Carcinogenic DNA Adducts: LC-MS for Exposure Assessment

A known carcinogen, 1,3-butadiene (BD) is linked to automobile exhaust, forest fires, cigarette smoke and industrial exposure. During the metabolism of BD, cytochrome P450 isozymes activate the compound to yield three epoxide metabolites, including 3,4-epoxy-1-butene (EB). These epoxides mediate BD’s carcinogenic and mutagenic activity by forming covalent nucleobase adducts that trigger DNA polymerase errors. Reliable quantification protocols for the sensitive, specific detection of in vivo BD-DNA adducts could enable clinicians to assess human cancer risk.

One BD-DNA adduct, EB-GII (N-7-(1-hydroxy-3-buten-2-yl) guanine), which forms when EB reacts with the N7-position of guanine, can serve as a mechanism-based biomarker for BD exposure. To this end, Sangaraju et al. applied nano-liquid chromatography with electrospray ionization coupled to high-resolution tandem mass spectrometry (nanoLC/ESI⁺ HRMS³) for the quantification of EB-GII adducts in vivo.¹

To do this, the team spiked extracted DNA with an isotopically labeled internal standard and released the EB-GII adducts from the DNA backbone by neutral thermal hydrolysis. After filtering, they subjected the product to off-line purification and enrichment before LC-MS on an LTQ Orbitrap Velos hybrid ion trap-Orbitrap mass spectrometer (Thermo Scientific). The researchers note that the high-resolution capabilities of the instrumentation were imperative to reduce matrix noise and attain detection at even sub-ppm levels with complex samples.

Sangaraju et al. verified the method using blank human buffy coat DNA (150 μg) spiked with EB-GII (0.2–10 fmol) and internal standard (3 fmol), with excellent results for accuracy and precision. In terms of sensitivity, they calculated the limits of detection and quantitation to be 0.05 fmol analyte in 150 μg DNA (0.1 adducts/10⁹ nucleotides) and 0.2 fmol analyte in 150 μg DNA (0.4 adducts/10⁹ nucleotides).

To evaluate the applicability of the protocol, the team used three sample types. First, they quantified EB-GII adducts in human fibrosarcoma (HT1080) cells treated with increasing concentrations of EB (0.5–10 μm). They report an adduct concentration range of 1.15±0.23 to 10.11±0.45 adducts/10⁸ nucleotides for the samples. 

Next, the researchers turned to rat liver tissue extracted from specimens exposed to low levels of BD via inhalation. At three levels of exposure (0.5 ppm, 1.0 ppm and 1.5 ppm), the recorded adduct concentration increased in a dose-dependent way: 0.17±0.05 adducts/10⁸ nucleotides, 0.33±0.08 adducts/10⁸ nucleotides, and 0.50±0.04 adducts/10⁸ nucleotides, respectively. 

Again using DNA extracted from rat liver tissue, the method described in this study and first-order kinetics analysis revealed the half-life of EB-GII adducts at 1,250 ppm BD exposure level: 2.20±0.12 days. Interestingly, this value is significantly shorter than the previously reported half-life of a structural analog (N7-THBG adducts, t1/2=3.6–4.0 days).

Finally, Sangaraju et al. evaluated the method’s applicability to human samples using blood leukocyte DNA drawn from volunteer smokers who had experienced routine exposure to relatively high concentrations of BD (8.5–48.2 μg/cigarette). They report successful detection of EB-GII adducts in human DNA at levels below the limit of quantitation. The team notes that future development of a nanoLC/ESI⁺ HRMS³ protocol for the quantification of EB-GII adducts in human urine would address the problem of small sample size, but that this would require additional cleanup before analysis. 

Overall, the team presents the method reported here as a sensitive and accurate approach for the quantification of trace levels of EB-GII adducts, mimicking occupational exposure. They posit that the protocol has potential for cancer risk assessment, with both research and clinical applications.

 

Reference

1. Sangaraju, D., et al. (2014, May) “NanoLC/ESI+ HRMS³ Quantitation of DNA Adducts Induced by 1,3-Butadiene,” Journal of the American Society for Mass Spectrometry, doi: 10.1007/s13361-014-0916-x [e-pub before print].

Post Author: Melissa J. Mayer. Melissa is a freelance writer who specializes in science journalism. She possesses passion for and experience in the fields of proteomics, cellular/molecular biology, microbiology, biochemistry, and immunology. Melissa is also bilingual (Spanish) and holds a teaching certificate with a biology endorsement.