Non-enzymatic and microsome-dependent binding of polycyclic hydrocarbons to DNA and polynucleotides

The binding of tritium-labelled 7,12-dimethylbenz[a]anthracene (DMBA), benzo[a]pyrene (BP) and 3-methylcholanthrene (MCA) to DNA or polynucleotides in vitro was re-examined both in the presence and in the absence of rat liver or human placental microsomes.A high level of non-enzymatic binding was evident when thymus DNA was used as acceptor. This non-enzymatic binding made it difficult to determine the effect of microsomes, except in the case of BP when induced rat microsomes were used. Better results were obtained using polynucleotides: a definite microsome-dependent binding occurred between all the polynucleotides and all the hydrocarbons tested.No clear evidence of binding catalysed by microsomes from human placenta was found except in polynucleotide-BP interactions: further studies are required to completely evaluate the ability of such nucleic acid-microsomal system for testing in vitro possible oncogenic substances in animals and humans.     

Xanthine oxidase-catalyzed DNA binding of dihydrodiol derivatives of nitro-polycyclic aromatic hydrocarbons

Xanthine oxidase, a mammalian nitroreductase, catalyzed the covalent binding of a series of nitro-polycyclic aromatic hydrocarbons (nitro-PAHs) trans-dihydrodiols to DNA. Some of the trans-dihydrodiols bound to DNA to a greater extent than their parent nitro-PAHs; however, when the dihydrodiol moiety was peri to the nitro substituent low levels of binding were observed. These data illustrate that ring-oxidation and hydrolysis of nitro-PAHs to their trans-dihydrodiols followed by nitroreduction is a potential metabolic pathway leading to DNA adducts in mammals.     

Characteristics of DNA during binding with polycyclic aromatic hydrocarbons in vitro]

Polycyclic aromatic hydrocarbons (PAH) were covalently bound to DNA by means of various activating systems. The following systems were used: the microsomal fraction of the rat liver, the system with I2, the system with ascorbic acid and FeSO4. Breaks in DNA due to the activating systems action appeared in all of these systems. Plateau of the PAH binding system curve in the microsomal system cannot be attributed either to the fall of the PAH metabolism rate to zero, or to the PAH binding sites in DNA. This plateau is the result of equalization of the rates of the two contrary-directed processes: the binding of metabolites and their removal due to DNA degradation. Because of the breaks in DNA caused by the activating systems, the authors failed to discover the changes in sedimentation data of DNA due to the covalently bound PAH.     

The relationship between ionization potential and prostaglandin H synthase-catalyzed binding of aromatic hydrocarbons to DNA

A series of polycyclic aromatic hydrocarbons (PAH) with ionization potentials (IP) ranging from 7.7 to 6.96 eV were examined for their ability to bind to DNA in vitro via the prostaglandin H synthase (PHS)-catalyzed co-oxidation of PAH. A relatively low IP was required for significant binding of PAH to DNA to occur. PAH with IP below approximately 7.35 eV were active in the binding reaction, while those with IP at or above 7.35 eV exhibited much lower levels of binding. Among the PAH which bound at high levels, there was no correlation between the extent of binding and IP. The results suggest that the prostaglandin H synthase-mediated binding of PAH to DNA occurs by one-electron oxidation, similarly to previously reported horseradish peroxidase (HRP)-catalyzed binding of PAH to DNA.     

Polycyclic aromatic hydrocarbons and PAH-related DNA adducts

Investigations on the impact of chemicals on the environment and human health have led to the development of an exposome concept. The exposome refers to the totality of exposures received by a person during life, including exposures to life-style factors, from the prenatal period to death. The exposure to genotoxic chemicals and their reactive metabolites can induce chemical modifications of DNA, such as, for example, DNA adducts, which have been extensively studied and which play a key role in chemically induced carcinogenesis. Development of different methods for the identification of DNA adducts has led to adopting DNA adductomic approaches. The ability to simultaneously detect multiple PAH-derived DNA adducts may allow for the improved assessment of exposure, and offer a mechanistic insight into the carcinogenic process following exposure to PAH mixtures. The major advantage of measuring chemical-specific DNA adducts is the assessment of a biologically effective dose. This review provides information about the occurrence of the polycyclic aromatic hydrocarbons (PAHs) and their influence on human exposure and biological effects, including PAH-derived DNA adduct formation and repair processes. Selected methods used for determination of DNA adducts have been presented.     

DNA damage in humans exposed to environmental and dietary polycyclic aromatic hydrocarbons

The paper describes recent research on human DNA damage related to environmental and dietary polycyclic aromatic hydrocarbon (PAH) exposures. The study populations either represent general populations of large geographical regions, or their exposure situation may have relevance to the general population. In Silesia, Poland, and Northern Bohemia, Czech Republic, where coal-based industry and domestic heating are the major sources of PAHs, significant differences have been observed in white blood cell DNA adducts and cytogenetic biomarkers between environmentally exposed and rural control populations, and significant seasonal variations of DNA damage have been detected. Bus drivers, traffic policemen and local residents have been involved in biomarker studies in Copenhagen, Athens, Genoa and Cairo, and differences have been measured in the level of DNA damage of urban and rural populations. Burning of smoky coal in unvented homes in Xuan Wei region, China, causes high PAH exposure of residents, which has been reflected in DNA adduct levels in different tissues. Indoor wood burning in open fireplaces did not increase human DNA adduct levels. Oil-well fires left burning in Kuwait after the Persian Gulf war created an unprecedented environmental pollution. However, insignificant environmental PAH levels were measured several miles from these fires. Aromatic and PAH-DNA adduct levels in white blood cells of US Army soldiers were lower during their deployment in Kuwait, than in Fulda, Germany, where they were stationed before and after serving in Kuwait. The contribution of dietary PAH exposure to blood cell DNA adduct levels had been demonstrated in studies in which volunteers consumed heavily charbroiled beef. Environmental tobacco smoke did not cause detectable changes, as measured by 32P-postlabelling, in DNA adduct levels in non-smokers. In the reviewed studies, observed DNA adduct levels were generally in the range of 1 to 10 adducts, and not higher than 40 adducts in 108 nucleotides. Typically, 1.5 to 3-fold differences have been detected in DNA adduct levels between the exposed and control groups.     

The electrical properties and stability of DNA to UV radiation and aromatic hydrocarbons

Studies of the d. c. conductive behavior of solid gels of DNA under high vacuum have shown that a reproducible energy gap of 2.4 eV and a photoelectric effect may be obtained following extensive evacuation procedures. Admission of air for short periods has a reversible effect on this photoelectirc effect and semiconductive behavior. After prolonged illumination in air the sample becomes permanently affected and will not reproduce the photoelectric effect or give rise to a semiconduction energy gap of 2.4 eV but give rise to a permanently lower energy gap of 1.6 eV. The effect of ultraviolet irradiation on the thermal denaturation of DNA in the presence of six aromatic hydrocarbons has been examined in order to determine the extent to which the presence of the hydrocarbon protects the DNA against ultraviolet damage.     

Photoproducts from DNA pyrimidine bases and polycyclic aromatic hydrocarbons

The major photoproduct formed between benzo[a]pyrene and thymine is identified as 1-(benzo[a]pyren-6-yl)-thymine by means of spectroscopic analysis and isotopic syntheses. Irradiation of 1-methylcytosine hydrochloride and anthracene gives two isolable photoproducts of which one is assigned the structure 5-(anthracen-9-yl)-1-methylcytosine.     

Repair of bulky DNA lesions deriving from polycyclic aromatic hydrocarbons

Genomic DNA is damaged by a variety of factors exerting an adverse effect on human health, such as environmental pollution, UV light, ionizing radiation, and toxic compounds. Air pollution with products of incomplete combustion of hydrocarbon fuels and wastes of various industries are main sources of polycyclic aromatic hydrocarbons, whose metabolites can damage DNA by forming bulky DNA adducts, which potentially lead to mutations and cancer. Nucleotide excision repair is the main pathway that eliminates these lesions in eukaryotic cells. The excision efficiency of bulky adducts depends on many factors, including the structure of a bulky substituent and the degree of DNA double helix distortion induced by a lesion. Clustered DNA lesions are the most dangerous for the cell. Several DNA repair systems cooperate to recognize and remove such lesions. The review focuses on the mechanisms that repair DNA with single and clustered bulky lesions, taking the natural carcinogen benzo[a]pyrene as an example.     

Applicability of the black slug Arion ater for monitoring exposure to polycyclic aromatic hydrocarbons and their subsequent bioactivation into DNA binding metabolites

The applicability of terrestrial black slugs Arion ater (Mollusca, Gastropoda) was studied for biomonitoring environmental exposure to polycyclic aromatic hydrocarbons (PAHs). In laboratory experiments, slugs were orally exposed to benzo[a]pyrene (BaP) for a short term (3 days) or a long term (119 days) period. Test animals were collected in the field, or were reared under laboratory conditions to ensure that they had no history of PAH-exposure. Benzo[a]pyrene hydroxylase (BPH) activity was measured in the digestive gland as a biomarker for BaP exposure. Bulky DNA adduct formation in kidney was measured as an effect biomarker for BaP bioactivation into DNA-binding metabolites. Although success of clutching was relatively low (5 out of 18 slugs produced egg packages), sufficient number of slugs were obtained to perform exposure experiments due to high hatching (89%) and survival rates (79%). After a short exposure to a relatively high BaP doses of 20 and 200 microg/g fresh feed, a dose-dependent and significant increase of BPH activity and bulky DNA adduct levels could be demonstrated in A. ater. Induction factors were low (two times control level), but optimization of the test conditions yielded a higher BPH induction factor of 4.8 times control level. BPH activity and bulky DNA adduct levels, however, did not increase after a long-term exposure to environmentally relevant BaP doses (upto 0.25 microg/g fresh feed). Based on this lack of response after realistic exposure it is concluded that A. ater is not sensitive to BaP exposure and, therefore, not suitable for monitoring environmental exposure to PAHs.     

Electrostatic contribution to the energy of binding of aromatic ligands with DNA

The method of solution of the nonlinear Poisson-Boltzmann equation was used to calculate electrostatic energy of binding of various aromatic ligands with DNA oligomers of different length. Analysis of the electrostatic contribution was made in terms of a two-step DNA binding process: formation of the intercalation cavity and insertion of the ligand. The total electrostatic energy was also partitioned into components: the energy of atom-atom coulombic interactions and the energy of interaction with surrounding water. The results indicate that electrostatic interactions are, as a whole, unfavorable to the intercalation process and that a correct analysis of structure-energy interrelation for Ligand-DNA interactions should only be accomplished at the level of the components rather than at the level of total electrostatic energy.     

Polycyclic aromatic hydrocarbons physically intercalate into duplex regions of denatured DNA

We have investigated the physical binding of pyrene and benzo[a]pyrene derivatives to denatured DNA. These compounds exhibit a red shift in their absorbance spectra of 9 nm when bound to denatured calf thymus DNA, compared to a shift of 10 nm when binding occurs to native DNA. Fluorescence from the hydrocarbons is severely quenched when bound to both native and denatured DNA. Increasing sodium ion concentration decreases binding of neutral polycyclic aromatic hydrocarbons to native DNA and increases binding to denatured DNA. The direct relationship between binding to denatured DNA and salt concentration appears to be a general property of neutral polycyclic aromatic hydrocarbons. Absorption measurements at 260 nm were used to determine the duplex content of denatured DNA. When calculated on the basis of duplex binding sites, equilibrium constants for binding of 7,8,9,10-tetrahydroxy-7,8,9,10-tetrahydro-benzo[a]pyrene to denatured DNA are an order of magnitude larger than for binding to native DNA. The effect of salt on the binding constant was used to calculate the sodium ion release per bound ligand, which was 0.36 for both native and denatured DNA. Increasing salt concentration increases the duplex content of denatured DNA, and it appears that physical binding of polycyclic aromatic hydrocarbons consists of intercalation into these sites.     

Comparison of in vivo and in vitro binding of polycyclic hydrocarbons to DNA

The in vivo binding of [³H]benzo(a)pyrene (BP) and 3-[³H]methylcholanthrene (3MC) to liver and lung DNA was studied in A/J mice. Only in liver was there any reduction in total DNA-bound radioactivity between 4 h and 24 h after administration of the hydrocarbon. DNA was fractionated on Sephadex LH-20 after enzymatic digestion. A single deoxyribonucleoside-BP adduct was detected whereas two major 3MC-adducts were observed. With both BP and 3MC, three additional peaks of radioactivity eluted rapidly in the lung DNA experiments while a fourth was noted with liver DNA. The nucleoside-bound adducts from lung represented a much larger proportion of the total radioactivity than with liver. In vitro analysis of 3MC binding to DNA showed the nucleoside-bound adducts to be predominantly deoxyguanosine-dependent but that the early peaks were independent of base suggesting binding to another part of the DNA molecule, perhaps phosphate, i.e., phosphotriesters.