Meeting on carcinogen testing is open.
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Carcinogens↗
Biomedical subjects
P Brookes
Publications and source records attributed to P Brookes.
Cell-mediated mutagenesis in cultured Chinese hamster cells by polycyclic hydrocarbons: mutagenicity and DNA reaction related to carcinogenicity in a series of compounds.
Three polycyclic hydrocarbons, benz(a)anthracene, 3-methylcholanthrene and 7,12-dimethylbenz(a)anthracene, have been studied in a cell-mediated mutagenesis system using BHK 21 cells to metabolize the hydrocarbons and V-79 cells as targets for detecting induced cytotoxicity and mutation. In large-scale experiments, the DNA of V-79 cells was analyzed by column chromatography to determine the nature and true extent of reaction of hydrocarbons with dexoyribonucleosides. Products with DNA formed by the two carcinogenic compounds were qualitatively very similar to those reported to occur in vivo and in primary cell cultures. Binding indices were calculated from the tritium content of DNA-hydrocarbon products, related to overall metabolism, for these two compounds together with benzo(a)pyrene and 7-methylbenz(a)anthracene using data from a previous study. These values reflected differences in carcinogenic potency between the compounds. Induced mutation frequencies were related to the extent of DNA reaction with each compound. At equivalent extents of DNA reaction with hydrocarbon products, levels of induced mutation were not significantly different.
9,10-Dimethyl-1,2-benzanthracene↗
A quantitative comparison of the mutagenicity of carcinogenic polycyclic hydrocarbon derivatives in cultured mammalian cells.
The mutagenicity of a series of reactive polycyclic hydrocarbon derivatives has been studied using Chinese hamster (V79) cells in culture and, as a mutational marker, resistance to the purine analogue 8-azaguanine. The compounds were compared by relating mutation frequency to the dose applied (mutagenic effectiveness) to induced cytotoxicity (mutagenic efficiency) and to the extent of reaction of the hydrocarbon with DNA (absolute mutagenic efficiency). In each case anti-benzo(alpha)pyrene (BP)-7,8 dihydrodiol-9,10 oxide, the suspected ultimate carcinogenic form of benzo(alpha)pyrene, was by far the most potent of the compounds tested. Furthermore, the mutagenicity of the syn- and anti-BP-diolepoxide isomers correlated positively with their documenrences in the ability of each derivative to form a carbonium ion. Variations in mutagenic efficiency and absolute mutagenic efficiency were more difficult to explain. The latter findings are discussed in relation to the types of hydrocarbon-DNA product obtained with each compound and also to the possibility of a variable cellular response to more subtle differences in the chemistry of the hydrocarbon-DNA interaction.
Animals↗
The in vitro and in vivo reaction at the N7-position of guanine of the ultimate carcinogen derived from benzolalpyrene.
The previously reported reaction at N2- and N7- of guanine following addition of 7 alpha,8 beta-dihydroxy-9 beta, 10 beta-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (anti-BPDE) to an aqueous solution of DNA has been studied in more detail. The extent of reaction and the relative yields of N2- and N7-products was measured over the range of pH 4--7. The depurination following reaction at the N7-position of guanine was found to have a half-life of 3 h. Reaction of the isomeric 7 alpha,8 beta-dihydroxy-9 alpha, 10 alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]-pyrene (syn-BPDE) with DNA gave the expected N2- and no N7-guanine product. When either benzo[a]pyrene or anti-BPDE was added to mouse embryo or Chinese hamster V79 cells respectively, a major N2-guanine product and a very minor adenine product were isolated from the DNA, but no N7-guanine product was detected.
Animals↗
The binding to mouse skin DNA of benzo[a]pyrene, its 7,8-diol and 7,8-diol-9,10-epoxides in relation to the tumorigenicity of these compounds.
Tritium labelled and carbon-14 labelled benzo[a]pyrene (PB), (+/-)trans 7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene (PB-7,8-diol), (+/-)-7 alpha,8 beta-dihydroxy-9 beta,10 beta-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene and (+/-)-7 alpha, 8 beta-dihydroxy-9 alpha, 10 alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]-pyrene were applied to the shaved backs of mice. After 24 h the DNA was isolated from the treated skin and the extent of hydrocarbon binding was determined. The level of DNA binding is considered in relation to the previously reported carcinogenicity of the various benzo[a]pyrene derivatives.
Animals↗
Lack of oestradiol-DNA binding in mouse embryo cell cultures or following rat-liver microsomal metabolism.
Oestradiol-17beta was compared to benzo[a]pyrene (BP) for its ability to bind to the DNA of mouse embryo cells in culture or to DNA added to a rat liver microsomal incubation mixture. No significant binding was found in embryo cells (at least 100-fold less than for BP). Microsomal incubation resulted in apparent oestradiol binding to a reisolated DNA-containing complex, but most of this was lost when the DNA was freed of RNA and protein.
Animals↗
The reaction of trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene with DNA involves attack at the N7-position of guanine moieties.
The reaction of trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene (anti-BPDE) with DNA prelabelled with [14C] and [3H]-purine precursors has indicated that in addition to the N2-position of guanine previously reported [10--12] reaction also involves the N7-position of guanine. The hydrocarbon-N7-guanine product was not detected earlier because it is lost from the DNA very readily at pH 7. The same N7-product was obtained by reaction of anti-BPDE with guanine in dimethylformamide.
Benzopyrenes↗
The identification of 3-methylcholanthrene-9,10-dihydrodiol as an intermediate in the binding of 3-methylcholanthrene to DNA in cells in culture.
Two metabolites of 3-methylcholanthrene (3MC), previously shown to be precursors of the DNA-bound form of 3MC observed in embryo cells in culture, were prepared from 3MC by microsomal metabolism and isolated by high pressure liquid chromatography (HPLC). From HPLC analysis of the metabolites of 3MC, from mass spectrometric analysis and from comparison with the fluorescence spectra of all 5 possible dihydrodiols of the alkylated benzanthracenes, it was deduced that one of the precursor metabolites was a 9,10-dihydrodiol of 3MC while the other was a 1 or 2-hydroxy derivative thereof.
Animals↗
Different tumours induced by benzo(a)pyrene and its 7,8-dihydrodiol injected into adult mouse salivary gland.
A comparison has been made between the carcinogenic activities of benzo(a)pyrene and the proposed proximate carcinogen, benzo(a)pyrene 7,8-dihydrodiol, in the adult C57BL mouse submandibular salivary gland. In preliminary studies using a range of doses, the dihydrodiol was slightly less active than the parent hydrocarbon in this system. There was a difference in the type of tumour induced by the 2 compounds. Benzo(a)pyrene induced tumours of the salivary glands at the site of injection, whereas the dihydrodiol induced malignant lymphosarcomas, particularly of the thymus, which were often metastatic to other orgnas. Possible reasons for the different sites of action of the 2 compounds are discussed.
Animals↗
The metabolism and DNA binding of 3-methyl-cholanthrene.
Tritiated 3-methylcholanthrene (3MC) was administered to mouse embryo cells in culture from which DNA was later isolated and hydrolysed to deoxyribonucleosides. The hydrolysate was analysed by LH20 chromatography, HPLC and fluorescence spectroscopy. The 3MC-nucleoside products were compared with the nucleoside derivatives obtained from 3MC-11,12-oxide-treated DNA and found to differ in both fluorescence spectra and chromatographic behaviour. Microsome-catalyzed DNA binding of primary metabolites of 3MC identified two metabolites for which DNA reaction was extensive. These metabolites were characterized by their chromatographic and spectrophotometric properties. The products of their microsome-induced DNA binding were isolated, and comparison with the products derived from cell-mediate 3MC-DNA reaction suggested that these metabolites might be precursors of this latter reaction. The chemical nature of the metabolites and of their DNA reaction products is discussed in relation to the "bay region" and carbonium ion concepts of the ultimate carcinogen of polycyclic hydrocarbons.
Animals↗
Cell-mediated mutagenesis in cultured Chinese hamster cells by carcinogenic polycyclic hydrocarbons: nature and extent of the associated hydrocarbon-DNA reaction.
A system of cell-mediated mutagenesis is described for the study of compounds which require metabolic activation to exert their cytotoxic and mutagenic effects. This system combines BHK21 cells for metabolism of the compounds and V79 cells as targets for mutagenesis. Using the two polycyclic hydrocarbon carcinogens benzo(a)pyrene and 7-methylbenz(a)anthracene we have shown that the hydrocarbon-DNA reaction which accompanies mutagenesis in the target cell is indistinguishable from that reported to occur in vivo and in primary cell cultures. Our results also support the view that a diol epoxide metabolite is responsible for the biological activity of benzo(a)pyrene. The application of cell-mediated mutagenesis to the routine testing of suspect environmental chemicals for biological activity is discussed.
Azaguanine↗
Mutagenicity of polycyclic aromatic hydrocarbons.
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Anthracenes↗
The role of 9-hydroxybenzo(a)pyrene in the microsome mediated binding of benzo(a)pyrene to DNA.
A study of the liver microsome-mediated binding to added DNA of the phenol metabolites of benzo(a)pyrene (BP-OH) and of 7,8-dihydro-7,8-dihydroxybenzo(a)pyrene (BP-7,8-diol) suggested that as in the case of BP itself the reaction was catalysed by the enzyme aryl hydrocarbon hydroxylase. The addition of glutathione to the microsomal incubation inhibited the binding of BP and BP-OH more than that of BP-7,8-diol. Analysis by LH20 chromatography of the deoxyribonucleoside products from BP-DNA showed greater inhibition by glutathione of formation of the major product believed to result from further metabolism of BP-OH, than of the product arising by metabolism of BP-7,8-diol. The chromatographic behaviour and fluorescence spectrum of this major product were consistent with its derivation from 9-hydroxybenzo(a)pyrene (BP-9-OH) and furthermore suggested that BP-9-OH-4,5-oxide was the derivative whose reaction with DNA yielded this microsome-mediated BP-DNA product.
Alkylation↗
The reaction of (+/-)-7alpha, 8beta-dihydroxy-9beta, 10beta-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene with DNA.
The reaction of (+/-)-7alpha,8beta-dihydroxy-9beta,10beta-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene (BP diol epoxide) with DNA, followed by enzymatic hydrolysis and LH 20 Sephadex column chromatography, yielded products containing a purine deoxyribonucleoside linked to a 7,8,9,10-tetrahydrobenzo(a)pyrene derivative. DNA containing tritiated adenine or pyrimidines gave little or no tritiated products, showing that reaction occurred chiefly with the guanine residues. Results with 8-(3H)-guanine-labelled DNA, and studies on the protonation and ionization of the products in acid or alkaline media, led to the formulation of the main product as an N2-substituted guanine derivative. The reaction of BP diol epoxide with deoxyadenosine and deoxyguanosine gave further nucleoside derivatives which were probably minor products in the reaction with DNA. Treatment with strong acid unexpectedly converted each of the isolated products to free guanine or adenine.
Acetates↗
Exceptional mutagenicity of a benzo(a)pyrene diol epoxide in cultured mammalian cells.
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Benzopyrenes↗
Rat liver microsome-mediated binding of benzo(a)pyrene metabolites to DNA.
Individual metabolites of benzo (a) pyrene were isolated from rat liver microsomal incubation of the parent hydrocarbon, and subsequently bound to DNA in separate incubations with microsomes. Of the metabolites examined, by far the greatest binding resulted with 7,8-dihydro-7,8-dihydroxy-benzo(a)pyrene (BP-7,8-diol); the binding od the benzo (a) pyrene phenols (BP-OH) was about 50% that of the 7,8-diol. Analysis of the hydrocarbon-deoxyribonucleoside derivatives obtained by enzymic degradation of these DNA samples revealed that the binding of BP-7,8-diol was accounted for mainly by a single product identical to one of the product identical to one of the products obtained from DNA with bound benzo(a)pyrene. Furthermore, the microsome-induced binding of BP-OH to DNA yielded mainly a single product identical in chromatographic behaviour to the major product derived from benzo(a)pyrene when bound to DNA during incubation with microsomes.
Animals↗
The 7-methylbenz(a)anthracene deoxyribonucleoside products isolated from DNA after metabolism of the carcinogen by rat liver microsomes in the presence of DNA.
Metabolism of 7-methylbenz[a]anthracene (7MeBA) by 3-methylcholanthrene-induced rat liver microsomes in the presence of added native or denatured DNA resulted in covalent binding of the hydrocarbon to the nucleic acid. Enzymatic degradation and column chromatographic fractionation showed that the hydrocarbon-deoxyribonucleoside products were separable from the products similarly obtained from DNA having 7MeBA bound following treatment of mouse embryo cells in culture with this hydrocarbon. Comparison of the microsome catalysed hydrocarbon-deoxyribonucleoside products with those obtained by reaction with DNA of 7MeBA-5,6-oxide suggested that this K-region epoxide made a significant contribution to the liver microsome-induced DNA binding.
Animals↗
Comparison of the metabolism of benzo[alpha]pyrene and binding to DNA caused by rat liver nuclei and microsomes.
Administration of 3-methylcholanthrene (3MC) to rats greatly enhanced the aryl hydrocarbon hydroxylase (AHH) activity of liver nuclei. However, the binding in vitro [3H]benzo[alpha]pyrene (BP) to DNA within the nuclei which occurred at the same time as hydroxylation of BP was much less enhanced. Thin layer chromatography of the metabolites of BP produced by these nuclei revealed the same metabolites in similar relative amounts as were produced by rat liver microsomes prepared from rats which had received 3MC. The binding to DNA was further analysed by hydrolysis of the DNA and fractionation on a Sephadex column. This analysis revealed that the binding to DAN in nuclei was very similar in nature to that which occurred when calf-thymus DNA was added to microsomes metabolising BP.
Animals↗