Induction of hepatic glutathione S-transferase activity by butylated hydroxyanisole and conjugation of benzo[a]pyrene diol-epoxide

Dietary administration of 2(3)-tert-butyl-4-hydroxyanisole (BHA)to mice caused an increase in the hepatic soluble glutathioneS-transferase activity towards (±)-7ß, 8-dihydroxy-9,10-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE) of 5-fold whereasthat towards 1-chloro-2,4-dinitrobenzene (CDNB) was increasedby 14-fold. Whereas with either substrate the catalytic capacityof the enzyme was elevated by BHA treatment, there was littleeffect on the K_m for CDNB but an increase in the K_m for BPDEas substrates. The results thus suggest that BHA-induced GSHS-transferase activity may be of limited importance for protectionfrom certain reactive intermediates of polycyclic aromatic hydrocarbons.     

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.     

Activated neutrophils induce prolonged DNA damage in neighboring cells

We have measured the capacity of highly-purified, paraffin oil-elicitedneutrophils to induce DNA single-strand breaks in a newly establishedplasmacytoma cell line, RIMPC 2304, which was induced by a retroviruscontaining the c-myc and V-Ha-ras oncogenes. This cell lineeffectively repairs DNA damage induced by -irradiation. DNAdamage induced by neutrophils was correlated with the oxidativeburst of the neutrophils. The levels of superoxide anion, H₂O₂and HOCl produced after stimulation of the neutrophils (6 x10⁵/cm³) with the tumor promoter phorbol myristate acetate (100nM) were 33.8 µM, 12.8µM and 1.7 µM respectivelyin 15 mm, and 98 µM, 20 µM and 8.7 µM respectivelyin 90 mm.The results of alkaline elution experiments revealedthat when the same concentration of neutrophils was co-incubatedfor15 min in serum-free medium with an equal number of radioactivelylabeled RIMPC 2304 cells, the latter incurred a level of damagethat approximated that caused by 300 rad equivalents of -irradiationor by a 1-min treatment with 20 µM H₂O₂ at 37C. Damagefrom neutrophils was coincident with the oxidative burst; itwas induced rapidly (within 5 min) but remained high for morethan 90 min. The level of damage achieved was dependent uponthe ratio of neutrophils: target cells and was clearly detectableat ratios as low as 0.25:1. Induction of single-strand breakswas completely inhibited by catalase and partially inhibitedby superoxide dismutase, mannitol, and reduced glutathione butnot by Na azide. Addition of the non-steroidal anti-inflammatorydrug indomethacin either enhanced (at 50 µM) or had noeffect (at 2 µM) on the damage detected. Finally, repairof strand breaks induced by neutrophils was significantly slower(half time 10 min) than that observed for repair of similarlevels of damage induced by H₂O₂ or -irradlatlon (half-times3 min, each). The results indicate that neutrophils cause prolongedDNA damage in neighboring cells. Moreover, they indicate thatalthough H₂O₂ produced in the oxidative burst is an essentialmediator of the damage observed, additional reactive oxygenintermediates including the superoxide anion are also implicated.The data are discussed in relation to the possible role of neutrophilsin chronic inflammation and in pristane-induced plasmacytomaformation in mice.     

Development of monoclonal antibodies specific for 1,N2-ethenodeoxyguanosine and N2,3-ethenodeoxyguanosine and their use for quantitation of adducts in G12 cells exposed to chloroacetaldehyde

Monoclonal antibodies specific for N²3-ethenodeoxyguanosine(N²,3-dGuo) and 1,N²-ethenodeoxyguanosine (1,N²-dGuo) were developed.In a competitive ELISA, 50% inhibition of binding of the N²,3-dGuospecific antibody (ETH1) was achieved with 18 fmol of N²,3-dGuo.Fifty per cent inhibition of the 1,N²-dGuo-specific antibody(ETH2) required 11 pmol 1,N²-dGuo. Immunoassays for N²,3-dGuoand 1,N²-dGuo in single-stranded DNA were developed using theseantibodies. The immunoassays could detect as little as 48 fmolof N²,3-dGuo or 340 fmol 1,N²-dGuo in 25 µg of singlestranded DNA. These assays and previously developed immunoassaysfor 1,N⁶-ethenodeoxyadenosine (1,N⁶-dAdo) and 3,N⁴-ethenodeoxycytidine(3,N⁴-dCyd) were used to measure etheno adduct levels in DNAof cells exposed to chloroacetaldehyde. The cells used wereV79 cells with an inactivated hprt gene and a single copy ofthe bacterial gpt gene (G12 cells). The most abundant ethenoadduct was 1,N⁶-dAdo, followed by 3,N⁴-dCyd and N²,3-dGuo. 1,N²-dGuowas not detected in chloroacetaldehyde-treated G12 cells. Chloroacetaldehydewas also shown to be mutagenic in these same cells.     

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.     

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.     

Specific binding of the tumor promoter TPA in various mouse organs as measured by a 'cold acetone-filter assay'

Rapid, specific, saturable and partially reversible bindingof the tumor promoter 12-O-tetradecanoylphorbol-13-acetate ([³H]TPA)to a particulate fraction of mouse brain can be demonstratedby means of a ‘cold acetone-filter assay’; by washingwith cold acetone at -20°C, nonspecific binding of the highlylipophilk [³H]TPA to membranes can be reduced to 10% of thetotal binding. A comparative study of homogenates of severalorgans with this test revealed specific [³H]TPA binding/ µgDNA in the order brain > lung spleen Over kidney thymus ovaries. Specific binding sites were also detected hi 600 xg supernatant fractions of homogenates from epidermis, forestomach,glandular stomach and small intestine. Competition experimentsshowed displacement of [³H]TPA by TPA > phorbol-12,13-didecanoate> 12-deoxyphorbol-13-tetradecano-ate > phorbol-12,13-dibutyrate(PDBu) > phorbol-12,13-diacetate > 4-O-methyl-TPA >4-phorbol-12,13-didecanoate in the brain particulate fraction.Specific [³H]TPA binding is sensitive to heat, periodate anddithiothreitol, but is relatively insensitive to urea or to1–5% solutions of several common detergents. It is estimatedthat the present binding test is 500 times more sensitive thanthe widely-accepted [³H]PDBu assay; perhaps more important,the present method employs TPA, which is extremely effectiveboth as a tumor promoter in vivo and as a pleiotropic effectorof cells in vivo and in vitro.     

Improvement of short-term tests for mutagenicity: on the optimal pH for the liver microsomal assay

The aim of this study was to optimize the pH in the liver microsomalassay (LMA) in processing short-term mutagenicity tests. pHoptimization would increase the sensitivity (i.e. decrease thepresence of false negatives) and increase the specificity (decreasefalse positives). Such optimization is a function of the relativeactivities and stabilities of the liver microsomal cytochromeP-450-and FAD-containing monooxygenase-dependent biotransformationenzymes present in the incubation mixtures used. The enzymeactivities ethoxyresorufin O-deethylase, dinemorphan N-demethylase,aminopyrine N-demethylase, p-nitroanisole O-demethylase andthiobenzamide S-oxidase (as phase-I markers), were examinedin terms of their exact incubation conditions for the LMA duringa period of pre-incubation (1 h) over the pH range 6–9.As a comparison, the behaviours of glutathione S-transferaseand epoxide hydrase activities (as phase-II markers) were alsostudied, Lipid peroxidation was also determined. Experimentswere carried out on S9 fractions derived from Na-phenobarbitaland ß-naphthoflavone induced mouse liver. The maximalvalue of the mean specific activity (_sp was found at pH 7.8for the phase-I drug metabolizing enzymes considered (30–45%increase). On the contrary, a lower increase of sp for epoxidehydrase and glutathione S-transferase (14%), was observed betweenpH 7.4 and 7.8. Lipid peroxidation was not changed appreciablyby varying pH. In vitro DNA binding of the well-known pre-mutagenicagent [¹⁴C]dimethylnitrosamine ([¹⁴C]DMNA), mediated by mousehepatic microsomal enzymes, showed a significant in crease ofspecific activity at pH 7.8 (2.8-fold) compared to the usualpH (7.4) employed. Additionalsupport for the above results hascome from mutagenesis experiments using DMNA on the diploldD7 strain of Saccharomyces cerevisiae as a biological test system.In fact, a significant enhancement of mitotic gene conversion(1.7-fold), mitotic cross-over (2.6-fold) and reverse pointmutation (2.3-fold) frequencies were observed at pH 7.8 comparedto pH 7.4. These data indicate that pH 7.8 provides a more favourablecondition for in vitro mutagenesis tests resulting in greaterrates of biotransformation (as measured by an increased _sp.phase-_sp phase-II ratio), DNA binding and genotoxic response.     

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.     

Fluoro-substituted N-nitrosamines. 7. Non-genotoxic N-nitrosobis(2,2,2-trifluoroethyl)amine and N-nitroso-bis(2,2,3,3,4,4,4-heptafluorobutyl)amine: binding to cytochrome P-450, acidity of {alpha}-protons and pharmacokinetic investigations

The biologically inactive fluorinated nitrosamines N-nitrosobis(2,2,2-trifluoroethyl)amine(NDEA-F₆) and N-nitroso-bis(2,2,3,3,4,4,4-heptafluorobutyl)amine(NDBA-F₁₄) were investigated for binding affinity to cytochromeP-450 and for ease of alkali-induced proton abstraction at the-C atom, in comparison with biologically active analogues (N-nitroso-diethylamine,NDEA; N-nitroso-2,2,2-trifluoroethylethylamine, NDEA-F₃; N-nitrosodibutylamine,NDBA; N-nitroso-4,4,4-trifluorobutylbutylamine, NDBA-F₃; N-nitroso-bis(4,4,4-trifluorobutyl)amine,NDBA-F₆). Binding to cytochrome P-450 was studied by spectroscopicmeasurements (optical difference spectra with microsomal fractions);base-catalyzed deuterium exchange of -hydrogen atoms was followedby ¹H n.m.r. measurements. Additionally the excretion of NDEA-F₆and NDBA-F₁₄ in expired air, urine and faeces was studied afteroral application to the rat. Compared with the biologicallyactive nitrosamine analogues, NDEA-F₆ and NDBA-F₁₄ showed higherbinding affinity to cytochrome P-450. N.m.r. spectroscopy showedthat NDEA-F₆, NDBA-F₁₄ and NDEA-F₃ (at the fluorinated alkylchain) were rapidly deprotonated at the -C-positlon in sodiumperdeutero methylate, in contrast to the other analogues tested.In vivo, NDEA-F₆ and NDBA-F₁₄ were excreted unchanged, mainlyvia exhalation. The biological inactivity of NDEA-F₆ and NDBA-F₁₄,together with the observed blocking of their microsomal activationcan be reconciled with the experimental findings which indicatethat homolytic -C-H bond fission is more likely to be involvedin -C-hydroxylation of dialkylnitrosamines, than -proton abstraction.     

Supplemental administration of 1{alpha}-hydroxyvitamin D3 inhibits promotion by intrarectal instillation of lithocholic acid in N-methyl-N-nitrosourea-induced colonic tumorigenesis in rats

The effect of 1-hydroxyvitamin D₃ [1(OH)D₃) on promotion byintrarectal instillation of lithocholic acid (LC) in N-methyl-N-nitrosourea(MNU)-induced colonic tumorigenesis was studied in a rodentmodel. Ninety-two female F344 rats received intrarectal injectionof 2.5 mg of MNU twice in one week followed by 1 mg of LC orits vehicle alone three times weekly for 48 weeks. Those whichreceived LC were given a concomitant intragastric administrationof 0.04 µg of 1(OH)D₃ or its vehicle alone three timesweekly. In the group receiving MNU alone (n=30) five rats borecolomc tumors; in the MNU + LC group (n=32) 15 and in the MNU+ LC + 1(OH)D₃ group (n=30) six rats bore colonic tumors (MNU+ LC versus MNU + LC + 1(OH)D₃ group, P<0.05). These resultsindicated that promotion of MNU-induced colonic tumorigenesisby LC was suppressed by supplemental administration of 1(OH)D₃.     

Effects of manganese compounds on carcinogenicity of nickel sub-sulfide in rats

The effects of manganese compounds upon the carcinogenicityof Ni₃S₂ were tested in male Fischer rats. In Experiment I,rats were given i.m. injections of Ni₃S₂ (2.5 mg) and Mn dust(2.0 mg), singly or in combination. By 100 weeks, sarcomas occurredat the injection site in 0 of 24 rats in the vehicle controlgroup, in 0 of 24 rats that received Mn dust alone, and in 23of 24 rats that received Ni₃S₂ alone. Combined administrationof Ni₃S₂ plus Mn dust as a single i.m. injection resulted insarcomas in 14 of 23 rats (p <0.05 versus Ni₃S₂ alone). Inrats that received injections of Ni₃S₂ in one thigh and Mn dustin the opposite thigh, the sarcoma incidence at the site ofNi₃S₂ injection was 24 of 24 rats. In Experiment II, rats weregiven i.m. injections of Ni₃S₂ (1.2 mg) and Mn compounds (MnS,Mn₂O₃, MnO₂ or MN₂(CO)₁₀, in dosages equivalent to 1.0 mg ofMn), singly or in combination. No sarcomas occurred at the injectionsite in rats that received the vehicle or any of the manganesecompounds alone. Sarcomas occurred in 13 of 27 rats that receivedNi₃S₂ alone; this sarcoma incidence was not reduced by admixtureof any of the Mn compounds. The median tumor latent period andthe me- Wan survival period were significantly longer (p <0.05)in rats that received MnS plus Ni₃S₂, compared with rats thatreceived Ni₃S₂ alone, suggesting that MnS may have weak anticarcinogeniceffect. These experiments demonstrate that inhibition of Ni₃S₂-carcinogenesisby Mn dust is a local rather than a systemic effect, and that,with the possible exception of MnS, the other manganese compoundsthat were tested are ineffective as inhibitors of Ni₃S₂-carcinogenesis.     

Recombinant rat and hamster N-acetyltransferases-1 and -2: relative rates of N-acetylation of arylamines and N,O-acyltransfer with arylhydroxamic acids

Genes for the 290 amino acid, 33–34 kDa cytosolic acetyltransferases(NAT1^* and NAT2^*) from rat and hamster were cloned and expressedin Escherichia coli. Active clones were selected by a simplevisual test for their ability to decolorize 4-aminoazobenzenein bacterial medium by acetylation. These recombinant acetyltransferaseswere analyzed for: (i) N-acetyltransferase, which was assayedby the rate of acetyl coenzyme A-dependent N-acetylation of2-aminofluorene (2-AF) or 4-aminoazobenzene (AAB); (ii) arylhydroxamicacid acyltransferase, assayed by N, O-acyltransfer with N-hydroxy-N-acetyl-2-aminofluorene.Both NAT2s showed first order increases in N-acetylation rateswith increasing 2-AF or AAB concentrations between 5 and 100µM, with apparent K_m values of 22–32 and 62–138µM respectively. Although under the same conditions theN-acetylation rates for the two NAT1s declined by >50%, below5 µM 2-AF or AAB, the NAT rate data fit Michaelis-MentenKinetics, and the apparent K_m values were 0.2–0.9 µM.For N, O-acetyltransferase, the apparent K_m values of the NAT1swere 6 µM, while the K_m values of the NAT2s were 20- to70-fold higher. SDS-PAGE/Western blot analysis of the recombinantacetyltransferases gave apparent relative molecular weights(MW_r) of 31 kDa for both NAT1s and rat NAT2 and 29 kDa for hamsterNAT2. Comparable MW_r values were observed for native hamsterliver NAT1 and NAT2 and for rat NAT1 under the same conditions.Although we did not detect NAT2-like activity in rat liver cytosolpreviously, the present data show that the rat NAT2^* gene doescode for a functional acetyltransferase, with properties similarto those of hamster liver NAT2. The data also indicate thatat low substrate concentrations, NAT1 would apparently playthe predominant role in vivo in N-acetylation and N, O-acyltransferof aromatic amine derivatives, including their metabolic activationto DNA-reactive agents.     

Inhibitory effect of {alpha}-tocopherol on the formation of nitrosomorpholine in mice treated with morpholine and exposed to nitrogen dioxide

The possibility that -tocopherol (vitamin E) inhibits the formationof nitrosomorpholine (NMOR) in vivo was investigated in miceorally pretreated with -tocopherol (2.5–100 mg/kg bodywt) once daily for 6 days. Twenty-four hours later, the animalswere injected i.p. with 2 mg of morpholine (MOR) per animalfollowed by exposure txo 47 p.p.m. of NO₂ for 2 h. Under theseconditions, low oral doses of -tocopherol (2.5–5 mg/kgbody wt) significantly decreased NMOR formation in vivo. Astotal body -tocopherol levels increased, in vivo NMOR formationdecreased, and a maximal 50–70% inhibition of NMOR formationoccurred at -tocopherol levels of 5 µg/g body wt. Additionalresults showed that NMOR was rapidly eliminated in mice, sothat studies which measure the levels of NMOR found in animalstreated with MOR and then exposed to NO₂ may underestimate theamount of NMOR that is actually formed. Finally, oral pretreatmentof up to 100 mg of -tocopherol/kg body wt had no effect on NMORelimination.     

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.     

Phorbol ester effects on splenic lymphocyte composition and cytotoxic T cell activities of SSIN mice: a strain deficient in CD8+ T cells

SSIN mice are considerably more sensitive to the effects of12-O-tetradecanoylphorbol-13-acetate (TPA) in two-stage skincarcinogenesis protocols than are most other strains and stocksof mice. Experiments were performed to determine whether therewas an immunological basis for this sensitivity. SSIN mice werehaplotyped and found to be H-2^q. T cells represented 31% ofthe splenic cellularity of non-treated SSIN mice, but 44% inBALB/c, C57BL/6, B₆C₃F₁ and SENCAR mice. Splenic CD4⁺/CD8⁺ Tcell ratios were 4.2, 2.9, 2.4, 1.8 and 1.7 in SSIN, SENCAR,BALB/c, B₆C₃F₁ and C57BL/6 mice, respectively. The unusuallyhigh ratio in SSIN spleens was the consequence of reductionsin CD8⁺ T cells. The ratio of CD4⁺/CD8⁺ T cells in SSIN thymocyteswas similiar to that measured in the spleen. The splenic cytotoxicT lymphocyte (CTL) activities of the various murine strainsinversely correlated with their splenic CD4⁺/CD8⁺ ratios andtheir sensitivities in two-stage skin carcinogenesis protocols.Repeated in vivo topical treatment of SSIN mice with TPA causedsignificant decreases in splenic T cell contents, but affectedneither the splenic CD4⁺/CD8⁺ T cell ratio nor the developmentof a CTL response upon allogeneic tumor challenge. SSIN micealso had very low splenic natural killer cell activities. Furthermore,relative to the other strains of mice, SSIN mice were poor respondersupon alloantigen challenge in mixed lymphocyte response assays.These findings demonstrate that SSIN mice differ markedly fromother murine stocks and strains in their splenic lymphocytecomposition and in their abilities to mount some MHC-restrictedand non-restricted immunosurveillance processes.     

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.     

Glutathione conjugation and DNA-binding of ({+/-})-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene and ({+/-})-7{beta},8{alpha}-dihydroxy-9{alpha},10{alpha}-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene in isolated rat hepatocytes

Isolated rat liver hepatocytes, previously depleted of glutathione(GSH) by treatment with diethylmaleate, were allowed to incorporate[³H]glycine into their GSH. Incubation of ³H-labelled cellswith ¹⁴C-labelled (±)-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene((±)-BP-7,8-dihydrodiol) or (±)7ß,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]-pyrene(()-BPDE) revealed the formation of double labelled products.This together with evidence from amino acid analysis indicatesformation of GSH-conjugates of the highly carcinogenic BP-derivatives.Incubation of hepatocytes isolated from 3-methylcholanthrene(MC) treated rats with ³H-labelled (±)-BP-7,8-dihydrodiolor (±)-BPDE resulted in binding of radioactivity to DNA.Reduction of the intracellular level of GSH to 40% of the normallevel resulted in an approximate 2-fold increase in the DNA-bindingof either substrate. In addition there was a concurrent decreasein the amount of GSH-conjugates formed. These data clearly demonstratethat GSH participates in conjugation reactions with carcinogenic(±)-BP-7,8-dihydrodiol and (±)-BPDE and that theintracelluilar level of GSH is important in preventing reactiveintermediates from reacting with the DNA in intact cells.     

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.