Analysis of the repair of benzo[a]pyrene- and dibenzo[a,l]pyrene-diol epoxide-DNA adducts in human and rodent cells in culture

Laura Jean Schild, Purdue University

Abstract

Polycyclic aromatic hydrocarbons (PAH) are carcinogenic combustion products readily linked to an increase in cancer incidence in humans. Benzo[ a]pyrene and dibenzo[a,l]pyrene are two ubiquitous pollutants that are metabolized into reactive anti-(R,S,S,R) diol epoxides that can covalently bind to DNA. It has been determined that human cells removed benzo[a]pyrene diol epoxide (BPDE)-DNA adducts more efficiently from the transcribed strand of the hypoxanthine phosphoribosyl transferase (HPRT) gene, while rodent cells did not utilize transcription-coupled repair (TCR) to remove the BPDE adducts from the dihydrofolate reductase (DHFR) gene. These studies were conducted with a bacterial repair complex to incise DNA at the sites of BPDE adducts. We have used a laser-induced cleavage procedure to quantitatively cleave at PAH-DNA adducts, combined with Southern blot analyses to assess the repair of BPDE- and (−)-anti-DB[ a,l]P-11,12-diol-13,14-epoxide (DB[a,l]PDE)-DNA adducts in the DHFR gene in human and rodent cells. Human embryonic kidney cells demonstrated a bias for repair of the transcribed strand of the DHFR gene at early times after 1 μM BPDE exposure. CHO-B11 cells exposed to 0.5 μM DB[a,l]PDE did not demonstrate any bias for repair in the transcribed strand of the DHFR gene. These results indicate a role for TCR in the response of human cells to BPDE exposure, while rodent cells do not utilize TCR for DB[a,l]PDE-DNA adducts. Xeroderma Pigmentosum (XP) and Cockayne's Syndrome (CS) are rare human genetic diseases associated with mutations in specific proteins involved in nucleotide excision repair. XPA cells lack repair, whereas XPC cells have TCR. CSA cells have global DNA repair. XP and CS cells were treated with 1 μM BPDE and the BPDE-DNA adducts were analyzed. Human diploid fibroblasts and CSA cells removed a majority of the BPDE-DNA adduct levels by 6 hours after exposure. In contrast, no change was detected in adduct levels in XPA cells during 12 hours after exposure. XPC cells treated with BPDE exhibited a 2-fold reduction of BPDE adduct levels by 4 hours, then the adduct levels remained constant, indicating that TCR is involved in the initial repair response to BPDE-induced DNA damage.

Degree

Ph.D.

Advisors

Baird, Purdue University.

Subject Area

Biochemistry|Molecular biology

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