Evaluation of DNA damage in the oral mucosa of tobacco users and non-users by ³²P-adduct assay

Abstract

Tobacco and its combustion products contain several known or potential human carcinogens and studies are now beginning to emerge for detecting DNA and protein adducts in tobacco users. A highly sensitive 32P-adduct assay, capable of measuring a wide spectra of aromatic and/or hydrophobic DNA adducts, was used to evaluate tobacco use-related adducts in human oral mucosal cells. Two volunteer groups of people participated: one, a random group of cigarette smokers (n = 28) with non-smoker controls (n = 13) from our Institute, and the other, a fisherfolk community of tobacco chewers (n = 19) with appropriate non-chewer controls (n = 15) from Bombay. The cells were dislodged from the oral cavity with either a cotton swab or a toothbrush. One to as many as 16 adduct spots in the range of 5-200 amol/micrograms DNA were detected in tobacco users as well as non-users. No adduct spot was detected in the tobacco users' samples that was not present in the controls, suggesting that tobacco use may not be responsible for the formation of these DNA adducts detected in the oral mucosa. The chromatographic behavior of the oral mucosal DNA adducts by selective PEI--cellulose TLC and their extractability in 1-butanol suggest that they have an aromatic and/or hydrophobic moiety and two of the major adducts have been tentatively assigned as aromatic amine derivatives. We have discussed various possibilities of endogenous and/or exogenous factors being involved in the formation of these adducts.

DNA Adducts in Human Tissues: Biomarkers of Exposure to Carcinogens in Tobacco Smoke

“…One of the adducts was at higher levels in some of the exposed groups, while two adducts were at reduced levels. Another study of tobacco smokers, tobacco chewers, and tobacco non-users found low levels of DNA adducts in oral mucosal cells from all three groups using the butanol extraction procedure of the 32P-postlabeling assay (27). None of the adducts detected were found to be specifically associated with tobacco smoking or chewing.…”

The analysis of DNA adducts: The transition from 32P-postlabeling to mass spectrometry

“…This extra step extended the detection limit to a single adduct in 10 9 –10 10 nucleotides while maintaining the extremely low sample requirements of only 1–10 μg of DNA [19]. In 1986, Reddy and Randerath [20] used Nuclease P1 enrichment to achieve similar sensitivity and in 1988 [21], reversed-phase high performance liquid chromatography (HPLC) was used for the separation of unadducted from adducted nucleotides, as well as for separation of adducts with different structures into individual fractions which were then visualized on TLC plates, significantly diminishing the detection complexity. Fig.…”

“…3 shows data comparing butanol and nuclease P1 enrichment in conjuction with 32 P-postlabeling in a study of DNA adducts from tobacco chewers [1,21]. In this case, butanol enrichment was proven to be superior to nuclease P1 enrichment in the isolation of adducts from oral mucosa [1,21]. While nuclease P1 treatment works well with PAH adducts, it does not work well with aromatic amines or C8-dG adducts [22–24].…”

Biomonitoring of tobacco smoke carcinogenicity by dosimetry of DNA adducts and genotyping and phenotyping of biotransformational enzymes: a review on polycyclic aromatic hydrocarbons

“…Initial reports on dosimetry of DNA adducts in the oral mucosa cells were inconclusive both in smokers versus non-smokers and in di erent groups of tobacco users, such as betel nut chewers, Khaini tobacco chewers and inverted smokers (Dunn andStich 1986, Chacko andGupta 1988). Subsequently, NP1-and butanol-enhanced 32 P-postlabelling analyses showed a higher level of adducts in the oral mucosa cells of smokers and suggested the aromatic amines and/or nitroaromatic nature of these adducts (Jones et al 1993, Stone et al 1995.…”