A new assay for albumin and hemoglobin adducts of 1,2- and 1,4-benzoquinones.
A new method has been developed to detect mono-S-substituted cysteinyl adducts of 1,2- and 1,4-benzoquinone (BQ) in hemoglobin (Hb) and albumin (Alb). After reacting the protein with trifluoroacetic anhydride and methanesulfonic acid, the resulting isomers of O,O',S-tris-trifluoroacetyl-hydroquinone and -catechol are extracted and detected by gas-chromatography-mass spectrometry in the negative-ion chemical ionization mode. The limit of detection of the assay is about 20 pmol adduct/g protein. This assay was employed to quantitate mono-S-substituted background adducts in human and rat Hb and Alb and benzene-specific adducts in Hb and Alb from F344 rats following a single oral dosage of 50-400 mg [13C6]benzene/kg body wt. In Alb, a dose-related increase in both [13C6]1,2- and [13C6]1,4-BQ adducts was observed with [[13C6]]1,4-BQ-Alb] >> [[13C6]1,2-BQ-Alb]. The formation of [13C6]1,2-BQ-Alb was linear with increasing dosage of benzene with a slope of 2.3 (pmol adduct/g protein)/(mg/kg body wt.) (S.E. = 0.18, R2 = 0.91). However, at dosages above about 100 mg [13C6]benzene/kg body wt., the levels of 1,4-BQ-Alb were greater than proportional to the dosage. Mono-S-substituted adducts of [13C6]1,2-BQ and [13C6]1,4-BQ were not detected in Hb. The background ([12C6]) adducts of 1,2- and 1,4-BQ in 20 F344 rats were estimated (in nmol adduct/g of protein) to be 3.9 (S.E. = 0.23) and 4.9 (S.E. = 0.30) in Hb and 2.7 (S.E. = 0.24) and 11.4 (S.E. = 0.60) in Alb. At the highest dosage of 400 mg [13C6]benzene/kg body wt., background levels of 1,2-BQ-Alb were about 4-fold higher than those of the benzene-specific adducts whereas the benzene-specific levels of 1,4-BQ-Alb were about 7-fold higher than those of the background adducts. Background levels of 1,2- and 1,4-BQ adducts in 10 portions of commercial human proteins were found to be (in nmol adduct/g of protein) 1.6 (S.E. = 0.05) and 0.85 (S.E. = 0.04) in Hb and 1.6 (S.E. = 0.06) and 8.9 (S.E. = 0.36) in Alb.
Waidyanatha
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S
Department of Environmental Science and Engineering, School of Public Health, University of North Carolina at Chapel Hill 27599-7400, USA.
Yeowell-O'Connell
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Rappaport
S M
SM
D000089:Acetic Anhydrides; D000818:Animals; D001554:Benzene / Q000737:chemistry / Q000633:toxicity; D016227:Benzoquinones / Q000032:analysis*; D002273:Carcinogens / Q000737:chemistry / Q000633:toxicity; D003510:Cyclohexanes / Q000032:analysis; D005463:Fluoroacetates; D008401:Gas Chromatography-Mass Spectrometry; D006454:Hemoglobins / Q000032:analysis / Q000737:chemistry*; D006801:Humans; D008297:Male; D008698:Mesylates; D051381:Rats; D011916:Rats, Inbred F344; D012709:Serum Albumin / Q000032:analysis / Q000737:chemistry*
D000089:Acetic Anhydrides; D016227:Benzoquinones; D002273:Carcinogens; D003510:Cyclohexanes; D005463:Fluoroacetates; D006454:Hemoglobins; D008698:Mesylates; D012709:Serum Albumin; C045880:methanesulfonic acid; C004532:quinone; C017958:trifluoroacetic anhydride; D001554:Benzene; C025225:2-benzoquinone; C023372:benzene oxide