This study is unique. It is the first to investigate human metabolism of a PAH specifically delivered by inhalation in cigarette smoke, without interference by other sources of exposure such as air pollution or the diet. The use of cigarettes containing [D10]Phe made this possible.
Our results clearly demonstrate that the formation of Phe diol epoxides occurs rapidly in smokers. The biomarker [D10
]PheT, which results from hydrolysis of [D10
]Phe diol epoxides, was readily detected in all plasma samples after subjects smoked a cigarette containing [D10
]Phe, and its levels were maximal 15–30 min after the subjects finished smoking the cigarette. These results are significant because PAH diol epoxides react readily with DNA, induce mutations, and are considered to be ultimate carcinogens of multiple PAH in cigarette smoke (6
). In fact, the concentration-time profiles looked similar to what one might have expected if an intravenous bolus dose had been administered, indicating how rapidly exposure to [D10
]PheT develops. This type of profile lacks both an absorptive and a distributive phase, and the highest concentration is at the first time of blood sampling. This concentration-time profile is even more surprising considering that it is the profile of the metabolite
rather than the parent compound. The pharmacokinetic interpretation of this profile is that the multi-stage metabolic processing of [D10
]Phe to its diol epoxide occurs extremely rapidly and supports the concept of the lung as an important “first-pass” metabolizing organ for compounds absorbed from cigarette smoke.
While previous studies indicate that PAH diol epoxides are formed in humans and that their levels may be higher in smokers than in non-smokers, their rates of formation in smokers were unknown (12
). As shown in , diol epoxide formation requires three steps catalyzed by P450s and epoxide hydrolase. Multiple competing reactions such as glutathione-S
-transferase-catalyzed detoxification of the initially formed epoxides, or glucuronidation of the diol metabolites could retard or even prevent diol epoxide formation (8
). Our results suggest that these competing reactions have relatively little impact on rates of diol epoxide formation.
One striking finding is the large variability in the exposure of the subjects to [D10]Phe diol epoxides. Subjects 2 and 4 had the lowest exposures (as measured by the AUC 0-∞) which resulted in very high apparent clearances, indicating that the mechanisms which remove [D10]PheT from the body of these subjects are relatively efficient. On the other hand, Subjects 1 and 6 had very high AUCs, indicating that these subjects have greater overall exposure to [D10]Phe diol epoxides. It is unlikely that this wide range of exposure is due to differences in dosing, considering the sophisticated and well-monitored means by which the cigarettes were smoked. It is much more likely that the difference in exposures is due to the subjects’ relative abilities to form and eliminate the metabolites of Phe.
Stable isotope labeling has been used previously in studies of nicotine and cotinine metabolism in tobacco users (28
). These studies have defined pharmacokinetic aspects of nicotine metabolism and have probed the utility of metabolic ratios in predicting nicotine dependence in smokers. A deuterium labeled tobacco-specific nitrosamine has also been added to cigarettes to investigate its metabolic products in smokers (30
). However, no previous studies have examined PAH metabolism in smokers using a deuterated probe compound.
The results reported here should serve as a stark warning to those who are considering starting to smoke cigarettes: PAH diol epoxide formation occurs immediately and is not a theoretical long term effect. PAH diol epoxides are DNA damaging compounds which can cause genetic damage and initiate the carcinogenic process.
This study has some limitations. We used [D10
]Phe as a representative PAH because Phe is generally considered non-carcinogenic. Addition of a carcinogenic PAH to a cigarette would be potentially hazardous and possibly unethical. Although there are similarities in the P450s that catalyze diol epoxide formation from Phe and BaP – P450s 1A1, 1A2, and 1B1 – as well as similarities in their detoxification by glutathione-S
), there are also some potential differences in the metabolism and toxicokinetics of Phe versus other PAH that could affect the interpretation of our results. First, metabolism of Phe by the angular ring diol epoxide pathway occurs mainly via Phe reverse diol epoxide (5
) in humans and to a much smaller extent by Phe bay region diol epoxide (2
), although our unpublished data indicate that measurements of PheT and diol epoxide 2
specifically are highly correlated. It is the bay region diol epoxide which, strictly speaking, would represent the potentially carcinogenic pathway. Second, the rates of tissue distribution and metabolism of Phe and higher molecular weight carcinogenic PAH such as BaP might be quite different, as highly lipophilic PAH such as BaP diffuse slowly into tissues (6
). Thus, the results for Phe might not be generalizable to higher molecular weight PAH.
In summary, we demonstrate that the formation of Phe diol epoxides, as represented by the biomarker PheT, is rapid in smokers. These results provide the first direct evidence for human metabolic activation of a PAH delivered in cigarette smoke and indicate the potential for immediate genetic damage in a cigarette smoker.