Bladder cancer (BC) is the most lethal cancer malignancy of the urinary system. Chronic infections, genetic susceptibilities, environmental and occupational exposure are BC risk factors. Smoking is the major risk factor for BC, with up to 50% of the cases attributed to it. However, the principal chemicals in tobacco smoke and their mechanisms of action leading to BC remain unknown. Among the 70 carcinogens produced by tobacco combustion, only 4-aminobiphenyl (4-ABP) and 2-naphthylamine (2-NA) are cited as associated with BC risk. This association is based on high occupational exposures of textile dye and rubber factory workers to these chemicals. However, 4-ABP and 2-NA occur at several ng per cigarette, levels which may not be sufficient to induce BC. In contrast, other pro-carcinogens including the heterocyclic aromatic amine (HAA) 2-amino-9H-pyrido[4,3-b]indole, and aldehydes including acrolein, occur at 100-fold or higher levels in tobacco smoke.
Employing the human RT4 bladder epithelial cell line, we characterized the cytotoxicity and genotoxicity of tobacco smoke condensate (TSC). TSC of Marlboro and 1R6F reference cigarettes induced a dose- and time-dependent cytotoxicity. The TSC was separated by liquid-liquid extraction into a neutral fraction containing polycyclic aromatic hydrocarbons (PAHs), nitro-PAHs, and hydrophobic aldehydes, and a basic fraction containing aromatic amines, HAAs, and N-nitroso compounds. The cytotoxicity ofTSC is mainly attributed to the neutral fraction. TSC and the neutral fraction induced oxidative stress marked by reactive oxygen species (ROS), decreased glutathione:oxidized glutathione (GSH:GSSG) ratio, and induction of AP-sites. The presence of ROS scavengers GSH and N-acetyl-cysteine, led to cytoprotective effects implicating oxidative stress as a mechanism of action.
Targeted and untargeted metabolomics-based approaches were employed to characterize glutathione-conjugates formed by TSC, neutral, and basic fractions. Over 50 GSH conjugates were detected, with the most formed by chemicals inTSC and neutral fraction or induced endogenous electrophiles. The major GSH-conjugates are formed with acrolein, crotonaldehyde, and isomeric p-benzoquinone, and o-benzoquinone.
We are currently investigating the DNA adducts formed by chemicals in the neutral and basic fractions of TSC. As the next step of this work, we will assess the role of hepatic metabolism to modulate TSC-induced cytotoxicity and DNA damage in RT4 cells.