Immediate consequences of cigarette smoking: rapid formation of polycyclic aromatic hydrocarbon diol epoxides

Abstract

Polycyclic aromatic hydrocarbons (PAH) are among the likely major causative agents for lung cancer in smokers. PAH require metabolic activation to exert their carcinogenic effects, and one important pathway proceeds through a three-step sequence resulting in the formation of diol epoxides, which react with DNA to produce adducts that can cause mutations and initiate the carcinogenic process. However, no previous published studies have examined this critical pathway in humans specifically exposed to PAH by inhalation of cigarette smoke. This study used a unique approach employing a stable isotope derivative of phenanthrene, the simplest PAH with a bay region, a feature closely associated with PAH carcinogenicity. Twelve subjects each smoked a cigarette to which [D(10)]phenanthrene had been added. Plasma was analyzed for [D(10)]r-1,t-2,3,c-4-tetrahydroxy-1,2,3,4-tetrahydrophenanthrene ([D(10)]PheT), the major end product of the diol epoxide metabolism pathway of phenanthrene. The analysis was performed by gas chromatography--negative ion chemical ionization--tandem mass spectrometry, using [(13)C(6)]PheT as internal standard. The results demonstrated that the three-step pathway resulting in the formation of diol epoxides, as monitored by [D(10)]PheT, occurred with remarkable rapidity. Levels of [D(10)]PheT in plasma of all subjects were maximal at the earliest time points examined, 15-30 min after smoking the cigarette containing [D(10)]phenanthrene, and decreased thereafter. These results demonstrate that the formation of a PAH diol epoxide occurs rapidly in smokers. Because PAH diol epoxides are mutagenic and carcinogenic, the results clearly demonstrate immediate negative health consequences of smoking, which should serve as a major warning to anyone contemplating initiating tobacco use.

Figure 1

Structures of BaP and BPDE

Figure 2

GC-NICI-MS/MS-SRM analysis of samples containing Phe and [D₁₀]Phe metabolites isolated from plasma of smokers: (A) 30 min prior to smoking a cigarette to which [D₁₀]Phe had been added; and (B) 15 min after smoking that cigarette. The transitions illustrated were monitored to analyze for PheT and [D₁₀]PheT in plasma, and internal standard [¹³C₆]PheT which was added to all plasma samples. Shaded peaks correspond to the target analytes. PheT is present in all samples due to exposure of the smoker to Phe from cigarette smoke and environmental or dietary sources. [D₁₀]PheT is present only after smoking the cigarette containing [D₁₀]Phe. The internal standard for the analysis is [¹³C₆]PheT.

Figure 3

Levels of [D₁₀]PheT (closed triangles) and PheT (open circles) in the plasma of subject 1 at various intervals after smoking a cigarette containing [D₁₀]Phe (N = 11).

Scheme 1

Formation of diol epoxides and tetraols in the metabolism of Phe. Three steps are required for diol epoxide formation, catalyzed sequentially by cytochrome P450s, epoxide hydrolase (EH), and cytochrome P450s. All metabolites have the absolute stereochemistry indicated. Compounds 3 + 6 comprise PheT, measured in this study.