Mass Spectrometry Based Quantitation of Carcinogen Metabolites and DNA Adducts

Carcinogen-DNA adducts

Humans are exposed to a wide range of carcinogens. Some of these are metabolically activated to reactive electrophiles which can form covalent adducts at nucleophilic sites in proteins and DNA. These DNA adducts can be repaired or lead to toxicity or carcinogenesis. The reactive electrophiles can also undergo further metabolism and detoxification to nontoxic products that are excreted from the body.

1,3-Butadiene

1,3-Butadiene (BD) is one of the most abundant carcinogens in cigarette smoke. BD is metabolized to the reactive epoxides 3,4-epoxy-1-butene (EB), 3,4-epoxy-1,2-butanediol (EBD), and 1,2,3,4-diepoxybutane (DEB), which are detoxified by glutathione S-transferase θ-1 (GSTT1) to form glutathione conjugates and further metabolized to form mercapturic acids that are excreted in urine. If not detoxified, these epoxides can react with DNA or proteins to form BD-DNA or BD-amino acid adducts. Our lab was the first to discover urinary biomarkers THBMA and bis-BDMA as well as the DNA adducts N6,N6-DHB-dA and 1,N6-HMHP-dA.

Previous studies in our lab have utilized urinary BD mercapturic acid metabolites as biomarkers of smoking exposure to 1,3-butadiene. Our results show significant ethnic differences in the excretion of BD mercapturic acid MHBMA, with African American smokers excreting the highest levels, followed by whites and Japanese Americans. These results correlate with the high lung cancer risk of African Americans and the low lung cancer risk of Japanese Americans, suggesting a role for BD in the lung cancer etiology seen among these groups. We also conducted a genome wide association study to identify genetic determinants of BD metabolism. Individuals with the GSTT1 gene deletion excreted the lowest levels of MHBMA, followed by those with one copy of the gene and those with two copies, further confirming the important role of GSTT1 in the detoxification of BD.

Urinary DNA adducts as biomarkers of human exposure to 1,3-butadiene

While the above mercapturic acid metabolites are useful biomarkers of BD exposure, they represent detoxified BD. An advantage of studying DNA adducts is that they represent the biologically relevant dose of BD and are directly associated with BD carcinogenesis.

Our lab developed extremely sensitive and specific methods for the detection of EB-Guanine II (EB-GII), an N7 guanine adduct formed from EB, from both DNA and urine . Using the stable isotope analog [15N5]-EB-GII, we have quantified EB-GII levels in urine from smokers participating in longitudinal and cessation studies at the University of Minnesota. We found that urinary EB-GII is stable over time and decreases upon smoking cessation, validating it as a biomarker of smoking induced DNA damage.

Ethnic differences in excretion of butadiene-DNA adducts by current smokers

Following the validation of urinary EB-GII as a biomarker of smoking induced DNA damage, we quantified urinary EB-GII in smokers and non-smokers from three ethnic groups with different risks of lung cancer: Native Hawaiians, Japanese Americans, and Whites, to investigate ethnic differences in 1,3-butadiene metabolism and adduct formation. Among smokers, levels of urinary EB-GII are higher in Japanese Americans as compared to whites and Native Hawaiians. Among non-smokers, low but detectable EB-GII levels that did not differ by ethnic group were observed. A genome wide association study (GWAS) is currently underway to identify potential genetic factors contributing to the ethnic differences in urinary EB-GII.

Our ongoing studies will quantify urinary EB-GII in smokers who have developed lung cancer and the corresponding smoker controls (who have not developed cancer) to investigate the associations between urinary EB-GII and cancer risk.

Endogenous DNA adducts in humans

Unexpectedly, we have observed significant amounts of EB-GII and N7-THBG adducts in non-smokers, untreated animals, and untreated cultured human cells. The presence of BD-DNA adducts originating from endogenous sources in humans complicates risk assessment and raises questions about the relative contributions of environmental, dietary, and endogenous sources of DNA damage to cancer development.

To quantify the contribution of endogenous EB-GII and N7-THBG adducts to the overall adduct load, we utilized stable isotope-labeling experiments. Laboratory rats were exposed to low ppm levels of BD-d6. In this experiment, any adducts originating from BD exposure will carry the d6-isotope tag, while endogenous adducts will be unlabeled. We observed a dose dependent increase in THBG-d6 and EB-GII-d6 in liver DNA of rats, while endogenous THBG and EB-GII were observed in quantities comparable to the exogenous adducts at the lower exposure levels. This data reveals that endogenous adducts represent a significant contribution to the overall adduct load, raising questions about the sources of endogenous adduct formation. In future studies, we will treat cells with stable isotope-labeled cellular metabolites to identify the pathways through which these endogenous adducts are forming.

Inter-individual differences in sensitivity to 1,3-butadiene

Furthermore, we are investigating BD metabolism, DNA adduct formation, and biological effects in human HapMap cells. These are human-derived lymphoblastoid cells that have been extensively genotyped and can be used to further investigate inter-individual and ethnic differences in susceptibility to BD in a cell culture model. In this project, GSTT1-/- or GSTT1 +/+ cells are treated with EB or DEB. After the treatment, DNA and metabolites are isolated from the cells to quantify BD-DNA adducts EB-Guanine II and bis-N7G-BD, and glutathione conjugates EB-GSH and DEB-GSH. Using this model, we can compare the extent of GSH conjugation, DNA adduct formation, and apoptosis in a range of genotypes following EB or DEB exposure.

Representative publications:

Jokipii Krueger, C. C.; et al. Ethnic Differences in Excretion of Butadiene-DNA Adducts by Current Smokers. In press at Carcinogenesis. Article
Boysen, G.; et al. Effects of GSTT1 Genotype on the Detoxification of 1,3-Butadiene Derived Diepoxide and Formation of Promutagenic DNA-DNA Cross-Links in Human Hapmap Cell Lines. Chemical Research in Toxicology 2021, 34 (1), 119–131. Article
Jokipii Krueger, C.C., et al., Urinary N7-(1-hydroxy-3-buten-2-yl) guanine Adducts in Humans: Temporal Stability and Association with Smoking. Mutagenesis 2020, 35 (1): p. 19–26. Article
Degner, A.; et al. Interindividual Differences in DNA Adduct Formation and Detoxification of 1,3-Butadiene-Derived Epoxide in Human HapMap Cell Lines. Chemical Research in Toxicology 2020, 33 (7), 1698–1708. Article
Boldry, E.J., et al., Genetic Determinants of 1,3-Butadiene Metabolism and Detoxification in Three Populations of Smokers with Different Risks of Lung Cancer. Cancer Epidemiol Biomarkers Prev, 2017. 26(7): p. 1034-1042. Article

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