Sharon E. Murphy

A tobacco-specific lung carcinogen in the urine of men exposed to cigarette smoke

BACKGROUND Environmental tobacco smoke has been classified by the Environmental Protection Agency as a carcinogen causally associated with lung cancer in adults, but there have been no reports of lung carcinogens or their metabolites in the body fluids or tissues of nonsmokers exposed to environmental tobacco smoke. METHODS Five male nonsmokers were exposed to sidestream cigarette smoke generated by machine smoking of reference cigarettes for 180 minutes on each of two days, six months apart. Sidestream smoke is the smoke that originates from the smoldering end of a cigarette between puffs. Twenty-four-hour urine samples were collected before and after exposure. The urine samples were analyzed for 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) and its glucuronide, which are metabolites of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), a powerful lung carcinogen in rodents. NNAL is also a lung carcinogen in rodents. RESULTS The urinary excretion of the metabolites increased after exposure to sidestream smoke in all the men. The mean (+/- SD) amount of NNAL and NNAL glucuronide was significantly higher after exposure than at base line (33.9 +/- 20.0 vs. 8.4 +/- 11.2 ng per 24 hours [127 +/- 74 vs. 31 +/- 41 pmol per day], P < 0.001) and was correlated with urinary cotinine excretion (r = 0.89, P < 0.001). The nicotine concentrations in the air to which the men were exposed were comparable to those in a heavily smoke-polluted bar. CONCLUSIONS Nonsmokers exposed to sidestream cigarette smoke take up and metabolize a lung carcinogen, which provides experimental support for the proposal that environmental tobacco smoke can cause lung cancer.

Effects of phenobarbital and 3-methylcholanthrene induction on the formation of three glucuronide metabolites of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, NNK

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a tobacco specific carcinogen believed to be a causative agent for human lung cancer. To exert its carcinogenic potential, NNK must be metabolically activated, by alpha-hydroxylation, at either the methyl or methylene carbons adjacent to the N-nitroso group. We recently reported the presence of a glucuronide conjugate of 4-(hydroxymethylnitrosamino)-1-(3-pyridyl)-1-butanone (alpha-hydroxymethylNNK-Gluc) in the urine of Phenobarbital (PB) treated rats, and in the media of PB induced hepatocytes incubated with NNK. PB induces the alpha-hydroxylation of NNK, which generates the aglycon, as well as several UDP-glucuronosyl transferases. In the study presented here, we compared the metabolism of NNK to alpha-hydroxymethylNNK-Gluc by PB induced, 3-methylcholanthrene (3-MC) induced and control rat hepatocytes. Media was analyzed for the products of alpha-hydroxylation, N-oxidation and glucuronidation by radioflow HPLC. PB induced both N-oxidation and alpha-hydroxylation of NNK. 3-MC did not induce N-oxidation but induced alpha-hydroxylation more than 10-fold. alpha-HydroxymethylNNK-Gluc was not detected (< 0.05% total metabolites) when control hepatocytes were incubated with 1 to 100 microM NNK. When 3-MC and PB induced hepatocytes were incubated with 1-100 microM NNK alpha-hydroxymethylNNK-Gluc, expressed as the average percent of metabolites, accounted for 0.725 +/- 0.27 and 1.35 +/- 0.24% (+/-S.D.) of the NNK metabolites, respectively. The percent of NNK metabolized to alpha-hydroxymethylNNK-Gluc is small. But this glucuronide is potentially important in NNK carcinogenesis, since its formation results in the direct conjugation of an active metabolite responsible for DNA adduct formation. When PB induced rats were injected with NNK the level of NNK hemoglobin adducts, which can serve as surrogates for DNA adducts, decreased 50% compared to control rats administered NNK. Hepatic microsomal metabolism increased 2-fold and urinary alpha-hydroxymethylNNK-Gluc increased more than 10-fold in PB treated rats. One explanation for the decrease in NNK hemoglobin adducts may be a PB induced increase in the glucuronidation of alpha-hydroxymethylNNK, the metabolite responsible for adduct formation.

Dual-label high-performance liquid chromatographic assay for femtomole levels of benzo[a]pyrene metabolites

A dual-label HPLC assay to measure femtomole quantities of ethyl acetate-extractable [3H]benzo[a]pyrene metabolites was developed. 14C-labeled metabolites of benzo[a]pyrene formed by rat liver 9000g supernatant were used as both internal standards and chromatographic markers. The percentage deviation between assays was determined to be between 11 and 13% for 9,10-dihydro-9,10-dihydroxybenzo[a]pyrene, 7,8-dihydro-7,8-dihydroxybenzo[a]pyrene, benzo[a]pyrene-3,6-quinone, benzo[a]pyrene-1,6-quinone, and 9-hydroxybenzo[a]pyrene, 22% for 4,5-dihydro-4,5-dihydroxybenzo[a]pyrene, and less than 5% for 3-hydroxybenzo[a]pyrene. The detection limit of this assay was between 3 and 10 fmol per metabolite. The application of this technique to the metabolism of [3H]benzo[a]pyrene by microsomes of hamster and human oral cavity tissue is described.

Detection of Polynuclear Aromatic Hydrocarbon Diol Epoxide-Derived DNA and Globin Adducts in Humans by Gas Chromatography-Mass Spectrometry

Abstract Gas chromatography-negative ion chemical ionization-mass spectrometry-selected ion monitoring (GC-NICI-MS-SIM) was employed to detect tetramethyl ether derivatives of tetraols released upon hydrolysis of DNA and globin adducts derived from diol epoxides of polynuclear aromatic hydrocarbons (PAH). The methodology involves the following steps: 1) isolation of DNA or globin; 2) mild acid hydrolysis under vacuum; 3) isolation of the resulting tetraols and derivatization to the corresponding tetramethyl ethers using CH3I; 4) HPLC purification followed by GC-NICI-MS-SIM analysis. Analysis of human globin samples by this method indicates the presence of adducts which release chrysene-1,2,3,4-tetraol and benzo[a]pyrene-7,8,9,10-tetraol in smokers and nonsmokers. The level of BP-diol epoxide globin adducts in smokers was significantly higher than that in nonsmokers (2.75 vs 0.96 fmol/mg globin). The concentration of chrysene diol-epoxide globin adducts in the same subjects was about two orders of magnitud...