TY - JOUR
T1 - Quantitation of acrolein–protein adducts
T2 - Potential biomarker of acrolein exposure
AU - Li, Hui
AU - Wang, Jianling
AU - Kaphalia, Bhupendra S.
AU - Ansari, G. A.S.
AU - Khan, M. Firoze
N1 - Funding Information:
This work was supported by the Texas Advanced Technology Program, Texas Higher Education Coordinating Board (THECB), under grant 004952-0020-2001, and by grant ES06476 from the National Institute of Environmental Health Sciences (NIEHS), National Institutes of Health (NIH). The contents of this article are solely the responsibility of the authors and do not necessarily represent the official view of THECB or NIEHS, NIH.
PY - 2004/3
Y1 - 2004/3
N2 - Acrolein, an α,β-unsaturated aldehyde, is a ubiquitous environmental toxic pollutant. Because of potential human exposure, there is a need for a sensitive, reliable, and specific method to monitor acrolein exposure. Acrolein is a potent electrophile and reacts with proteins mainly through Michael addition reaction, leading to acrolein–protein adducts (APA). The present study aimed to develop a competitive enzyme-linked immunosorbent assay (ELISA) method for the quantitation of APA in biological samples. Antibody to acrolein–keyhole limpet hemocyanin adduct was raised in rabbits, and the specificity of the antibody was determined by ELISA using acrolein–albumin adduct (AAA) or native albumin. A dose-dependent response was observed with AAA, but no immunoreactivity with native albumin. Further, lack of cross-reactivity of anti-acrolein antibody with formaldehyde–, malondialdehyde–, or 4-hydroxynonenal–albumin adducts indicates its specificity for acrolein. For the competitive ELISA, 1:16,000 diluted antisera was used with varying concentrations of AAA, which provided a linear detection range between 250 and 10,000 pg. To test the efficacy of the method for possible use as a biomarker of acrolein exposure, SD rats were orally administered 1 or 7 doses of 9.2 mg/kg/d acrolein. APA levels, quantitated in the serum, showed significantly greater formation (32% and 58% after 1 and 7 doses, respectively) in acrolein-treated rats as compared to the controls. Western blot analyses of APA in the sera from acrolein-treated rats showed APA bands (especially 29, 31, and 100 kD) with greater intensity in comparison to controls, further supporting our ELISA results. These results suggest that quantitation of APA has potential to be used as biomarker of acrolein exposure and eventually for molecular dosimetry and risk assessment.
AB - Acrolein, an α,β-unsaturated aldehyde, is a ubiquitous environmental toxic pollutant. Because of potential human exposure, there is a need for a sensitive, reliable, and specific method to monitor acrolein exposure. Acrolein is a potent electrophile and reacts with proteins mainly through Michael addition reaction, leading to acrolein–protein adducts (APA). The present study aimed to develop a competitive enzyme-linked immunosorbent assay (ELISA) method for the quantitation of APA in biological samples. Antibody to acrolein–keyhole limpet hemocyanin adduct was raised in rabbits, and the specificity of the antibody was determined by ELISA using acrolein–albumin adduct (AAA) or native albumin. A dose-dependent response was observed with AAA, but no immunoreactivity with native albumin. Further, lack of cross-reactivity of anti-acrolein antibody with formaldehyde–, malondialdehyde–, or 4-hydroxynonenal–albumin adducts indicates its specificity for acrolein. For the competitive ELISA, 1:16,000 diluted antisera was used with varying concentrations of AAA, which provided a linear detection range between 250 and 10,000 pg. To test the efficacy of the method for possible use as a biomarker of acrolein exposure, SD rats were orally administered 1 or 7 doses of 9.2 mg/kg/d acrolein. APA levels, quantitated in the serum, showed significantly greater formation (32% and 58% after 1 and 7 doses, respectively) in acrolein-treated rats as compared to the controls. Western blot analyses of APA in the sera from acrolein-treated rats showed APA bands (especially 29, 31, and 100 kD) with greater intensity in comparison to controls, further supporting our ELISA results. These results suggest that quantitation of APA has potential to be used as biomarker of acrolein exposure and eventually for molecular dosimetry and risk assessment.
UR - http://www.scopus.com/inward/record.url?scp=1442286490&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=1442286490&partnerID=8YFLogxK
U2 - 10.1080/15287390490276539
DO - 10.1080/15287390490276539
M3 - Article
C2 - 14742096
AN - SCOPUS:1442286490
SN - 1528-7394
VL - 67
SP - 513
EP - 524
JO - Journal of Toxicology and Environmental Health - Part A
JF - Journal of Toxicology and Environmental Health - Part A
IS - 6
ER -