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Chem. Res. Toxicol. 2006, 19, 150-155
Reaction of the Tobacco Alkaloid Myosmine with Hydrogen Peroxide Wolfgang Zwickenpflug* and Stefan Tyroller Walther Straub Institute of Pharmacology and Toxicology, Ludwig-Maximilians UniVersity of Munich, Goethestrasse 33, D-80336 Munich, Germany ReceiVed October 7, 2005
Myosmine is not only one of the minor tobacco alkaloids but is also present in various foods. Therefore, research on myosmine metabolism and activation has been intensified. 3-Pyridylacetic acid, 4-oxo-4(3-pyridyl)butanoic acid (keto acid), 3-pyridylmethanol, 3′-hydroxymyosmine, and 4-hydroxy-1-(3-pyridyl)1-butanone (HPB) have been identified as urinary metabolites after oral administration to female Wistar rats. Although N-nitrosation of myosmine, yielding N‘-nitrosonornicotine (NNN) and HPB, was considered as a possible in vivo activation route, the formation pathways of most metabolites could not be explained until now. Therefore, under consideration of its high reactivity due to its imine structure, peroxidation of myosmine seemed to be a promising additional activation pathway. In vitro peroxidation using myosmine (8.9 µmol in 200 µL methanol) with a mixture of hydrogen peroxide (57.6 µmol, 5 µL of a 35% solution) and acetic acid anhydride (106 µmol, 10 µL) already showed high yields of reaction products after 30 min ultrasonic treatment. The product pattern was analyzed by HPLC/UV and GC/MS. Besides unchanged myosmine, 3-pyridylacetic acid, keto acid, 3-pyridylmethanol, HPB, and nornicotyrine have been identified as myosmine peroxidation products. Different product patterns were obtained after 24 h and 4 days due to a time-dependent degradation, formation, and conversion of the reaction products. Therefore, peroxidation reaction of myosmine might explain the in vivo formation of 3-pyridylacetic acid, keto acid, 3-pyridylmethanol, and HPB in rats. In addition, because of acetylating conditions using acetic acid anhydride, N-(4-oxo-4-pyridin-3-yl-butyl)acetamide was rapidly formed during the first 30 min of the reaction. Introduction The minor tobacco alkaloid myosmine has been quantified in tobacco samples as 48-189 µg/g (1). Its content in snuff is between 0.001 and 0.003% (2). Studies on the smoke composition of unblended cigarettes yielded myosmine at 13-33 µg/ cigarette in the mainstream smoke and 73-224 µg/cigarette in the sidestream smoke (3). In the mainstream smoke of plain nonfilter cigarettes, myosmine is present at 13-300 µg/cigarette (4). Myosmine was one of the first structural identified tobacco alkaloids besides nicotine. Both nicotine and myosmine have been detected in plants other than tobacco. However, occurrence of myosmine is not limited to solanaceae like nicotine (5). After detection of myosmine in Duboisia hopwoodii (6), it was also found in nuts and nut products (7). In a follow-up study, myosmine has been identified not only in various foods including staple foods like wheat, maize, rice, and milk but also in different fruits and vegetables at concentrations between 0.02 and 6.1 ng/g (8). The LD50 value for rats is 190 mg/kg (ip) and 1875 mg/kg (po) (9). Myosmine is not mutagenic in the Ames assay but induced DNA damage in the test system Escherichia coli polA+/polA- (10). Studies on nasal mucosal cells and human lymphocytes showed DNA damage in the Comet assay after incubation with myosmine (11). Myosmine is easily nitrosated yielding the esophageal carcinogen N-nitrosonornicotine (NNN)1, classified as “carcinogenic to humans” by the IARC (12), and 4-hydroxy-1-(3-pyridyl)-1-butanone (HPB) (13). Myosmine activation by nitrosation is assumed to be a one-step reaction * Corresponding author. Phone: +49-89-2180-75744. Fax: +49-892180-75701. E-mail:
[email protected].
leading to a reactive intermediate, an unstable diazotate with the ability to interact with DNA and proteins (14). Under nitrosation conditions, adduct formation of myosmine with DNA in vitro has been shown in preliminary studies (13). The in vivo metabolism of myosmine after po administration to female Wistar rats has been investigated (15) (Scheme 1). The main metabolites have been identified as 3-pyridylacetic acid (20-26%) and 4-oxo-4-(3-pyridyl)butanoic acid (keto acid, 50-63%), whereas 3-pyridylmethanol, 3′-hydroxymyosmine, and HPB each only contributed
