Philip L. Grover

Formation of K-region epoxides as microsomal metabolites of pyrene and benzo[a]pyrene

Abstract Epoxides of pyrene and benzo[a]pyrene have been detected for the first time as microsomal metabolites of these polycyclic hydrocarbons. The epoxides, which have been identified as the K-region derivatives, pyrene 4,5-oxide and benzo[a]pyrene 4, 5-oxide respectively, were formed by the NADPH-dependent mixed function oxidase of a rat-liver microsomal incubation system where the epoxide hydrase was inhibited. In the absence of hydrase inhibition, benzo[a]pyrene was converted into a metabolite with the Chromatographic properties of a K-region dihydrodiol, trans-4,5-dihydro-4,5-dihydroxybenzo[a]pyrene, which has not previously been described as a metabolite of this hydrocarbon. Pyrene 4,5-oxide and benzo[a]pyrene 4,5-oxide rearrange in acid to compounds with the properties of 4-pyrenol and 4-benzo[a]pyrenol respectively, are converted by microsomal epoxide hydrase into compounds indistinguishable from the corresponding 4, 5-dihydro-4,5-dihydroxy derivatives and react with glutathione to yield conjugates. Both epoxides are reactive towards polyguanylic acid. The significance of these results is discussed in relation to our hypothesis that polycyclie hydrocarbon carcinogenesis results from somatic mutations caused by epoxides that are formed from the hydrocarbons by metabolism.

Fluorescence spectral evidence that benzo(a)pyrene-DNA products in mouse skin arise from diol-epoxides

When carcinogenic polycyclic hydrocarbons like benzo(a)pyrene (I) are metabolized, derivatives are formed that react with cellular macromolecules [ 1,2] . The positive identification of the reactive metabolites concerned has been hindered by the very low levels of reaction with nucleic acids that occur in tissues treated with these chemical carcinogens, although some progress has been made using radioactivelylabelled hydrocarbons [3]. A fresh approach to this problem has been made possible by the construction of a highly sensitive spectrophotofluorometer that incorporates a photon-counting device [4]. The instrument allows the fluorescence characteristics of polycyclic hydrocarbon-modified nucleic acids that have been isolated from treated tissues to be investigated directly without prior degradative or chromatographic procedures [5] . We report here the results of the first studies on benzo(u)pyrene undertaken with this spectrophotofluorometer in which the fluorescence spectral characteristics of DNA from benzo(a)pyrenetreated mouse skin have been compared with those of DNA treated in solution with reactive benzo(a)pyrene derivatives.

Mutagenicity of non-K-region diols and diol-epoxides of benz(a)anthracene and benzo(a)pyrene in S. typhimurium TA 100

Abstract When incubated with a 9,000 x g rat-liver supernatant, benzo(a)pyrene 7,8-diol and benz(a)anthracene 8,9-diol were more active than the parent hydrocarbons in inducing his+ revertant colonies of S. typhimurium TA 100. Benzo(a) pyrene 9,10-diol was less active than benzo(a)pyrene; the K-region diols, benz(a)anthracene 5,6-diol and benzo(a)pyrene 4,5-diol, were inactive. None of the diols was active when the cofactors for the microsomal mono-oxygenase were omitted. The diol-epoxides benzo(a)pyrene 7,8-diol 9,10-oxide, benz(a)anthracene 8,9-diol 10,11-oxide and 7-methylbenz(a)anthracene 8,9-diol 10,11-oxide and the K-region epoxides, benzo(a)pyrene 4,5-oxide and benz(a)anthracene 5,6-oxide, were mutagenic without further metabolism.

Epoxides as microsomal metabolites of polycyclic hydrocarbons.

The main metabolites of polycyclic hydrocarbons are hydroxylated derivatives [ 1] . These could theoretically arise from epoxide intermediates [2] formed by the NADPHdependent microsomal mixed function oxidase. K-region epoxides of polycyclic hydrocarbons that have been prepared synthetically [3] have been shown to be alkylating agents that react covalently with nucleic acids [4] and with the constituents of cells in culture [S] . These K-region epoxides are also mutagenic to T, bacteriophage [6] , to bacteria [7] , to Drosophila [8] and to Chinese hamster cells [9] and are active in producing malignant transformation of cells in culture [lo]. Consequently, it seemed important to establish that epoxides are in fact involved in the metabolism of polycyclic hydrocarbons. In the experiments described here, direct evidence has been obtained that epoxides are formed as microsomal metabolites of phenanthrene, benz[a] anthracene and dibenz[a,hh] anthracene.

Monitoring occupational exposure to carcinogens: detection by 32P-postlabelling of aromatic DNA adducts in white blood cells from iron foundry workers

Blood samples were volunteered by workers in a Finnish iron foundry who were occupationally exposed to polycyclic aromatic hydrocarbons and from control subjects not known to be occupationally exposed to this class of chemical carcinogens. DNA was isolated from peripheral white blood cells and digested with micrococcal nuclease, spleen phosphodiesterase and nuclease P1. The DNA digest was then incubated with [gamma-32P]ATP and polynucleotide kinase. Aromatic adducts present in the digest that were resistant to nuclease P1 were thus 32P-labelled while unmodified nucleotides were not. The 32P-labelled adducts were resolved by t.l.c. and detected by autoradiography. Foundry workers were classified as belonging to high, medium or low exposure groups according to their exposure to airborne benzo[a]pyrene (high greater than 0.2, medium 0.05-0.2, low less than 0.05 microgram BP/m3 air). Aromatic adducts were found to be present in DNA from 3/4 samples from the high exposure group, 8/10 samples from the medium exposure group. 4/18 samples from the low exposure group and 1/9 samples from the unexposed controls. The levels of adducts found in the high and medium group samples ranged up to 1 adduct in 10(7) nucleotides but the levels formed in the low exposure group samples were not significantly different from those in unexposed controls. No differences related to the smoking habits of the subjects were observed. Most of the DNA adducts detected had chromatographic mobilities distinct from those formed when the 7,8-diol 9,10-oxide of BP reacted with DNA. The results indicate that highly-exposed individuals are more likely to contain aromatic DNA adducts in their white blood cells, but large interindividual variations were evident. In addition, multiple samples from the same subjects indicate that qualitative and quantitative changes in adduct patterns occur with time. This pilot study suggests that 32P-postlabelling may be useful in monitoring human exposure to known and to previously unidentified environmental genotoxic agents.

The metabolic activation of 7-methylbenz(a)anthracene in mouse skin

The metabolism of 7-methylbenz(a)anthracene by rat-liver preparations and by mouse skin has been studied using a combination of thin-layer and high pressure liquid chromatography and all five possible trans-dihydrodiols have been detected as metabolites but in different proportions. The roles of these dihydrodiols and of the related vicinal diol-epoxides in the metabolic activation of 7-methylbenz(a)anthracene in mouse skin has been studied using Sephadex LH-20 column chromatography. The results show that the hydrocarbon-nucleic acid products formed in mouse skin in vivo most probably arise from 3,4-dihydro-3,4-dihydroxy-7-methylbenz(a)anthracene 1,2-oxide which, on the basis of this and other evidence, appears to be the reactive intermediate involved in the metabolic activation of 7-methylbenz(a)anthracene in this tissue.

Polycyclic hydrocarbon epoxides: The involvement of 8,9-dihydro-8,9-dihydroxybenz (a) anthracene 10,11-oxide in reactions with the DNA of benz (a) anthracene-treated hamster embryo cells

Carcinogenic polycyclic hydrocarbons are metabolized to epoxides by mammalian microsomal monooxygenases [l-4] and it has been suggested that this type of reactive intermediate is responsible for the. biological effects, including carcinogenesis, attributed to the parent hydrocarbons [5] . Although synthetic K-region epoxides possess many properties relevant to the initiation of tumours [6 ] , some previous work has indicated that K-region epoxides are not the actual epoxides involved in reactions with the nucleic acids of cells treated with the parent hydrocarbons [7]. 8,9-Dihydro-8,9-dihydroxy-benz(a)anthracene lO,lloxide, a new type of epoxide that is formed on the isolated 10,l l-double bond of the 8,9-diol of benz(a)anthracene, has recently been detected as a metabolite of the dihydrodiol and has also been prepared by synthesis [8]. The experiments described here provide evidence that when hamster embryo cells in culture are treated with benz (a) anthracene, the DNA products formed result from the reaction of 8,9dihydroxybenz(a)anthracene 10,l l-oxide with this nucleic acid. Similar results have been obtained when rat-liver microsomal preparations are incubated with the 8,9-dihydrodiol of benz(a)anthracene and DNA. As an important corollary, these results also provide some of the strongest evidence so far obtained in support of the hypothesis that epoxides are the metabolically-activated intermediates formed from polycyclic hydrocarbons.

Mutagenicity of isomeric diol-epoxides of benzo[a]pyrene and benz[a]anthracene in S. typhimurium TA98 and TA100 and in V79 Chinese hamster cells.

Pairs of isomeric vicinal diol-epoxides derived from benzo[a]pyrene 7,8- and 9,10-dihydrodiols and from benz[a]anthracene 8,9-dihydrodiol were tested for their abilities to revert salmonella typhimurium strains TA98 and TA100 to histidine prototrophy and to induce the formation of 8-azaguanine- or of ouabain-resistant V79 Chinese hamster cells. All six diol-epoxides were active in both bacterial strains, but 7beta,8alpha-dihydroxy-9beta,10beta-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (the syn isomer) was considerably more mutagenic than the other diol-epoxides. Within the three pairs of stereo-isomeric diol-epoxides, the ratio of the mutagenic potencies of the syn over the related anti isomers varied bothwith the chemical structure and the bacterial strain. The half lives of hydration of these diol-epoxides at pH 7.4 were inversely related to their mutagenic potencies in bacteria. In V79 cells, the two benzo[a]pyrene 7,8-diol 9,10-oxides were mutagenic and the anti isomer was more active than the syn isomer; a reversed order of mutagenic potency with these stereo isomers was observed in S. typhimurium. The other four diol-epoxides were non-mutagenic in V79 cells at the concentrations tested.

Fluorescence spectral studies on the metabolic activation of 3-methylcholanthrene and 7,12-dimethylbenz[a]anthracene in mouse skin.

The metabolic activation of the polycyclic hydrocarbons benzo [a] pyrene and 7-methylbenz [a] anthracene in mouse skin appears to involve the formation of vicinal diol-epoxides [l] that react with nucleic acids in this tissue [2-51. This mechanism of activation may be a general one for this class of chemical carcinogens and efforts are now being made to extend the hypothesis to include other hydrocarbons. The examination, using photon-counting spectrofluorimetry, of nucleic acids isolated from tis’sues treated in vivo with polycyclic hydrocarbons, has already proved to be of value in identifying the sites of activation in benzo [a] pyrene [2] and in 7-methylbenz [a] anthracene [4,6] . In this paper we present data from fluorescence studies obtained with the potent carcinogens 3-methylcholanthrene (I) and 7,12-dimethylbenz[a] anthracene (II). These data show that the hydrocarbon moieties, bound to the DNA of mouse skin treated in vivo with either of the parent hydrocarbons, have spectral characteristics consistent with the retention of a substituted anthracene nucleus in

Binding of K-region epoxides and other derivatives of benz[a]anthracene and dibenz[a,h]anthracene to DNA, RNA and proteins of transformable cells

Abstract The firm binding of benz[a]anthracene and dibenz[a,h]anthracene and their K-region epoxides, cis -dihydrodiols, and phenols to the DNA, RNA, and proteins of exponentially growing cells has been studied. In hamster embryonic cells that undergo malignant transformation, the epoxide of benz[a]anthracene was bound to all macromolecules to a much greater extent than the hydrocarbon and other derivatives. The binding to DNA reached its maximum at 3 h. In the dibenz[a,h]anthracene series, the epoxide was highly bound to RNA and protein, but only to a small extent to DNA. The extent of binding of all compounds to DNA, RNA, and proteins of transformable G23 cells was less than to hamster cels, and the binding to malignant T24 cells was very much lower. The relationship of metabolism to binding and carcinogenesis is discussed.