P. Sims

Qualitative and quantitative studies on the metabolism of a series of aromatic hydrocarbons by rat-liver preparations.

Abstract Qualitative and quantitative investigations on the metabolism in rat-liver preparations of eleven tritiated hydrocarbons, phenanthrene, pyrene, chrysene, benz[a]-anthracene, dibenz[a,h]anthracene, dibenz[a,c]anthracene, benzo[a]pyrene, benzo[e]-pyrene, 3-methylcholanthrene, 7-methylbenz[a]anthracene and 7,12-dimethylbenz[a]-anthracene were carried out. The qualitative studies, using homogenates of the livers of rats that had been pretreated with 3-methylcholanthrene, showed that all the major ethyl acetate-soluble metabolites of these hydrocarbons have been recognised: in most cases the structure of the metabolites have been confirmed. Quantitative studies using microsomes or homogenates from the livers of normal or 3-methylcholanthrene-treated rats showed increases in the amounts of metabolites formed from the liver preparations of the treated animals as compared with those from the normal animals. The amounts of dihydrodiols formed on the so-called ‘K-region’ varied among the hydrocarbons: with benzo[e]pyrene, the K-region dihydrodiol was the major metabolite, whereas with chrysene and benzo[a]pyrene no products of this type were detected. Intermediate amounts of K-region products were formed with other hydrocarbons. Large differences in the rates at which the individual hydrocarbons were metabolised were also found.

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.

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.

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

Metabolism of polycyclic hydrocarbons by rat-lung preparations

Abstract The metabolism of benz[a]anthracene, 7-methylbenz[a]anthracene and benzo[a]pyrene has been studied in homogenates and microsomal fractions prepared from rat-lung. The results have been compared with those obtained in parallel experiments where rat-liver preparations were used and they show that, qualitatively, the hydrocarbons are metabolized to similar ethylacetate soluble products by both tissues. Quantitative estimations of the metabolism of the hydrocarbons indicated that rat-liver was more active than rat-lung when compared on the basis of the weight of tissue involved. Comparisons made on the basis of the protein content of the tissue preparations showed, however, that rat-lung was at least as active as rat-liver in the metabolism of polycyclic hydrocarbons. The metabolites found are mainly ring-hydroxylated products; this implies, therefore, that epoxides are formed from polycyclic hydrocarbons by the enzymes present in rat-lung.

The metabolism of a series of polycyclic hydrocarbons by mouse skin maintained in short-term organ culture

Investigations on the metabolism of 3H-labelled chrysene, benz[a]anthracene, 7-methylbenz[a]anthracene, 7,12-dimethylbenz[a]anthracene, 3-methylcholanthrene, benzo[a]pyrene, dibenz[a,c]anthracene and dibenz[a,h]anthracene by mouse skin maintained in short-term organ culture were carried out. Estimations of the distribution of the metabolites of each hydrocarbon present after 24 h showed that there were wide variations both in the rates at which the hydrocarbons were metabolised and in the amounts of metabolites covalently bound to skin macromolecules. All the hydrocarbons were metabolised to dihydrodiols, which were identified by comparison on high pressure liquid chromatography (HPLC) with the authentic compounds, and these were the same diols as those that were formed in previous experiments with rat-liver microsomal fractions. However, free dihydrodiols represented only relatively small proportions of the total amounts of metabolites formed. All the hydrocarbons yielded dihydrodiols of the type that could give rise to bay-region diol-epoxides, when further metabolised, some of which are thought to be involved in hydrocarbon carcinogenesis.

Metabolic activation of polycyclic hydrocarbons Fluorescence spectral evidence is consistent with metabolism at the 1,2- and 3,4-double bonds of 7-methylbenz[a]anthracene

When photon-counting spectrophotofluorimetry was first applied to studies on the in vitro metabolic activation of the carcinogenic polycyclic hydrocarbons [l-3] , the initial studies showed that the K-region epoxide of 7-methylbenz [a] anthracene did not appear to be the species that reacted with nucleic acid in cells treated with the parent hydrocarbon [l] . This technique was later used to confirm that the metabolic activation of benzo [a] pyrene involved a vicinal diol-epoxide formed on the adjacent 7,8and 9,10double bonds [3]. We now wish to report the results of further fluorescence spectral studies on the metabolic activation of 7-methylbenz [a] anthracene (see formula). These show that the hydrocarbon moieties,

The metabolism of benz[a]anthracene and dibenz[a,h]anthracene and their related “K-region” epoxides, cis-dihydrodiols and phenols by hamster embryo cells

Abstract The nature of some of the metabolites present in the cell media from hamster embryo cells that had been grown in the presence of benz[a]anthracene, dibenz[a,h]anthracene and their related K-region epoxides, phenols and cis -dihydrodiols has been investigated. Small amounts of phenols, dihydrodiols and unidentified water-soluble metabolites were detected in the media from the incubations with the hydrocarbons. Benz[a]anthracene 5,6-oxide yielded mainly the related dihydrodiol, whereas dibenz-[a,h]anthracene 5,6-oxide yielded mainly the related phenol and only small amounts of the dihydrodiol. The “K-region” phenols were metabolized to a small extent and both they and the epoxides yielded unidentified water-soluble metabolites. Much of the cis -dihydrodiols were recovered unchanged from the cell media, whereas with the other substrates large proportions of the material originally added to the media were not present in the media at the end of the incubation periods. These results are discussed in relation to those obtained in an examination of the levels of binding of the substrates to the protein, DNA and RNA of the cells that had been grown in the media.