Further metabolism of diol-epoxides of chrysene and dibenz[a,c]anthracene to DNA binding species as evidenced by 32P-postlabelling analysis

Incubation of r-1t-2-dihydroxy-t-3,4-oxy-1,2,3,4-tetrahydro-chrysene(anti-chrysene-1,2-diol 3,4-oxide), the bay-region diol-epoxideof chrysene, with rat liver microsomes in the presence of NADP⁺and DNA, followed by ³²P-postlabelling analysis of the DNA,revealed the presence of at least two adducts not detected whenanti-chrysene-1,2-diol 3,4-oxide was incubated with DNA alone.The formation of these adducts was not blocked by the epoxidehydrolase inhibitor 1,1,1-trichloropropane-2,3-oxide. One ofthe adducts cochromatographed with the adduct spot obtainedwhen authentic 9-hydroxy-r-1,t-2-dihydroxy-t-3,4-oxy-1,2,3,4-tetra-hydrochrysene(anti-9-OH-chrysene-1,2-diol 3,4-oxide) was reacted with DNA.Evidence suggested that a second adduct could also be formedby further metabolism of anti-9-OH- chrysene-1,2-diol 3,4-oxide.In addition, evidence was obtained for the further metabolismof the syn-isomer of chrysene 1,2-diol 3,4-oxide and the anti-isomersof a non-bay-region diol epoxide of dibenz[a,c]anthracene toDNA binding species, but not for that of either the anti- orsyn- isomers of the bay-region diol-epoxide of benzo[a]pyrene,the anti-isomers of the bay-region or a non-bay-region diol-epoxideof benz[aanthracene, or the anti-isomer of the bay-region diol-epoxideof benzo[bfluoranthene.     

Metabolic activation of chrysene by hamster embryo cells: evidence for the formation of a 'bay-region' diol-epoxide-N2-guanine adduct in RNA

Ribonudeoside-hydrocarbon adducts present in hydrolysates ofRNA isolated from hamster embryo cells treated with ³H-labelledchrysene were examined by chromatography on Sephadex LH20 andby h.p.l.c. on Zorbax ODS. Two adducts formed in cells had chromatographicproperties identical to those of two synthetic adducts formedwhen r-1,t-2-dihydroxy-t-3,4-oxy-1,2,3,4-tetrahydrochrysene(anti-chrysene 1,2-diol 3,4-oxide) reacted with poly G in vitro.Another adduct formed in cells had chromatographic propertiesidentical to those of a synthetic adduct formed when anti-chrysene1,2-diol 3,4-oxide reacted with poly A. In addition to the characterizedadducts, other minor adducts were detected whose structuresare not known. The structure of the more abundant guanosine-hydrocarbonadduct formed in cells was investigated by determining its pKvalues and stability in 1 M KOH. The structures of the syntheticguanosine-hydrocarbon adducts were investigated by ¹H-n.m.r.spectroscopy. The data show that, in the hydrocarbon-guanosineadducts studied, the hydrocarbon moiety is attached to the exocyclicamino group of guanine.     

Reactions of 'bay-region' and non-'bay-region' diol-epoxides of benz(a)anthracene with DNA: evidence indicating that the major products are hydrocarbon-N2-guanine adducts

The rates of reaction and the products formed when two vicinaldiol-epoxides derived from benz(a)an-thracene, anti-BA-3, 4-dioI1, 2-oxide (t-3, r-4-dihydroxy-t-1, 2-oxy-l, 2, 3, 4-tetrahydrobenz(a)anthracene)^* and anti-BA-8, 9-diol 10, 11-oxide (r-8, t-9-dihydroxy-t-10,ll-oxy-8, 9, 10, ll-tetrahydro-benz(a)anthracene) reacted withDNA were studied in vitro and the results were compared withthose obtained in similar experiments using anti-BP-7, 8-diol9, 10-oxide (r-7, t-8-dihydroxy-t-9, 10-oxy-7, 8, 9, 10-tetrahydrobenzo(a)pyrene).The reactivities appeared to decrease in the order anti-BP-7,8-diol 9, 10-oxide > anti-BA-3, 4-diol 1, 2-oxide anti-BA-8,9-diol 10, 11-oxide. The diol-epoxides reacted to a similarextent with single- and with double-stranded DNA but reactionswith dGMP, at equivalent concentrations, were much slower thanwith DNA. With the diol-epoxides of benz(a)anthracene, two principaladducts were present in DNA hydrolysates and evidence was obtained,based on pK determinations before and after nitrous acid treatment,consistent with their being N²-guanine derivatives, analogousto the known DNA-reaction products of benzo(a)-pyrene 7, 8-diol9, 10-oxide.     

The metabolism of the 10,11-dihydrodiol of benz[a]-anthracene to a vicinal diol-epoxide that is not involved in metabolic activation

Benz[a]anthracene-10,11-diol, a major metabolite of benz[a]anthracene, is metabolized by a rat-liver microsomal system to form anti-BA-10,11-diol 8, 9-oxide (t-10,r-11-dihydroxy-t-8,9-oxy-8,9,10,11 -tetrahydrobenz[a]anthracene) and, to a lesser extent, syn-BA-10,11-diol 8,9-oxide (t-10,r-11-dihydroxy-c-8,9-oxy-8,9,10,11-tetrahydrobenz[a] anthracene). However, when benz[a]anthracene is incubated with DNA in a rat-liver microsomal system, anti-BA-10,11-diol 8,9-oxide does not contribute to the covalent binding of this hydrocarbon to DNA.     

The involvement of a 'bay-region' and a non-'bay-region' diol-epoxide in the metabolic activation of benz[a]anthracene in mouse skin and in hamster embryo cells

The principal nucleoside-hydrocarbon adducts present in hydrolysatesof DNA that had been isolated either from mouse skin or fromhamster embryo cells treated with benz(a)anthracene have beenexamined by chromatography on Sephadex LH20 and by high pressureliquid chromatography on Spherisorb 5 ODS. The results showthat one of the major adducts prepared from the DNA of mouseskin and of hamster embryo cells has chromato-graphic propertiessimilar to those of an adduct formed when anti-BA-8, 9-diol10, 11-oxide (r-8,t-9-dihydroxy-t10, 11-oxy-8, 9, 10, 11-tetrahydro-benz(a)anthracene)reacts with DNA, whilst a second major adduct has chromatographicproperties similar to those of an adduct prepared by reactinganti-BA-3, 4-diol 1, 2-oxide (t-3, r-4-dihydroxy-t-1, 2-oxy-1,2, 3, 4-tetrahydrobenz(a)anthracene) with DNA. On the basisof this and other evidence, it appears that both of these diol-epoxidesmay contribute to the biological activity of benz(a)anthracene.     

The metabolic activation of benz[a]anthracene in three biological systems

The 3,4- and 8,9-dihydrodiols of benz[a]anthracene (BA) are formed as metabolites of the parent hydrocarbon by rat-liver microsomes, by mouse skin and by hamster embryo cells. In incubations with rat-liver microsomal fractions, only small amounts of the 3,4-dihydrodiol of BA were detected relative to other dihydrodiol metabolites and only small amounts of BA-deoxyribonucleoside adducts derived from the related diol-epoxide, t-3, r-4-dihydroxy-t-1,2-oxy-1,2,3,4-tetrahydrobenz[a]anthracene (anti-BA-3,4-diol 1,2-oxide), were detected relative to adducts derived from r-8,t-9-dihydroxy-t-10,11-oxy-8,9,10,11-tetrahydrobenz[a]anthracene (anti-BA-8,9-diol 10,11-oxide). However, in studies with mouse skin and hamster embryo cells, larger amounts of free 3,4-dihydrodiol were detected and a larger proportion of the hydrocarbon-deoxyribonucleoside adducts resulted from the reaction of anti-BA-3,4-diol 1,2-oxide with DNA.     

Morphological transformation and DNA adduct formation by dibenz[a,h]anthracene and its metabolites in C3H10T1/2CL8 cells

The major routes of metabolic activation of dibenz[a,h]-anthracene(DBA) have been studied in transformable C3H10T1/2CL8 (C3H10T1/2)mouse embryo fibroblasts in culture. The morphological transformingactivities of three potential intermediates formed by metabolismof DBA by C3H10T1/2 cells, trans-3,4-dihydroxy-3,4-dihydro-DBA-(DBA-3,4-diol),trans-dihydroxy-3,4-dihydro-DBA-anti-1,2-oxide (DBA-3,4-diol-1,2-oxide)and DBA-5,6-oxide were determined. DBA-3,4-diol-1,2-oxide wasa strong morphological transforming agent giving a mean of 73%dishes with Type II or III foci and 1.63 Type II and HI fociper dish at 0.5 µg/ml. DBA-3,4-diol produced a mean of42% dishes with Type II or III foci and 0.81 Type II and IIIfoci per dish at 2.5 µg/ml. DBA gave a mean of 24% disheswith Type II or III foci and 0.29 Type II and III foci per dishat 2.5 µg/ml. DBA-5,6-oxide was found to be inactive.DNA adducts of DBA, DBA-3,4-diol, DBA-3,4-diol-1,2-oxide, DBA-1,4/2,3-tetroland DBA-5,6-oxide in C3H10T1/2 cells were analyzed by ³²P-postlabelingmethod. DBA gave 11 adducts, nine of which were observed inthe DNA of cells treated with DBA-3,4-diol and seven from cellstreated with DBA-3,4-diol-1,2-oxide. Two of these adducts thatappear in each of the treatment groups have been identifiedas the product of the interaction of DBA-3,4-diol-1,2-oxidewith 2-deoxyguanosine. Furthermore, there is evidence for DBA-DNAadducts in cells treated with DBA, DBA-3,4-diol and DBA-3,4-diol-1,2-oxidearising from metabolism to (+,-)-trans,trans-3,4,10,11-tetrahydroxy-3,4,10,11-tetrahydro-DBA(DBA-3,4,10,11-bis-diol). These results are based on co-migrationof C3H10T1/2 DNA adducts with skin DNA adducts formed aftertopical treatment of mice with DBA-3,4,10,11-bis-diol. In C3H10T1/2cells, DBA is metabolically activated through DBA-3,4-diol,which is further activated via the DBA-3,4-diol-1,2-oxide andDBA-3,4,10,11-bis-diol pathways. No evidence is provided forthe metabolism of DBA by the Kregion pathway.     

The metabolic activation of dibenz[a,c]anthracene

In rat liver microsomal preparations, the 10, 11-dihydro-diolof dibenz[a, c]anthracene (DBA) is metabolized to r-10, t-11-dihydroxy-t-12,13-oxy-10, 11, 12, 13-tetrahydro-dibenz[a, c]anthracene (anti-DBA10, 11-diol 12, 13-oxide), the anti isomer of a non-bay-regiondiol-epoxide of DBA. When ³-labelled DBA or trans-10, 11-di-hydro-10,ll-dihydroxydibenz[a, c]anthracene were metabolized in thissystem in the presence of DNA or when ³H-labelled DBA was addedto primary cultures of hamster embryo cells, covalent reactionsof hydrocarbon metabolites with DNA occurred. The chromato-graphiccharacteristics of the radioactive hydrocarbon — deoxyribonucleosideadducts formed in these reactions were examined using SephadexLH20 column chromatography and high pressure liquid chromato-graphy.The results showed that whilst some of the radioactive hydrocarbon— deoxyribonucleoside adducts formed were indistinguishablefrom adducts that were formed when anti-DBA 10, 11-diol 12,13-oxide reacted with DNA, other, unidentified adducts, whichdid not apparently arise from reactions of this diol-epoxidewith DNA, were also present. Hydrocarbon — nucleosideadducts were not detected in hydrolysates of nucleic acids thatwere isolated from mouse skin that had been treated in vivowith DBA.     

Genotoxicity characteristics of reverse diol-epoxides of chrysene

Trans-3,4-dihydroxy-3,4-dihydrochrysene (chrysene-3,4-diol),a major metabolite of chrysene, is further metabolized by ratliver enzymes to products which effectively revert the his^–Salmonella typhimurium strain TA98 to histidine prototrophy,but are only weakly mutagenic in strain TA100 and in Chinesehamster V79 cells (acquisition of resistance to 6-thioguanine).The liver enzyme mediated mutagenicity of chrysene-3,4-diolis substantially enhanced in the presence of 1,1,1-trichloropropene2,3-oxide, an inhibitor of microsomal epoxide hydrolase. Thepredominant metabolites of chrysene-3,4-diol, namely the anti-and syn-isomers of its 1,2-oxide (termed reverse diol-epoxides),proved to be extraordinarily effective mutagens in S.typhimuriumstrain TA98, but were only moderately active in strains TA100and TA104, and in the SOS induction in Escherichia coli PQ37.These genotoxicity spectra in bacteria are completely differentfrom those observed with the bay-region diol-epoxides of chryseneand 3-hydroxychrysene. In V79 cells, the reverse diol-epoxidesformed low levels of DNA adducts and were very weak inducersof gene mutations. In M2 mouse prostate cells, however, highnumbers of transformed foci were induced by chrysene-3,4-dioland its diastereomeric 1,2-oxides. Chrysene-3,4-diol was somewhatmore potent than chrysene-1,2-diol. The potency of both reversediol-epoxides was similar to that of the syn-diastereomers ofthe bay-region diol-epoxides of chrysene and 3-hydroxychrysene,but lower than that of their anti-diastereomers. The reversediol-epoxides of chrysene, unlike the bay-region diol-epoxides,were inactivated by purified microsomal epoxide hydrolase. Noteworthyfindings were also made with regard to the chemical stabilityof the diol-epoxides in buffer, determined from the declinein mutagenicity after preincubation in the absence of the targetcells. Despite its lower     

Metabolic activation of chrysene in mouse skin: evidence for the involvement of a triol-epoxide

All three possible dihydrodiols of chrysene and a chrysene triol,formed from the further metabolism of the chrysene-1,2-diol,were detected when ether extracts of mouse skin that had beentreated with ³-labelled chrysene were examined by h.p.l.c. Themajor deoxyribonucleoside-hydrocarbon adducts present in hydrolysatesof DNA isolated from the mouse skin were examined by chromatographyon Sephadex LH20 and by h.p.l.c. on Zorbax ODS. One adduct hadchromato-graphic properties identical to those of the majoradduct formed when r-1,t-2-dihydroxy-t-3,4-oxy-1,2,3,4-tetrahydrochrysenereacts with DNA. A second major adduct was present that hadchromatographic properties that were indistinguishable fromthose of an adduct that was formed when either chrysene-1,2-diolor 3-hydroxychrysene were incubated with DNA in a rat livermicrosomal metabolising system. The results provide evidencethat this new adduct is formed via the reaction of a ‘triol-epoxide’,that appears to be 9-hydroxychrysene-1,2-diol 3,4-oxide, withDNA in mouse skin.     

The enzyme-catalysed conversion of anti-benzo[a]pyrene-7,8-diol 9,10-oxide into a glutathione conjugate

Anti-BP-7,8-diol 9,10-oxide (r-7,t-8-dihydroxy-t-9, 10-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene) was converted in the presence of a rat-liver supernatant fraction and glutathione into a water-soluble metabolite that was identified as a glutathione conjugate. The formation of the glutathione conjugate appears to be catalysed by glutathione S-transferases, present in the rat-liver supernatant, because the amount of conjugate formed was reduced considerably when anti-BP-7,8-diol 9,10-oxide was incubated with glutathione either in the absence of the supernatant fraction or in the presence of heat-denatured supernatant fraction.     

The enzymatic conjugation of glutathione with bay-region diol-epoxides of benzo[a]pyrene, benz[a]anthracene and chrysene

The kinetics of the enzymatic conjugation of glutathione (GSH)with the anti-diastereoisomers of trans-7, 8-dihydroxy-9, 10-epoxy-7,8, 9, 10-tetrahydrobenzo[a]pyrene (BPDE), trans-3, 4-dihydroxy-1,2-epoxy-1, 2, 3, 4-tetrahyd-robenz[a] anthracene (BADE) andtrans-1, 2-dihydroxy-3, 4-epoxy-1, 2, 3, 4-tetrahydrochrysene(CDE) catalyzed by transferase 4-4 from rat liver have beencompared. When the concentration of these diol-epoxides wasvaried (using 2mM GSH) the apparent V_max values were 560, 2100and 1500 nmol/ing/min for (?)-anti-BPDE, (?)-anti-BADE and (?)-anti-CDE,respectively, with corresponding apparent K_m values of 11, 125and 105 µM. The catalytic efficiency of transferase 4–4in the GSH conjugation of (?)-anti-BADE and (?)-anti-CDE isthus approximately one-third of (?)-anti-BPDE (0.014 and 0.012s^-1 µM^-1 respectively versus 0.042 s^-1µM^-1). Similarnon-linear Lineweaver-Burk plots were obtained with each diol-epoxidewhen the concentration of GSH was varied, and two apparent.K_m values of 0.02–0.04 and 0.4–0.9 mM GSH were estimated.The GSH-conjugates formed with the individual enantiomers ofthe recemic substrates used were resolved by h.p.l.c. The dataindicate that with each diol-epoxide transferase 4–4 ishighly selective(95%) towards the biologically most active (+)-enantiomer.     

Metabolic activation of benz[a]anthracene in hamster embryo cells: the structure of a guanosine-anti-BA-8, 9-diol 10, 11-oxide adduct

The structures of two guanosine-hydrocarbon adducts preparedfrom polyG that had been incubated with anti-BA-8, 9-diol 10,11-oxide (r-8,t-9-dihydroxy-t-10, 11-oxy-8, 9, 10, 11-tetrahydrobenz[a]anthracene)were investigated by examining their ¹H-n.m.r. spectra, theirpK values before and after treatment with nitrous acid and theirstabilities in 1M KOH. The data show that both of the adductswere formed by reaction between the exocyclic amino group ofguanine and the 11-position of the diolepoxide. One of theseadducts is indistinguishable from an adduct isolated from hamsterembryo cells that had been treated with benz[a]anthracene andthat may contribute to the biological activity of this weakcarcinogen.     

In vivo and in vitro formation of glutathione conjugates from the K-region epoxides of 1-nitropyrene

4,5-Epoxy-4,5-dihydro-1-nitropyrene (1-nitropyrene 4,5-oxide)and 9,10-epoxy-9,10-dihydro-1-nitropyrene (1-nitro-pyrene 9,10-oxide),which are electrophilic metabolites formed during the metabolismof the environmental pollutant, 1-nitropyrene, reacted slowlywith glutathione. The rate of conjugation was greatly enhancedby the addition of purified rat liver glutathione (GSH) transferases,with transferases 3-3 and 4-4 exhibiting higher catalytic activitiesthan transferases 1-1, 2-2 and 7-7. Two GSH conjugates wereformed from each of the oxides: 1-nitropyrene 4,5-oxide gavea 1:1 mixture of 4-(glutathion-S-yl)-5-hydroxy-4,5-dihydro-1-nitro-pyreneand 5-(glutathion-S-yl)-4-hydroxy-4,5-dihydro-1-nitro-pyrenewhile 1-nitropyrene 9,10-oxide gave a 2:1 mixture of 9-(glutathlon-S-yl)-10-hydroxy-9,10-dihydro-1-nitropyrene and 10-(glutathion-S-yl)-9-hydroxy-9,10-dihydro-1-nitro-pyrene. Both K-region oxides were converted to trans-di-hydrodiolsby hepatic microsomal epoxide hydrase, and faster rates wereobserved with 1-nitropyrene 4,5-oxide. In subsequent experiments[4,5,9,10-³H]1-nitropyrene was administered to Sprague-Dawleyrats by intravenous and intraperitoneal injections. HPLC analysisof biliary metabolites indicated the presence of four GSH conjugatesthat were identical to those obtained from reactions of theK-region oxides with GSH. In addition, glucuronide conjugateswere detected from trans-4,5-dihydroxy-4,5-dihydro-1-nitropyrene(1-nitropyrene trans-4,5-dihydrodiol) but not trans-9,10-di-hydroxy-9,10-dihydro-1-nitropyrene(1-nitropyrene trans 9,10-dihydrodiol). These data combinedwith earlier studies indicate that 1-nitropyrene is oxidizedpreferentially to 1-nitropyrene 4,5-oxide and that, while themain detoxification pathway of 1-nitropyrene 9,10-oxide is GSHconjugation, 1-nitropyrene 4,5-oxide is excreted via both GSHconjugation and dihydrodlol formation followed by O-glucuronidation.     

Metabolism of benzo[a]pyrene-7,8-dihydrodiol and benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide to protein-binding products and glutathione conjugates in isolated rat hepatocytes

Isolated hepatocytes from 3-methylcholanthrene (MC)-treatedrats metabolized trans-7, 8-dihydroxy-7, 8-dihydrobenzo[a]pyrene(BP-7, 8-diol) and (± )-7ß, 8-dihydroxy-9,10-oxy-7, 8, 9, 10-tetrahydrobenzo[a]pyrene (anti-BPDE) to watersoluble conjugates including glutathione (GSH) conjugates. Underthe conditions employed 35% of total water soluble productsderived from BP-7, 8-diol could be accounted for by GSH conjugates.The corresponding figure for anti-BPDE was estimated to be >80%.Isolated hepatocytes metabolized BP-7, 8-diol and anti-BPDEto GSH conjugates at maximal rates of 0.5 and 9 nmol per 106cells per min, respectively. Thus, identifying the rate limitingstep in the reaction sequence as the metabolism of BP-7, 8-diolto the GSH conjugating intermediates. In addition to the directconjugation of anti-BPDE with GSH, anti-BPDE but not the correspondingBP-tetraols, was further metabolized to reactive intermediatesthat subsequently bound to cellular proteins or reacted withGSH forming water soluble conjugates. The identity or identitiesof these novel reactive intermediates is discussed.     

Metabolism and DNA adduct formation of benzo[a]pyrene and 7,12-dimethylbenz[a]anthracene in fish cell lines in culture

The metabolic activation of the carcinogens benzo[a]anthracene(BP) and 7,12-dimethylbenz[a]anthracene (DMBA) was examinedin cell lines derived from bluegill fry (BF-2), rainbow trout(RTG-2) and brown bullhead (BB). All three cell lines metabolizedBP (0.5 µg/ml medium) almost completely to water-solublemetabolites within 120 h, but the maximum amount of BP boundto DNA ranged from only 5 pmol/mg DNA in the BF-2 cells to 17in the BB cells and 44 in the RTG-2 cells. The major BP-DNAadduct in the BB and BF-2 cells was that formed by reactionof (+)-anti-BP-7,8-diol-9,10 epoxide [(+)anti-BPDE] with deoxyguanosine.This adduct was also present in the RTG-2 cell DNA, but therewere larger amounts of unidentified polar BP-DNA adducts. Exposureof the cells to [³H]BP-7,8-diol, a metabolic precursor of (+)anti-BPDE,resulted in binding of 1.5, 12 and 35 pmol BP per mg DNA inthe BF-2, BB and RTG-2 cells, respectively. More than 90% ofthe BP-7,8-diol added to the BF-2 cultures was recovered asa glucuronic acid conjugate, but the RTG-2 cells formed moreglutathione conjugates than glucuronide conjugates. The BB cellsformed both types of conjugates at a slower rate for more than75% of the 7,8-diol was recovered unchanged after 24 h. Thethree cell lines differed in the proportion of a 0.1 µg/mldose of DMBA metabolized in 48 h: the values ranged from 47%in the BF-2 cells to 78% in the BB cells and 97% in the RTG-2cells. The amount of DMBA bound to DNA ranged from 4.7 to 8.6pmol/mg DNA in the three cell lines: DMBA-3,4-diol-1,2-epoxide(DMBADE) adducts were present in the BB cell DNA, but no significantamounts of DMBADE-DNA adducts were detected in the RTG-2 orBF-2 cell DNA. These results demonstrate that fish cell culturescan activate BP to an ultimate carcinogenic metabolite, (+)anti-BPDE,but the level of binding of this metabolite to DNA is much lowerthan that which occurs in rodent embryo cell cultures. In BF-2cell cultures formation of BP-7,8-diol-glucuronide effectivelyprevents the activation of this diol to (+)anti-BPDE. A substantialproportion of the BP-7,8-diol is also metabolized to glucuromdeand glutathione conjugates in BB and RTG-2 cells. DMBA alsobinds to DNA at very low levels in these fish cell cultures.Thus effective conjugation of diols and their metabolites byfish cell lines appears to greatly reduce metabolic activationof hydrocarbons through the bay-region diol epoxide pathwaythat predominates in mammalian cell cultures.     

Mutagenic potential of DNA adducts formed by diol-epoxides, triol-epoxides and the K-region epoxide of chrysene in mammalian cells

The syn- and anti-tsomers of chrysene-l,2-diol-3,4oxide (syn-diol-epoxideand anti-diol-epoxide) and of 9-hydroxychry-sene-l, 2-diol-3,4-oxide (syn-triol-epoxide and anti-triol-epoxide) and chrysene-5,6-oxide,the K-region epoxide, were tested for their ability to induce6-thioguanine-resistant mutants in V79 Chinese hamster cells.The levels of DNA adducts formed by each compound in the V79cells were determined by ³²P-post-labelling analysis. The mostpotent mutagen, in terms of the mutation frequency/nmol compoundadministered, was the anti-triol-epoxide, which was 1.7 timesas active as the anti-triol-epoxide. The anti-diol-epoxide was10 times more active than both the syn-triol-epoxide and thesyn-diol-epoxide, which in turn were several times more activethan the K-region epoxide. However, when the results were expressedas mutations/pmol total adducts formed, the anti-triol-epoxideand anti-diol-epoxide were shown to be of similar potency andapproximately twice as active as the other three compounds.Thus differences in the conformation of adducts formed withDNA by syn- and anti-isomers may be responsible for their differentmutagenic potentials; the presence of a phenolic OH-group atthe 9-position of a chrysene-l,2-diol-3,4-oxide appears to increaseits chemical reactivity.     

The lack of transformation activity of 9-hydroxybenzo[a]pyrene related to the absence of modified deoxyribonucleosides in DNA of C3H/10T1/2 cells

Sephadex LH-20 chromatography of DNA digests of C3H/10T1/2 cellstreated with [³H]9-hydroxybenzo[a]pyrene (9-OH-BaP) and [¹⁴C]BaP-7,8-diol showed: (1) the presence only of uncharacterized ³H radioactivityeluted in the early portion of the gradient; [³H]9-OH-BaP-4,5-oxide-modifieddeoxyribonucleosides were not observed. (ii) The total amountof [¹⁴C]-labelled radioactivity corresponded to nucleoside moietieswhich were modified by anti- and syn-benzo[a]pyrene diol-epoxide.The absence of nucleosides modified by 9-OH-BaP-4,5-oxide inC3H/10T1/2 cells observed in this study may account in partfor the relative resistance of these cells to the mutagenicand transforming action of 9-OH-BaP.     

Dibenz[a,j]acridine: distributions of metabolites formed by liver and lung microsomes from control and pretreated rats

The structures of many dibenz[a,j]acridine (DBAJAC) metabolitesformed in vitro in incubations with liver microsomes preparedfrom 3-methyicholanthrene-pretreated male Wistar rats have previouslybeen determined; they were trans-DBAJAC-3,4-dihydrodiol, trans-DBAJAC-5,6-dihydro-diol,DBAJAC-5,6-oxide, 3-hydroxy-DBAJAC, 4-hydroxy-DRAJAC and severalmultiply oxidized secondary metab olites. Herein are reported[14³]DBAJAC metabolite distributions obtained by h.p.l.c. separationof products pro duced in incubations with liver and lung microsomesprepared from untreated, phenobarbital-pretreated and 3-methyl-cholanthrene-pretreatedmale Wistar rats. Liver microsomal metabolites were also quantitatedin preparations from trans stilbene oxide-pretreated rats. Forall preparations trans DBAJAC-3,4-dihydrodiol, the candidateproximate car cinogen according to the bay-region theory ofcarcinogenesis, was the major metabolite (30–40%) whileDBAJAC-5,6-oxide and phenols were also quantitatively important.In incuba tions conducted in the presence of 3,3,3-trichloropropene-1,2-oxide (1.5 mM) formation of dihydrodiol was inthibited byabout 85%. DBAJAC-N-oxide was also identified as a minor metabolite(1%) formed in incubations with pheno barbital-induced and controlliver microsomes.     

Tumor-initiating activity on mouse skin of bay region diol-epoxides of 5,6-dimethylchrysene and benzo[c]phenanthrene

Previous metabolism and DNA-binding studies indicated that 5,6-dimethylchrysene(5,6-diMeC) is metabolically activated in mouse skin throughformation of its 1,2-dihydrodiol (5,6-diMeC-1,2-diol) and bayregion diol-epoxide (anti-5,6-diMeC-1,2-diol-3,4-epoxide). Thesemetabolites were tested as tumor initiators on mouse skin. Includedfor comparison were syn-5,6-diMeC-1,2-diol-3,4-epoxide and anti-4,3-di-hydroxy-2,1-epoxy-4,3,2,1-tetrahydrobenzo[c]phenanthrene(anti-B[c]Ph-4,3-diol-2,1-epoxide). At an initiating dose of100 nmol/mouse, 5,6-diMeC-1,2-diol and anti-5,6-diMeC-1,2-diol-3,4-epoxidewere significantly more tumorigenic than 5,6-diMeC, inducing7.1 and 3.9 skin tumors per mouse respectively compared to 1.1induced by 5,6-diMeC. Similar results were obtained at an initiatingdose of 33 nmol/mouse. This is the first example of a methylatedpolynuclear aromatic hydrocarbon bay region diol-epoxide whichis more tumorigenic than its parent hydrocarbon on mouse skin.syn-5,6-diMeC-1,2-diol-3,4-epoxide was only weakly tumorigenic.Comparisons of anti-5,6-diMeC-1,2-diol-3,4-epoxide and anti-B[c]Ph-4,3-diol-2,1-epoxidedemonstrated that the latter was a stronger tumor initiator.The results of this study confirm the bay region diol-epoxidemetabolic activation pathway of 5,6-diMeC but do not providean explanation for the relatively weak tumorigenicity of thishydrocarbon on mouse skin.