Identification of a Potential Direct-Acting DNA Ethylating Agent Originating from the Synthetic Cooling Agent...

Previous studies have reported the existence of a direct-acting DNA ethylating agent in tobacco products; it is known that ethyl DNA adducts such as 7-ethylguanine (7-Et-Gua) are elevated in tissues of smokers, but the identity of the ethylating agent is unknown. In the study reported here, we investigated the possibility that N-ethyl-5-methyl-2-(1-methylethyl)cyclohexanecarboxamide (WS-3), a synthetic cooling agent used in tobacco and other products as a menthol alternative, could be nitrosated to produce the reactive DNA ethylating agent N-nitroso-N-ethyl-5-methyl-2-(1-methylethyl)cyclohexanecarboxamide (NO-WS-3). We synthesized NO-WS-3 and investigated its formation and stability, as well as its ability to ethylate DNA. Formation of NO-WS-3 is favored under acidic conditions that are likely to be found in the gastrointestinal tract, along with endogenous nitrite. The half-life of NO-WS-3 under biological conditions was about 16 h, ample time for its reaction with DNA to create potentially mutagenic ethyl DNA adducts which have been associated with tobacco use. Reaction of NO-WS-3 with calf-thymus DNA resulted in the formation of 7-Et-Gua (167 adducts per 10⁸ nucleotides), 3-Et-Ade (437 adducts per 10⁸ nucleotides), and O⁶-Et-Gua (148 adducts per 10⁸ nucleotides) well above background DNA levels in control incubations without NO-WS-3. This is the first identification of an agent potentially present in consumer products or formed in vivo that can directly ethylate DNA, providing a novel source of ethyl DNA adducts present in humans.