Methemoglobin Formation and Characterization of Hemoglobin Adducts of Carcinogenic Aromatic Amines and Heterocyclic Aromatic Amines

Khyatiben V. Pathak, Ting Lan Chiu, Elizabeth Ambrose Amin, Robert J. Turesky

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21 Scopus citations


Arylamines (AAs) and heterocyclic aromatic amines (HAAs) are structurally related carcinogens formed during the combustion of tobacco or cooking of meat. They undergo cytochrome P450 mediated N-hydroxylation to form metabolites which bind to DNA and lead to mutations. The N-hydroxylated metabolites of many AAs also can undergo a co-oxidation reaction with oxy-hemolgobin (HbO2) to form methemoglobin (met-Hb) and the arylnitroso intermediates, which react with the β-Cys93 chain of Hb to form Hb-arylsulfinamide adducts. The biochemistry of arylamine metabolism has been exploited to biomonitor certain AAs through their Hb arylsulfinamide adducts in humans. We examined the reactivity of HbO2 with the N-hydroxylated metabolites of 4-aminobiphenyl (ABP, HONH-ABP), aniline (ANL, HONH-ANL), and the HAAs 2-amino-9H-pyrido[2,3-b]indole (AαC, HONH-AαC), 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP, HONH-PhIP), and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx, HONH-MeIQx). HONH-ABP, HO-ANL, and HONH-AαC induced methemoglobinemia and formed Hb sulfinamide adducts. However, HONH-MeIQx and HONH-PhIP did not react with the oxy-heme complex, and met-Hb formation and chemical modification of the β-Cys93 residue were negligible. Molecular modeling studies showed that the distances between the H-ON-AA or H-ON-HAA substrates and the oxy-heme complex of HbO2 were too far away to induce methemoglobinemia. Different conformational changes in flexible helical and loop regions around the heme pocket induced by the H-ON-AA or H-ON-HAAs may explain the different proclivities of these chemicals to induce methemoglobinemia. Hb-Cys93β sulfinamide and sulfonamide adducts of ABP, ANL, and AαC were identified, by Orbitrap MS, following the proteolysis of Hb with trypsin, Glu-C, or Lys-C. Hb sulfinamide and sulfonamide adducts of ABP were identified in the blood of mice exposed to ABP, by Orbitrap MS. This is the first report of the identification of intact Hb sulfinamide adducts of carcinogenic AAs in vivo. The high reactivity of HONH-AαC with HbO2 suggests that the Hb sulfinamide adduct of AαC may be a promising biomarker of exposure to this HAA in humans.

Original languageEnglish (US)
Pages (from-to)255-269
Number of pages15
JournalChemical research in toxicology
Issue number3
StatePublished - Mar 21 2016

Bibliographical note

Funding Information:
This work was supported by NIH grants RO1CA134700 and R01CA134700-03S1 of the Family Smoking Prevention and Tobacco Control Act, and Cancer Center Support grant no. CA-77598 (to R.J.T.). We thank the Mass Spectrometry core facility, a subsidiary of "The Analytical Biochemistry shared resource" at Masonic Cancer Center, University of Minnesota, Minneapolis and Dr. Peter Villalta, Core Facility Coordinator for helpful discussions on MS instrument operation. We also thank the Dr. David Tabb Laboratory at Department of Biomedical informatics (Nashville, TN), for providing the MyriMatch software program. We acknowledge Mrs. Lihua Yao for providing blood samples of mice treated with ABP. Grateful acknowledgment is also given to the Minnesota Supercomputing Institute for Advanced Computational Research (MSI). We thank Dr. Gabriele Sabbioni, Institute of Environmental and Occupational Toxicology, Casella Postale 108, CH-6780 Airolo, Switzerland, for critical comments on the manuscript.

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