Lung DNA Adducts Detected in Human Smokers Are Unrelated to Typical Polyaromatic Carcinogens

Abstract

Several studies have reported the presence of DNA adducts derived from benzo(a)pyrene and other polyaromatics by 32P-postlabeling/TLC by measuring diagonal radioactive zones (DRZs) in lung tissues of human smokers. However, our experimental studies in rodent models, which used modified chromatographic conditions to obtain distinct adduct spots, suggested that cigarette smoke-related lipophilic DNA adducts may not be derived from polycyclic aromatic hydrocarbons (PAHs) or aromatic amines. In the present study, we have performed similar analysis of human lung tissues to study the chemical nature of DNA adducts. Fifty human lung tissues from cancer patients (ages 42-83 years) with active, ex-, or never-smoking status were analyzed for highly lipophilic DNA adducts by nuclease P1- and n-butanol enrichment-mediated 32P-postlabeling assay. All DNA samples yielded low to highly intense adduct DRZs when adducts were resolved by PEI-cellulose TLC in standard high-salt, high-urea solvents. Adduct burden ranged from 6.6 to 2930 per 10(10) nucleotides. However, when adducts were resolved in a different solvent system comprising of high-salt, high-urea in direction 3 and dilute ammonium hydroxide in direction 4, which retained adducts derived from PAHs and aromatic amines on the chromatograms, this yielded no detectable adducts from human lung DNAs. Furthermore, analysis of human lung DNAs mixed with reference adducted DNAs in multisolvent systems confirmed an absence of PAH- and aromatic amine-derived adducts in human smoker lung DNA. To determine the origin of cigarette smoke-associated DNA adducts, calf thymus DNA was incubated with formaldehyde and acetaldehyde, which are known to be present in cigarette smoke in significant quantities. Analysis of purified DNAs by 32P-postlabeling resulted in adduct DRZs in the aldehyde-modified DNAs when adducts were resolved in standard urea-containing solvents, but no adducts were detected when the ammonium hydroxide-based solvent was used, suggesting that even nonpolyaromatic electrophiles can result in adduct DRZs on the chromatograms similar to those from PAH metabolites. Taken together, our data demonstrate that cigarette smoke-associated lung DNA adducts appear on chromatograms as DRZs, consistent with the literature, but they are not related to PAHs and aromatic amines.

Mutagenicity and Sequence Specificity of Acrolein-DNA Adducts

“…Gupta laboratory (35) recently reported that the majority of diagonal radioactive zones (DRZs) detected in cigarette smokers using 32 P post-labeling and 2 dimensional thin layer chromatograph (2D-TLC) method are not PAHs and aromatic amines. Interestingly, these workers also found that DNA modified with acetyl aldehydes and formaldehydes produced the same DRZs as DNA from lung tissues of cigarette smokers.…”

Progress and Challenges in Selected Areas of Tobacco Carcinogenesis

“…Disadvantages include lack of chemical specificity, particularly in 32 P-postlabeling analyses, and difficulty in quantitation. Although the adducts detected using this method are often referred to in the literature as "aromatic DNA adducts", there is strong evidence that they are not related to PAH (63). The application of these methods to tissues obtained from smokers has been extensively reviewed (61).…”

Correlation of CLPTM1L polymorphisms with lung cancer susceptibility and response to cisplatin-based chemotherapy in a Chinese Han population

“…They found that the "G/G" genotype of rs402710 was associated with significantly higher levels of DNA adducts (p=0.02). High levels of bulky aromatic and/or hydrophobic DNA adducts in lung tissue from smokers are formed by DNA-binding polycyclic aromatic hydrocarbons (PAHs) and by aldehydes found in cigarette smoke [34,35]. Smokingrelated DNA adducts can lead to mutation of key genes, such as TP53, and ultimately result in tumor development [36].…”