Binding Of Polycyclic Aromatic Hydrocarbons Research Articles

Polycyclic aromatic hydrocarbon binding macromolecules. Identification, characterization and temperature activation of a 4.5 S binding nucleoprotein.

A macromolecule binding 3H-methylcholanthrene (3H-MCA) and 3H-benzo(a)pyrene (3H-BaP) and sedimenting in the 4-5 S region of sucrose gradient (4.5 S) was identified in rat liver cytosol. The binding was displaced by 100-fold molar excess unlabeled ligands whereas 2,3,7,8-tetrachloro-dibenzo-p-dioxin (TCDD) was ineffective. The dissociation constant for both polycyclic aromatic hydrocarbons (PAHs) was of the order of 10(-8) M or lower. Both 3H-MCA and 3H-BaP bound to 4.5 S in a non covalent manner, since 92% of the bound radioactivity was extractable with ethyl ether. Furthermore the binding was strongly reduced by urea 8 M and by guanidine. HCl 4 M (99 and 70% respectively). Thin layer chromatography of the ethyl ether-solubilized radioactivity showed a peak comigrating with PAHs used as standards. When chromatographed on Sephadex G-200, 4.5 S was eluted as a sharp peak with an apparent molecular weight of 50-60,000 daltons. Enzyme treatment of liver cytosol showed that the 4.5 S binding sites were destroyed by micrococcal nuclease (92% of inhibition). Papain and phosphodiesterase I and II reduced the binding to 50%, whereas DNase I, DNase II, RNase, phospholipase A2 and C and trypsin were ineffective. These data suggest that the PAHs binding macromolecule of rat liver cytosol is a protein associated with a polynucleotide. The binding of both PAHs was enhanced by increasing the incubation temperature, the maximum being reached after 20-30 min at 37 degrees C. After 2.5 min at 65 degrees C, binding sites were completely destroyed. The same temperature-induced "activation" was obtained also by prewarming the cytosol at 37 degrees C in the absence of ligands.

Brain microsomal enzyme mediated covalent binding of benzo[a]pyrene to DNA

The covalent binding of benzo[a]pyrene (BP) to calf thymus DNA by brain microsomes isolated from control and 3-methylcholanthrene (3-MC) treated rats was investigated. The influence of incubation time, pH, and concentrations of protein, BP and NADPH on covalent binding was investigated to obtain optimum conditions for the in vitro binding of [3H]BP to DNA. Treatment of rats to 3-MC resulted in a 1.53-fold increase in the brain microsomal mediated covalent binding of [3H]BP to DNA. Inhibitors of monooxygenase enzyme activity such as alpha-naphthoflavone, metyrapone, 1-benzylimidazole and ellagic acid significantly inhibited the binding of [3H]BP to DNA from control and 3-MC stimulated brain microsomes. Our results indicate that inhibitors and inducers of monooxygenases may modulate brain enzyme-mediated binding of polycyclic aromatic hydrocarbons (PAHs) to DNA.

The physico-chemical speciation of polycyclic aromatic hydrocarbons (PAH) in aquatic systems

‘Physico-chemical speciation’ is presented as a concept to describe the environmental chemistry of organic compounds in aquatic systems, using polycyclic aromatic hydrocarbons as an example. Survey data and experimental results are reported which demonstrate the presence of different physico-chemical species of PAH in the Tamar Estuary, UK. The degree, and hence chemistry of binding of PAH to particulates in the Tamar is shown to be incompatible with current sorption modelling techniques. This finding has extensive repercussions on prediction of the aquatic chemistry of PAH, including availibility of the compounds for fates such as photo-oxidation and biological uptake/ toxicity.

The relationship between ionization potential and horseradish peroxidase/hydrogen peroxide-catalyzed binding of aromatic hydrocarbons to DNA

The ionization potentials (IP) of 91 alternant polycyclic aromatic hydrocarbons (PAH) were determined from the absorption maximum of the chargetransfer complex of each hydrocarbon and chloranil in chloroform. The extent of horseradish peroxidase (HRP)-catalyzed binding to DNA of 14 hydrocarbons of varying IP was measured. Only hydrocarbons with IP < approx. 7.35eV were significantly bound to DNA. These results provide further evidence that HRP-mediated binding of PAH to DNA occurs by one-electron oxidation and indicate that hydrocarbons must have IP < approx. 7.35eV to be activated by one-electron oxidation. Thus, determination of IP and HRP-catalyzed binding to DNA can offer some guidelines for selecting aromatic hydrocarbons which might undergo carcinogenic activation by this mechanism.

Perturbation of hydrogen bonds in the adenine . thymine base pair by Na+: A quantum chemical study.

Ab initio self-consistent field calculations with 4-31G and STO-3G basis sets show that Na+ are able to damage the H bonds in the formamidine . formamide complex, modeling the adenine . thymine base pair (both H bonds in the complex investigated are the same as in adenine . thymine). If a water molecule is placed between the complex and Na+, the H bonds of the model are fully protected. Covalent as well as noncovalent binding of polycyclic aromatic hydrocarbons to nucleic acids should decrease the local concentration of water as a result of the insertion of the hydrophobic hydrocarbon into the DNA and thereby increase the exposure of the DNA H bonds to Na+ attack. Thus, Na+ reaching the base pair could provoke or interfere with cell division.

Effect of vitamin A palmitate on mutagenesis induced by polycyclic aromatic hydrocarbons in human cells.

The effect of vitamin A palmitate (VAP) on mutagenesis induced by polycyclic aromatic hydrocarbons (PAH) was examined in a human epithelial-like cell line (EUE). Cultures were exposed to benzo[a]pyrene (BP), 3-methylcholanthrene (MCA), 7,12-dimethylbenz[a]anthracene, with or without VAP, and mutation frequencies were determined by selection against diphtheria toxin. A strong inhibition of mutagenesis was observed particularly with MCA and BP. VAP, in the same cell line, reduced 3H-labeled PAH binding to DNA.

The binding of benzo(a)pyrene to subcellular structures of rat liver

1. 1. The development of the polycyclic aromatic hydrocarbon binding protein in cytosol of rat liver has been studied. The highest amount was observed in the cytosol of the 11 day old rat (60 fmoles/mg protein). Adult levels (20 fmoles/mg protein) were reached at the age of 25 days. The fetal rat liver contains only minute amounts of the binding protein. 2. 2. Benzo(a)pyrene (BP) proved to be an inducer of the binding protein. This induction was age dependent, the highest induction (about 10-fold) being observed in liver of rats older than 30 days. No induction could be observed in the fetal liver, while during the period of high endogenous content of the binding protein, the content of the binding protein was lower after treatment of the rat with BP. A high endogenous amount of binding protein was found to parallel a high degree of induction of cytosolic NAD(P)H dehydrogenase (DT-diaphorase) by BP. 3. 3. The induction of the binding protein proved to be sensitive to SKF-525A. Since SKF-525A does not induce the binding protein, it is concluded that metabolic conversion of BP is required for the induction. 4. 4. BP bound to the 100,000 g supernatant of rat liver was bound to nuclei, nuclear envelopes, and microsomes. The dissociation constant for this binding was found to be 0.3 nM. The highest amount of binding was observed in nuclei isolated from the adult rat liver. Fetal rat liver nuclei demonstrated about 40 × less binding of BP bound to 100,000 g rat liver supernatant than adult rat liver nuclei.

The stimulation of murine hepatic aryl hydrocarbon hydroxylase activity in vitro by caffeine.

The activity of aryl hydrocarbon hydroxylase (AHH) of murine liver is increased by caffeine. However, other analogs of caffeine, such as theophylline, theobromine xanthine, hypoxanthine and uric acid, do not significantly alter AHH activity. The maximal stimulation is observed at a caffeine concentration of approx. 3 mM. The Km of AHH for benzo[a]pyrene is decreased in the presence of caffeine. Thus, the inhibition by caffeine of the binding of polycyclic aromatic hydrocarbons (PAH) to DNA may be related to its effect on AHH.

Covalent binding of polycyclic aromatic hydrocarbons to adenine correlates with tumorigenesis in mouse skin

Mouse epidermal homogenates were utilized to convert various polycyclic aromatic hydrocarbons to metabolites capable of binding covalently with nucleic acids. Poly(G) showed the highest capacity to bind covalently with the hydrocarbons; however, there was no correlation between binding to poly(G) and mouse skin tumorigenicity. On the other hand, covalent binding to poly(A) correlated well with values obtained for binding to DNA and mouse skin tumorigenicity. The order of binding to poly(A) was; 7,12-dimethylbenza[a]anthracene greater than benzo[a]pyrene greater than dibenz[a,h]anthracene greater than dibenz[a,c]anthracene.

Binding of polycyclic aromatic hydrocarbons to DNA of cells in culture: A rapid method for its analysis using hydroxylapatite column chromatography

A rapid procedure to study the interaction of carcinogens with DNA in cultured cells has been developed. The cells, which are labeled with 7,12-[3H]dimethylbenz[a] anthracene ([3H]DMBA), are lysed with 0.24 M phosphate buffer (pH 6.8), 1% sodium dodecyl sulfate (SDS), 8 M urea and 0.01 M ethylenediamine-tetraacetate (EDTA) and sonicated. The cell lysates are fractionated on columns of hydroxylapatite. Proteins and RNA are removed with 8 M urea in 0.24 M phosphate buffer (pH 6.8). DMBA-bound DNA is eluted with 0.4 M phosphate buffer (pH 6.8). DMBA-DNA isolated by this procedure is virtually free from proteins and RNA. Thermal stability, ultraviolet spectra and the density of DNA is not altered by DMBA binding. The uptake of DMBA by mouse epidermal cells is rapid and the binding of DMBA to DNA is linear for the first 8 h of exposure. DMBA binds to DNA in all phases of the cell cycle. However, the highest binding occurs immediately following maximum DNA synthesis.

Characteristics of DNA during binding of polycyclic aromatic hydrocarbonsin 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.

Dose-dependent preferential binding of polycyclic aromatic hydrocarbons to reiterated DNA of murine skin cells in culture.

The distribution of active metabolites of polycyclic aromatic hydrocarbons bound to reiterated or unique regions of murine DNA has been studied by a DNA-DNA renaturation technique. Murine skin cells were exposed to different doses of radioactive polycyclic aromatic hydrocarbons for 24 hr; then the hydrocarbon-labeled DNA was isolated, fragmented, and denatured. Renaturation kinetics and thermal stabilities of DNA-DNA duplexes were studied. At high carcinogen doses, polycyclic aromatic hydrocarbon adducts seem to be distributed equally among the DNA of all reiteration frequencies. At low carcinogen doses, however, a dose-dependent preferential binding to reiterated DNA sequences occurs. An inverse linear relationship appears to exist between the enrichment of hydrocarbon adducts in reiterated DNA sequences and the logarithm of the amount of total hydrocarbon bound to DNA.

The mechanism of binding of polycyclic aromatic hydrocarbons to nucleic acids: A theoretical investigation

AbstractA kinetic model employing calculated atom and bond localization energies to approximate relative activation energies of reaction is used to analyse previously obtained experimental results for in vitro and in vivo chemical binding of polycyclic aromatic hydrocarbons to nucleic acids. It is found that in vitro linkage of hydrocarbons to DNA induced by a microsomal hydroxylating system is compatible with mechanisms involving either attack at the most reactive hydrocarbon center or attack at the most reactive hydrocarbon bond. Independent evidence leads us to favor the former mechanism. Further, the limited experimental data for in vivo linkage of hydrocarbons to DNA is found to be consistent with a model involving attack at the most reactive bond of the molecule: the “K region”. This model is supported by a close parallelism found between extent of hydrocarbons bound to DNA in vivo and the experimentally determined relative reactivities of their K regions.

Binding of polycyclic aromatic hydrocarbons to polyadenylic acid.

A number of polycyclic aromatic hydrocarbons bind to the double-stranded, acid form of polyadenylic acid (poly A). Model building shows that these hydrocarbons may intercalate in the helix, and be well protected from contact with the aqueous medium. Hydrocarbons that are too large to be so protected are found not to bind. A size criterion for the binding of hydrocarbons to poly A therefore exists. This criterion differs from one that was previously found for DNA. The size criteria for DNA and poly A, together, serve as strong evidence for the intercalation model for hydrocarbon complexes.Model-building experiments show that only a small portion of the hydrocarbon need extend into the medium to prevent binding. This finding implies that in two cases (1,2,5,6-dibenzanthracene.poly A and 3,4-benzpyrene.DNA) the structure of the complex is almost completely determined by the size criterion alone.