Ethnic differences in the metabolism of 1,3-butadiene and lung cancer risk

Abstract

Cigarette smoking remains one of the most preventable causes of death in the world, and is the leading cause of lung cancer. Epidemiological studies show inherent differences in lung cancer risk among smokers of different ethnic groups, with Native Hawaiian and African Americans have the highest risk, European Americans having an intermediate risk, and Latinos and Japanese Americans having the lowest risk. It has been proposed that these disparities in risk are due to ethnic differences in the metabolism, and ultimately bioactivation, of carcinogens in present in cigarette smoke. 1,3-butadiene (BD) is one of the most abundant and potent carcinogens present in cigarette smoke. BD is metabolically activated to the reactive species 3,4-epoxy-1-butene (EB), hydroxymethylvinylketone (HMVK), 3,4-epoxy-1,2-butanediol (EBD), and 1,2,3,4-diepoxybutane (DEB), which have the ability to form pro-mutagenic DNA adducts. These species can be detoxified through glutathione conjugation and excreted in urine; EB and HMVK are excreted as 2-(N-acetyl-L-cystein-S-yl)-1-hydroxybut-3-ene/ 1-(N-acetyl-L-cystein-S-yl)-2-hydroxybut-3-ene (MHBMA) and 4-(N-acetyl-L-cystein-S-yl)-1,2-dihydroxybutane (DHBMA). The research presented in this thesis focuses on ethnic differences in BD metabolism and the initial development of a DEB specific biomarker. A high throughput HPLC-ESI--MS/MS method for the simultaneous quantitation of MHBMA and DHBMA in humans previously developed in our laboratory was applied to quantify these mercapturic acids in smokers of different ethnic groups. In a large multi-ethnic study composed of African American, European American, and Japanese American smokers (N = 1,072). Urinary MHBMA and MHBMA/MHBMA+DHBMA were highest in African Americans, followed by European Americans, and Japanese Americans, and strongly influenced by GSTT1 genotype. A genome wide association study (GWAS) revealed strong associations between MHBMA and GSTT1: associations with 136 SNPs were detected, and all of them were located between 24.2—24.4 Mb near the GSTT1 gene on chromosome 22q11. Additional experiments with recombinant human GSTT1 and GSTT2 confirmed EB as a substrate for the first time. The same method was also applied to a separate smaller study of African American and European American smokers (N = 151). In contrast to the previous work, urinary MHBMA was higher in European Americans than African Americans in this study; this is likely due to decreased sample size. Statistical analyses revealed no correlation between urinary MHBMA or DHBMA and urinary N7-(1-hydroxy-3-buten-2-yl)guanine (EBGII), as well no significant associations between urinary EBGII, MHBMA, or DHBMA and various specific SNPs from BD-metabolizing genes (EPHX1 and CYP2E1) and DNA repair genes (FANCE). GSTT1 copy number was also included in this analysis, and showed a significant association with urinary MHBMA. Urinary MHBMA and DHBMA were also quantified in smokers (N=79) receiving treatment with the chemopreventative agent 2-phenethyl isothiocyanate (PEITC). Overall, PEITC treatment resulted in only slight increases in MHBMA and DHBMA as compared to treatment with a placebo, but was found to significantly increase MHBMA in individuals null for GSTT1 or both GSTT1 and GSTM1, indicating a potential protective effect of PEITC in these individuals. Lastly, an HPLC-ESI+-MS/MS method for the of detection of a novel DEB-specific biomarker, Nε, Nε-(2,3-dihydroxybutan-1,4-diyl)-L-lysine (DHB-Lys) was explored. Initial development focused on the use of an ion-pairing agent, perfluoroheptanoic acid (PFHA), which was chosen to increase HPLC retention of DHB-Lys. Though addition of PFHA to the aqueous mobile phase during HPLC-ESI+-MS/MS analysis did result in increased retention of the analyte, its use also presented additional challenges with analyte carryover, sample contamination, and ion suppression. Methodology utilizing derivatization of DHB-Lys through the addition of a 6-aminoquinolyl group (6-AQ) at the alpha nitrogen was tested on DEB-treated O6-alkylguanine DNA alkyltransferase (AGT), and showed a dose dependent increase in DHB-Lys formed.