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DNA synthesis inhibition in response to benzo[a]pyrene dihydrodiol epoxide is associated with attenuation of p(34)cdc2: Role of p53.

ABSTRACT: Our previous findings demonstrated that DNA damage by polynuclear aromatic hydrocarbons (PAHs) triggers a cellular protective response of growth inhibition (G1-S cell cycle arrest and inhibition of DNA synthesis) in human fibroblasts associated with accumulation of p53 protein, a growth-inhibitory transcription factor. Here, we report that BPDE (the ultimate carcinogenic metabolite of the PAH benzo[a]pyrene) treatment triggers a variable extent of inhibition of DNA synthesis/cell growth, which does not correspond to the extent of increased p53 accumulation. BPDE treatment of cells significantly attenuates expression of p(34)cdc2, a cell cycle activating protein. Although the role of cdc2 down-regulation in inhibition of cell cycle progression is well known, cdc2 down-regulation in response to cellular insult by PAHs has not been reported. Unlike p53 accumulation, there is a correspondence between DNA synthesis/cell growth inhibition and cdc2 down-regulation by BPDE. BPDE-induced cdc2 down-regulation is p53 dependent, although there is no correspondence between p53 accumulation and cdc2 down-regulation. BPDE-induced cdc2 down-regulation corresponded with accumulation of the cell cycle inhibitor protein p21 (transactivation product of p53). DNA synthesis/cell growth inhibition in response to DNA-damaging PAHs may involve down-regulation of cdc2 protein mediated by p53 activation (transactivation ability), and the extent of p53 accumulation is not the sole determining factor in this regard.

SUBMITTER: Mukherjee JJ

PROVIDER: S-EPMC3743414 | biostudies-literature | 2013 Jul

REPOSITORIES: biostudies-literature

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DNA synthesis inhibition in response to benzo[a]pyrene dihydrodiol epoxide is associated with attenuation of p(34)cdc2: Role of p53.

Mukherjee Jagat J JJ Kumar Subodh S

Mutation research 20130518 1

Our previous findings demonstrated that DNA damage by polynuclear aromatic hydrocarbons (PAHs) triggers a cellular protective response of growth inhibition (G1-S cell cycle arrest and inhibition of DNA synthesis) in human fibroblasts associated with accumulation of p53 protein, a growth-inhibitory transcription factor. Here, we report that BPDE (the ultimate carcinogenic metabolite of the PAH benzo[a]pyrene) treatment triggers a variable extent of inhibition of DNA synthesis/cell growth, which d ...[more]

PMID: 23692869

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Project description:Benzo[a]pyrene (B[a]P) is a polycyclic aromatic hydrocarbon (PAH) commonly found in cigarette smoke, automobile exhaust fumes, grilled meat, and smoked food among others. Exposure to B[a]P is associated with a range of toxic effects including developmental, neurological, oxidative, inflammatory, mutagenic, carcinogenic and mortal. Efficient and more affordable experimental models like Drosophila melanogaster could provide more insight into the mechanism of PAH toxicity and help develop new strategies for prevention, diagnosis and treatment of PAH-related conditions. In this study, we examined the induction of some biochemical changes along with mortality and functional senescence by B[a]P and its metabolite, benzo[a]pyrene- 7,8-dihydrodiol-910-epoxide (BPDE) in the Canton-S strain of Drosophila melanogaster, with the aim to establish an alternative assay medium for B[a]P toxicity in flies. Flies were exposed to 2-200 μM of B[a]P and 1-10 μM of BPDE through diet for a seven-day survival assay followed by a four-day treatment to determine the effects of the compounds on negative geotaxis, fecundity and some biochemical parameters of oxidative damage. BPDE significantly reduced the survival rate of flies along the 7 days of exposure whereas B[a]P did not cause any significant change in the survival rate of flies. B[a]P and BPDE significantly reduced the climbing ability of flies after 4 days of exposure. Rate of emergence of flies significantly reduced at 10-200 μM of B[a]P and 5-10 μM of BPDE. Both compounds caused various levels of alterations in the values of reduced glutathione (GSH), total thiol (T-SH), glutathione-S-transferase (GST), catalase (CAT), hydrogen peroxide (H2O2), nitric oxide (NO) and acetylcholinesterase (AChE) of the flies. The compounds also exhibited high binding affinities and molecular interactions with the active site amino acid residues of Drosophila GST and the inhibitor binding site of Drosophila AChE in an in silico molecular docking analysis, with BPDE forming stable hydrogen bonds with AChE. Hence, the Canton-S strain of Drosophila melanogaster could offer a simple and affordable assay medium to study B[a]P toxicity.

Project description:This study demonstrates that benzo[g]chrysene-11,12-dihydrodiol (B[g]C-11,12-dihydrodiol) derived from the fjord-region parent hydrocarbon B[g]C is oxidized by rat AKR1C9 with a k c a t/ K m 100 times greater than that observed with the commonly studied bay-region benzo[ a]pyrene-7,8-dihydrodiol (B[a]P-7,8-dihydrodiol). Conversely, despite its strikingly similar structure to B[ g]C-11,12-dihydrodiol, benzo[ c]phenanthrene-3,4-dihydrodiol (B[ c]Ph-3,4-dihydrodiol) is consumed by AKR1C9 at sluggish rates comparable to those observed with B[ a]P-7,8-dihydrodiol. CD spectroscopy revealed that only the (+)-B[ g]C-11,12-dihydrodiol stereoisomer was oxidized, while AKR1C9 oxidized both stereoisomers of B[a]P-7,8-dihydrodiol and B[ c]Ph-3,4-dihydrodiol. The (+)- S, S- and (-)- R, R-stereoisomers of B[g]C-11,12-dihydrodiol were purified by chiral RP-HPLC. The 11 S,12 S-stereoisomer was oxidized at the same rate as the racemate. The 11 R,12 R-stereoisomer did not act as an inhibitor to AKR1C9, indicating that the (-)- R, R-stereoisomer was excluded from the active site. To understand the basis of stereochemical preference, we screened alanine-scanning mutants of active site residues of AKR1C9. These studies revealed that in comparison to the wild type, F129A, W227A, and Y310A enabled the oxidation of both the B[g]C-11 S,12 S-dihydrodiol and the B[g]C-11 R,12 R-dihydrodiol. Molecular modeling revealed that unlike B[a]P-7,8-dihydrodiol and B[ c]Ph-3,4-dihydrodiol, B[g]C-11,12-dihydrodiol enantiomers are significantly bent out of plane. As a consequence, the (-)- R, R-stereoisomer was prevented from binding to the active site because of unfavorable interactions with F129, W227, or Y310. Additionally, LC/MS validated that the product of the reaction of B[g]C-11,12-dihydrodiol oxidation catalyzed by AKR1C9 was B[g]C-11,12-dione, which was trapped in vitro with the nucleophile 2-mercaptoethanol. The similarity between rates of trans-dihydrodiol oxidation by the rat and human liver specific AKRs (AKR1C9 and AKR1C4) implicate these enzymes in hepatocarcinogenesis in rats observed with the fjord-region PAH.

Dataset Information

DNA synthesis inhibition in response to benzo[a]pyrene dihydrodiol epoxide is associated with attenuation of p(34)cdc2: Role of p53.

Publications

DNA synthesis inhibition in response to benzo[a]pyrene dihydrodiol epoxide is associated with attenuation of p(34)cdc2: Role of p53.

Similar Datasets

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