Metabolism of benzo[a]pyrene and benzo[a]pyrene-7,8-diol by human cytochrome P450 1B1

Benzo[a]pyrene (B[a]P), a ubiquitous environmental, tobacco and dietary carcinogen, has been implicated in human cancer etiology. The role of human cytochrome P450 1B1 in the metabolism of B[a]P is poorly understood. Using microsomal preparations of human P450 1A1, 1A2 and 1B1 expressed in baculovirus-infected insect cells, as well as human and rat P450 1B1 expressed in yeast, we have determined the metabolism of B[a]P, with and without the addition of exogenous epoxide hydrolase, and B[a]P-7,8-dihydrodiol (7,8-diol), each substrate at a concentration of 10 microM. HPLC analysis detected eight major metabolites of B[a]P and four metabolites of the 7,8-diol. The results of these studies indicate that cytochrome P450 1B1 carries out metabolism of B[a]P along the pathway to the postulated ultimate carcinogen, the diol epoxide 2, at rates much higher than P450 1A2 but less than P450 1A1. The rates of formation of the 7,8-diol metabolite in incubations with epoxide hydrolase are 0.17 and 0.38 nmol/min/nmol P450 for human P450 1B1 and 1A1, respectively, and undetectable for 1A2. The rates of total tetrol metabolite formation from the 7,8-diol, which are indicative of diol epoxide formation, are 0.60, 0.43 and 2.58 nmol/min/nmol P450 for 1B1, 1A2 and 1A1 respectively. In agreement with other reports of rat P450 1B1 activity, our data show this rat enzyme to be very active for B[a]P and 7,8-diol, with rates higher than human P450 1B1. In addition to the established role of P450 1A1 in B[a]P metabolism, P450 1B1 may significantly contribute to B[a]P and 7,8-diol metabolism and carcinogenesis in rodent tumor models and in humans.      

Metabolic activation of benzo[a]pyrene by a human hepatoma cell line

The liver-derived human cell line, Hep G2, has high benzo[a]pyrene-metabolizing activity and converts benzo[a]pyrene to intermediates that are mutagenic and that bind to DNA. This cell line will be useful for studying metabolic activation of polycyclic aromatic hydrocarbons and other xenobiotics by human tissue and as an activation system in short-term screening assays for identifying compounds with carcinogenic potential for humans.     

Relative contribution of various forms of cytochrome P450 to the metabolism of benzo[a]pyrene by human liver microsomes

A variety of cytochromes P450 have been implicated in the hepaticmetabolism of benzo[a]pyrene (BP), including forms that areconstitutively expressed and those that are highly inducible.In the present study the metabolism of BP to organic solvent-solublederivatives by eight forms of cytochrome P450 isolated fromrat liver and by a series of 11 human liver microsomal sampleswas investigated. The relative contribution of specific P450forms to the human hepatic metabolism was evaluated. A 4-foldvariation in formation of total organic solvent-soluble BP metaboliteswas observed, as well as differences in the regio- and stereo-selectivityof this metabolism between the three individuals studied. Thelevels of expression of cytochromes P450 from five gene sub-families,as determined by Western blot analysis, did not show any correlationwith the rate of BP metabolism to organic solvent-soluble derivativesin these livers. No reduction in metabolism was observed inthree livers in which either the debrisoqulne P450 (P450IID1)was not expressed or bufuralol 1-hydroxylase activity was low.Of six different antibodies to forms of rat liver P450 tested,only those to P450s MC_la (P450IA2), MC_lb (P450IA1) and UT (P450IIA1)consistently inhibited BP metabolism. This inhibition was generallylimited and rarely exceeded 30%. An antibody to cytochrome P450PB (P450 did, however, inhibit the formation of metabolitesat the 4,5- and 9,10-positions of BP by microsomal fractionsof livers from one individual who had been receiving the drugphenytoin. These data indicate that several forms of P450 inhuman liver are involved in the metabolism of BP and that bothconstitutively expressed as well as inducible forms are importantin its disposition in man.     

Inhibition of benzo[a]pyrene-dependent mutagenesis and cytochrome P-450 reductase activity by copper complexes

The superoxide dismutase (SOD) biomimetic copper(ll) (3, 5-diisopropylsalicylate)₂(CuDIPS) has been previously reported to inhibit the tumorigenicityof a polycyclic aromatic hydrocarbon (PAH) requiring metabolicactivation. We have used the Ames Salmonella typhimurium revertantassay to survey the effects of CuDIPS and its analogs on themetabolic activation of the PAH benzo[a]pyrene (BP) by liverhomogenates. Supplementation of homogenates from normal andAro-clor 1254-treated SENCAR mice with varied concentrationsof CuDIPS resulted in a dose-dependent noncompetitive inhibitionof BP mutagenesis. Cytochrome P-450 reductase activity in liverhomogenates and microsomal preparations was also inhibited byconcentrations of CuDIPS possessing antimutagenic activity.Neither DIPS nor ZnDIPS, analogs of CuDIPS lacking SOD activity,inhibited mutagenesis or P-450 reductase activity. CuSO₄, whichhas SOD activity, was almost as effective as CuDIPS on a molarbasis in inhibiting mutagenesis and P-450 reductase activity.The inhibitory effects of CuDIPS and CuSO₄ could not be attributedto their SOD activity since bovine liver superoxide dismutase,at a 100-fold excess of CuDIPS-SOD activity, had no effect ontheir activity. Collectively these findings suggest that thein vitro antimutagenic activity of CuDIPS is independent ofits salicylate structure and is mediated through a copper-dependentbut non SOD-associated inhibition of P-450 reductase activity.     

Binding and metabolism of benzo[a]pyrene and 7,12-dimethylbenz[a]anthracene by seven purified forms of cytochrome P-450

Seven different forms of cytochrome P-450 have been purifiedfrom rat liver microsomes. The major 3-methylcholanthrene (MC)inducible cytochrome P-450 (form c) exhibits the greatest activitytoward both benzo[a]pyrene (BP) (58 min^-1) and 7,12-dimethylbenz[a]anthracene(DMBA) (29 min^-1) and forms substantially high spin, high affinitycomplexes (K_d = 10 nM) with both hydrocarbons. Cytochrome P-450d,a minor MC-inducidble form, has far lower activity for metabolismof both polycyclic aromatic hydrocarbons (PAH), yet also formshigh affinity complexs (K_d {small tilde} 100 nM) with both PAH,retaining the full high spin state of the free cytochrome. Althoughtwo phenobarbital (PB)-induced forms (P-450's b and e) differby only 13 amino acids, they exhibit significant differencesin metabolism of PAH and in complex formation. Whereas P-450bis only active in metabolism of DMBA (9.8 min^-1 versus 1.9 min^-1for BP), P-450e has low activity for both substrates (3.3 and1.2 min^-l). Nevertheless, P-450e forms a high affinity complex(K_d {small tilde} 100 nM) with both PAH that enhances the proportionof the high spin state (from 30% to 70%). Failure to displacen-octylamine (NOA) suggests binding that is removed from theheme. P-450b remains low spin in the presence of PAH and NOAis again not displaced. In addition, the two forms can be distinguishedby their regioselectivities for both PAH. P-450' a, h, and pregnenolone-l6-carbonitrile(PCN) exhibit little activity toward BP or DMBA, but P-450 PCNdoes form a low spin complex with BP (not DMBA). Regioselectivityin metabolism of DMBA by PB-induced microsomes does not agreewith that of the major constituent forms. Only the minor, lessactive purified forms (e and a) mediate substantial 12-hydroxylationand 3,4-epoxidation of DMBA. Thus, additional factors in microsomalreactlons must contribute to these differences.     

Effects of synthetic and naturally occurring flavonoids on benzo[a]pyrene metabolism by hepatic microsomes prepared from rats treated with cytochrome P-450 inducers

Activity-directed fractionation of Trifolium pratense resulted in isolation of the isoflavone biochanin A, a potent inhibitor of metabolic activation of the carcinogen benzo[a]pyrene (B[a]P) in cells in culture. To determine the structural features required for maximal inhibition of cytochrome P-450 mediated metabolism of B[a]P, the inhibitory potencies of 23 flavonoids on metabolism of B[a]P to water-soluble derivatives were examined in liver S-9 homogenate from rats induced with Aroclor 1254. Flavones were much more efficient inhibitors than their corresponding isoflavone or flavanone analogs. Most flavonols were as effective inhibitors as their flavone analogs with the exception of kaempferide. Flavones with two hydroxyl or two methoxyl groups at positions 5 and 7 were the most active. Although all eight flavonoids tested effectively inhibited B[a]P metabolism by beta-naphthoflavone-induced microsomes, none were very effective inhibitors of B[a]P metabolism by phenobarbitol-induced microsomes, and only three were effective inhibitors of B[a]P metabolism by microsomes from non-induced rats. These results indicate that flavones or flavonols that contain free 5- and 7-hydroxyls are potent inhibitors of P-450 induced by beta-naphthoflavone (P-450IA1 and/or P-450IA2) and may potentially be useful as chemopreventive agents against hydrocarbon-induced carcinogenesis.     

Metabolic activation to a mutagen of 3-hydroxy-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene, a secondary metabolite of benzo[a]pyrene

3-Hydroxy-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene (3-OH-BP-7,8-diol)wag isolated from arylsulfatase/ß-glucuronidase-treatedbile of rats to which 3-hydroxybenzo[a]pyrene (3-OH-BP) hasbeen administered. This triol was investigated for mutagenicityin Salmonella typhimurium (reversion to histidine prototrophyof strains TA 97, TA 98, TA 100 and TA 1537) and in V79 Chinesehamster cells (acquisition of resistance to 6-thioguanine).When no exogenous metabolizing system was added the triol wasinactive, while 3-OH-BP showed weak mutagenic effects with allfour bacterial strains. In the presence of NADPH-fortified postmitochondrialsupernatant fraction (S9 mix) of liver homogenate from Aroclor1254-treated rats, the mutagenicity of 3-OH-BP was potentiated,and the triol was activated to a mutagen(s). In the presenceof S9 mix, the triol was 5—18 times more mutagenic than3-OH-BP in strains TA 97, TA 100 and TA 1537, but both compoundsshowed similar mutagenic potencies with strain TA 98. Thesestrain differences strongly suggest that the mutagenicity of3-OH-BP in the S9 mix-mediated test was not exclusively dueto metabolites of 3-OH-BP-7, 8-diol. Trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene(BP-7,8-diol), like the triol, showed mutagenic effects onlyin the presence of S9 mix. Strain TA 1537 was reverted by thetriol but not by the diol. In the other bacterial strains thediol was more mutagenic than the triol, the difference in potencybeing largest in strain TA 100 (2.5-to 10-fold, depending onthe experimental conditions). In V79 cells, the diol was a potentmutagen, while the triol showed only very weak mutagenic effects.However the triol was more cytotoxic than the diol. High cytotoxicityof the triol was observed even in the absence of S9 mix. Theresults of the present study demonstrate that metabolites of3-OH-BP-7, 8-diol) are biologically-active derivatives of benzo[a]pyrene.Comparison of the mutagenic effectiveness in different bacterialstrains also reveals that metabolites of 3-OH-BP-7, 8-diol andof BP-7, 8-diol substantially differ in the kind of geneticalterations they evoke.     

Role of hepatic cytochrome p450s in the pharmacokinetics and toxicity of cyclophosphamide: studies with the hepatic cytochrome p450 reductase null mouse

Cyclophosphamide (CPA) is an anticancer prodrug that is dependent on cytochrome P450 (CYP) metabolism for its therapeutic effectiveness. In spite of the use of CPA in the clinic for over 50 years, little is known about the relationship between its toxicokinetics and therapeutic response. We have employed a powerful new model, the Hepatic Cytochrome P450 Reductase Null (HRN) mouse, which has almost no hepatic cytochrome P450 activity, to study the toxicokinetics of CPA and to establish in vivo the role of hepatic P450 metabolism in its pharmacokinetics. In HRN mice the in vitro metabolism and intrinsic clearance of CPA was over 6-fold lower than in wild-type animals. This change in CPA metabolism was also reflected in vivo, with a profound difference in the pharmacokinetics of both CPA and its metabolites. At a CPA dose of 100 mg/kg, the Cmax, plasma area under the curve (AUC) and half-life were increased by 2.6-, 6.2-, and 3.2-fold, respectively, in the HRN mice. Similar changes were also observed at a dose of 300 mg/kg. These data confirm that hepatic metabolism is the major route of CPA elimination and disposition. The primary metabolites of CPA, 4-hydroxycyclophosphamide (4-OH-CPA) and 3-dechloroethylcyclophosphamide, were still formed, but at altered rates in the HRN mice. At 100 mg/kg the t1/2 for 4-OH-CPA was increased 1.8-fold, the Cmax reduced 1.7-fold, and the AUC remained unchanged. This latter finding shows that P450-mediated oxidative metabolism is essential for the clearance of this compound. Toxicokinetic analysis of CPA-induced myelosuppression and granulocytopenia showed that at high doses (> or =100 mg/kg) there was no difference in myelotoxicity between the wild-type and HRN mice. However, at lower doses (< or =70 mg/kg) a significant difference was observed, with little toxicity seen in HRN mice but at least a 45% reduction in the bone marrow granulocyte population in wild-type mice. Meta-analysis of the toxicity experiments showed the myelotoxicity of CPA was found to be closely correlated with the Cmax of 4-OH-CPA (r2= 0.80, P = 0.002). As the therapeutic effectiveness of CPA has been linked to the AUC for 4-OH-CPA, the finding that 4-OH-CPA Cmax may determine its level of myelotoxicity indicates that the therapeutic index could be altered by changing the method of CPA administration. Furthermore, monitoring 4-OH-CPA Cmax may identify individuals at most risk of CPA side effects.     

Cell specific activation of benzo[a]pyrene by fibroblasts and hepatocytes

The cell specific activation of benzo[a]pyrene (BP) by embryonicfibroblasts and by mature hepatocytes to intermediates thatcan interact with DNA, or cause mutations in Chinese hamsterV79 cells has been investigated. At BP concentrations of upto 15 ;µM, BP was activated to mutagenic intermediatesfor the V79 cells by embryonic fibroblasts but not by hepatocytes.However, hepatocytes from rats that had been pretreated withan inducer of the mixed function ox-idases, 3-methylcholanthrene,did metabolize higher doses of BP (> 15 µM) to mutagenicintermediates. BP was extensively metabolized by both cell types,but the hepatocytes and fibroblasts showed differences bothin the profiles of BP metabolites and the nature of the BP-DNAadducts formed. Hepatocytes metabolized BP principally to 4,5-dihydro-4,5-dihydroxybenzo[a]pyrene,phenols, and quinones, which underwent further metabolism towater-soluble metabolites. Metabolism of BP to 7,8-dihydro-7,8-dihydroxybenzo[a]-pyrene(BP-7,8-diol) occurred but proceeded rapidly to the formationof triols and tetraols. Fibroblasts metabolized BP predominantlytoward the formation of BP-7,8-diol. The proportion of primarymetabolites undergoing further metabolism to conjugates wasless extensive than in the hepatocytes. Hepatocytes bound moreBP to their DNA than the fibroblasts. In the hepatocytes themajor DNA adducts formed were hydrophilic derivatives, and no[±]7ß,8-di-hydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene(BPDE) adducts were detected even after treatment with BP-7,8-diol.In the fibroblasts, the major BP-DNA adduct was derived fromthe reaction of BPDE with deoxyguanosine. These results suggestthat the differences in the response of embryonic fibroblastsand mature hepatocytes in the activation of BP to a mutagenfor mammalian cells is determined at least in part by the overallbalance of oxidation and detoxification processes in the cellsand, hence, by the levels of critical oxidative intermediatesthat interact with DNA.     

Topical treatment of mice with benzo[a]pyrene or parenteral administration of benzo[a]pyrene diol epoxide--DNA to rats results in faecal excretion of a putative benzo[a]pyrene diol epoxide--deoxyguanosine adduct

The administration of [³H]BPDE-DNA, whether by i.p. or i.v.injection, to male Wistar rats resulted in the majority of theradioactivity being recovered in the faeces. Excretion was rapid:within 24 h post-injection, 45% of the applied dose was recoveredin the faeces. H.p.l.c. analysis of radioactive material extractedfrom the faeces by methanol showed that it contained a singlecomponent which co-chromatographed with [³H]BPDE-dGuo and whichwas not affected by treatment with alkaline phosphatase, arylsulphatase or ß-glucuronidase. To determine if thisphenomenon occurs after topical application of BP to a targettissue, such as mouse skin, animals were treated with [³H]BPand their faeces collected. After an extensive extraction procedureinvolving differential solubility in organic solvents, SephadexLH-20 chromatography and h.p.l.c, a product was isolated frommice faeces which had characteristics consistent with a [³H]BPDE-dGuoadduct. These findings are discussed in relation to detectionof BPDE adducts in human populations.     

The time-dependent increase in the binding of benzo[a]pyrene to DNA through (+)-anti-benzo[a]pyrene-7,8-diol-9,10-epoxide in primary rat hepatocyte cultures results from induction of cytochrome P450IA1 by benzo[a]pyrene treatment.

The proportion and amount of benzo[a]pyrene (B[a]P) that binds to DNA through the carcinogenic (+)-anti-benzo[a]pyrene-7,8-diol-9,10-epoxide [(+)-anti-BPDE] increases with time of exposure to B[a]P in cell cultures derived from a number of species. Pretreatment of primary rat hepatocyte cultures for 12 h with 1 microgram B[a]P/ml medium increased the subsequent metabolism of [3H]B[a]P by 47% and [3H]B[a]P-DNA binding by 53% compared with acetone-pretreated hepatocytes. The amount of (+)-anti-BPDE bound to DNA in the B[a]P-pretreated hepatocytes increased 175%. B[a]P pretreatment also increased DNA-binding 2-fold in hepatocytes treated with [3H]7,8-dihydroxy-7,8-dihydro-B[a]P but had no effect on DNA binding in cells treated with anti-B[a]P-7,8-diol-9,10-epoxide. Western blotting showed that cytochrome P450IA1, which was not detectable prior to B[a]P treatment, was selectively increased by B[a]P treatment. A monoclonal antibody that specifically inhibits cytochrome P450IA1 reduced the binding of B[a]P to DNA by greater than 90% in microsomal preparations from B[a]P-pretreated hepatocytes. These results indicate that the time-dependent increase in the formation of (+)-anti-BPDE-DNA adducts results from an increase in the amount and proportion of B[a]P metabolized to this ultimate carcinogen by P450IA1 that is induced by the B[a]P treatment. The importance of P450IA1 induction by the B[a]P for its activation to this ultimate carcinogenic metabolite suggests that long-term exposure of cells to B[a]P could result in activation of a higher proportion of the B[a]P to the carcinogenic (+)-anti-BPDE.     

Metabolism of [14C]benzo[a]pyrene in vivo in the rat

[¹⁴C]Benzo[a]pyrene ([¹⁴C]B[a]P) injected intraperitoneally into rats appeared rapidly in liver, lung and kidney, and remained detectable in these tissues for at least 7 days. A large proportion (7–13%) of the ¹⁴C became covalently bound to tissue macromolecules, probably primarily proteins. Subcellular organelles of the liver were all found to bind the carcinogen, the microsomes most rapidly and the light mitochondrial fraction taking up ¹⁴C later. Nuclear bound ¹⁴C was detected in both liver and lung.Purification of the cytosolic ¹⁴C from liver revealed specific binding to the same cytosolic proteins purified from the in vitro reaction of [¹⁴C]B[a]P.     

Binding of benzo[a]pyrene and intracellular transport of a bound electrophilic benzo[a]pyrene metabolite by lipoproteins

Human serum lipoproteins were isolated by means of size exclusionh.p.l.c. Non-covalent uptake of [³H]benzo[a]pyrene was quantitatedfor fractions collected from the effluent of a liquid chromatographicseparation of human serum, and was found to directly correlatewith the lipoprotein concentration. An electrophilic benzo[a]pyrenemetabolite, [³H]trans 7,8-dihydrodiol-9,10-epoxybenzo[a]pyrene,non-covalently associated with low density lipoproteins wastransferred to human lymphocytes in vitro and bound acid-precipitablenucleic acids of the lymphocytes as a function of time. Benzo[a]-pyrenemetabolite binding to lymphocyte DNA was demonstrated by meansof CaCl density gradient analysis. Non-mitogen-stimulated lymphocytesexposed to very low concentrations of carcinogen in the presenceof low density lipo-protein demonstrated [³H]thymidine incorporation;without the concomitant addition of low density lipoproteinthe low concentrations of carcinogen did not stimulate [³H]thymidineincorporation.     

Specificity of rat liver cytochrome P-450 isozymes in the mutagenic activation of benzo[a]pyrene, aromatic amines and aflatoxin B1

The ability of three purified forms of rat liver cytochromeP-450 to metabolically activate benzo[a]pyrene, trans-benzo-[a]pyrene-7,8-dihydrodiol,2-aminofluorene, afiatoxin B₁, dimethylnitrosamine, and a pyrolysisproduct of tryptophan(3-amino-l-methyl-5H-pyrido(4,3-b)indole)(Trp-P-2) to muta-genic products was examined using Salmonellatyphimurium strains TA98 and G46 in a reconstituted monooxygenasesystem. The isozymes examined were cytochrome P-450-PB (themajor phenobarbital inducible form), and the two major 3-MCinducible forms (cytochromes P-448₅₂ and P-448₅₅). CytochromesP-448₅₂ and P-448₅₅ preferentially metabolize 2-aminofluoreneand Trp-P-2 to mutagenic products. However, only cytochromeP-448₅₅ metabolizes benzo[a]pyrene and its 7,8-dihydrodiol derivativeto mutagenic products. Both cytochrome P-448₅₂ and P-448₅₅ metabolizeafiatoxin B, to mutagenic products at a much faster rate thancytochrome P-450-PB. Dimethylnitrosamine was not activated byany of the isozymes tested.     

Peroxidase-mediated glutathione conjugation of benzo[a]pyrene-7,8-dihydrodiol is enhanced by benzo[a]pyrene phenols in vitro.

We reported previously that glutathione (GSH) is oxidized by peroxidases to a thiyl radical that can react with a number of chemicals, including the penultimate carcinogenic metabolite benzo[a]pyrene-7,8-dihydrodiol (7,8-B[a]PD), to give GSH conjugates. Here, we report that phenolic metabolites of benzo[a]pyrene (B[a]P) enhance the peroxidase-mediated formation of glutathione conjugates of 7,8-B[a]PD. The GSH conjugation of 7,8-B[a]PD in a horseradish peroxidase/peroxide system was increased over control values as follows: 9-OH-B[a]P by 4-fold, 7-OH-B[a]P by 3-fold, 1-OH-B[a]P by 2-fold. In contrast 3-OH-B[a]P was ineffective. A phenolic derivative of another polycyclic aromatic hydrocarbon (PAH), benz[a]anthracene, also enhanced GSH conjugation of 7,8-B[a]PD. The enhancement was dependent upon the presence of the phenol, horseradish peroxidase and peroxide. The phenolic compounds, including 3-OH-B[a]P, were also efficient reducing cofactors for the peroxidase. With the exception of 3-OH-B[a]P, the phenolic metabolites of PAH enhanced peroxidase-mediated formation of thiyl radical as detected by electron spin resonance spectrometry. Since both phenols and dihydrodiols are metabolites of B[a]P catalyzed by the cytochromes P450 system, enhancement of peroxidase-dependent 7,8-B[a]PD-GSH conjugation by phenols suggests a possible interaction between peroxidases and cytochromes P450 systems. This interaction may contribute to the detoxication of the penultimate carcinogenic PAH-dihydrodiols and other chemicals.     

Synthesis of a novel fluorinated benzo[a]pyrene: 4,5-difluorobenzo[a]pyrene

The synthesis of 4,5-difluorobenzo[a]pyrene, as a fluorinated probe to investigate the involvement of the K-region in the further metabolic activation of benzo[a]pyrene metabolites, is described. Benzo[a]pyrene-4,5-dione obtained from 2,3-dichloro-5,6-dicyano-1,4-benzoquinone oxidation of cis-4,5-dihydro-4,5-dihydroxybenzo[a]pyrene was fluorinated with dimethylaminosulfur trifluoride to give 4H,5H,4,4,5,5,-tetra-fluorobenzo[a]pyrene. Defluorination using lithium aluminum hydride in tetrahydrofuran gave 4,5,-difluorobenzo[a]pyrene.     

Metabolic inactivation of N-nitrosamines by cytochrome P-450 in vitro and in vivo

Nitrite was formed on incubation of N-nitrosamines with both microsomal systems and a reconstituted system consisting of cytochrome P-450 and NADPH P-450 reductase from pig liver. Nitrite was not obtained when the nitrosamines were incubated with NADPH P-450 reductase alone or when molecular oxygen or NADPH was omitted. Various inhibitors of the microsomal monooxygenase decreased nitrite generation. Furthermore, nitrite and a substantially higher amount of nitrate could be found in the urine of rats given N-nitrosodiphenylamine. Diphenylamine was also detected. From in-vitro studies, it is concluded that denitrosation of N-nitrosamines is a cytochrome P-450-dependent process, which also occurs in vivo.     

Metabolic activation of the potent carcinogen dibenzo[a, l]pyrene by human recombinant cytochromes P450, lung and liver microsomes

The metabolic activation of dibenzo[a, l]pyrene (DB[a, l]P),recently considered the most potent carcinogen among all polycyclicaromatic hydrocarbons, to the 11, 12-dihydrodiol, a precursorof the ultimate carcinogens, the 11, 12-diol-13, 14-epoxides,was investigated using eleven human recombinant cytochrome P450s,as well as human lung and liver microsomes. Of all human P450s,1A1 was the most active in the metabolism of DB[a, l]P (310pmol/min, nmol P450) and had 5–23-fold higher catalyticactivity than other P450s examined. The order of activity inthe formation of the 11, 12-dihydrodiol was as follows: 1A1(116 pmol/min, nmol P450) > 2C9 (29) > 1A2 (22) > 2B6(18) > 3A4 (16) > others (     

Comparative 32P-postlabeling analysis of benzo[a]pyrene--DNA adducts formed in vitro upon activation of benzo[a]pyrene by human, rabbit and rodent liver microsomes

An interspecies comparison was made of the DNA-adducts formedin vitro upon incubation of rat liver DNA (RL-DNA) with benzo[a]pyrene(BP) in the presence of liver microsomes. Incubations were carriedout with RL-DNA, BP (100 µM) and liver microsomes fromhamsters, mice, rabbits, rats, 3-methylcholanthrene (3MC) pretreatedrats and from humans. To analyse the adduct profiles, the ³²P-postlabelingtechnique with the nuclease P1-enhancement procedure was used.The total amount of adduct formed varied greatly with the species;also the number of adduct spots detected was different, rangingfrom 1 to 5. In all incubations the BP-N²-deoxyguanosine adductwas formed. Relative to the total adduct level, the level ofthis adduct varied from 26% with rat, 54% with hamster, 56%with 3MC-pretreated rat, 58% with mouse and 75% with rabbit,to 100% with human liver microsomes. In human liver microsomesboth the total amount of cytochrome P-450 per mg microsomalprotein and the ethoxyresorufin O-deethylation (EROD) activitywere low compared to that in animal liver microsomes. In microsomesfrom 3MC-pretreated rats the EROD activity was strongly induced.There was no correlation between EROD activity in non-inducedmicrosomes and total adduct level. To compare BP—DNA adductformation in human white blood cells (WBC) with that in RL-DNA,WBC were incubated with BP and 3MC-pretreated rat microsomes.The adduct profile in WBC-DNA differed from that observed afterincubation of RL-DNA: the BP-N²-deoxyguanosine adduct in WBC-DNAaccounted for 97% of the total adduct level. It is concludedthat the ³²P-postlabeling method is a suitable technique toinvestigate both qualitative and quantitative differences inBP — DNA adduct formation between species. Furthermore,the incubation of microsomes from the liver (or other sources)with a genotoxic agent and isolated DNA or cells can be a usefulapproach to study the formation and stability of reactive intermediatesthat are able to bind to DNA, also with respect to differencesbetween species or tissue.