Modulation of novel DNA adducts during human uterine cervix cancer progression

Abstract

Progressive accumulation of DNA lesions leads to genetic mutations that are central to the process of tumorigenesis. Human cervix provides an ideal system to determine progressive accumulation of DNA adducts in the target tissue because of its accessibility during routine diagnostic checkups. Uterine cervix samples from various pathologies, i.e. normal (n=13), inflammation (n=9), dysplasia (n=5) and different stages of invasive cancer (n=47), were analyzed for DNA adduct burden by modified 32 P-postlabeling/TLC systems. Six subgroups of adducts were detected in the following descending order of polarities: P-1, P-2, PL-1, PL-2, L-1 and L-2 (P, polar; L, lipophilic; PL, between polar and lipophilic). No qualitative differences were observed in adduct profiles in the various cervix pathologies analyzed. However, significant quantitative differences were found. Previously known lipophilic adducts increased significantly from normal to cancer (144±61 to 503±51 adducts/10 9 nucleotides). Interestingly, the newly discovered polar adducts were present at 61-to 527-fold higher levels than lipophilic adducts. Of all the polar adducts, the known mutagenic lesion, 8-oxodeoxyguanosine, predominated in all cervix conditions. Notably, this lesion was elevated 27-fold in inflammation compared with normal cervix (51,058±9,863 versus 1,886±507 adducts/10 9 nucleotides). The P-1, PL-1, PL-2 and L-1 adducts were elevated 3-to 13-fold in inflammation compared with normal cervix, and were also higher in dysplasia and cancer. Our data suggest that inflam-mation may be involved in directing the course of disease progression by accumulating higher levels of DNA lesions. The data further suggest the biomarker potential of the newly detected array of DNA adducts. Abbreviations: 8-OxodG, 8-oxodeoxyguanosine; CIN, cervical intraepithelial neoplasia; DRZ, diagonal radioactive zone; HPV, human papillomavirus Key words: DNA adducts, oxidative DNA damage, 32 P-postlabeling, cervical cancer, inflammationFigure 3. Scatter plots highlighting the inter-individual variability of different subgroups of adducts in various cervical pathology.

Potent Chemopreventive/Antioxidant Activity Detected in Common Spices of the Apiaceae Family

“…After adding 4E 2 (50 μM; 2% ethanol) and CuCl 2 (50 μM), the reaction mixture was further incubated at 37 °C for 4 h, and the DNA was isolated by solvent- extraction and ethanol precipitation as described (18, 20). The isolated DNA was tested for oxidative DNA adducts.…”

“…One of them is oxidative DNA damage contributed by redox activity of endogenous and exogenous species. To assess the DNA damage caused by ROS, 32 P-postlabeling followed by 2-dimensional polyethyleneimine-cellulose thin-layer chromatography (PEI-cellulose TLC) is commonly used (18). This method has an advantage of detecting a variety of polar adducts in addition to the oxidative DNA lesion 8-oxo-2’-deoxyguonosine (8-oxodG) (19) .…”

Prevention of Oxidative DNA Damage by Bioactive Berry Components

“…Remaining 2 µg of the digest was enriched for 8-oxo-7,8-dihydroguanine (8-oxodG) by polyethyleneimine (PEI)-cellulose TLC and 0.5-1 µg was labeled as described (31). The 5 -32 P-labeling of both enriched DNA adducts and normal nucleotides were done in parallel by T4-polynucleotide kinase and molar excess of [γ − 32 P] adenosine triphosphate as described earlier (26,27). Labeled adducts were separated by 2-D PEIcellulose TLC using 50 mM sodium phosphate, pH 6.0, and 1 M formic acid in the D1 direction.…”

“…D2 was perpendicular to D1 using a solvent mixture of isopropanol:4M ammonium hydroxide:8M urea (3.3:1:1.6). Adducts with decreasing polarities in tissue DNA were eluted by increasing the sodium phosphate concentrations (50 mM-1,000 mM) in the presence of 1 M formic acid (D1) but maintaining the same D2 solvent (27). The adduct subgroups were classified based on their polarity (P) or lypophillicity (L), with the most polar subgroups named P-1 and the most lipophillic subgroups as L-1 and the adducts in between as PL-1, 2, etc.…”

“…All chemicals used were >95% pure and were used without further purification. st-DNA was freed from contaminating RNA and protein prior to use as described previously (26,27).…”