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Binding of [3H]benzo(a)pyrene to natural and synthetic nucleic acids in a subcellular microsomal system
Abstract:
Several carcinogens are bound covalently to cellular nucleic acids. This is also the case with polycyclic hydrocarbon carcinogens, but their precise mechanism of in vivo activation to reactive forms and the structure(s) of the nucleic acid adducts are not known. This study demonstrates that in the presence of rat liver microsomes and reduced nicotinamide adenine dinucleotide phosphate there is covalent attachment of tritiated benzo(a)pyrene (BP) to transfer RNA, DNA, certain synthetic polyribonucleotides, and an RNA species endogenous to the microsomal fraction. Evidence has been obtained that the binding occurs mainly to guanine and, to a lesser extent, adenine residues and is not simple an artifact of tritium exchange. The microsomal-mediated binding of [3H]BP to nucleic acids requires reduced nicotinamide adenine dinucleotide phosphate and in inhibited by 7,8-benzoflavone, glutathione, and magnesium. It is enhanced somewhat by the addition of styrene oxide, cyclohexene oxide, and trichloropropylene oxide. These results provide the first evidence that: (a) the microsome-mediated binding of [3H]BP to nucleic acids is not just due to tritium exchange; (b) a derivative of the hydrocarbon is covalently bound to the nucleic acid, and not simply intercalated; (c) there is a preferential binding to guanine residues; and (d) in addition to binding to exogenous nucleic acids, [3H]BP is bound to an RNA species present in the microsomes. Our data are consistent with but do not prove that nucleic acid binding of this polycyclic hydrocarbon proceeds via an epoxide intermediate.
Insights
Polycyclic aromatic hydrocarbons like benzo(a)pyrene covalently bind to DNA and RNA. This study shows microsomes activate these carcinogens, forming adducts primarily on guanine residues, not just tritium exchange artifacts.
Area of Science:
- Biochemistry
- Molecular Biology
- Carcinogenesis
Background:
- Carcinogens, including polycyclic hydrocarbons, form covalent bonds with cellular nucleic acids.
- The exact in vivo activation pathways and resulting nucleic acid adduct structures for polycyclic hydrocarbon carcinogens remain largely unknown.
Purpose of the Study:
- To investigate the mechanism of in vivo activation of benzo(a)pyrene (BP) by rat liver microsomes.
- To identify the nucleic acid adducts formed by BP and determine the binding sites on nucleic acids.
Main Methods:
- Incubation of tritiated benzo(a)pyrene ([3H]BP) with rat liver microsomes and reduced nicotinamide adenine dinucleotide phosphate (NADPH).
- Analysis of covalent binding of [3H]BP to exogenous nucleic acids (tRNA, DNA, synthetic polyribonucleotides) and endogenous microsomal RNA.
- Investigation of factors affecting binding, including inhibitors (7,8-benzoflavone, glutathione, magnesium) and enhancers (epoxides).
Main Results:
- Demonstrated covalent attachment of [3H]BP to various nucleic acids and endogenous microsomal RNA in the presence of microsomes and NADPH.
- Evidence indicates preferential binding to guanine residues, with some binding to adenine residues.
- Microsomal-mediated binding requires NADPH and is inhibited by 7,8-benzoflavone, glutathione, and magnesium, suggesting a metabolic activation process.
Conclusions:
- Microsomal activation leads to covalent binding of a BP derivative to nucleic acids, not merely tritium exchange or intercalation.
- Binding predominantly occurs at guanine residues.
- The findings support, but do not confirm, an epoxide intermediate in the nucleic acid binding of polycyclic hydrocarbons.