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.     

Gamma interferon induction depresses murine hepatic promutagen/procarcinogen activation

In vivo induction of gamma interferon (IFN-) by sensitizationof mice with Mycobacterium bovis strain BCG and subsequent challengewith tuberculin depressed the ability of liver homogenates fromtreated animals to metabolically activate promutagens. The AmesSalmonella typhimurium revertant assay was used for analysesof metabolic conversion of promutagens by liver homogenates.Relative to the mutant frequencies determined with control liverbomogenates, induction of IFN- depressed the abilities of homogeoatesfrom treated animals to activate N-acetylaminofluorene (AAF),aflatoxin B₁ (AFB₁), and benzo[a]pyrene (BP) by 55%, 44% and95%, respectively. Within 18–24 h of Aroclor 1254 treatment,liver P-450 content had increased 43%, and the relative mutantyields per unit protein for all three promutagens had approximatelydoubled. In vivo induction of IFN- suppressed the Aroclor 1254-dependeotincreases in mutagenesis by AAF (63%), AFB₁ (90%), and BP (reducedto a level 23% below non-Araclor 1254 treatment). In all cases,the levels of depression of promutagen activation qualitativelycorrelated with cytochrome P-450 content and the induction ofIFN-.     

Facilitated expression of adaptive responses to phenobarbital in putative pre-stages of liver cancer

Female rats received N-nitrosomorpholine to produce alteredcell foci (potential cancer pre-stages) in the liver, followedby phenobarbital (PB) for 2 weeks. As indicators of adaptiveresponses we measured DNA synthesis cumulatively by infusionof [₃H]thymidine for the entire period of PB treatment, andcytochrome P450-PB by immunocytochemistry on histo-logical liversections. Altered cell foci were identified by expression of-glutamyltransferase (-GT), and/or altered morphology. The followingresults were obtained, (i) In normal parts of the liver PB inducedDNA replication only in association with expression of P450-PB;both responses were restricted to the pericentral area of theliver lobule, (ii) In foci, PB treatment led to enhanced DNAsynthesis. Furthermore, PB caused a 3-fold increase in the numberof foci expressing cytochrome P450-PB, -GT or both. Cumulativedetermination of DNA synthesis showed that this increase didnot result from selective proliferation of single initiatedcells. It is concluded that in foci also PB can induce expressionof the adaptive increases in cytochrome P450-PB and DNA synthesis,(iii) Foci show the responses to PB more readily than normalhepatocytes, and irrespective of their position within the liverlobule. These findings suggest that expression of adaptive responsesto inducing tumor promoters is facilitated in altered foci.     

Hepatocarcinogenic heterocyclic aromatic amines that induce cytochrome P-448 isozymes, mainly cytochrome P-448H (P-450IA2), responsible for mutagenic activation of the carcinogens in rat liver

Male F344 rats were treated with hepatocarcinogenic heterocydicaromatic amines such as amino acid-and protein-pyrolysate components(Trp P-1, Trp P-2, Glu P-1, Glu P-2, AC, MeAC, IQ and MeIQx)and changes in microsomal cytochrome P-450 isozymes in the liverswere examined by means of immuno-Western blotting using anti-ratcytochrome P-450 monoclonal antibodies. The results suggestedthat all chemicals tested induce cytochrome P-448 isozymes,particularly cytochrome P-448H (P-450IA2), which efficientlymediate mutagenic activation of the carcinogens. This was substantiatedby the enzymatic analyses with the substrates showing differentcharacters to rat cytochrome P-450 isozyme-mediated mutagenesis.     

Relative merits of immunohistochemical demonstrations of placental, A, B and C forms of glutathione S-transferase and histochemical demonstration of {gamma}-glutamyl transferase as markers of altered foci during liver carcinogenesis in rats

The values of the immunohistochemical demonstrations of glutathioneS-transferases (GSTs) A, B, C and P and histo-chemical demonstrationsof -glutamyl transpeptidase (-GT) for detection of enzyme alteredfoci in F344 rat liver were compared. Rats were given a singlei.p. injection of 200 mg/kg body weight of diethylnitrosamine(DENA), from 2 weeks later they were given 0.02% N-2-fluorenylacetamide(2-FAA), phenobarbital (PB), butylated hydroxyanisole (BHA)or butyl-ated hydroxytoluene (BHT) in their diet for 6 weeksand then they were given basal diet and tap water for 4 weeks.They were subjected to partial hepatectomy at the end of week3. Results showed that immunohistochemical demonstration ofGSTs A, B and C for detection of foci were only effective whenthe administration of 2-FAA, PB, BHA or BHT in the diet wasdiscontinued, because these GSTs were induced in surroundinghepatocytes by these compounds in the diet. -GT was inducedin periportal hepatocytes strongly by BHA and BHT and slightlyby PB, and -GT positive foci in periportal areas were not distinguishablefrom -GT positive peri-portal hepatocytes. GST-P was also inducedmoderately by BHA and slightly by BHT in periportal hepatocytes,but all GST-P positive foci were clearly distinguishable. Inaddition, almost all -GT positive foci gave a positive reactionfor GST-P, but 5–10% of the GST-P positive foci were not-GT positive.     

Differential regulation of two microsomal epoxide hydrolases in hyperplastic nodules from rat liver

Two microsomal epoxide hydrolases of the rat liver were foundto be differentially regulated in hyperplastic nodules. Whilstthe activity for substrates of the well-known microsomal epoxidehydrolase with a broad substrate specificity (EHb), benzo[a]pyrene4,5-oxide and androstene oxide (16,17-epoxyandrosten-3-one),was greatly (5-fold) increased in the nodule microsomes andmoderately (2-fold) increased in the surrounding tissue, thatfor the substrate of the novel microsomal epoxide hydrolase,cholesterol 5, 6-oxide (EH_ch) remained unchanged. Since bothenzymes convert endogenous steroid epoxides but with distinctstructural features, this differential regulation may indicatea role of endogenous steroid epoxide(s) of a defined structureduring hepatocarcinogenesis. Alternatively, this differentialregulation may serve as a marker during hepatocarcinogenesis.     

Promotion of preneoplastic lesions and induction of CYP2B by unleaded gasoline vapor in female B6C3F1 mouse liver

An initiation-promotion protocol was used to test the hypothesisthat unleaded gasoline (UG) vapor acts as a liver tumor promoterin female mice under exposure conditions in which UG was hepatocarcinogenicin a cancer bioassay. Twelve day old female B6C3F1 mice wereinjected with N-nitrosodiethylamine (DEN, 5 mg/kg, i.p.) orvehicle. Starting at 5–7 weeks of age, mice were exposedby inhalation 6 h/day, 5 days/week for 13 weeks to 0 or 2039p.p.m. of PS-6 blend UG, the same gasoline blend used in thecancer bioassay. Putative preneoplastic lesions in liver, characterizedmainly as basophilic foci in H&E-stained liver sections,were found exclusively in mice treated with DEN. While similarnumbers of altered hepatic foci were found in DEN-initiatedmice treated with 0 or 2039 p.p.m. UG, UG treatment significantlyincreased both the mean volume (3.2-fold) and the volume fraction(3.6-fold) of the foci. To determine if UG induced CYP2B, asubfamily of cytochrome P450 commonly induced by liver tumorpromoters in rodents, pentoxyresorufin-O-dealkylase (PROD) activitywas assayed in hepatic microsomes derived from the above livers.UG vapor increased hepatic PROD activity 8-fold, while increasingcytochrome P450 content only 30%. To ascertain if a more recentblend of UG, API 91-1, would have similar biological effectsas PS-6, female B6C3F1 mice were gavaged for 3 days with cornoil or 1800 mg/kg/day PS-6 or API 91–1 blend UG. PS-6and API 91-1 blend UG induced similar increases in relativeliver weight (25%), PROD activity (9-fold) and hepatocyte labelingindex (8-fold) relative to controls. These data demonstratethat PS-6 blend UG vapor promotes preneoplastic lesions andinduces CYP2B in female mouse liver under exposure conditionsin which it causes liver tumors, and suggest that a more recentblend of UG may have similar effects.     

1,N6-etheno-2' -deoxyadenosine and 3,N4-etheno-2'-deoxycytidine detected by monoclonal antibodies in lung and liver DNA of rats exposed to vinyl chloride

1,N⁶-Etheno-2'-deoxyadenosine (dAdo) and 3,N⁴-Etheno-2'-deoxycytidine(dCyd) are formed in vitro by reaction of DNA with the electrophilicmetabolites of vinyl chloride (VC), chloroethylene oxide andchloroacetaldehyde. To detect and quantitate these DNA adductsin vivo, we have raised a series of specific monoclonal antibodies(Mab). Among those, Mab EM-A-1 and Mab EM-C-1, respectively,were used for detection of dAdo and dCyd by competitive radioimmunoassay(RIA), following pre-separation of the etheno adducts from DNAhydrolysates by high perfonnance liquid chromatography. At 50%inhibition of tracer-antibody binding, both Mab had a detectionlimit of 187 fmol and antibody affinity constants (K) of 2 x10⁹ l/mol. The levels of dAdo and dyd were quantitated in theDNA of lung and liver tissue of young Sprague-Dawley rats exposedto 2000 p.p.m. of VC for 10 days. The dAdo/2'-deoxyadenosineand dCyd/2'-deoxycytidine molar ratios were 1.3 x 10^–7and 3.3 x 10^–7 respectively, in lung DNA, and 5.0 x 10^–8and 1.6 x 10^–7 in liver DNA. When hydrolysates of 3 mgof DNA were analyzed by RIA at 25% inhibition of tracer-antibodybinding, dAdo and dCyd were not detected in liver DNA from untreatedrats above the limiting dAdo/2'-deoxyadenosine and dCyd/2'-deoxycytidinemolar ratios of 2.2 x 10^–8 and 3.1 x 10^–8, respectively.     

Existence of multiple forms of microsomal epoxide hydrolases with radically different substrate specificities

Evidence for the existence in rat and rabbit liver of two microsomalepoxide hydrolases with radically different substrate specificitieswas obtained, one with a broad specificity (EH_b), whilst theother catalyzed the hydrolysis of cholesterol 5,6-oxide (EH_ch),a reaction taken as diagnostic since it was not observed withpure fractions of EH_b. The two enzymes were physically separatedby immunoprecipitation using antibodies which had been raisedagainst EH_b purified to apparent homogeneity. The substratespecificity of the two enzymes is radically different and mutuallycomplementary. Cholesterol 5, 6-oxide has a trisubstituted oxiranering. All epoxides of this nature tested to date were not, orvery poor, substrates of EH_b. The two enzymes can also effectivelybe discriminated by inhibitors, in that 5, 6-imino-5-cholestane-3ß-olpotently inhibits EH_ch but not EH_b whilst 1, 1, 1-trichloropropeneoxide has the opposite specificity. The cytosolic EH did notsignificantly contribute to the catalysis of the hydrolysisof cholesterol 5, 6-oxide.     

Utilization of 1, N6-Etheno-2'-deoxyadenosine 5'-triphosphate during DNA synthesis on natural templates, catalyzed by DNA polymerase I of Escherichia coli

To test whether vinyl chloride-induced mutagenesis might involveambiguous base pairing of 1, N⁶-etheno-adenine (A) during DNAsynthesis, we examined the base pairing potential of dATP duringDNA synthesis catalyzed by Escherichia coli DNA polymerase I(Klenow fragment). An electrophoretic assay of chain elongationwas used to assess the degree to which dATP could substitutefor each of the normal dNTPs during elongation of a primer annealedto a bacteriophage template. Despite the fact that the ethenobridge completely blocks normal Watson-Crick pairing of A withT, we observed that dATP could substitute for dATP during primerelongation (although inefficiently). In addition, detectablesubstitution of dATP for dGTP and dCTP occurred, indicatingthat A exhibits ambiguous base pairing properties. The relativeease of dAMP incorporation (opposite template T, C and G) appearedto vary considerably at different positions along the template.The major, form of eA incorporation (replacement of A) was confirmedby measurements of dATP-dAMP turnover (a commonly used methodfor detecting misincorporation), and also by the demonstrationthat A was present in enzymatic hydrolysates prepared from DNAthat was synthesized with dATP replacing dATP. A model for ambiguousbase pairing of dATP is proposed, in which incorporation occursvia the protonated, syn form of dATP.     

Studies by fluorescence and n.m.r. spectroscopy of the major product formed after further metabolism of ({+/-})-7{beta}, 8{alpha}-dihydroxy-9{alpha}, 10{alpha}-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene

Racemic 7ß, 8-dihydroxy-9, 10-epoxy-7,8,9,10-tetrahydro-benzo[a]pyrene(anti-BPDE) is further metabolized by liver microsomes obtainedfrom 3-methylcholanthrene pretreated rats in the presence ofNADPH to at least four products as revealed by h.p.l.c. Dataobtained from measurements by fluorescence spectroscopy underneutral and alkaline conditions and high resolution two-dimensional¹H n.m.r. spectroscopy on the major metabolite derived fromanti-BPDE are consistent with aromatic hydroxylation at the3-position either directly or indirectly via transient epoxideintermediates.     

Typical signature of DNA damage in white blood cells: a pilot study on etheno adducts in Danish mother-newborn child pairs

The impact of DNA damage commonly thought to be involved inchronic degenerative disease causation is particularly detrimentalduring fetal development. Within a multicenter study, we analyzed77 white blood cell (WBC) samples from mother–newbornchild pairs to see if imprinting of DNA damage in mother andnewborn shows a similar pattern. Two adducts 1,N⁶-ethenodeoxyadenosine(dA) and 3,N⁴-ethenodeoxycytidine (dC) were measured by ourultrasensitive immunoaffinity ³²P-post-labeling method. Thesemiscoding etheno-DNA adducts are generated by the reaction oflipid peroxidation (LPO) end products such as 4-hydroxy-2-nonenalwith DNA bases. Mean dA and dC levels when expressed per 10⁹parent nucleotides in WBC-DNA from cord blood were 138 and 354,respectively; in maternal WBC-DNA, the respective values were317 and 916. Thus, the DNA-etheno adduct levels were reliablydetectable and about two times lower in child cord blood, thedifference being significant at P < 0.0004. Analysis of dAand dC levels in cord versus maternal blood WBC showed strongpositive correlations (R² 0.9, P < 0.00001). In conclusion,LPO-induced DNA damage arising from endogenous reactive aldehydesin WBC of both mother and newborn can be reliably assessed bydA and dC as biomarkers. The high correlation of etheno adductlevels in mother and child WBC suggests that a typical signatureof DNA damage is induced similarly in fetus and mother. Prospectivecohort studies have to reveal whether these two WBC-DNA adductscould serve as risk indicator for developing hematopoietic cancersand other disorders later in life. Abbreviations: dA, 1,N⁶-ethenodeoxyadenosine; dC, 3,N⁴-ethenodeoxycytidine; LPO, lipid peroxidation; M₁dG, 3-(2-deoxy-D-erythro-pentofuranosyl)pyrimido[1,2-]purin-10(3H)-one; WBC, white blood cell Received September 19, 2008; revised November 13, 2008; accepted November 18, 2008.     

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.     

The biochemical basis for the species difference in hepatic microsomal 4-vinylcyclohexene epoxidation between female mice and rats

Mice but not rats are susceptible to 4-vinylcyclohexene (VCH)-inducedovarian toxicity and carcinogenicity. This is due in part toa 4- to 6-fold greater rate of hepatic microsomal bioactivationof VCH to the ovotoxicant VCH-1,2-epoxide. The biochemical basisfor this difference was investigated in microsomes using enzymeinduction, enzyme inhibition with chloramphenicol or specificinhibitory antibodies, and correlation with marker steroid hydroxylaseactivities to associate VCH epoxidation with particular cytochromeP450 forms. Testosterone 6ß- and 15-hydroxylase activitiesand VCH epoxidation were decreased in microsomes from chloramphenicol-treatedmice, initially suggesting the possible involvement of P450IIIAand P450IIA forms in VCH metabolism. Although both testosterone6ß-hydroxylase and VCH epoxidase activities were increasedby dexamethasone treatment (P450IIIA inducer), anti-rat P450IIIAIgG inhibited testosterone 6ß-hydroxylase (68%) butnot VCH epoxidase activity. These latter results do not supportthe involvement of mouse P450IIIA forms in VCH epoxidation.However, results were obtained which indicated that mouse P450IIIAforms are involved in VCH epoxidation. In microsomes from untreatedfemale mice VCH epoxidase activity was inhibited 48% by antibodiesto mouse P450₁₅ (P450IIA3) at a concentration that inhibitedtestosterone 15-hydroxylase activity by 86%. No protein immunochemicallyrelated to mouse P450₁₅ was detected in female rat hepatic microsomes.VCH epoxidation by hepatic microsomes was increased in femalemice and rats by phenobarbital treatment and was inhibited byapproximately one-third by anti-rat-P450IIB1 IgG in microsomesfrom untreated animals of both species. Furthermore, microsomalVCH epoxidase and testosterone 16-hydroxylase activities werelower (34%) in female 129/J mice (deficient in constitutiveexpression of P450IIB forms) than in B6C3F1 mice. These resultssuggested partial involvement of P450IIB forms in the microsomalepoxidation of VCH. Therefore, P450 forms IIA and IIB accountfor the majority of VCH bioactivation in female mouse liver,which explains in part the susceptibility of mice to VCH-inducedovarian toxicity and carcinogenicity.     

Intercellular communication in primary cultures of putative preneoplastic and 'normal' hepatocytes

Gap junctional intercellular communication (IC) was studiedin -glutamyltranspeptidase (-GT)-positive and -negative hepatocytesisolated from carcinogen-treated rats. Putative preneoplastic-GT-positive hepatocytes were visualized in monolayer culturesby indirect immunofluorescence using anti--GT-antibodies. ICwas evaluated by studying dye coupling of the cells. -GT-positivehepatocytes showed a significantly lower dye coupling than did-GT-negative liver cells. Spread of the dye Lucifer Yellow CHto neighboring cells was decreased further by the tumor-promotingchemical phenobarbital in both cell types in vitro. Also treatmentin vivo with the barbiturate significantly reduced dye couplingof hepatocytes. The findings suggest that as a result of theirdecreased ability to communicate, preneoplastic hepatocytesmay escape from growth control and differentiation signals givenout by surrounding ‘normal’ cells.     

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.     

A combination of palytoxin with 1-oleoyl-2-acetyl-glycerol (OAG) or insulin or interleukin-1 synergistically stimulates arachidonic acid metabolism, but combinations of 12-O-tetradecanoyl-phorbol-13-acetate (TPA)-type tumor promoters with OAG do not

The combination of palytoxin and 1-oleoyl-2-acetyl-glycerol(OAG) synergistically stimulates production of 6-keto-PGF₁ andPGF₂ by rat liver cells (the C-9 cell line). In contrast, thecombination of 12-O-tetradecanoylphorbol-13-acetate (TPA)-typetumor promoters (TPA, dihydroteleocidin B, aplysiatoxin, phorbol-12,13-didecanoate)and OAG does not. Production of 6-keto-PGF₁ by palytoxin addedwith recombinant murine interleukin-1 (IL-1) or with insulinis also greater than the sum of the two effects taken independently.Palytoxin and OAG individually stimulate the release of radio-labeledcompounds from the rat liver cells pre-labeled with [³H]arachidonicacid and also act synergistically to release labeled metabolites.After separation by h.p.l.c., these materials co-chromatographwith authentic 6-keto-PGF₁ and arachidonic acid. The synergisticstimulation by palytoxin and OAG is biphasic; a rapid synergisticproduction of 6-keto-PGF₁ or release of radiolabel from [³H]arachidonicacid prelabeled cells is followed, after 2 –4 h, by aprolonged synergistic response.     

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.