control diet containing p-XSC (10 ppm selenium), either during or after carcinogen administration. Dietary p-XSC significantly reduced lung tumor multiplicity, regardless of whether it was given during or after carcinogen administration. p-XSC was also an effective inhibitor of tumor development in the forestomach. To provide some biochemical insights into the protective role of p-XSC, its effect on selected phase I and II enzyme activities involved in the metabolism of NNK and B(a)P was also examined in vivo in this animal model. Dietary p-XSC significantly inhibited the activities of the phase I enzymes, methoxyresorufin O-dealkylase (MROD) and N-nitrosodimethylamine N-demethylase (NDMAD), in mouse liver, but it had no effect on ethoxyresorufin O-dealkylase (EROD), pentoxyresorufin O-dealkylase (PROD), and erythromycin N-demethylase (ERYTD). Total glutathione S-transferase (GST) enzyme activity, as well as GST-pi and GST-mu enzyme activities, were significantly induced by dietary p-XSC in both the lung and liver. Glutathione peroxidase (GPX) activity was also induced by p-XSC in mouse lung, but not in the liver. Dietary p-XSC had no effect on selenium-dependent glutathione peroxidase (GPX(Se)), GST-alpha, and UDP-glucuronosyl transferase (UDPGT) enzyme activities in either the lung or the liver. These studies suggest that the chemopreventive efficacy of p-XSC, when fed during carcinogen administration, may be, in part, due to the inhibition of certain phase I enzymes involved in the metabolic activation of these carcinogens, and the induction of specific phase II enzymes involved in their detoxification. The mechanisms that account for the effect of p-XSC when fed after carcinogen administration remain to be determined.     

Modulation by phytochemicals of cytochrome P450-linked enzyme activity

Compounds derived from plant sources with putative anticancer properties were studied for their effects on alkoxyresorufin O-dealkylase activity, a measure of cytochrome P450 activity. The phytochemicals investigated included benzyl isothiocyanate, caffeic acid, chlorogenic acid, diosmin, ferulic acid, indole-3-carbinol, phenethyl isothiocyanate and resveratrol. Each phytochemical at concentrations of 0.25 and 0.5 microM was incubated with 0.2 mg hamster liver microsomal protein and 0.5 microM concentrations of benzyloxyresorufin, ethoxyresorufin and methoxyresorufin. Three of the phytochemicals tested, namely benzyl isothiocyanate, phenethyl isothiocyanate and resveratrol, exhibited potent inhibition of alkoxyresorufin O-dealkylase activity. Benzyl isothiocyanate inhibited benzyloxyresorufin O-dealkylase (BROD) activity, ethoxyresorufin O-deethylase (EROD) activity and methoxyresorufin O-demethylase (MROD) activity by 90% at both the 0.25 and 0.5 microM concentrations. Phenethyl isothiocyanate inhibited BROD activity by 69%, EROD activity by 90% and MROD activity by 94% at both concentrations tested. Resveratrol inhibited BROD activity by 69% at the 0.25 microM concentration and by 78% at the 0.5 microM concentration. It inhibited EROD activity by 60% at the 0.25 microM concentration and by 80% at the 0.5 microM concentration. Resveratrol exhibited the greatest inhibitory action toward MROD, i.e. 76% and 84% at the two concentrations tested. Chlorogenic acid significantly affected BROD, EROD and MROD activity only at the 0.5 microM concentration inhibiting by 51%, 47% and 54%, respectively. Caffeic acid affected BROD and MROD activity at 0.5 microM only inhibiting BROD activity by 46% and MROD activity by 40%. Diosmin inhibited EROD activity by 11% at the 0.25 microM concentration and by 61% at 0.5 microM. It inhibited MROD by 47% and 54% at the two concentrations tested but did not significantly alter BROD activity. Ferulic acid significantly inhibited EROD and MROD activity at the 0.5 microM concentration by 28% and 32%, respectively. Indole-3-carbinol significantly inhibited BROD activity by 26% at 0.25 microM and by 42% at 0.5 microM. It inhibited EROD and MROD activity by 28% and 29% at 0.5 microM, respectively. The alkoxyresorufin O-dealkylase reactions are selective for various isoforms of cytochrome P450. Our results suggest that the phytochemicals we tested have varied effects on the enzymatic activity of isoforms of cytochrome P450 that dealkylate benzyloxyresorufin, methoxyresorufin and ethoxyresorufin and therefore may have varied effects on the metabolism of substrates for these isoforms.     

Enzymes involved in the bioactivation of 4-(methylnitrosamino)-1-(3- pyridyl)-1-butanone in patas monkey lung and liver microsomes

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a potent tobacco-specific carcinogen in animals. Our previous studies indicated that there are differences between rodents and humans for the enzymes involved in the activation of NNK. To determine if the patas monkey is a better animal model for the activation of NNK in humans, we investigated the metabolism of NNK in patas monkey lung and liver microsomes and characterized the enzymes involved in the activation. In lung microsomes, the formation of 4-oxo-1-(3-pyridyl)-1-butanone (keto aldehyde), 4-(methylnitrosamino)-1-(3-pyridyl-N-oxide)-1-butanone (NNK- N-oxide), 4-hydroxy-1-(3-pyridyl)-1-butanone (keto alcohol), and 4- (methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) was observed, displaying apparent Km values of 10.3, 5.4, 4.9, and 902 microM, respectively. NNK metabolism in liver microsomes resulted in the formation of keto aldehyde, keto alcohol, and NNAL, displaying apparent Km values of 8.1, 8.2, and 474 microM, respectively. The low Km values for NNK oxidation in the patas monkey lung and liver microsomes are different from those in human lung and liver microsomes showing Km values of 400-653 microM, although loss of low Km forms from human tissue as a result of disease, surgery or anesthesia cannot be ruled out. Carbon monoxide (90%) significantly inhibited NNK metabolism in the patas monkey lung and liver microsomes by 38-66% and 82-91%, respectively. Nordihydroguaiaretic acid (a lipoxygenase inhibitor) and aspirin (a cyclooxygenase inhibitor) decreased the rate of formation of keto aldehyde and keto alcohol by 10-20 % in the monkey lung microsomes. Alpha-Napthoflavone and coumarin markedly decreased the oxidation of NNK in monkey lung and liver microsomes, suggesting the involvement of P450s 1A and 2A6. An antibody against human P450 2A6 decreased the oxidation of NNK by 12-16% and 22-24% in the patas monkey lung and liver microsomes, respectively. These results are comparable to that obtained with human lung and liver microsomes. Coumarin hydroxylation was observed in the patas monkey lung and liver microsomes at a rate of 16 and 4000 pmol/min/mg protein, respectively, which was 5-fold higher than human lung and liver microsomes, respectively. Immunoblot analysis demonstrated that the P450 2A level in the individual patas monkey liver microsomal sample was 6-fold greater than in an individual human liver microsomal sample. Phenethyl isothiocyanate, an inhibitor of NNK activation in rodents and humans, decreased NNK oxidation in the monkey lung and liver microsomes displaying inhibitor concentration resulting in 50% inhibition of the activity (IC50) values of 0.28-0.8 microM and 4.2-6.8 microM, respectively. The results demonstrate the similarities and differences between species in the metabolic activation of NNK. The patas monkey microsomes appear to more closely resemble human microsomes than mouse or rat enzymes and may better reflect the activation of NNK in humans.      

Induction of cytochrome P450 1B1 in lung, liver and kidney of rats exposed to diesel exhaust.

We have shown previously that diesel exhaust particle (DEP) extracts (DEPE) and 1-nitropyrene were genotoxically activated by human cytochrome P450 1B1 in SOS/umu assay. In this study, the in vivo induction of P450 family 1 enzymes in rats by exposure to diesel exhaust was investigated with regard to mRNA levels, P450 enzyme content, drug oxidation activities in the microsomes and umu gene expression of typical P450 substrates and DEPE itself catalyzed by the microsomes. Male Fischer 344 rats (4 weeks old) were exposed to 0.3 and 3.0 mg/m(3) DEP for 12 h per day for 4 weeks; the former dose corresponded to the typical daily airborne particle concentration. The levels of mRNA of rat P450 1B1 and P450 1A1 in the lung and liver were significantly increased 1.1-1.4-fold by exposure to 0.3 mg/m(3) DEP. Diesel exhaust particle extracts induced umu gene expression in Salmonella typhimurium TA1535/pSK1002 in the absence of a functional P450 system and were further activated by human recombinant P450 1B1. Using an O-acetyltransferase overexpressing Salmonella strain, genotoxic activation of P450 1B1 marker chemicals (1-nitropyrene, 1-aminopyrene and DEPE) by lung, liver and kidney microsomes was increased 1.7-4.2-, 1.4-1.5- and 1.0-1.3-fold, respectively, by exposure to 0.3 mg/m(3) DEP. Activation of 3-amino-1,4-dimethyl-5H-pyrido [4,3-b]indole (Trp-P-1; marker for P450 1A1) by lung microsomes and the P450 1A2 content in liver microsomes were slightly increased by exposure to 3.0 mg/m(3) DEP. This is the first report to suggest that typical daily contaminant levels (0.3 mg particle/m(3)) of diesel exhaust can induce P450 1B1 in rats and that the induced P450 1B1 may catalyze the genotoxic activation of DEP.     

Effects of long term dietary phenethyl isothiocyanate on the microsomal metabolism of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone and 4- (methylnitrosamino)-1-(3-pyridyl)-1-butanol in F344 rats

Phenethyl isothiocyanate (PEITC), a cruciferous vegetable component, inhibits lung tumor induction by the tobacco specific nitrosamine, 4- (methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). To gain insight into the mechanism of PEITC lung tumor inhibition, we examined, in male F344 rats, the effects of dietary PEITC (3 micromol/g NIH-07 diet) in combination with NNK treatment (1.76 mg/kg, s.c., three times a week) for 4, 12 and 20 weeks on liver and lung microsomal metabolism of NNK and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), a major metabolite of NNK and also a lung carcinogen. This was compared with rats fed NIH-07 diet, without PEITC, and treated with NNK alone or saline. The protocol was identical to that employed for inhibition of lung tumorigenesis by PEITC. We observed decreased rates of alpha- hydroxylation of NNK and NNAL in lung microsomes of 4-, 12- and 20-week PEITC + NNK treated rats compared with those treated with NNK or saline. NNK treatment alone also decreased lung alpha-methylene hydroxylation of NNK. Long-term NNK + PEITC administration did not significantly affect liver oxidative metabolism of NNK or NNAL, and did not affect the rate of glucuronidation of NNAL in liver microsomes when compared with rats treated with NNK or saline. Thus, PEITC selectively inhibited lung metabolic activation of NNK and NNAL. These results support the hypothesis that PEITC inhibits NNK-induced lung tumors by inhibiting metabolic activation of NNK in the lung. This study also demonstrated that PEITC inhibits lung alpha-hydroxylation of NNAL; this may play a role in PEITC inhibition of lung tumorigenesis by NNK.      

Biotransformation of aflatoxin B1 in rabbit lung and liver microsomes

Aflatoxin B1 (AFB1) is a potent hepatotoxic and hepatocarcinogenic mycotoxin that requires bioactivation to AFB1-2,3-oxide for activity. In addition to epoxidation, microsomal monooxygenases biotransform AFB1 to the less toxic metabolites, aflatoxin M1 (AFM1) and aflatoxin Q1 (AFQ1). The lung is at risk from AFB1 both via inhalation and via the circulation. In the present study, we have characterized rabbit lung and liver microsomal AFB1-DNA binding (an index of AFB1-2,3-oxide formation), AFM1 formation and AFQ1 formation. Vmax values for AFB1-DNA binding were not different between lung and liver when expressed per mg microsomal protein (1.06 +/- 0.13 and 2.12 +/- 1.30 nmol/mg/h for lung and liver respectively), but lung values were greater than liver when expressed per nmol cytochrome P450 (3.64 +/- 0.31 and 1.29 +/- 0.70 nmol/nmol P450/h for lung and liver respectively). Km values for this reaction were not different between lung and liver. Vmax values for AFM1 formation in liver microsomes were greater than in lung when expressed per mg protein, but not when expressed per nmol P450. No differences were detected for the Km for AFM1 formation between lung and liver microsomes. For AFQ1 formation, no differences were detected between Vmax values of lung and liver, regardless of whether results were expressed per mg protein or per nmol P450, while the Km for AFQ1 formation was lower in liver. SKF-525A inhibited these reactions by 63-74% in lung microsomes and 90-96% in liver microsomes. These results indicate that the lung is capable of activating AFB1, and that rabbit lung microsomes contain high activity for this reaction. Furthermore, little AFM1 and AFQ1 are formed in lung microsomes, leading to minimal shunting of AFB1 from the activation pathway.     

Mechanistic Insights into Chemopreventive Effects of Phenethyl Isothiocyanate in N-Nitrosobis(2-oxopropyl)amine-treated Hamsters

The influence of phenethyl isothiocyanate (PEITC) on cell kinetics in the target organs of N -nitroso-bis(2-oxopropyl)amine (BOP) tumorigenicity and on xenobiotic-metabolizing enzymes was investigated in hamsters. Female 5-week-old Syrian hamsters were given a single s.c. dose of 0, 20 or 50 mg/ kg of BOP 2 h after receiving PEITC by gavage at a dose of 0, 100 or 250 μmol/animal (0, 16.3 or 40.8 mg/animal). Six and 22 h after the BOP administration, hamsters were killed and tissues were sampled. Proliferating cell nuclear antigen immunohistochemistry demonstrated significant reduction (P< 0.05–0.001) by PEITC of the labeling indices in the pancreatic acini and ducts, bronchioles, and renal tubules of the BOP-treated animals in a dose-dependent manner. In the lungs, the PEITC pre treat merit significantly (P< 0.001) reduced the 0⁶-methyldeoxyguanosine levels as compared to the BOP-alone value. Immunoblot analysis of liver cytochrome P450 isoenzymes showed CYP 2B1 to be mainly involved in the metabolic activation of BOP. PEITC significantly ( P <0.05) inhibited the induction of several isoenzymes, including CYP 2B1, while lowering the hepatic glutathione S- transferase activity as well as glutathione levels, regardless of BOP administration. Our results thus suggest that PEITC exerts its chemopreventive activity against BOP initiation of carcinogenesis in hamsters by decreasing cell turnover and DNA methylation in the target organs, and by influencing hepatic xenobiotic-metabolizing phase I enzymes, although the relationship, if any, of the latter with the former events remains to be investigated.     

Chloramphenicol and actinomycin-d-induced modifications in the carcinogen-metabolizing capacities of mouse-liver microsomes

The effect of antibiotics chloramphenicol (CML) and actinomycin-D (AMD) on the modification of the carcinogen metabolizing capacity was studied in vivo in mouse liver microsomes at different durations of treatment, for one day and three and six consecutive days. Following the administration of CML, a significant increase was observed in the activity of the low and high substrate levels of the hepatic microsomal N-nitrosodimethylamine demethylases (NDMAd I and II, respectively). On the contrary, AMD reduced the activity of NDMAd I and II in a time-dependent manner up to 3 days of treatment while no effect was observed when the drug was administered for 6 consecutive days. No effect was observed in the activity of the arylhydrocarbon (benzo(alpha)pyrene) hydroxylase either at the single or the repeated doses of CML only for 6 days of treatment with AMD. The expression of the hepatic content of cytochrome P450 revealed a significant induction at the various treatment intervals with CML. AMD, however, reduced the cytochrome P450 at 1 and 6 days treatment with induction at 3 days of repeated treatment.     

Metabolism of aflatoxin B1 by rabbit and rat nasal mucosa microsomes and purified cytochrome P450, including isoforms 2A10 and 2A11

The nasal mucosa of some mammalian species are susceptible tothe toxicity of aflatoxin B1 (AFB1), a potent hepato-carcinogen,but little is known about the nasal enzymes involved in themetabolic activation of AFB1 or the metabolites produced. Inthe present study, the metabolism of AFB1 was studied with nasalmicrosomes from rats and rabbits and with several purified isozymesof rabbit P450 in a reconstituted enzyme system. The rates ofAFB1-N7-guanine DNA adduct formation with rabbit and rat nasalmicrosomes are over 3- and 10-fold higher, respectively, thanwith liver microsomes from the same species. On the other hand,the rates of formation of AFM1 (9a-hydroxy-AFB1) and AFQ1 (3-hydroxy-AFB1)products known to be less toxic, are lower with nasal than withliver microsomes. Of particular interest, nasal microsomes producehigh levels of six unidentified polar metabolites that are notformed by microsomes from liver or several other tissues. Thesesame products are also generated by P450 NMa purified from rabbitnasal microsomes in a reconstituted system, but not by fiveother isozymes of cytochrome P450 (1A2, 2B4, 2E1, 2G1, 3A6)that are known to be present in nasal microsomes. AFB1-DNA adductsare formed by P450 NMa at a rate 3-fold higher than that bynasal microsomes. The DNA adducts are formed at much slowerrates by P450s 2G1, 2B4, and 1A2, and adducts are not formedat measurable rates by P450s 2E1 and 3A6. Moreover, AFB1—     

A new selective and potent inhibitor of human cytochrome P450 1B1 and its application to antimutagenesis.

Human cytochrome P450 (P450) 1B1 is found mainly in extrahepatic tissues and is overexpressed in a variety of human tumors. Metabolic activation of 17beta-estradiol (E(2)) to 4-hydroxy E(2) by P450 1B1 has been postulated to be a factor in mammary carcinogenesis. The inhibition of recombinant human P450 1B1 by 2,4,3',5'-tetramethoxystilbene (TMS) was investigated using either bacterial membranes from a human P450/NADPH-P450 reductase bicistronic expression system or using purified enzymes. TMS showed potent and selective inhibition of the ethoxyresorufin O-deethylation (EROD) activity of P450 1B1 with an IC(50) value of 6 nM. TMS exhibited 50-fold selectivity for P450 1B1 over P450 1A1 (IC(50) = 300 nM) and 500-fold selectivity for P450 1B1 over P450 1A2 (IC(50) = 3 microM). The inhibitory effects of TMS on EROD activity of human liver microsomes were determined. TMS inhibited EROD activity of human liver microsomes at the same concentration as with recombinant human P450 1A2. TMS also strongly inhibited 4- and 2-hydroxylation of E(2) by P450 1B1-expressing membranes or purified P450 1B1. TMS was a competitive inhibitor of P450 1B1 with a K(i) of 3 nM. The inhibition by TMS was not mechanism-based, and the loss of activity was not blocked by the trapping agents glutathione, N-acetylcysteine, or dithiothreitol. Using purified histidine-tagged P450 1B1, the binding kinetic analysis was performed with TMS, yielding a K(d) of 3 microM. The activation of 2-amino-3,5-dimethylimidazo[4,5-f]quinoline in an Escherichia coli lac-based mutagenicity tester system containing functional human P450 1B1 was strongly inhibited by TMS. Our results indicate that TMS is a very selective and potent competitive inhibitor of P450 1B1. TMS is selective for inhibiting P450 1B1 among other human P450s including 1A1, 1A2, and 3A4 and warrants consideration as a candidate for preventing mammary tumor formation by E(2) in humans.     

Metabolism of nitrosamines by purified rabbit liver cytochrome P-450 isozymes

The metabolism of nitrosamines by microsomal cytochrome P-450 (P-450) isozymes was studied in a reconstituted monooxygenase system. P-450 LM2, LM3a, LM3b and LM3c, LM4, and LM6 were purified, respectively, from the livers of phenobarbital-treated, ethanol-treated, untreated, isosafrole-treated, and imidazole-treated rabbits. Of these isozymes, LM3a had the highest N-nitrosodimethylamine demethylase (NDMAd) activity with a Km of 2.9 mM and Vmax of 9.3 nmol/min/nmol. LM2, LM4, and LM6 exhibited NDMAd activity only at high N-nitrosodimethylamine concentrations, and isozymes LM3b and LM3c had poor activity even at the highest substrate concentrations examined. LM2, however, was more active than LM3a in the metabolism of N-nitrosomethylaniline. With each isozyme (LM3a or LM4), only one Km for NDMAd was observed, whereas with rabbit liver microsomes, multiple Km of 0.07, 0.27, and 36.8 mM were obtained. P-450 isozymes also catalyzed the denitrosation of nitrosamines at rates comparable to or lower than the demethylation, and the ratio of these two reactions was different with different nitrosamines. 2-Phenylethylamine and 3-amino-1,2,4-triazole, which were believed previously to affect NDMAd by mechanisms independent of P-450, were shown to be potent inhibitors of P-450-dependent NDMAd. These results further establish the role of P-450 isozymes in the metabolism of nitrosamines and indicate that LM3a is apparently responsible for the increased N-nitrosodimethylamine metabolism associated with ethanol treatment.