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.     

Phenols as toxic metabolites of benz(a)pyrene

The effect of benzo(a)pyrene and two its phenolic metabolites 3-hydroxybenzo(a)-pyrene and 6-hydroxybenzo(a)pyrene--on the cultures of normal and transformed fibroblasts has been studied. In was shown that unlike the parent carcinogen its phenolic metabolites exerted only toxic (but not transforming) effect on cultured cells, and this effect has been developed at a faster rate than that produced by benzo(a)pyrene. 6-hydroxybenzo(a)pyrene was more toxic than 3-hydroxybenzo(a) pyrene. It was concluded that both metabolites produced their effects without preliminary activation by microsomal enzymes.     

Sequence-specific modification of DNA by 6-hydroxybenzo[a]pyrene

6-Hydroxybenzo[a]pyrene cleaved phi X174 supercoiled DNA to open circular DNA in the presence of heavy metal ions. It induced an alkali-labile modification in DNA via an oxygen-radical-mediated reaction; the most frequent alkali-labile sites were on the 3' side of the pyrimidine residues of the pyrimidine cluster.     

High ratio of alkali-sensitive lesions to total DNA modification induced by benzo(a)pyrene diol epoxide

The ratio of alkali-labile lesions to total DNA adducts for DNA modified by an active metabolite of benzo(a)pyrene was investigated using DNA sequencing methodology. About 40% of the adducts formed result in alkali-labile sites. About 25% of the lesions were alkali-labile at positions of guanine, 10% at adenine, and 5% at cytosine. This study highlights the potential role of adducts other than the N2-substituted guanine in mutagenic and carcinogenic effects of benzo(a)pyrene.     

Preferential association of newly synthesized histones with replicating SV40 DNA.

The assembly of newly synthesized histones into nucleosomes during replication of SV40 minichromosomes in vivo was studied. Infected cells were labeled with 35S-methionine for a time shorter than that required to complete a round of viral DNA replication. Mature and replicating SV40 minichromosomes were extracted and separated by zonal sedimentation, and their histone content was analyzed by polyacrylamide gel electrophoresis (SDS and acidic urea). We show that the pulse-labeled histones associate preferentially with the replicating DNA.     

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.     

Preferential modification of guanine bases in DNA by dimethyldioxirane and its application to DNA sequencing

From gel sequencing experiments with 32P-end-labelled oligodeoxyribonucleotides, it is shown that treatment of DNA with the powerful oxidant dimethyldioxirane, followed by heating in piperidine, causes selective strand scission at the sites of guanine bases. The same specificity for cleavage at guanine was observed with a 45-mer labelled at either the 3'- or 5'-end and with a single and double stranded 34-mer. On account of its speed and operational simplicity, modification with dimethyldioxirane is proposed as a practicable alternative to conventional chemical sequencing procedures for locating guanine bases in DNA.     

Reactivity with DNA of three pyrenofuran analogues of benzo(a)pyrene and benzo(e)pyrene

Three pyrenofurans, the pyreno[1,2-b]furan (FP1), the pyreno[2,1-b] furan (FP2) and the pyreno[4,5-b]furan (FP3) have been synthesized as analogues of the mutagenic and carcinogenic benzo(a)pyrene (FP1 and FP2) and of its non-carcinogenic isomer benzo(e)pyrene (FP3). For each of the pyrenofurans, the reactivity with DNA has been tested in presence of liver microsomes of rats induced with 3-methylcholanthrene. Fluorescence spectroscopy showed that only FP2 and FP3 which possess a "bay region" react with DNA. In both cases, metabolites bound to DNA have a fluorescence emission comparable to that of the "bay region" dihydrodiols obtained after the "in vitro" metabolism of initial molecules. FP2 is shown to react similarly to benzo(a)pyrene whereas the reactivity of FP3 is different from that of benzo(e)pyrene, in spite of their structural similarities. This is probably due to reasons of three-dimensional space configuration. The peculiar reactivity of FP3 is predicted by calculations of the bond order values.     

Role of poly(ADP-ribose) glycohydrolase silencing in DNA hypomethylation induced by benzo(a)pyrene

Benzo(a)pyrene (BaP) is a known carcinogen cytotoxic which can trigger extensive cellular responses. Many evidences suggest that inhibitors of poly(ADP-ribose) glycohydrolase (PARG) are potent anticancer drug candidates. However, the role of PARG in BaP carcinogenesis is less understood. Here we used PARG-deficient human bronchial epithelial cell line (shPARG cell) as an in vitro model, and investigated the role of PARG silencing in DNA methylation pattern changed by BaP. Our study shows, BaP treatment decreased global DNA methylation levels in 16HBE cells in a dose-dependent manner, but no dramatic changes were observed in shPARG cells. Further investigation revealed PARG silencing protected DNA methyltransferases (DNMTs) activity from change by BaP exposure. Interestingly, Dnmt1 is PARylated in PARG-null cells after BaP exposure. The results show a role for PARG silencing in DNA hypomethylation induced by BaP that may provide new clue for cancer therapy.     

Early replicating DNA in heterochromatin ofPlagiochila ovalifolia (Liverworts)

The timing of DNA replication of heterochromatin in malePlagiochila ovalifolia was investigated by the use of³H-thymidine autoradiography. The estimated duration of the mitotic cycle was as follows: S period, 19 hr: G₂+prophase, 10 hr; G₁+meta-, ana-, telophase, 5 hr; total mitotic cycle, 34 hr. The first appearance of silver grains over the chromosomes was observed at 8 hr after the beginning of pulse labelling at which time the silver grains were only over the euchromatic regions, not over the heterochromatic regions. This labelling pattern was also observed at 10 to 15 hr. The heterochromatic regions having more grains than the euchromatic regions were observed at 20 to 25 hr. These results show that the DNA of the heterochromatin of this species is replicated earlier than the euchromatin.     

Cellular binding of benzo(a)pyrene to DNA characterized by low temperature fluorescence

A new approach has been developed to detect ultra low concentrations of benzo(a)pyrene products bound to nucleic acids $\text{in}\text{vivo}$. The binding to DNA of hamster embryo cell cultures was characterized by low temperature fluorescence spectroscopy. The method can detect less than one polycyclic hydrocarbon residue per 50,000 nucleotides. The fluorescence spectra indicate that the benzo(a)pyrene derivative bound to DNA has a pyrene-like chromophore and resembles that obtained when DNA is reacted $\text{in}\text{vitro}$ with the 7,8-diol-9,10-oxide of benzo(a)pyrene. This confirms that metabolism of the 7,8,9,10 ring on benzo(a)pyrene precedes reaction with DNA. The method should be useful for detecting and characterizing the $\text{in}\text{vivo}$ binding of other fluorescent carcinogens to nucleic acids.     

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.     

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.     

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.