ABSTRACT
The potent environmental carcinogen benzo[a]pyrene (BaP), following enzymatic activation to enantiomeric pairs of bay-region 7,8-diol 9, 10-epoxides (the benzylic 7-hydroxyl group and epoxide oxygen are cis for DE-1 diastereomers and trans for DE-2 diastereomers), reacts with DNA to form covalent adducts predominately at the exocyclic amino groups of purines. Specific adducts, corresponding to the trans opening of each of the four optically active BaP DE isomers at C-10 by the N 2-amino group of dG, were synthesized as appropriately blocked phosphoramidites and were incorporated at either the first or second G of codon 12 within the G-rich sequence of human K-ras codons 11-13: GCT G1G2T GGC. The adducted oligonucleotides were incorporated into plasmids by primer extension, followed by purification of the covalently closed circular constructs. Adducts derived from either (+)- or (-)-DE-2, placed at either G1 or G2, presented strong blocks to in vitro transcription elongation by bacteriophage T3 RNA polymerase, but only moderately blocked transcription elongation by human RNA polymerase II in nuclear extracts. Adducts derived from all four DEs, placed on either G1 or G2, were used as substrates in a DNA repair synthesis assay using human whole cell extracts. Adducts derived from three of the DE stereoisomers exhibited significant amounts of repair synthesis, but the (-)-DE-2 adduct experienced no repair synthesis above that of the control. Constructs containing a pre-existing nick at the sixth phosphodiester bond 3' to either (+)-DE-2 or (-)-DE-2 adducts exhibited increased repair synthesis.
Subject(s)
7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide/chemistry , Codon , DNA Adducts/chemistry , DNA Repair , Genes, ras , Oligonucleotides/chemical synthesis , Oligonucleotides/metabolism , Bacteriophage T3/enzymology , Base Sequence , Cell Line, Transformed , Cell Nucleus/drug effects , Cell Nucleus/genetics , DNA-Directed RNA Polymerases/genetics , Humans , Lymphocytes/metabolism , Molecular Sequence Data , Oligonucleotides/chemistry , Stereoisomerism , Transcription, Genetic/drug effectsABSTRACT
The carcinogen (+)-(1R,2S,3R,4S)-3,4-dihydroxy-1,2-epoxy-1,2,3,4- tetrahydrobenzo[c]phenanthrene (in which the 4-OH group and epoxide oxygen are cis) was reacted with duplexes formed from self-complementary oligodeoxyribonucleotides, producing 1S or 1R configured adducts through trans or cis epoxide ring opening, respectively, by the exocyclic amino group of a central target A. Sequences containing 5'-AT-3' generated much higher S vs R ratios than the average of 3.38 observed with calf thymus DNA samples, while sequences containing 5'-TA-3' generated much lower ratios. Sequences with G in the position immediately 5' to the central AT or TA, and C in the position immediately 3', generated moderately higher ratios than did sequences with adjacent 5'C and 3'G. When thymidine was replaced by deoxyuridine in several sequences, the ratios of S vs R configured dA adducts and dA adducts vs dG adducts were substantially and uniformly reduced, but otherwise varied with the choice of nearest neighbors in patterns similar to those observed with the T containing sequences. Two hypothetical mechanisms are proposed to explain the effect of nearest neighbors on the S vs R dA adduct ratio; in both, diol epoxide intercalation precedes covalent bonding. In one mechanism, intercalation to the 5' side of the target A yields an S adduct while intercalation to the 3' side yields an R adduct, and the extent of adduct formation follows the nearest neighbor series G > C > T.(ABSTRACT TRUNCATED AT 250 WORDS)
Subject(s)
Carcinogens/chemistry , DNA Adducts/chemistry , DNA/chemistry , Deoxyadenosines/chemistry , Nucleic Acid Conformation , Phenanthrenes/chemistry , Animals , Base Sequence , Cattle , Macromolecular Substances , Molecular Sequence Data , Oligodeoxyribonucleotides/chemistry , Structure-Activity RelationshipABSTRACT
Although the solution chemistry of diol epoxides is now fairly well understood, a great deal remains to be elucidated regarding their reaction in the presence of DNA. Not only DNA but also small molecules are capable of sequestering diol epoxides in aqueous solutions with equilibrium constants on the order of 10(2)-10(4) M-1. In the case of DNA, at least two major families of complexes are presently recognized, possibly the result of groove binding vs. intercalation. As is the case for diol epoxides free in solution, the complexed diol epoxides undergo solvolysis to tetraols and in some cases possibly to keto diols as well. Fractionation between covalent bonding and solvolysis from within the complex(s) is determined more by the nature of the parent hydrocarbon from which the diol epoxide is derived than any other factor. Studies of a wide variety of alkylating and arylating agents have show that practically every potentially nucleophilic site on DNA can serve as a target for modification. In the case of the diol epoxides, practically all of the modification occurs at the exocyclic amino groups of the purine bases. In contrast to the diol epoxides, other epoxides such as those derived from aflatoxin B1, vinyl chloride, propylene, 9-vinylanthracene, and styrene preferentially bind to the aromatic ring nitrogens N-7 in guanine and N-3 in adenine (cf. Chadha et al., 1989). Molecular modeling as well as the spectroscopic evidence suggests that the hydrocarbon portion of the diol epoxides lies in the minor groove of DNA when bound to the exocyclic 2-amino group of guanine and in the major groove when bound to the exocyclic 6-amino group of adenine. Detailed conformational analysis of adducted DNA should prove to be extremely valuable in developing mechanistic models for the enzymatic processing of chemically altered DNA. At present, the critical lesion or lesions responsible for induction of neoplasia remains obscured by the large number of apparently noncritical adducts which form when polycyclic hydrocarbon diol epoxides bond to DNA.
Subject(s)
DNA/metabolism , Epoxy Compounds/metabolism , Animals , Base Sequence , Binding Sites , DNA/chemistry , Epoxy Compounds/chemistry , Epoxy Compounds/pharmacology , Kinetics , Molecular Structure , Mutagens , Polycyclic Compounds/chemistry , Polycyclic Compounds/metabolism , Polycyclic Compounds/pharmacologyABSTRACT
The restricted ability of deoxyribonuclease I/snake venom phosphodiesterase digestion to liberate deoxyadenosine (dA) nucleotide adducts of polycyclic aromatic hydrocarbons from DNA, first observed by Dipple and Pigott with the bay-region diol epoxide adducts of 7,12-dimethylbenz[a]anthracene, has been observed with the dA adducts of benz[a]anthracene and benzo[c]phenanthrene diol epoxides. The micrococcal nuclease/spleen phosphodiesterase digestion used in the original 32P postlabeling procedure developed by Randerath to determine DNA adducts also failed to liberate dA nucleotide adducts quantitatively. Thus either method can potentially lead to an underestimation of the extent to which dA has been modified in DNA. The two digestion procedures exhibit systematic and mostly opposite stereoselectivity in the pattern of which dA adducts are resistant to digestion, which suggest that these adducts may have preferred orientations within modified DNA that are determined by whether they have the R or S configuration at C-1, the point of attachment between the exocyclic amino group of dA and the hydrocarbon; this in turn is dictated by the configuration about the precursor benzylic epoxide carbon and the cis versus trans nature of epoxide opening during adduct formation.
Subject(s)
DNA/metabolism , Deoxyadenosines/metabolism , Deoxyribonuclease I/pharmacology , Phosphoric Diester Hydrolases/pharmacology , Polycyclic Compounds/metabolism , Micrococcal Nuclease/pharmacology , Phosphodiesterase I , Phosphorus Radioisotopes , StereoisomerismABSTRACT
Mesityl oxide and isophorone, two beta-methylated-alpha,beta-unsaturated industrial solvent ketones, were found to be converted to mutagens by aqueous chlorination under conditions of pH and reactant concentration that may be relevant to waste water and drinking water chlorination. Chlorination of millimolar concentrations of isophorone generated mutagens at a pH as low as 8.5, while mutagens were formed from submillimolar concentrations of mesityl oxide at pH 8.5, or millimolar concentrations at pH 7.5. It is suggested that mutagen formation can occur via a haloform reaction at such low pH levels because of extended resonance stabilization of an intermediate carbanion.
Subject(s)
Chlorine , Hydrocarbons, Chlorinated , Ketones , Mutagens , Chemical Phenomena , Chemistry , Chlorine/adverse effects , Hydrocarbons, Chlorinated/toxicity , Hydrogen-Ion Concentration , Ketones/toxicity , Mutagenicity TestsABSTRACT
XAD resins are commonly used to recover and concentrate organics from chlorinated water. It was found that the action of residual chlorine on XAD-4 resin produced mutagenic artifacts in a dose dependent manner. The production of mutagenic artifacts could be suppressed at least ten-fold by converting free chlorine to monochloramine. Kinetic studies of the reaction between free chlorine and XAD-4 resin showed a reaction rate dependence upon pH and chloride ion concentration that suggests participation of species besides hypochlorous acid.
Subject(s)
Chlorine , Mutagens/chemical synthesis , Polystyrenes , Polyvinyls , Chemical Phenomena , Chemistry , Chloramines/chemical synthesis , Hydrogen-Ion Concentration , Kinetics , TemperatureABSTRACT
A mutagenesis assay employing the frog, Rana pipiens, is currently under development [McKinnell et al, 1979]. A question that must be answered is whether the frog is metabolically capable of activating a large number of promutagens. The Ames assay offers a simple means of comparing the metabolism of mutagens by different animal species. The Ames response obtained with frog-liver S-9 was compared to the response with rat-liver S-9, using the following compounds: Benzo(a)pyrene, 7,12-dimethylbenz(a)anthracene, 2-amino-fluorene, azobenzene, Sudan II, dibutylnitrosamine, hydrazine sulfate, hydroxyethylhydrazine, cyclophosphamide, 1,2-dichloroethane, tris(2,3 dibromopropyl)phosphate, diallate, quinoline, quercetin, aflatoxin B 1, emodin, and safrole. Of these compounds, activation by rat S-9 was observed for all except hydrazine sulfate and safrole. All except Sudan II, 1,2-dichloroethane, quinoline, and safrole gave positive Ames responses with frog S-9. In general, the responses with frog S-9 were quantitatively lower than those obtained with Aroclor-induced rat S-9; however, the optimum procedure for frog-liver induction has not been determined. The response to dichloroethane is very sensitive to the amount of activating enzyme present; it might be positive with optimally induced frog S-9. Thus, only two of the 15 compounds positive with rat S-9 were definitely missed when tested with frog S-9. We feel that the frog assay appears to be promising from the standpoint of false-negatives.
Subject(s)
Liver/metabolism , Mutagens/pharmacology , Animals , Carcinogens/pharmacology , Female , Humans , Infant, Newborn , Male , Mutagenicity Tests , Rana pipiens , Rats , Salmonella typhimurium/drug effects , Species SpecificityABSTRACT
In concentrates of water produced in a laboratory simulation of a drinking water treatment process, direct-acting, nonvolatile mutagens were readily detected by means of the Ames Salmonella test. The mutagens were shown to be produced by the chlorination process. Treatment of the water with chloramine resulted in less mutagenic activity than treatment with free chlorine. Dechlorination of drinking water with sulfite sharply reduced the mutagenic activity. Treatment with sulfur dioxide is proposed as an effective, inexpensive method of reducing the direct-acting mutagenic activity of drinking water and of aqueous industrial effluents.