Safrole-DNA adduct in hepatocellular carcinoma associated with betel quid chewing

Betel quid chewing, which contributes high concentration of safrole in saliva, is a popular oral habit in Taiwan. Safrole is a documented rodent hepatocarcinogen, yet its hepatocarcinogenic potential in human is not known. Here, we used LC/ESI-ITMSn and LC/QTOF-MS confirmed safrole-dGMP as reference standard to detect the safrole-DNA adduct in hepatic tissues from HBsAg-/HCV-seronegative hepatocellular carcinoma patients by 32P-postlabeling. We first synthesized and confirmed safrole-dGMP by LC/MS. Two isomeric safrole-dGMPs were characterized as N2-(trans-isosafrol-3′-yl) deoxyguanosine and N2-(safrol-1′-yl) deoxyguanosine. This technique was able to detect hepatic safrole-DNA adduct in mice that were treated with safrole but not sensitive enough to detect safrole-DNA adduct in human samples. Using the nuclease P1 version of the 32P-postlabeling technique, we detected the presence of safrole-DNA adduct in two out of 28 hepatic tissues from hepatocellular carcinoma patients, and only these two patients had a history of betel quid chewing lasting more than 10 years. From co-chromatography with the mass confirmed safrole-dGMPs, this safrole-DNA adduct was identified as N2-(trans-isosafrol-3′-yl) deoxyguanosine. These results suggest that betel quid-containing safrole might be involved in the pathogenesis of hepatocellular carcinoma in human beings and LC/MS has the potential to identify DNA adducts in clinical samples.