Binding of benzo(a)pyrene metabolites and derivatives to DNA : characterization using optical spectroscopy techniques

• DNA with covalently bound benzo(a)pyrene (BP) derivatives was studied with fluorescence spectroscopy, fluorescence decay measurements, fluorescence quenching measurements, light absorption and linear dichroism of flow oriented samples. The modified DNA was prepared in three different ways: (1) by incubating calf thymus DNA with rat liver micromomes, necessary cofactors and benzo(a)pyrene or derivatives of benzo(a)pyrene, (2) by isolating DNA.from rat hepatocytes after incubation with the hydrocarbon, or (3) by reacting benzo(a)pyrene-diolepoxides directly with calf thymus DNA in aqueous solution. Among the benzo(a)pyrene metabolites and derivatives tested only BP, BP-7,8-dihydrodiol and the two phenols 9-OH-BP and 2-OH-BP gave any significant binding to DNA after metabolism as revealed by the fluorescence measurements.Our data show that the strong carcinogen BP-7,8-dihydrodiol is metabolized to a DNA-binding BP-dihydrodiol-9,10- epoxide, in agreement with previous studies. This epoxide is generally considered to be the major carcinogenic metabolite of BP itself. We found that the weak carcinogen 9-OH-BP is probably also metabolized to a DNA-binding epoxide, with the oxygen bound to carbon atoms 4 and 5. Further studies showed that also 2-OH-BP may be metabolized in a similar way as BP itself, i.e. with diol and epoxide formation in the angular benzo ring.The covalent adducts of calf thymus DNA with the two diastereomers of benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE), the strong carcinogen anti-BPDE and the weak carcinogen syn-BPDE, were studied in some detail. Fluorescence spectroscopy and quenching as well as linear dichroism studies showed that the DNA adduct of the major metabolite of anti-BPDE exhibits moderatly strong interaction with DNA and has its molecular plane close to parallel with the undisturbed axis of the DNA helix. The DNA adduct of the major metabolite of syn-BPDE has significantly different properties, close to those expected from an intercalated complex in the classical sence.Taken together, the results presented in this thesis show that fluorescence and other optical spectroscopic techniques can be used to characterize the DNA binding metabolites of BP and to describe many physico-chemical properties of their DNA complexes. The aim is of course to find out if and in what way the properties of the complexes between DNA and a potential chemical carcinogen determine its carcinogenic potency in a living organism. The present studies have given at least a few descriptive answers to some questions in this field of knowledge.

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NATURVETENSKAP  -- Kemi (hsv//swe)

NATURAL SCIENCES  -- Chemical Sciences (hsv//eng)

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