Preferential modification of replicating DNA by benz(a)pyrene)

The nuclei of cells from regenerating rat liver were incubated with benzo(a)pyrene and the concentrations of the metabolites that covalently bound to DNA of different nuclear fractions were compared. It appeared that DNA associated with nuclear matrix (containing replicating DNA) is modified most intensively. The synchronized mouse embryo cells were incubated with benzo(a)pyrene during S phase and the levels of modifications in short and long single-stranded DNA fragments were compared. It has been observed that replicating DNA is represented in short fragments. These short DNA fragments were found to be modified by benzo(a)pyrene 4-9 times more intensively than total DNA. The possible mechanisms of both the increase in the number of DNA modifications in proliferating cells and the reason for the enhancement of carcinogenic effect on dividing cells are being discussed.     

Repair in the nuclear matrix of DNA damaged by benz(a)pyrene

The confluent culture of hamster embryo cells was incubated with benzo(a)pyrene for 24 hours. Then the medium was replaced by maximal lacking both the serum and benzo(a)pyrene. The process of DNA repair was observed in four nuclear fractions according to two indexes: the disappearance of metabolites of benzo(a)pyrene covalently bound to DNA and the incorporation of 3H-thymidine to DNA in the period from I min to 72 hours. Hydroxyurea at the concentration of 5 mM was added 2-19 hours before 3H-thymidine. The highest concentration of benzo(a)pyrene metabolites was found in the DNA of nuclear matrix fraction throughout all the experiment. The initial concentration of 3H-thymidine right after its addition into the cell culture medium was the highest in DNA of nuclear matrix fraction and the lowest in DNA fraction soluble in the buffer with low ionic strength. Later on, the concentration of 3H-thymidine was decreased in matrix-bound fractions and increased in other fractions up to the total DNA level. The results suggest that the repair process requires joining of benzo(a)pyrene damaged DNA region to the nuclear matrix with the following reverse transition into the fraction where the fragment was initially located.     

Effect of benz(a)pyrene on a monolayer culture of normal and malignant mouse liver cells]

Both the cells of monolayer culture of mouse embryonic liver and that of highly malignant hepatoma 22A transplanted for 20 years actively metabolized the carcinogenic hydrocarbon benz(a)pyrene and were highly sensitive to iits toxic action. Since hepatic tissue was resistant in vivo to carcinogenic carbohydrates it is suggested that the resistance depended on factors acting at the organ or the organism but not as the cellular level. The mechanism of retention of hepatoma 22A sensitivity to the toxic action of benz(a)pyrene is also discussed.     

UDP-glucuronosyl transferase and the conjugation of benzo(a)pyrene metabolites to DNA.

Addition of UDP-glucuronic acid to microsomal incubations containing benzo(a)pyrene caused a dose-dependent conjugation of principally quinone and phenol metabolites. Total benzo(a)pyrene oxidation was also stimulated with a maximum increase at 2 nM UDPGA. In the presence of calf thymus DNA, UDPGA caused a 2.7-fold increase in benzo(a)pyrene diol-oxide modification of DNA, as analyzed by Sephadex LH-20 chromatography. Maximum DNA modification by diol-oxides occurred at a UDPGA concentration which gave the highest level of free benzo(a)pyrene 7,8-dihydrodiol; likewise, the amount of DNA adduct derived from benzo(a)pyrene phenols declined in parallel with levels of free phenol metabolites. The UDPGA-induced increase in benzo(a)pyrene oxidation and concomitant increase in diol-oxide modification of DNA is consistent with removal of product inhibition by glucuronide conjugation of an inhibitory benzo(a)pyrene metabolite.     

Mutagenicity of non-K-region diols and diol-epoxides of benz(a)anthracene and benzo(a)pyrene in S. typhimurium TA 100

When incubated with a 9,000 x g rat-liver supernatant, benzo(a)pyrene 7,8-diol and benz(a)anthracene 8,9-diol were more active than the parent hydrocarbons in inducing his⁺ revertant colonies of S. typhimurium TA 100. Benzo(a) pyrene 9,10-diol was less active than benzo(a)pyrene; the K-region diols, benz(a)anthracene 5,6-diol and benzo(a)pyrene 4,5-diol, were inactive. None of the diols was active when the cofactors for the microsomal mono-oxygenase were omitted. The diol-epoxides benzo(a)pyrene 7,8-diol 9,10-oxide, benz(a)anthracene 8,9-diol 10,11-oxide and 7-methylbenz(a)anthracene 8,9-diol 10,11-oxide and the K-region epoxides, benzo(a)pyrene 4,5-oxide and benz(a)anthracene 5,6-oxide, were mutagenic without further metabolism.     

Repair of DNA damaged by mutagenic metabolites of benzo(a)pyrene in human cells

The repair of human DNA after damage by known and potential metabolites of benzo(a)pyrene has been examined utilizing the bromodeoxyuridine photolysis assay. Repair was characterized as either ultraviolet (“long”) or ionizing radiation type (“short”) repair utilizing normal cells and cells deficient in ultraviolet-type repair endonuclease from a patient with xeroderma pigmentosum (XP). We have found that only (±)-7β,8-dihydroxy-9β,-10β-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene (BP diol epoxide 1) and its disastereomer, (±)-7β,8,-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene (BP diol epoxide 2) elicit damage to DNA which is recognizable by the ultraviolet excision repair system in normal human cells. Benzo(a)pyrene 4,5-, 9,10-, 11,12-oxides do not elicit damage which is repairable by this repair system. The 1,2-diol-3,4-epoxides from naphthalene have no measurable activity in our assay. These results indicate that both the benzo(a)pyrene ring structure and the diol epoxide groups are important in causing the damage to DNA which is repairable by the ultraviolet excision repair system. These results parallel the reported high mutagenic activity of these compounds and support the concept that benzo(a)pyrene 7,8-diol-9,10-epoxides may be the ultimate, metabolically activated forms of benzo(a)pyrene.     

The dose-effect of benz(a)pyrene in organ cultures of embryonic mouse lungs resistant and predisposed to pulmonary blastomogenesis

A direct effect of benz(a)pyrene (BP) was studied on organ cultures from embryonic lungs of C57Bl and A mice. The toxic effect of 3, 6, 12 micrograms/ml BP was observed in alveolar epithelium and mesenchymal cells after 7 days of cultivation. After 14-21 days diffuse and focal hyperplasia of the epithelium was discovered in 35.5, 20.0 and 36.1% of treated cultures (explants) from embryonic lungs of C57Bl mice and in 45.5, 56.3 and 53.2% of treated explants from embryonic lungs of A mice. Squamous epithelial metaplasia was observed in 3.0, 2.2 and 4.2% of treated explants from C57Bl mice and in 2.4, 10.4 and 23.4% of treated explants from A mice. Total papillary adenomatous growth of the epithelium was discovered in 10.0, 18.3 and 36.1% of treated explants from embryonic lungs of A mice only. Thus, a direct effect of BP on organ cultures from embryonic lungs of C57B1 and A mice depended on BP doses and the mouse line.     

Metabolism of benzo(a)pyrene by isolated hepatocytes and factors affecting covalent binding of benzo(a)pyrene metabolites to DNA in hepatocyte and microsomal systems

Covalent binding of benzo(a)pyrene (BP) metabolites to DNA was investigated in hepatocytes and liver microsomes (MC-microsomes) isolated from 3-methylcholanthrene-treated rats. The major DNA adducts formed during BP metabolism in both hepatocytes and incubations of calf thymus DNA with MC-microsomes were adducts of anti and syn isomers of trans-7,8,-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene (diol-epoxides) and of epoxide derivatives of BP-9-phenol (phenol-oxides). Diol-epoxide adducts predominated over phenol-oxide adducts in hepatocytes, while the reverse was found in microsomal incubations. In hepatocytes, both diol-epoxide and phenol-oxide adducts increased with increasing BP concentration; the ratio of diol-epoxide adduct to phenol-oxide adduct decreased from 6:1 to 3:1 between 30 and 100 μm BP. In microsomal incubations, decreases in DNA concentration or addition of the hepatocyte L15 medium produced larger decreases in phenol-oxide adducts than in diol-epoxide adducts. The effects of the inhibitors salicylamide, diethylmaleate, and 3,3,3,-trichloropropene oxide on formation of BP-DNA adducts are interpreted in terms of changes in precursor formation and metabolism and reductions in hepatocyte glutathione levels. Addition of 1.5 mg/ml exogenous DNA to hepatocyte incubations produced no change in covalent binding to cellular DNA, even though extracellular BP-DNA adducts accounted for 97% of the total adducts formed. Both the relative amounts of diol-epoxide and phenol-oxide adducts and the total adducts per milligram of DNA were indistinguishable with respect to extracellular and intracellular DNA. Modification of extracellular DNA by diol-epoxides was at least as efficient as modification of calf thymus DNA in incubations with MC-microsomes. It is concluded that BP diol-epoxides and phenol-oxides can leave the cell or enter the nucleus with equal facility but are more effective in binding to DNA in the cell in which they are generated.     

Potassium cyanate modification of the toxic and mutagenic effects of gamma radiation and benzo(a)pyrene

In experiments with CHO-AT3-2 cell culture, a study was made of the effect of potassium cyanate (KNCO) on the effect of gamma radiation and benzo(a)pyrene (BP) by the following tests: cell viability, induction of cells with micronuclei and fragmented nuclei and mutations by thymidine kinase (TK) and Na+/K+-ATPase loci. Some tests have revealed the increase in the effect of gamma radiation and BP produced by potassium cyanate. It is suggested that the sensitizing effects are related to repair system inhibition and/or changes in the cell chromatin structure produced by KNCO.     

Benzo(a)pyrene diolepoxide-haemoglobin and albumin adducts at low levels of benzo(a)pyrene exposure

A biomonitoring study was conducted to simultaneously measure individual benzo(a)pyrene (BaP) exposure in 50 office employees, not occupationally exposed to polycyclic aromatic hydrocarbons (PAH), using personal samplers and the formation of (+) r-7, t-8-dihyroxy-t-9,t-10-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene (BPDE) adducts to haemoglobin (BPDE-Hb) and serum albumin (BPDE-SA). The population enrolled was exposed to an average of 0.58 ± 0.46 ng BaP m^-3 (mean ± SD). The concentration of BaP collected from smokers' samples was double that from non-smokers (P = 0.007). BPDE adducts to Hb and SA were quantified as BaP tetrols released from hydrolysis of macromolecules and measured by high-resolution gas chromatography-negative ion chemical ionization-mass spectrometry. BPDE-Hb adducts were detected in 16% of the population and BPDE-SA adducts in 28%. Smoking did not affect adduct formation. When BaP personal monitoring data were used as the criterion of exposure, no correlation was found with the presence and the levels of BPDE-Hb and BPDE-SA adducts. Undetected sources of PAH, such as the diet, might markedly alter the exposure profile depicted by individual air sampling and affect the frequency and levels of protein biomarkers. This is the first comparative analysis of BPDE-Hb and BPDE-SA adducts, providing reference values for these biomarkers in a general urban population. However it is difficult to establish which biomarkers would be the more relevant in assessing low BaP exposure, due to undetectable factors such as dietary PAHs, that might have influenced the results to some degree.     

Interaction of benz[a]pyrene diol epoxide with chromatin studied by flow linear dichroism

Chromatin isolated from Ehrlich ascites cells was incubated with the tumourigenic compound (+)-7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10- tetrahydrobenz[a]pyrene [(+)-anti-BPDE] at low ionic strength and the modified chromatin was analysed using flow linear dichroism (LD). The results confirm that (+)-anti-BPDE preferentially binds to the DNA in the linker regions, and furthermore show that the long axis of the bound pyrenyl chromophore is oriented parallel or close to parallel to the average orientation of the chromatin fiber axis. The data indicate that the binding geometry of (+)-anti-BPDE in chromatin is similar to that in pure DNA and deoxyguanosine-containing double-helical oligonucleotides.