Protein Adducts: Biomarkers for Carcinogenic Exposure

Chemical changes that occur during combustion can produce the structurally similar environmental carcinogens known as aromatic amines (AAs) and heterocyclic aromatic amines (HAAs). Once activated by cytochrome P450 oxidation, these compounds form N-hydroxylated intermediates, which can react with DNA directly or further biotransform into unstable esters, ultimately forming adducts and producing mutations. Common sources of human exposure to AAs and HAAs include industrial applications (occupational exposure), tobacco smoking, meat grilling and general atmospheric pollution.

Unfortunately for exposure monitoring, DNA lesions rapidly repair, and even advanced mass spectrometry (MS) platforms may struggle to detect trace levels of DNA adducts remaining in human tissues. Conversely, protein adducts do not repair and instead accumulate with chronic exposure. Thus, scientists and clinicians may be able to use stable carcinogen protein adducts—specifically hemoglobin (Hb) and serum albumin carcinogen adducts—to biomonitor for AAs and HAAs over time.

In the interest of developing biomarkers for these compounds, Pathak et al. (2016) evaluated reactivity between oxyhemoglobin (HbO₂) in red blood cells and N-hydroxylated intermediates of ABP (4-aminobiphenyl, HONH-ABP), ANL (aniline, HONH-ANL), AαC (2-amino-9H-pyrido[2,3-b]indole, HONH-AαC), MeIQx (2-amino-3,8-dimethylimidazo[4,5- f]quinoxaline, HONH-MelQx) and PhIP (2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine, HONH-PhIP).¹ For this purpose, the team relied on the biochemical propensity of arylhydroxylamine metabolites to undergo multiple rounds of co-oxidation with HbO₂ to produce methemoglobinemia and, upon acid or base treatment, release parent AAs suitable for measurement by MS. They used an UltiMate 3000 ultra-performance liquid chromatography system and an Orbitrap Elite Hybrid Ion Trap-Orbitrap mass spectrometer (both Thermo Scientific) to separate and acquire spectral data.

Overall, the research team reported the following observations:

Three compounds reacted with hemoglobin and formed sulfinamide adducts: HONH-ABP, HONH-ANL and HONH-AαC. The other two compounds (HONH-MelQx and HONH-PhIP) did not. AαC was the most potent inducer of methemoglobinemia.
Based on molecular modeling, variations in location (distance between the substrates and the oxyheme complex of HbO₂) and conformation (changes in the helical and loop regions near the heme pocket) may account for different predispositions to induce methemoglobinemia among the analyzed compounds.

In terms of proteomics advances, the researchers were able to apply proteolysis of Hb (trypsin, Glu-C or Lys-C) and bottom-up Orbitrap-based MS to identify sulfinamide and sulfonamide adducts of ABP, ANL and AαC in vitro. They also employed this technology to detect sulfinamide and sulfonamide adducts of ABP in mouse blood following exposure to ABP; this represents a novel in vivo identification of intact sulfinamide adducts of carcinogenic AAs.

The observed high reactivity between HONH-AαC and HbO₂ indicates that the sulfinamide adduct of AαC may emerge as a novel biomarker for HAA exposure in humans. Pathak et al. offer this finding, along with the other data mentioned here, as a first step in the development of biomarkers suitable to human risk assessment. Future research endeavors include enhancing the understanding of chemical reactivity between HbO₂ and N-hydroxy AA and HAA substrates, including redox potential, electronic properties and induced conformational changes.

Reference
1. Pathak, K.V., et al. (2016) “Methemoglobin formation and characterization hemoglobin adducts of carcinogenic aromatic amines and heterocyclic aromatic amines,” Chemical Research in Toxicology, 29(3) (pp. 255–269), doi: 10.1021/acs.chemrestox.5b00418.

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.