Sg. Carmella et al., EVIDENCE THAT A HEMOGLOBIN ADDUCT USED FOR DOSIMETRY OF 4-(METHYL-NITROSAMINO)-1-(3-PYRIDYL)-1-BUTANONE IS A CARBOXYLIC ESTER, Environmental health perspectives, 99, 1993, pp. 203-205
Hemoglobin adducts of the carcinogenic tobacco-specific nitrosamine 4-
(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) release 4-hydroxy-1
-(3-pyridyl)-1-butanone (HPB) upon mild base or acid hydrolysis. HPB h
as been detected in hydrolysates of hemoglobin from smokers and snuff
dippers and has been proposed as a dosimeter of exposure to and metabo
lic activation of NNK in people exposed to tobacco products. In this s
tudy, labeling experiments were carried out with [O-18]NaOH that provi
de strong evidence that the globin adduct that releases HPB upon hydro
lysis is a carboxylic ester. Globin was isolated from rats treated wit
h [5-H-3]NNK. This globin was reacted with NaCNBH3, followed by hydrol
ysis at room temperature with 0.2 N NaOH. Analysis of the products dem
onstrated the presence of 4-hydroxy-1-(3-pyridyl)-1-butanol, but not H
PB. These results demonstrate that the adduct in globin has a free car
bonyl group and is not a Schiff base. This sequence of reactions was t
hen carried out with [O-18]NaOH under conditions that were shown to re
sult in incorporation of O-18 if nucleophilic displacement at C-4 had
occurred. Analysis by GC-MS of the 4-hydroxy-1-(3-pyridyl)-1-butanol f
ormed in this experiment demonstrated that there was no incorporation
of 0-18. These results are consistent only with the hydrolysis of an e
ster by a B(AC)2 mechanism. Therefore, the adduct releasing HPB upon m
ild base hydrolysis must be a 4-(3-pyridyl)-4-oxobutyl ester of aspart
ate, glutamate, or a terminal carboxylate. Futher support for this con
clusion was obtained by investigating the chemistry of a model ester,
-pyridyl)-4-oxobutyl-N-carbobenzyloxy-L-aspartate. The hydrolysis prop
erties of this compound were similar to those of the HPB-releasing hem
oglobin adduct.