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.     

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.     

Tissue and species distribution and developmental variation of specific receptors for biologically active phorbol and ingenol esters

A new and rapid binding assay for [³H]phorbol-12,13-dibutyrate(PDBu) binding to its receptors in cell or tissue homogenatesor subcellular fractions is described. PDBu binding sites arewidely distributed in mouse tissues. Brain and spleen containexceptionally high concentrations of these receptors. PDBu receptorsare present in all regions of the brain. The choroid plexusand pituitary bind markedly lower amounts of PDBu than otherbrain regions. Brains or spleens from different murine strainsbind almost the same amounts of PDBu while those organs fromembryonic mice bind very little PDBu. Binding increases withage and reaches a maximum at 4 weeks of age. Young and maturebrains from various species contain these specific binding sites.These data suggest a structural or enzymatic role for PDBu receptors.     

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.     

Comparative effects of a complete tumor promoter, TPA, and a second-stage tumor promoter, RPA, on intercellular communication, cell differentiation and cell transformation

The biological activities in vitro of the incomplete (second-stage)tumor promoter, 12-O-retinoyl phorbol-13-acetate (RPA), andthe complete tumor promoter, 12-O-tetradecanoyl phorbol-13-acetate(TPA) were compared. The doses of TPA and RPA necessary to inhibitthe specific binding of [³H]-phorbol-12,13-dibutyrate ([³H]PDBu)to BALB/c 3T3 cells (50% inhibition doses; ID₅₀; 8–13ng/ml) were very similar; however, RPA was less potent thanTPA in inhibiting [³H]-PDBu binding to Friend erythroleukemiacells (FELC). Intercellular communication between BALB/c 3T3cells, measured by transfer of microinjected fluorescent dye(Lucifer Yellow), was inhibited by RPA as well as by TPA; TPAwas about five times more potent than RPA. RPA also inhibitedFELC differentiation induced by hexamethylene bisacetamide (HMBA)but not the differentiation of a TPA-resistant clone. The dose-responsesof these two compounds in inhibiting differentiation of bothTPA-sensitive and resistant FELC were very similar. When TPAand RPA were compared in their promoting activity of in vitrocell transformation of BALB/c 3T3 cells initiated with 3-methykholanthrene(MCA, 0.1 µg/ml), both TPA and RPA significantly increasedthe yield of morphologically transformed foci, and RPA was 10times more potent than TPA. These results suggest that RPA andTPA share many common in vitro biological effects and that thesein vitro studies do not allow us to delineate clearly the effectof a second-stage tumor promoter from that of complete tumorpromoters such as TPA.     

Activation of protein kinase C by tumor promoting phorbol esters, teleocidin and aplysiatoxin in the absence of added calcium

We have found that maximum stimulation (> 10-fold) of kinaseactivity of a bovine brain preparation of calcium- and phospholipid-dependentprotein kinase (PKC) by 12-O-tetradecanoylphorbol-13-acetate(TPA) occurs in the presence of phosphollpid, but in the absenceof added Ca²⁺. In effect, nM concentrations of TPA substitutefor mM concentrations of added Ca²⁺, and the two agents arenot synergistic. Biologically active analogs of TPA such asphorbol-12,13-dibutyrate (PDBu), 12-O-hexadecanoyl-16-hydroxyphorbol-13-acetate(HHPA) or mezerein were also effective activators of PKC, aswere the chemically unrelated tumor promoters teleocidin andaplysiatoxin, when tested at nM concentrations in the absenceof added Ca²⁺. On the other hand, the biologically inactivecompounds phorbol, 4--phorbol-12,13-didecanoate (4--PDD), HHPA-13,20-diacetateand 1,2-dihydro-20-deoxy-HHPA did not affrect PKC activity inthe absence or presence of Ca²⁺. Our results are consistentwith a stereochemical model in which the hydrophilic domainsof certain diterpenes, teleoddin and aplysiatoxin interact specificallywith PKC apoenzyme, while their hydrophobic domains interactwith phospholipid, thus forming an enzymatically active ternarycomplex.     

Tumor promoter 12-O-tetradecanoylphorbol-13-acetate receptors in normal human transitional epithelial cells

As a prelude to study the promotion with TPA of in vitro transformationof human urothelial cells (HUC) in culture, we characterizedtumor promoter TPA receptors in primary cultures of HUC. [³H]TPAbound specifically to intact living HUC; maximum specific bindingwas attained in 30 min at 37°C. [³H]TPA bound to HUC ina saturable and competitive manner. Scatchard analysis of specificbinding to intact cells displayed a single slope correspondingto an equilibrium dissociation constant (Kd) of 0.56 nM; atsaturation TPA-binding capacity was 2.37 pmol/10⁶ HUC (1.43x 10⁶ sites per cell). [³H]TPA bound specifically and with highaffinity to the particulate fractions of HUC; binding was bothsaturable and reversible. Saturation of the specific bindingof [3H]TPA occurred at 1 nM at 4°C. Scatchard analysis ofspecific binding to the particulate fraction displayed a singleslope corresponding to a Kd of 1.08 nM; at saturation TPA-bindingcapacity was 2.05 pmol/mg protein (750 000 molecules per HUC).[³H]TPA binding was inhibited by the biologically active phorbolester, phorbol didecanoate, whereas inactive phorbol did notcompete for TPA binding. Binding was not affected by sodiumsaccharin, epidermal growth factor, retinoic acid or dexamethasone.[³H]TPA bound specifically to the HUC cytosolic fraction butonly in the presence of calcium and phosphatidylserine. Calcium-activatedand phospholipid-sensitive protein kinase activity was detectedin HUC fractions. These results indicate the presence of high-affinityspecific receptors for TPA in HUC.     

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.     

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.     

Methylation of nuclear proteins of Novikoff hepatoma

When the nuclei isolated from Novikoff hepatomas were incubatedin vitro with S-adenosyl-L-[³H-methyl]methionine and the nuclearproteins were subsequently fractionated, histones were foundto have incorporated the radioactivity of a four-to-five timeshigher rate than those of normal rat or tumor-bearing host ratliver nuclei. This observed increase of histone methylationrate in Novikoff hepatoma nuclei was mainly due to the methylationof two histone subfractions H3 and H4. On amino acid analysis,it was found that tumor histones had much higher proportionof -N-trimethyllysine in comparison with normal or host liverhistones, thus shifting the relative ratio of the amounts of-N-mono, -N-di- and -N-trimethyllysine. This was also reflectedin the observation of increased -N-trimethyllysine levels inthe serum of the Novikoff hepatoma-bearing animal.     

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.     

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.     

Phorbol esters stimulate the rapid release of choline from prelabelled cells

Evidence implicating the cell surface membrane as the primarysite of action of phorbol ester tumor promoters has led us tocharacterize early changes in phospholipid metabolism whichoccur in response to these compounds. When C3H10T mouse embryofibroblasts were incubated with [³H]choline for 24 h, {smalltilde}78% of the cell associated material was found in phosphatidylcholine and the remainder in sphingomyelin and the acid solublepool. Within 5 min of exposure of these prelabelled cells tothe potent tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate(PPD) the release of water soluble [³H] metabolites from cellsinto the medium was enhanced 2-fold and by 60–120 minthe release was 2–5 times that found in control cultures.The released material was identified by chromatography as cholineand phosphoryl choline. Evidence was obtained that this materialwas not derived exclusively from the acid soluble pool or fromthe degradation of labelled sphingomyelin. Choline metaboliterelease was concentration dependent between 10^–8 and 10^–7M TPA and was not associated with cell toxicity. Phorbol-12,13-didecanoate(PDD) was also active but 4PDD, which is not a tumor promoter,was inactive. Neither cyclohexlmide (4–40 µg/ml)nor cordycepln (4–40 µg/ml) blocked the TPA inducedrelease. The release was, however, temperature sensitive anddid not occur at 4°C. TPA did not induce the release of[³H]inositol from prelabelled phosphatidyl inositol. Although,the calcium ionophore A23187 [GenBank] induced the release of arachidonicacid from prelabelled cells it did not induce choline release.In addition, 5,8,11,14-eicosatetraynoic acid, which inhibitsboth lipoxygenase and cyclooxygenase activity, did not inhibitTPA stimulation of choline release. We propose that the bindingof TPA to cell surface receptors leads to a rapid activationof membrane associated phospholipase(s) that specifically degradephosphatidyl choline. This effect may be analogous to the abilityof other agonists to activate degradation of phosphatidyl inositol.     

32P-postlabeling analysis of non-radioactive aromatic carcinogen -- DNA adducts

A newly developed enzymatic ³²P-postlabeling method was appliedto the analysis of DNA's containing non-radioactive arylamine,arylamide, and polycyclic aromatic hydrocarbon adducts. DNAreacted in vitro with N-hydroxy-2-amino-fluorene, N-acetoxy-2-acetylaminofluorene,and 7ß,8-di-hydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene,respectively, as well as DNA preparations from the livers ofrats treated with N-hydroxy-2-acetylaminofluorene and benzo[a]pyrene,respectively, were enzymatically digested to deoxyribonudeoside3'-monophosphates, which were then converted to [5'-³²P]deoxyribonucleoside3',5' -bisphosphates by T4 polynucleotide kinase-catalyzed [³²P]phosphatetransfer from [-³²P]ATP. The ³²P-labeled nucleotides were resolvedby anion-exchange t.l.c. on polyethylenelmine-cellulose anddetected by autoradiography. Aromatic adduct nucleotides werefound to be retained at the origin in aqueous electrolyte solutions,but to migrate as distinct spots in solvents containing 7–8.5M urea. Advantage was taken of this observation to remove ³²P-labelednormal DNA nudeotides from adduct nudeotides. This purificationenabled the detection of a single adduct in 10⁷–10⁸ normalnucleotides. The method appears applicable to the ultrasensitivedetection of a large number of carcinogen - DNA adducts of diversestructure without requiring radioactive carcinogens or specificantibodies.     

UVR induction of TGF{alpha}: a possible autocrine mechanism for the epidermal melanocytic response and for promotion of epidermal carcinogenesis

The occurrence of the epidermal growth factor homologue, transforminggrowth factor (TGF), in embryonic and neoplastic tissues suggeststhat it may he an oncofetal version of epidermal growth factor.A strong case is developing for TGF to have an autocrine modeof action in sustaining the autonomous growth of several typesof tumour. We propose that TGF normally has an autocrine rolenot only in stimulating the growth of some fetal tissues butalso with postnatal epidermal cells in response to local stimuli—inparticular ultraviolet radiation (UVR). As a first step to testthis hypothesis we have checked whether UVR will induce theproduction of TGF, measured by radioimmunoassay, using a highlyspecific monodonal antibody which recognizes native, biologicallyactive human TGF. We found that cultures of normal foreskinmelanocytes do not produce detectable amounts of TGF when grownunder routine conditions, but, within 12 h of exposure to lowdoses of short-wavelength UVR, significant quantities of TGFare produced. The UVR-induced TGF is both cell associated andreleased into the medium of these cultures. Also, UVR has apromoting action on epidermal cells which have been initiatedby carcinogenic activity. A significant part of the promotingactivity may be due to autocrine stimulation of multiplicationof partially transformed epidermal cells. In this regard wefound that UVR induced TGF in HeLa cells and all human melanomalines so far tested. Induction was complete within 24 h of asingle exposure. Dose-response curves of TGF induction in amalignant melanoma cell line showed a distinctive peak of factorinduced by low (2 J/m²) doses of UVR. Higher doses which inhibit[³H]thymidine incorporation resulted in lower levels of inducedTGF. These findings are consistent with the participation ofTGF as an autocrine mediator of UVR-induced tumour promotion,as well as cell multiplication, in sun-exposed skin.     

Covalent binding of isomeric benzo[a]pyrene diol-epoxides to DNA

We have compared the abilities of two diol-epoxide derivativesof benzo[a]pyrene (B[a]P) to bind covalently to DNA in a simplein vitro system. Purified DNA in aqueous solution was allowedto react with (±)-7, 8ß-dihydroxy-9ß,10ß-oxy-7,8,9,10-tetrahydroB[a]P (BPDE) or with (±)-9,10ß-dihydroxy-7,8-oxy-7,8,9,10-tetrahydroB[a]P (reverseBPDE) to completion. After repurification of the DNA, bindingwas detected by fluorescence spectroscopy or by absorbance spectroscopy.Both BPDE and reverse BPDE but not their hydrolysis productsexhibited binding which increased linearly with increasing diolepoxide concentration. When DNA modified by reverse BPDE wasenzymatically hydrolysed, two major fluorescent deoxyribonucleoside-adductswere detected by reverse phase h.p.l.c. These were separablefrom the major adduct obtained from BPDE-modified DNA and fromthe major products obtained by hydrolysis of reverse BPDE inthe absence of DNA. Absorbance and fluorescence spectroscopyof modified native DNA suggested that the pyrene nucleus ofreverse BPDE but not of BPDE was intercalated in the DNA doublehelix. This suggestion was supported by fluorescence-quenchingstudies. In the presence of increasing DNA concentrations, covalentbinding of both diol epoxides increased towards an apparentmaximum. Double reciprocal analysis of the data indicated amaximum binding level of 5% of the total dose for BPDE and 4%for reverse BPDE. This suggests that for both diol epoxidesthe ratio of the rate constants for covalent binding and forDNA-enhanced hydrolysis are nearly the same. Covalent bindingof reverse BPDE to DNA was effectively blocked by low concentrationsof Mg²⁺, suggesting that formation of a non-covalent intercalationcomplex may be a prerequisite for covalent reaction.     

The relevance of covalent binding to mouse liver DNA to the carcinogenic action of hexachlorocyclohexane isomers

[³H]Hexachlorocyclohexane (HCH) was synthesized by chlorinationof [³H]benzene prepared by catalytic tritiation of benzene withtritiated water. The isomers of HCH were separated by adsorptionchromatography on silica gel. In order to determine the covalentbinding to DNA, [³H]HCL was administered to male mice by oralgavage, and liver DNA was isolated via chromatin. The specificradioactivity of the DNA was normalized by the dose administeredand expressed in the molar units of the Covalent binding index,CBI = DNA damage/dose = (µmol bound HCH/mol DNA nucleotide)/(mmolHCH administered/kg body weight). CBI values of 0.2 were found10 h after the administration of alpha- and gamma-HCH. Enzymaticdigestion of the DNA to the nucleosides and h.p.l.c. analysisrevealed that 40% of the radioactivity co-migrated with thenatural nucleosides. At elution volumes known to contain themore lipophilic carcinogen-nucleoside adducts, 10% of the radioactivitycould be detected. The remaining 50% of the radioactivity elutedwith the front, representing a mixture of oligonucleotide-HCHadducts and/or hydrophilic degradation products which were stronglybut not covalently associated with intact DNA. Therefore, atrue CBI of 0.02–0.1 must be expected both for alpha-and gamma-HCH. This CBI is by a factor of 10⁵–10⁶ belowthe value found with the strongest DNA-binding carcinogens likeaflatoxin B₁ or dimethylnitrosamine and is unlikely to be decisivefor the liver tumor induction in mice because of the followingadditional findings: (i) Both isomers gave rise to similar levelsof DNA damage although the alpha-isomer is a much more potenttumor inducer. This similarity was seen not only at the timeof maximum binding but up to 10 days after oral administration;(ii) three mouse strains with apparently different susceptibilityto tumor induction by gamma-HCH could not be distinguished withrespect to DNA binding; (iii) the level of DNA binding of alpha-HCH(CBI = 0.02–0.1) is more than three orders of magnitudelower than would be expected if the mechanism of tumor inductionwas by genotoxicity mediated by DNA-binding. For a preliminaryinvestigation on a potential stimulatory effect on liver DNAreplication and cell division, [¹⁴C]thymidine was administeredi.p. 3.5 h before sacrifice of the [³H]HCH-trated mice. Thealpha-isomer was found to be more potent than the gamma-isomerin this respect. Taken together, our data allow the conclusionthat the non-mutational processes must be more important forthe carcinogenicity of HCH.     

Sensitivity of rat and mouse peripheral blood lymphocytes to BaP adduction and SCE formation

Both mice and rats were injected i.p. with doses of benzo[a]pyrene(BaP) ranging from 10 to 100 mg/kg to and compare species sensitivityto and the relationship between sister chromatid exchange (SCE)induction and DNA adduct formation. Twenty-four hours afterinjection, blood was removed by cnrdiac puncture and the peripheralblood lymphocytes (PBLs) were analyzed for both DNA adduct formationby ³²P-postlabeling and SCE induction following lymphocyte culture.BaP induced similar, but not identical, SCE dose-response curvesfor each species. After BaP administration, the major DNA adduct,N²-[10ß-(7ß, 8, 9-trihydroxy-7, 8, 9, 10-tetrahydrobenzo[a]pyrene)y1]deoxy-guanosine(BPDEI-dGuo), was 10-fold more prevalent in the PBLs of themouse than those of the rat. Thus, for equivalent amounts ofBPDEI-dGuo, a greater number of SCEs are induced in the ratthan the mouse.     

Structures of covalent adducts derived from the reactions of the 9,10-epoxides of 7,8,9,10-tetrahydrobenzo [a] pyrene and 9,10,11,12-tetrahydrobenzo [e] pyrene with DNA

The reaction of a racemic mixture of 7ß, 8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (B[a]PDE) and itsenantiomer with DNA is highly stereoselective. About 90% ofthe adducts are derived from the former enantiomer reactingwith the amino group of guanine residues. To investigate thisstereoselectively we compared the reactions of 9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyreneand 9,10-epoxy-9,10,11,12-tetrahydrobenzo[e]pyrene with DNA.Most of the stereoselectivity seen with B[a]PDE is lost. Bothepoxides give mainly adducts on the N² group of guanine by bothcis and trans additions to the epoxide. Other adducts, tentativelyidentified as deoxyadenosine derivatives, were also detected.