Aflatoxin B1 hydroxylation by the pregnenolone-16{alpha}-carbonitrile-inducible form of rat liver microsomal cytochrome P-450

The effects of treating rats with various pregnenolone-16-carbonitrile(PCN)-type inducers of cytochrome P-450p on the liver microsomalmetabolism of aflatoxin B₁ (AFB₁) were investigated. Treatmentof male rats with PCN resulted in a 6-fold increase in the 9-hydroxylationof AFB₁ to aflatoxin Q₁ (AFQ₁). Treatment of female rats withPCN resulted in a 16-fold increase in the formation of AFQ₁.The age-dependent decline in constitutive cytochrome P-450plevels in female but not male rats resulted in a sex differencein the formation of AFQ₁ in liver microsomes from untreatedrats (male: female 3: 1). The formation of AFQ₁ was stimulatedup to 5.4-fold when liver microsomes from triacetyloleandomycin(TAO)-treated rats were treated with potassium ferricyanide,which dissociates the complex between cytochrome P-450p andTAO. Treatment of male rats with the cytochrome P-450p inducer,dexamethasone, increased ( 7-fold) the 9-hydroxylation of AFB₁to AFQ₁ by liver microsomes, and also enhanced ( 2-fold) themicrosomal activation of AFB₁ to metabolites that were mutagenicto Salmonella typhimurium TA98 and TA100. These results indicatethat the 9-hydroxylation of AFB₁ to AFQ₁ is catalyzed by ratliver microsomal cytochrome P-450p.     

Selective interactions of cytochromes P-450 with the hydroxymethyl derivatives of 7, 12-dimethylbenz[a]anthracene

Competition between a hydroxylated metabolite and the parentpolycyclic aromatic hydrocarbon (PAH) for metabolism at cytochromesP-450 may result in the generation of hydroxylated dihydrodiolepoxides. The effectiveness of the competition between 7-hydroxymethyI-12-methylbenz[a]-anthracene(7HOMMBA) or 12-hydroxymethyl-1–7-methyl-benz[a]anthracene(12HOMMBA) and 7, 12-dimethylbenz[a]-anthracene (DMBA) is highlydependent on the form(s) of cytochrome P-450 in the microsomes.The inhibitory effects of exogenously added 7HOMMBA or 12HOMMBAon DMBA metabolism were 30- to 50-fold greater in 3-methyl-cholanthrene(MC-induced rat liver microsomes (K_i = 0.4 µM) comparedto either uninduced or phenobarbital (PB-induced liver microsomes(K_i = 14 and 11 µM, respectively). Similarly, productinhibition of total DMBA metabolism by metabolites generatedin situ was significant only in MC-induced liver microsomes(K'_i = 2.5 µM). Metabolism of 7HOMMBA in these microsomeswas strongly restricted by an unusual substrate inhibition derivedfrom the inhibitory binding of a second molecule of 7HOMMBA.This same phenomenon was observed with reconstituted cytochromeP-450c but not with PB-induced or uninduced microsomes. Complexformation by binding of DMBA, 7HOMMBA, and 12HOMMBA to purifiedP-450c reconstituted in phospholipid micelles was determinedby optical spectroscopy and fluorescence quenching. Bindingaffinities of both the 7HOMMBA and 12HOMMBA (K_d = 95 and 110nM, respectively), were 2.5-fold higher compared to that ofDMBA (K_d = 265 nM). These results provide a first demonstrationthat hydroxylation of a PAH can lead to preferential metabolismthrough an increased affinity for cytochrome P-450.     

Activation of the food-derived mutagen 2-amino-1-methyl-6-phenylimidazo[4, 5-b]pyridine by rabbit and human liver microsomes and purified forms of cytochrome P-450

The specificity of rabbit cytochrome P-450 involved in the mutagenicactivation of 2-amino-1-methyl-6-phenylimid-azo[4, 5-b]pyridine(PhIP) was assessed using control and induced rabbit liver andlung microsomes, and six purified forms of cytochrome P-450.The number of revertants produced/2.5µg PhIP by controlrabbit liver was 260 ± 196/10 µg of microsomalprotein (mean ± SD; n = 3), and this increased to 1265± 248 when 2,3,7,8-tetrachloro-dibenzo-p-dioxin (TCDD)-inducedliver microsomes were used as the activation source in the Amestest. Microsomes form phenobarbital-, rifampicin-and acetone-pretreatedrabbits showed no increase in activity over controls. Controllung microsomes did not activate PhIP to a mutagen, whereasTCDD-induced lung microsomes produced 1443 ± 136 (mean± SD; n=4) Ames/Salmonella revertants/100 µg protein.In reconstitution experiments cytochrome P450 forms 4 and 6were found to be efficient activators of PhIP to a mutagen.Form 6 was 3.1-fold more active than form 4 and produced 4577revertants/10 pmol with a 20-min preincubation step in the Amestest. Cytochrome form 5 produced 17 revertants/10 pmol and forms2, 3b and 3c were not active in metabolizing PhIP to a mutagen.A highly significant statistical correlation existed betweenthe capacity of control and induced liver microsomes to activatePhIP to a mutagen and their cytochrome P-450 form 4 (r = 0.97,r² = 0.94) and form 6 (r = 0.95, r² = 0.90) content. These datastrongly support the involvement of polycyclic hydrocarbon-inducibleforms of cytochrome P450 in the activation of PhIP in the rabbit.Anti-rabbit forms 4 and 6 IgGs recognized proteins in sevenhuman liver microsomes of comparable mol. wt to rabbit cytochromeP-450 forms 4 and 6. However, no correlation existed betweenthe content of these proteins and the capacity of human livermicrosomes to activate PhIP.     

Inhibition of 2-aminofluorene mutagenesis in bacteria by inducers of cytochrome P-450d

Certain chemical inducers of rat liver microsomal cytochromeP-450d are tightly bound to the cytochrome. We investigatedthe ability of two inducers of cytochrome P-450d, Aroclor 1254and isosafrole, to inhibit the microsomal activation of 2-aminofluoreneto a mutagen as measured in Salmonella typhimurium. Butanoltreatment of microsomes from isosafrole-treated rats removedan inhibitory metabolite of isosafrole and increased 2-aminofluorenemutagenesis by {small tilde}2-fold over controls. Butanol treatmentof microsomes from Aroclor 1254-treated rats failed to eitherremove any of the Aroclor 1254 associated with microsomal cytochromeP-450 or affect 2-aminofluorene-induced mutagenesis. However,addition of Aroclor 1254 to butanol-treated microsomes fromisosafrole-treated rats almost completely inhibited 2-aminofluorenemutagenesis. Aroclor 1254 completely inhibited the cytochromeP-450d-dependent estradiol 2-hydroxylase activity of butanoi-treatedmicrosomes from isosafrole-treated rats. Thus, we suspect thatcertain congeners from Aroclor 1254, a widely used mixture forinduction of cytochrome P-450 activities, could inhibit cytochromeP-450d and partially mask its ability to metabolize some chemicalsto mutagens.     

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.     

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.     

Metabolic processing of 2-acetylaminofluorene by microsomes and six highly purified cytochrome P-450 forms from rabbit liver

Kinetic analysis of oxidative metabolism of 2-acetylaminofluorene(AAF) was studied in control and 2,3,7,8-tetrachlorodibenzo-p-dioxin(TCDD)-induced microsomes and with six highly purified cytochromeP-450 isoenzymes from rabbit liver. Kinetic parameters weredefined for 7-, 5-, 3-, 1- and N-hydroxylations of AAF. 7-Hydroxylationwas best defined by a two enzyme system, displaying a high affinityand relatively low capacity and a low affinity high capacitycomponents in both control and TCDD induced microsomes. Allthe purified cytochrome P-450 isoenzymes were capable of catalyzingthe 7-hydroxylation of AAF and, with the exception of form 4,this was the only oxidation on the AAF molecule catalyzed bythese forms. It is probable that forms 1, 4 and 6 accountedfor a substantial part (25%) of total metabolic capacity correspondingto the high affinity component of 7-hydroxylation, whereas forms3b and 3c accounted for <5% of the metabolic capacity displayedby the low affinity component in control microsomes. However,forms 4 and 6 could account for >90% of the metabolic capacityof the high affinity component of 7-hydroxylation in TCDD microsomes,whereas the form(s) responsible for the metabolic capacity ofthe low affinity component were not identified. Each of the1-, 3-, 5- and N-hydroxylations were best defined by a singleenzyme system in both control and TCDD microsomes (3- and 5-hydroxylationscould not be defined in TCDD microsomes). Close agreements werefound between the apparent Km for N-hydroxylation in control,TCDD induced microsomes and with form 4. -Naphthoflavone inhibitedAAF N-hydroxylation to a similar extent in control and TCDDmicrosomes and in form 4. These date indicate that: (i) a subpopulationof cytochrome P-450 isoenzymes, which includes all the purifiedP-450 forms tested in the present study, is solely involvedin detoxification (i.e., 7-hydroxylation) of AM, and as suchprobably behave as a functional unit in vivo; (ii) modulationof cytochrome P-450 content by inducers such as TCDD resultsin emergence of relatively few cytochrome P-450 isoenzymes thatcan account for most of the oxidative metabolism of AAF; and(iii) a single cytochrome P-450 isoenzyme (i.e., form 4) isresponsible for catalyzing N-hydroxylation of AAF, the firstand the obligatory step in the metabolic activation of thiscarcinogen.     

Immunochemical study on the contributions of two molecular species of cytochrome P-450 in mutagenesis by selected aminoazo dyes

The relative contributions of two species of cytochrome P-450,the major cytochrome P-450 components of liver microsomes ofphenobarbital-treated rats (PB-P-450) and of 3-methylcholanthrene-treatedrats (MC-P-448), in the mutagenic activation of 3'-methyl-N,N-dimethyl-4-aminoazobenzene(3'-Me-DAB) and its eight metabolites were studied in the Salmonellaassay system using specific antibodies and inhibitors. The antibodyagainst MC-P-448 considerably inhibited the mutagenicities of3'-Me-DAB, 3'-CH₂OH-DAB, their three N-demethylated compounds,3'-CHO-DAB, and 3'-Me-4'-OH-DAB in strain TA98, whereas theantibody against PB-P-450 inhibited their mutagenicities <29%.In contrast, the antibody against MC-P-448 caused no or slight(29%) inhibition of the mutagenicities of 3'-hydoxymethyl-N-methyl-4-aminoazobenzene(3'-CH₂OH-MAB) and 3'-COOH-DAB. However, the mutagenicitiesof both compounds were considerably inhibited by 7,8-benzo-flavone,like those of other seven aminoazo dyes. These results demonstratethat rat liver cytochrome P-450, especially MC-P-448, is involvedin mutagenic activation of the aminoazo dyes. Participationof a second form of cytochrome P-448 in mutagenic activationof 3'-CH₂OH-MAB and 3'-COOH-DAB is discussed.     

Rat CYP1B1: an adrenal cytochrome P450 that exhibits sex-dependent expression in livers and kidneys of TCDD-treated animals

The broad spectrum of biological responses associated with exposureto 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, dioxin) is believedto be due to the alteration in expression of TCDD-induciblegenes. The aim of this study was to investigate the effectsof TCDD on the in vivo tissue-specific expression of the recentlyidentified TCDD-inducible cytochrome P450 CYP1B1 [Sutter etal. (1994) J. Biol. Chem., 269, 13092–13099] in Sprague-Dawleyrats. We cloned the 5.0 kb rat homolog of CYP1B1 from a TCDD-treatedrat liver cDNA library and showed that the rat and human CYP1B1predicted amino acid sequences are 80% identical. RNA hybridizationanalysis snowed that CYP1B1 is constitutively expressed in theadrenal glands and also in the testes of untreated rats. Thistissue distribution suggests that CYP1B1 may be a physiologicalsteroid hydroxylase. Seventy-two hours post-administration of25 µg/kg body wt TCDD by gavage, steady-state levels ofthe 5.1 kb CYP1B1 RNA were increased > 50-fold in liver,and to a lesser extent in kidneys, lung, heart and ovaries.Average CYP1B1 RNA levels were significantly higher in the kidneysand livers of TCDD-treated females than in those from similarlytreated males. In contrast, no significant sex-difference wasobserved in the levels of CYP1A1 in these tissues in TCDD-treatedanimals. In Sprague-Dawley rats, TCDD is a more potent hepatocarcinogenin females than in males. The induction of CYP1B1 in TCDD ratliver may be a contributing factor to the carcinogenic actionof this persistent environmental pollutant.     

Species, sex and organ differences in induction of a cytochrome P-450 isozyme responsible for carcinogen activation: effects of dietary hepatocarcinogenic tryptophan pyrolysate components in mice and rats

Both sexes of BALB/cxDBA/2 F₁ mice and F344 rats were treatedfor 1 week with a diet containing 0.02% of hepatocarcinogenictryptophan pyrolysate component (Trp P-1 or Trp P-2), and changesin the carcinogen activation enzyme activity in various organswere examined comparatively using a mutation test with Salmonellatyphimurium TA98 as a tester bacterium. Hepatic enzymes fromuntreated mice and rats showed a definite catalytic activityfor mutagenic activations of Trp P-1 and Trp P-2, whereas theactivities of other organs —such as lung, kidney, smallintestine and colon—were undetectable or very low. Inboth mice and rats either the Trp P-1 or Trp P-2 feeding resultedin induction of cytochrome P-450 isozyme(s), which could mediatein the liver but not in other organs the mutagenic activationof the carcinogen itself. As to the sex difference, the inductionof the activation enzyme(s) was greater in the female animalsthan in the males. Species difference in the activity of hepaticenzymes catalyzing the Trp P-1 and Trp P-2 mutageneses was alsoobserved in animals treated with the basal diet; the activitywas higher in mice than in the sex-matched rats (Trp P-1, {smalltilde}1.5-fold; Trp P-2, {small tilde}7-fold). When diet containingTrp P-1 or Trp P-2 was fed for 1 week, the activity of the ratliver for Trp P-1 mutagenesis was of a level similar to thatof the sex-matched mice, but for Trp P-2 mutagenesis it wasless than half that in the mice. The induced hepatic enzymesin mice and rats were suggested to be 3-methylcholanthrene-induciblecytochrome P-448 isozymes as determined by mutation tests withTrp P-1, Trp P-2 and two other substrates and by immunochemicalanalyses of rat hepatic cytochrome P-450 using monoclonal antibodiesagainst rat cytochrome P-448 isozymes. These results indicatethat a form of cytochrome P-450 responsible for activation ofTrp P-1 and Trp P-2 is inducible by dietary treatment of miceor rats with these carcinogens and that the amount of the cytochromeP-450, including resident and induced forms, is related to thespecies, sex and organ differences in their carcinogenic susceptibilityto these chemicals.     

Selective expression and induction of cytochrome P450PB and P450MC during the development of hereditary hepatitis and hepatoma of LEC rats

The Long-Evans rat with a cinnamon-like coat color (LEC rat)is a mutant strain displaying hereditary hepatitis with severejaundice. The age related difference in microsomal dealkylationof pentoxyresorufin and ethoxyresorufin was examined. The enzymeactivity levels of pentoxyresorufin O-depentylase in LEC ratswere decreased to 25% of the levels in control [Long-Evans ratswith an agouti coat color (LEA rats)]. In contrast, ethoxyresorufinO-deethylase exhibited a much less marked difference betweenthe strains. In parallel with these strain differences in enzymeactivities, a decrease in phenobarbital (PB) indudble P450 isozymes,mainly P450b and P450e, was observed by Western blot analysis.The level of P450_PB in LEC rats was more markedly depressedthan in the LEA strain. On the other hand, microsomes from uninducedLEC rat liver had more 3-methylcholanthrene (MC) inducible P450_MC,mainly P450c and P450d, than microsomes from LEA rat liver andthese isozymes in the LEC were markedly induced by 3-methylcholanthrenetreatment. The great difference in cytochrome P450_PB contentof the liver microsomes between LEC and LEA rats and the maintainedconstitutive levels of hepatic cytochrome P450_MC in the LECrats suggest a possible role of these cytochrome isozymes inthe onset of spontaneous hepatitis and hepatoma.     

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.     

Metabolic deactivation of furylfuramide by cytothrome P450 in human and liver microsomes

Metabolic deactivation of furylfuramide by human and rat livermicrosomal cytochrome P450 enzymes has been investigated ina system measuring induction of umu gene expression responsein Salmonella typhimurium TA1535/pSK1002. Both human and ratliver microsomes catalyzed the metabolism of furylfuramide toinactive form(s) that are incapable of inducing umu gene expressionin the tester strain. The reaction required an NADPH-generatingsystem and molecular oxygen and was inhibited by carbon monoxide,suggesting that a cytochrome P450-linked monooxygenase systemis prerequisite for the deactivation reaction. With liver microsomesfrom variously pretreated rats, 3-methylcholanthrene was foundto be a powerful inducer for the furylfuramide-metabolizingactivity, and antibodies raised against rat P450IA1(BNF-B, c)and P450IA2(ISF-G, d) inhibited the microsomal activity. Humanliver microsomal furylfuramide-metabolizing activity was alsoinhibited significantly by anti-P450IA2 lgG but weakly by anti-P450IA2IgG. In liver microsomes prepared from seven different humansamples, the activities of deactivation of furylfuramide werefound to correlate with the amounts of immunoreactive proteinrelated to rat P450IA2 and with the monooxygenase activitiesof metabolic activation of 2-amino-3,4-dimethyl-imidazo [4,5-]quinoline(MeIQ) and of ethoxyresorufin O-deethylation. These resultssuggest that P450IA1 and P450IA2 in rats, and P45OPA (IA2, thephenacetin O-deethylase and ortholog of rat P450IA2) in humansare the major enzymes involved in the deactivation of furylfuramidein liver microsomes. The metabolic studies involving HPLC analysisof products followed by spectrophotometric examination havealso suggested that furylfuramide can be degraded very rapidlythrough the aerobic metabolism by liver microsomes.     

Effect of modifying agents on the phenotypic expression of cytochrome P-450, glutathione S-transferase molecular forms, microsomal epoxide hydrolase, glucose-6-phosphate dehydrogenase and {gamma}-glutamyltranspeptidase in rat liver preneoplastic lesions

The expression of A and P forms of glutathione S-transferase(GST-A and P), two cytochrome P-450 isoenzymes (P-450 PB_3a andP-450 MC₂), microsomal epoxide hydrolase (mEHb), glucose-6-phosphatedehydrogenase (G6PD) and -glutamyltranspeptidase (-GT) was comparedin preneo-plastic liver lesions and background parenchyma ofF344 rats post-treated with butylated hydroxyanisole (BHA),ethoxyquin (EQ) or acetaminophen (AAP). These latter three compoundshave been shown to inhibit hepatocarcinogenesis after initialtreatment with N-ethyl-N-hydroxyethylnitrosamine (EHEN) anda significant decrease in the number of enzymealtered foci andnodules positive for GST-P, GST-A, G6PD and -GT and negativefor P-450 PB_3a, P-450 MC₂ was associated with their administration.Whereas in the foci case the decrease was most prominent fornon-discrete (heterogeneous) type lesions, the results of quantitationof nodules revealed a most significant alteration in the discretehomogeneously staining population. This indicates that BHA,EQ and AAP have the potential to inhibit the growth of the phenotypicallystable lesions thought most likely to be the immediate precursorsof hepatocellular carcinomas. The two anti-oxidants were associatedwith periportal increase of all enzymes investigated, whereasAAP induced GST species and mEHb in the perivenular zone. Irrespectiveof slightly elevated enzyme levels in surrounding parenchyma,mEHb antibody binding levels within lesions showed a reciprocalshift from positive to negative in rats treated with BHA, EQand AAP.     

Induction of alkoxyresorufin O-dealkylases, epoxide hydrolase, and liver weight gain: correlation with liver tumor-promoting potential in a series of barbiturates

The effects of a series of barbiturates, of known and varyingliver tumor-promoting ability, on several short-term endpointsincluding liver weight and liver-to-body weight ratio increasesand induction of cytochromes(s) P-450 and epoxide hydrolaseactivities were examined. Male F344 rats (3 months of age) wereadministered barbiturates in the drinking water for 12 days.At the end of the treatment period they were killed, body andliver weights were taken, microsomal p-nitroanisole O-demethylationand epoxide hydration, and liver S-9 O-dealkylation of ethoxy-,pentoxy-andbenzyloxyresonifin were measured. The latter two substrateshave been shown to be preferentially metabolized by the majorphenobarbital inducible form of cytochrome(s) P-450 (P-450_band were employed since they offered a means of differentiatingmore dearly varying levels of P-450 induction. Exposure to sodiumbarbital (SB) and sodium phenobarbital (PB) resulted in significantincreases in liver weight and liver-to-body weight ratios. Inductionof cytochrome(s) P-450 and epoxide hydrolase activities by thevarious barbiturates depended on the functional groups on C₅.When ranked in terms of decreasing induction potency, the followingorder was obtained for each enzyme activity quantitated: PB,SB, sodium pentobarbital, amobarbital, hexobarbital and theC₅ substituted parent compound (barbituric acid). Thus, thebarbiturates were found to exhibit a spectrum of induction potendes,with PB and SB, the most potent liver twnor promoters, yieldingthe greatest degree of liver weight increase and induction ofcytochrome(s) P-450 and epoxide hydrolase activities.     

Characterization of covalent binding of N'-nitrosonornicotine in rat liver microsomes

The metabolism of the carcinogenic nitrosamine, N'-nitrosonornicotine(NNN), to reactive intermediates which bind covalently was assessedusing male Sprague-Dawley rat liver microsomes. The NADPH-dependentcovalent binding of [¹⁴C]NNN was linear with time up to 90 minand protein concentration up to 3.0 mg/ml. The apparent K_m andV_max of the binding were determined from the initial velocitiesand found to be 0.91 mM and 4.7 pmol/min/mg protein, respectively.Although NNN is not a hepatocarcinogen, this amount of NADPH-dependentcovalent binding is 7-fold greater than that reported for dimethylnitrosamine,a potent hepatocarcinogen. Extensive covalent binding of [¹⁴C]NNNto liver and muscle microsomal protein was also present in theabsence of an NADPH-generating system and in the presence of50% methanol, indicating a non-enzymatically mediated reaction.Addition of the nucleophiles glutathione, cysteine and N-acetylcysteinesignificantly decreased (p <0.01) the non-NADPH-dependentbinding, but did not affect NADPH-dependent binding. In vitroaddition of the cytochrome P-450 inhibitors metyrapone, piperonylbutoxide and SKF-525A significantly decreased (p <0.05) NADPH-dependentbinding of [¹⁴C]NNN by 27–40%. NADH did not replace NADPHin supporting covalent binding. Replacement of an air atmospherewith nitrogen or CO: O₂(8: 2) significantly decreased (p <0.05)NADPH-dependent binding of [¹⁴C]NNN by 40 and 27%, respectively.Aroclor 1254 pre-treatment of the rats did not enhance the NADPH-dependentbinding of [¹⁴C]NNN. These data indicate that cytochrome P-450is at least in part responsible for the metabolic activationof the carcinogen NNN but also suggest additional mechanismsof activation.     

Ovarian hormones enhance 2,3,7,8-tetrachlorodibenzo-p-dioxin-mediated increases in cell proliferation and preneoplastic foci in a two-stage model for rat hepatocarcinogenesis.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a potent hepatocarcinogen in rodents. However, liver tumor incidence is increased by TCDD in female Sprague-Dawley rats but not male rats in chronic carcinogen bioassays. Our studies have investigated this finding by evaluating histological and biochemical parameters in a two-stage model for hepatocarcinogenesis in female Sprague-Dawley rats (intact and ovariectomized), using diethylnitrosamine (DEN) as the initiating agent and TCDD as the promoting agent. Increases in gamma-glutamyl transpeptidase-positive foci were greater in intact female rats than in ovariectomized (OVX) animals. For example, in intact rats receiving both DEN and TCDD, the percentage of liver occupied by gamma-glutamyl transpeptidase-positive foci was 0.37, compared to 0.08 in OVX rats. Values for intact or OVX rats receiving either DEN or TCDD only were 0.04 or less. Similar results were obtained when using placental glutathione S-transferase to detect hepatic preneoplastic lesions. Cell proliferation data, obtained using bromodeoxyuridine in osmotic minipumps, were consistent with preneoplastic foci data in that the hepatocyte labeling index was increased in DEN/TCDD intact rats but not in DEN/TCDD OVX rats. Analysis of data from individual animals revealed a strong correlation (P less than 0.01) between cell proliferation and placental glutathione S-transferase-positive foci/cm3 in liver. These findings did not reflect effects of ovariectomy on TCDD tissue distribution, since livers of OVX rats contained more TCDD than livers of intact rats, although both groups of rats received a dose of 1.4 micrograms TCDD/kg once every 2 weeks for 30 weeks. Hepatic cytochrome P-450d (IA2) was induced approximately 6-8-fold in all TCDD-treated groups, and the magnitude of induction was not influenced by ovariectomy. This cytochrome efficiently catalyzes metabolism of 17 beta-estradiol to catechol estrogens. Our data suggest that ovarian hormones (probably estrogen) play a significant role in the hepatocarcinogenic actions of TCDD.     

Effect of a Prudhoe Bay crude oil on hepatic and renal peroxisomal {beta}-oxidation and mixed-function oxidase activities in rats

The effect of oral administration of a Prudhoe Bay crude oil(PBCO) to male rats (PBCO, 2.6 g/kg body weight, daily) for5–12 days on hepatic and renal microsomal mono-oxygenaseactivities and peroxisomal ß-oxidation has been investigated.PBCO administration leads to liver enlargement. This is associatedwith induction of microsomal cytochrome P-450 levels (1.6- to2.0-fold) and dependent mixed-function oxidase activities (7-ethoxyresorufin-O-deethylaseand 7-pentoxyresorufin-O-depentylase, representing cytochromeP-450I and cytochrome P-450IIB isoenzymes respectively, 9-to15-fold; -oxidation of lauric acid representing the cytochromeP-450IVA1 isoenzyme, 1.4- to 1.5-fold) along with peroxisomalß-oxidation (palmitoyl CoA oxidation, 2- to 5-fold).It was observed that rats exposed to PBCO showed an increasein renal microsomal cytochrome P-450 content (1.6- to 2.3-fold),cytochrome P-450I activity (5- to 8-fold) and -oxidation activity(1.3- to 1.4-fold). However, renal peroxisomal ß-oxidationwas unaltered. Serum total triglycerides were lowered by 41–46%after PBCO exposure. These results suggest that induction ofperoxisomal ß-oxidation and possibly mono-oxygenasesmay be related to the carcinogenic/tumorigenic potential ofcrude oil.     

Specificity of hepatic cytochrome P-450 isoenzymes from PCB-treated rats and participation of cytochrome b5 in the activation of aflatoxin B1

Employing six forms of cytochrome P-450s fractionated from thehepatic microsomes of PCB-treated rats, the activation of aflatoxinB₁ (AFB₁) was examined in the reconstituted cytochrome P-450system. AFB₁ was specifically activated into DNA-binding formby cytochrome P-450 I-a, which is one of P-450 type cytochromesand possesses an absorption peak at 450.0 nm in its carbon monoxidedifference spectrum. This activation was enhanced by cytochromeb₅ and the maximal enhancement (1.6-fold of the control) wasobserved with the molar ratio of 0.25 cytochrome b₅:1.0 cytochromeP-450.     

DNA binding by 1-nitropyrene and 1,6-dinitropyrene in vitro and in vivo: effects of nitroreductase induction

1-Nitropyrene, the predominant nitropolycyclic aromatic hydrocarbonfound in diesel exhaust,is a mutagen and tumorigen. 1,6-Dinitropyreneis present in diesel exhaust in much smaller quantities thanl-nitropyrene, but is much more mutagenic and carcinogenic.In an attempt to understand this difference in biological potencies,we have compared the extent of DNA binding by these two nitropyrenesin vivo. We have also determined the effect of 1-nitropyrenepretreatment upon the induction of nitroreductases and the subsequentDNA binding by both 1-nitropyrene and 1,6-dinitropyrene. CovalentDNA binding by 1-nitropyrene could not be detected in vivo;however, 1,6-dinitropyrene formed N-(deoxyguano-sin-8-yl)-1-amino-6-nitropyreneas the major DNA adduct in rat liver, kidney, urinary bladderand mammary gland, with the highest levels being found in thebladder. The capability of liver microsomes to catalyze theoxidative metabolism of 1-nitropyrene was unchanged after treatingrats with 8 mg/kg 1-nitropyrene. Cytochrome P-450, NADPH-cytochromeP-450 reductase and cytochrome b₅ levels were also unchanged,while slight increases were detected in NADH-cytochrome b₅ reductaseand epoxide hydrase activities. Liver cytosolic and microsomalnitroreductase activities toward both 1-nitro-pyrene and 1,6-dinitropyrenewere increased 2-fold, and cytosolic nitrosoreductase activitytoward 1-nitrosopyrene and 1-nitro-6-nitrosopyrene was elevatedby {small tilde}20%. DNA binding of both 1-nitropyrene and 1,6-dinitropyrenein vitro was 2-fold higher when using cytosol from 1-nitropyrene-pretreatedrats. However, pretreatment of rats with l-nitropyrene onlyslightly increased the amount of in vivo DNA binding by 1,6-dinitropyreneexcept in kidney where there was a 60% increase. These resultsindicate that although nitroreduction is involved in DNA adductformation by 1,6-dinitropyrene, additional factors (e.g. O-acetylation)limit the extent of DNA binding in vivo.