Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Acrolein”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 19 recordsLinked to original sources

Quantitation of acrolein-protein adducts: potential biomarker of acrolein exposure.

Acrolein, an alpha,beta-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.

Acrolein↗

Acrolein is a product of lipid peroxidation reaction. Formation of free acrolein and its conjugate with lysine residues in oxidized low density lipoproteins.

Lipoprotein peroxidation, especially the modification of apolipoprotein B-100, has been implicated to play an important role in the pathogenesis of atherosclerosis. However, there have been few detailed insights into the chemical mechanism of derivatization of apolipoproteins during oxidation. In the present study, we provide evidence that the formation of the toxic pollutant acrolein (CH2=CH-CHO) and its conjugate with lysine residues is involved in the oxidative modification of human low density lipoprotein (LDL). Upon incubation with LDL, acrolein preferentially reacted with lysine residues. To determine the structure of acrolein-lysine adduct in protein, the reaction of acrolein with a lysine derivative was carried out. Employing Nalpha-acetyllysine, we detected a single product, which was identified to be a novel acrolein-lysine adduct, Nalpha-acetyl-Nepsilon-(3-formyl-3,4-dehydropiperidino )lysine. The acid hydrolysis of the adduct led to the derivative that was detectable with amino acid analysis. It was revealed that, upon in vitro incubation of LDL with acrolein, the lysine residues that had disappeared were partially recovered by Nepsilon-(3-formyl-3, 4-dehydropiperidino)lysine. In addition, we found that the same derivative was detected in the oxidatively modified LDL with Cu2+ and that the adduct formation was correlated with LDL peroxidation assessed by the consumption of alpha-tocopherol and cholesteryl ester and the concomitant formation of cholesteryl ester hydroperoxide. Enzyme-linked immunosorbent assay that measures free acrolein revealed that a considerable amount of acrolein was released from the Cu2+-oxidized LDL. Furthermore, metal-catalyzed oxidation of arachidonate was associated with the formation of acrolein, indicating that polyunsaturated fatty acids including arachidonate represent potential sources of acrolein generated during the peroxidation of LDL. These results indicate that acrolein is not just a pollutant but also a lipid peroxidation product that could be ubiquitously generated in biological systems.

Acrolein↗

Application of an immunoassay for cyclic acrolein deoxyguanosine adducts to assess their formation in DNA of Salmonella typhimurium under conditions of mutation induction by acrolein.

Acrolein has been shown to form cyclic deoxyguanosine adducts when it reacts with DNA in vitro. In this study, we have used a recently developed immunoassay for these adducts to study their formation in DNA from Salmonella typhimurium exposed to acrolein. Acrolein--deoxyguanosine adducts were formed in a dose-dependent fashion in Salmonella tester strains TA100 and TA104, reaching levels as high as 5 mumol adduct per mol deoxyguanosine. Using the liquid pre-incubation assay, acrolein-induced mutations were also found in strains TA100 and TA104. The correlation between acrolein--deoxyguanosine adduct concentration and acrolein-induced mutations in TA100, which contains GC base pairs at the site of reversion, suggests that the acrolein--deoxyguanosine adduct is a promutagenic lesion. That mutations are also seen in TA104 which contains AT base pairs at the site of reversion suggest that adducts of bases other than deoxyguanosine may also be important in the mutagenic activity of acrolein.

Acrolein↗

Depletion of nasal mucosal glutathione by acrolein and enhancement of formaldehyde-induced DNA-protein cross-linking by simultaneous exposure to acrolein.

Incubation of homogenates of rat nasal mucosa with acrolein resulted in the apparent formation of DNA-protein cross-links. However, inhalation exposure of male Fischer-344 rats to acrolein (2.0 ppm, 6 h) did not cause detectable DNA-protein cross-linking in the nasal respiratory mucosa. Simultaneous exposure of rats to both acrolein (2.0 ppm) and formaldehyde (6.0 ppm) for 6 h resulted in a significantly higher yield of DNA-protein cross-links than was obtained following exposure to formaldehyde (6.0 ppm) alone. Acrolein exposure at concentrations of 0.1, 0.5, 1.0, or 2.5 ppm resulted in a concentration-dependent depletion of nonprotein sulfhydryl groups in the nasal respiratory mucosa. A plausible explanation for the enhancement of DNA-protein cross-links by simultaneous exposure to formaldehyde and acrolein may be that depletion of glutathione by acrolein inhibited the oxidative metabolism of formaldehyde, leading to an increase of formaldehyde-induced DNA-protein cross-links.

Acrolein↗

DNA adducts of acrolein: site-specific synthesis of an oligodeoxynucleotide containing 6-hydroxy-5,6,7,8-tetrahydropyrimido[1,2-a]purin-10(3H)-one, an acrolein adduct of guanine.

3-(2-Deoxy-beta-D-erythro-pentofuranosyl)-6-hydroxy-5,6,7,8-tetrahydropyrimido[1,2-a]purin-10(3H)-one is formed in low yield by the reaction of acrolein with 2'-deoxyguanosine. The nucleoside and an oligodeoxynucleotide containing it have been synthesized. For preparation of the nucleoside 2'-deoxyguanosine was alkylated at the N1 position using 1-bromo-3-butene to give 1-(3-butenyl)-2'-deoxyguanosine. Oxidation with OsO(4) and N-methylmorpholine-N-oxide to give the 3,4-dihydroxybutyl adduct followed by oxidation with NaIO(4) gave the 1-(3-oxopropyl) adduct which cyclized spontaneously to yield the title compound as a rapidly epimerizing mixture of two diastereomers. Reduction of the nucleoside with NaBH(4) gave the unfunctionalized compound plus 1-(3-hydroxypropyl)-2'-deoxyguanosine showing that epimerization was occurring via both the imine and the 1-(3-oxopropyl) adduct. Reduction with NaCNBH(3) gave exclusively unfunctionalized 3-(2-deoxy-beta-D-erythro-pentofuranosyl)-5,6,7,8-tetrahydropyrimido[1,2-a]purin-10(3H)-one. The phosphoramidite reagent needed for preparation of oligonucleotides was prepared from 1-(3-butenyl)-2'-deoxyguanosine by glycolation after protection of the 3' and 5' hydroxyl groups as silyl derivatives. Acetylation of the vicinal hydroxyl groups and the exocyclic amino group followed by removal of silyl protection gave the protected nucleoside. Protection of the 5' hydroxyl group as the 4,4'-dimethoxytrityl ether followed by phosphitylation with 2-cyanoethyl-N,N,N',N'-tetraisopropylphosphorodiamidite gave the prosphoramidite reagent which was used to prepare a 12-mer oligodeoxynucleotide.

Acrolein↗

Acrolein activates mitogen-activated protein kinase signal transduction pathways in rat vascular smooth muscle cells.

Acrolein, a major component of cigarette smoke, an environmental pollutant and an endogenous lipid peroxidation product, has been implicated in the development of atherosclerosis. Although a link between vascular injury and acrolein has been indicated, the exact molecular mechanism of acrolein-induced toxicity to vasculature is unknown. In an effort to elucidate the molecular basis of acrolein-induced vascular toxicity, the possibility of the intracellular signaling system as one of the targets of acrolein-induced toxicity is investigated in the present study. Exposure of cultured rat vascular smooth muscle cells (VSMCs) to different doses of acrolein not only causes cytotoxicity but also alters cellular morphology in a concentration and time-dependent manner. VSMCs exhibit cytotoxicity to a narrow concentration range of 5-10 microg/ml and display no toxicity to 2 microg/ml acrolein even after 24 h of exposure. Furthermore, exposure to acrolein results in activation of members of the mitogen-activated protein kinase (MAPK) family and protein tyrosine kinases. The extracellular signal-regulated kinases 1 and 2 (ERK1/2), stress-activated protein kinases/c-jun NH2-terminal kinases (SAPK/JNK) and p38MAPK are effectively and transiently activated by acrolein in a concentration and time-dependent fashion. While all three MAPKs exhibit significant activation within 5 min of exposure to acrolein, maximum activation (ERK1/2 and p38MAPK) or close to maximum activation (SAPK/JNK) occurs on exposure to 5 microg/ml acrolein for 2 h. Acrolein-induced activation of MAPKs is further substantiated by the activation of transcription factors, c-jun and activator transcription factor-2 (ATF-2), by acrolein-activated SAPK/JNK and p38MAPK, respectively. Additionally several cellular proteins exhibit spectacular protein tyrosine phosphorylation, particularly in response to 2 and 5 microg/ml of acrolein. Interestingly, the acrolein-induced activation of MAPKs precedes acrolein-stimulated protein tyrosine phosphorylation, which occurs after 2 h of exposure to acrolein. However, the time course of maximum protein tyrosine phosphorylation profile corresponds to the peak activation profile of MAPKs. The activation of MAPKs and protein tyrosine phosphorylation by acrolein appears to be independent of acrolein-induced toxicity. VSMCs exposed to 2 microg/ml acrolein exhibit no toxicity but stimulates significant activation of MAPKs and protein tyrosine phosphorylation. Although acrolein-induced VSMC toxicity is not blocked by MAPK inhibitors, PD98059, an inhibitor of MAPK kinase and SB203580, an inhibitor of p38MAPK, eitheralone or in combination, each MAPK responds differently to the inhibitors. Most prominently, although SB203580, an inhibitor of both SAPK/JNK and p38MAPK, significantly inhibited acrolein-induced activation of p38MAPK, it also stimulated SAPK/JNK activation by acrolein alone and in combination with PD98059. These results provide the first evidence that the activation of both growth-regulated (ERK1/2) and stress-regulated (SAPK/JNK and p38MAPK) MAPKs as well as tyrosine kinases are involved in the mediation of acrolein-induced effects on VSMC, which may play a crucial role in vascular pathogenesis due to environmentally and endogenously produced acrolein.

Acrolein↗

Protection of rat embryos in culture against the embryotoxicity of acrolein using exogenous glutathione.

The aldehyde acrolein is embryotoxic in vivo and in vitro. Since acrolein is reactive towards thiols, glutathione was evaluated for its protective effects against the in vitro embryotoxicity of acrolein. Day 10 rat embryos were cultured in the presence of acrolein and glutathione, either concurrently or sequentially, and evaluated for embryo deaths, malformations, growth retardation, and content of glutathione and protein. Acrolein, added alone at the initiation of the culture period, was embryolethal to 64 and 100% of the embryos at 120 and 160 microM respectively. At acrolein concentrations of 80 and 120 microM, 50 and 100%, respectively, of the surviving embryos were malformed. In addition, both of these concentrations of acrolein produced growth retardation manifested by significant decreases in the yolk sac diameter, crown-rump and head lengths, number of somites, and morphological score. Concurrent exposure to 100 or 500 microM glutathione markedly protected embryos against all of these effects. To study the mechanism of the protective effect of glutathione against the embryotoxicity of acrolein, the effects of sequential addition of acrolein and glutathione were determined. When rat embryos were cultured in the presence of acrolein for 2 hr prior to exposure to glutathione, even 500 microM glutathione could not offer any protection against the embryolethality, teratogenicity, and growth retardation induced by acrolein. However, a 6-hr preincubation with 500 microM glutathione, prior to exposure to acrolein (in the absence of exogenous glutathione), significantly decreased the incidence of embryo deaths at 160 microM acrolein and brought the number of deaths and malformations among embryos exposed to 120 microM acrolein down to a level not significantly different from control; unlike the embryos exposed concurrently to acrolein and glutathione, however, the sequential treatment with glutathione and acrolein did not protect against growth retardation. While there were some changes in the total glutathione and protein content of embryos and yolk sacs with acrolein exposure, none of the treatments had any overall effect on the glutathione concentration per mg protein. Thus, exogenous glutathione can protect against the in vitro embryotoxicity of acrolein. We propose that this protection is mediated in part by a direct interaction between glutathione and acrolein, added concurrently to the serum medium, and in part by an indirect effect on the embryo of glutathione added prior to acrolein.

Abnormalities, Drug-Induced↗

NTP Technical Report on the comparative toxicity studies of allyl acetate (CAS No. 591-87-7), allyl alcohol (CAS No. 107-18-6) and acrolein (CAS No. 107-02-8) administered by gavage to F344/N rats and B6C3F1 mice.

Allyl acetate, allyl alcohol, and acrolein are used in the manufacture of detergents, plastics, pharmaceuticals, and chemicals and as agricultural agents and food additives. Male and female F344/N rats and B6C3F(1) mice received allyl acetate, allyl alcohol, or acrolein by gavage for 14 weeks. Genetic toxicology studies were conducted in Salmonella typhimurium, Drosophila melanogaster, cultured Chinese hamster ovary cells, rat bone marrow erythrocytes, and mouse peripheral blood erythrocytes. Groups of 10 male and 10 female rats were administered 0, 6, 12, 25, 50, or 100 mg allyl acetate/kg body weight, 0, 1.5, 3, 6, 12, or 25 mg/kg allyl alcohol, or 0, 0.75, 1.25, 2.5, 5, or 10 mg/kg acrolein in 0.5% methylcellulose by gavage, 5 days per week for 14 weeks. Groups of 10 male and 10 female mice were administered 0, 8, 16, 32, 62.5, or 125 mg/kg allyl acetate, 0, 3, 6, 12, 25, or 50 mg/kg allyl alcohol, or 0, 1.25, 2.5, 5, 10, or 20 mg/kg acrolein in 0.5% methylcellulose by gavage, 5 days per week for 14 weeks. In the allyl acetate rat study, all males and females in the 100 mg/kg groups died or were killed moribund by day 8; there were no other deaths. In the allyl alcohol study, all rats survived to the end of the study except one 6 mg/kg female. In the acrolein rat study, eight males and eight females in the 10 mg/kg groups died by week 9 of the study. Two males in the 2.5 and 5 mg/kg groups and one or two females in the 1.25, 2.5, and 5 mg/kg groups also died early; two of these deaths were gavage accidents. In the allyl acetate mouse study, all males and females in the 125 mg/kg group died during the first week of the study. All other early deaths, except five 62.5 mg/kg males and one 32 mg/kg female, were gavage accidents. In the allyl alcohol mouse study, one 50 mg/kg female died due to a gavage accident; all other animals survived to the end of the study. In the acrolein mouse study, all males and females administered 20 mg/kg died during the first week of the study. All other early deaths, except one male and one female administered 10 mg/kg, were unrelated to chemical administration. The concentration of 3-hydroxypropyl mercapturic acid (3-HPM) in the urine of rats and mice was determined after the first dose of chemical and at the end of the 14-week study. At both time points, the concentrations of 3-HPM in the urine of animals that received allyl acetate or allyl alcohol increased linearly with dose. In animals dosed with acrolein, the concentrations of 3-HPM exhibited a nonlinear increase with dose at the first time point. At the end of the study, the concentration of 3-HPM in the urine of animals dosed with acrolein was linear with dose except at the highest concentration administered. Since urine volumes were not recorded during the urine collection, complete quantitation of these data was not possible. The final mean body weights and mean body weight gains of male rats administered 12 or 50 mg/kg allyl acetate and of male and female rats administered 10 mg/kg acrolein were significantly less than those of the vehicle controls. The mean body weight gain of male mice in the 50 mg/kg group in the allyl alcohol study was also less than that of the vehicle controls. Final mean body weights and mean body weight gains of dosed female rats and male and female mice in the allyl acetate studies, male and female rats and female mice in the allyl alcohol studies, and male and female mice in the acrolein studies were generally similar to those of the respective vehicle controls. Clinical findings related to allyl acetate administration included pallor, eye or nasal discharge, ruffled fur, lethargy, diarrhea, and thinness among rats in the 100 mg/kg groups and lethargy, abnormal breathing, thinness, and ruffled fur among mice that died early. In the acrolein study, clinical findings included abnormal breathing, eye or nasal discharge, ruffled fur, thinness, and lethargy in rats in the 10 mg/kg groups. The liver weights of male rats administered 25 mg/kg allyl alcohol, female rats administered 50 mg/kg allyl acetate or 5 or 10 mg/kg acrolein, and male mice administered 10 mg/kg acrolein were significantly greater than those of the vehicle controls. Female rats administered 10 mg/kg acrolein had significantly lower absolute and relative thymus weights than did the vehicle controls. Female rats administered 25 mg/kg allyl alcohol spent more time in diestrus and less time in metestrus than the vehicle controls. The estrous cycles of female mice dosed with 16 or 32 mg/kg allyl acetate were significantly longer than that of the vehicle controls. Gross lesions related to allyl acetate treatment were observed in the liver, forestomach, and thorax/abdomen of male and female rats in the 100 mg/kg groups. Microscopically, the incidences of forestomach squamous epithelial hyperplasia were significantly increased in male rats administered 12 mg/kg or greater, female rats administered 25 or 50 mg/kg, male mice administered 32 or 62.5 mg/kg, and female mice administered 16, 32, or 62.5 mg/kg. Forestomach necrosis, hemorrhage, and inflammation were present in most rats in the 100 mg/kg groups, and the incidence of hemorrhage in 125 mg/kg male mice was increased; male mice in the 62.5 and 125 mg/kg groups and 125 mg/kg female mice had significantly increased incidences of glandular stomach hemorrhage. Increased incidences of several liver lesions occurred in male or female rats administered 50 or 100 mg/kg, and to a lesser extent in 25 mg/kg rats, 62.5 mg/kg male mice, and 125 mg/kg male and female mice. Bone marrow hyperplasia, hemorrhage or depletion in the mediastinal, mandibular, and mesenteric lymph nodes, hemorrhage and necrosis of the thymus, and hematopoietic cell proliferation of the red pulp were also observed in 100 mg/kg rats. Increased incidences of necrosis in the mandibular and mesenteric lymph nodes, spleen, and thymus were observed in 62.5 and 125 mg/kg mice. Male and female rats administered 6 mg/kg allyl alcohol or greater and male and female mice administered 12 mg/kg allyl alcohol or greater had significantly increased incidences of squamous hyperplasia of the forestomach epithelium. Female rats in the 25 mg/kg group had significantly increased incidences of bile duct hyperplasia and periportal hepatocyte hypertrophy in the liver. Incidences of portal cytoplasmic vacuolization were significantly increased in 50 mg/kg male mice and female mice in the 25 and 50 mg/kg groups. Gross lesions related to acrolein treatment were observed in the forestomach and glandular stomach of male and female rats in the 10 mg/kg groups and 20 mg/kg female mice. Microscopically, the incidences of squamous hyperplasia of the forestomach epithelium were significantly increased in male rats in the 5 and 10 mg/kg groups, female rats administered 2.5 mg/kg or greater, and male and female mice administered 2.5, 5, or 10 mg/kg. Male and female rats in the 10 mg/kg groups and 20 mg/kg male and female mice had significantly increased incidences of glandular stomach hemorrhage. Female mice in the 20 mg/kg group also had significantly increased incidences of glandular stomach inflammation and epithelial necrosis. Allyl acetate was mutagenic in S. typhimurium strains TA100 and TA1535, in the absence of S9 activation. With S9, no mutagenicity was detected in these two strains; negative results were obtained in strains TA97 and TA98, with and without S9. Allyl alcohol was not mutagenic in four strains of S. typhimurium, with or without S9 metabolic activation. Acrolein, tested in a preincubation protocol, was weakly mutagenic in S. typhimurium strain TA100 in the presence of 10% induced rat liver S9. Equivocal results were obtained in strains TA100 and TA1535 with 10% induced hamster liver S9. Negative results were obtained with TA97, TA98, and TA1538 under all test conditions, and acrolein gave negative results in all four S. typhimurium strains tested for mutation induction under a vapor protocol. No induction of micronuclei was noted in bone marrow erythrocytes of male rats administered allyl acetate by gavage three times at 24-hour intervals. No significant increases in micronucleated erythrocytes were noted in bone marrow samples from male rats administered allyl alcohol by intraperitoneal injection for 3 days. A small, but significant increase in the frequency of micronucleated normochromatic erythrocytes was observed in the peripheral blood of female mice administered allyl acetate by gavage for 14 weeks; no increase was observed in male mice. No increases in the frequencies of micronucleated normochromatic erythrocytes were observed in the peripheral blood of male or female mice administered allyl alcohol or acrolein by gavage for 14 weeks. Acrolein induced sister chromatid exchanges in cultured Chinese hamster ovary cells in the absence, but not the presence, of S9; it did not induce chromosomal aberrations, with or without S9. Results of three independent Drosophila melanogaster sex linked recessive lethal tests in which acrolein was administered to adult flies via feeding or injection and to larvae via feeding were negative.

Acetates↗

Relationships between cell density, glutathione and proliferation of A549 human lung adenocarcinoma cells treated with acrolein.

Acrolein is a highly electrophilic alpha,beta-unsaturated aldehyde to which humans are exposed in various situations. Acrolein reacts rapidly with and depletes cellular glutathione (GSH), and is toxic to various types of cells. In the current study, the ability of acrolein to alter proliferation of A549 cells was found to be dependent on cell density as well as total cell number. Thus, 'doses' must be expressed per cell rather than as a concentration, and all related studies need to be performed by plating a constant number of cells. A549 cells were plated at various densities and treated with acrolein after 48 h. Acrolein doses up to 47 fmol/cell at the time of treatment did not cause cell lethality. However, growth of A549 cells (as shown by thymidine incorporation, alamarBlue and total protein) was inhibited at acrolein levels > 34 fmol/cell in 6-well plates seeded at 5000 cells/cm2 48 h prior to treatment. Cellular GSH levels were decreased 34% by 2 h at acrolein levels of 6.7 fmol/cell and by 65% at 47 fmol/cell. Recovery of GSH was rapid at 6.7-47 fmol/cell acrolein, returning to control levels or above by 12 h post-treatment. These data show a strong correlation between cellular GSH and proliferation. The apparent conflict with a previous study of Ramu et al., suggesting that sublethal concentrations (up to 10 microM) of acrolein inhibited the proliferation of A549 cells without a decline in total cellular GSH, arose because, while the acrolein concentration was the same in cells used for proliferation and GSH assays, GSH measurements were done in cells plated at a higher density, resulting in a much lower acrolein dose per cell. Interestingly, very low dose levels of acrolein with cells seeded at low densities stimulated cell growth despite an initial decline in GSH content. Preliminary studies with the stress genes hsp70 and gadd153 suggest that acrolein at 35 fmol/cell does not stimulate formation of their mRNA beyond the level stimulated by a 2 h incubation in serum-free medium but may actually delay or decrease the induced expression. The mechanism(s) of the inhibitory and mitogenic effects of acrolein remains to be determined, but could be due to changes in gene expression induced by this electrophile, perhaps mediated by changes in GSH.

Acrolein↗

N(epsilon)-(3-methylpyridinium)lysine, a major antigenic adduct generated in acrolein-modified protein.

Acrolein, a representative carcinogenic aldehyde, that could be ubiquitously generated in biological systems under oxidative stress shows facile reactivity with a nucleophile such as a protein. In this study, to gain a better understanding of the molecular basis of acrolein modification of protein, we characterized the acrolein modification of a model peptide (the oxidized B chain of insulin) by electrospray ionization-liquid chromatography/mass spectrometry method and established a novel acrolein-lysine condensation reaction. In addition, we found that this condensation adduct represented the major antigenic adduct generated in acrolein-modified protein. To identify the modification site and structures of adducts generated in the acrolein-modified insulin B chain, both the acrolein-pretreated and untreated peptides were digested with V8 protease and the resulting peptides were subjected to electrospray ionization-liquid chromatography/mass spectrometry. This technique identified nine peptides, which contained the acrolein adducts at Lys-29 and the N terminus, and revealed that the reaction of the insulin B chain with acrolein gave multiple adducts, including an unknown adduct containing two molecules of acrolein per lysine. To identify this adduct, we incubated N(alpha)-acetyllysine with acrolein and isolated a product having the same molecular mass as the unknown acrolein-lysine adduct. On the basis of the chemical and spectroscopic evidence, the adduct was determined to be a novel pyridinium-type lysine adduct, N(epsilon)-(3-methylpyridinium)lysine (MP-lysine). The formation of MP-lysine was confirmed by amino acid analysis of proteins treated with acrolein. More notably, this condensation adduct appeared to be an intrinsic epitope of a monoclonal antibody 5F6 that had been raised against acrolein-modified protein.

Acrolein↗

Acrolein-induced injury to cultured pulmonary artery endothelial cells.

We evaluated the specific effects of acrolein on sulfhydryl status and plasma membrane-dependent functions of cultured pulmonary artery endothelial cells. Acrolein exposure caused a dose-dependent increase in lactate dehydrogenase (LDH) release and decreases in reduced glutathione (GSH) and protein sulfhydryl (P-SH) content, whereas oxidized glutathione (GSSG) content was not altered. Exposure to 4.5 microM, but not 1.5 or 3.0 microM, of acrolein caused significant (p < 0.05) LDH release. With increasing concentrations (25 microM) of acrolein, LDH release was increased to 66% (p < 0.001). Acrolein (3.0-25 microM) resulted in 36 to 100% reductions in GSH content, whereas reductions in P-SH content at these concentrations of acrolein ranged from 11 to 37%. Uptake of amino acids (cystine, glycine, and glutamic acid) and incorporation of valine into the protein fraction were significantly reduced in a dose-dependent fashion in acrolein (1.5-4.5 microM)-exposed cells. Reductions in cystine, glycine, and glutamic acid uptakes were maximal in cells exposed to 3 and 4.5 microM acrolein (p < 0.001). Similarly, maximum reductions (p < 0.001) in both uptake and incorporation of valine into the protein fraction were observed at 3.0 and 4.5 microM acrolein. Acrolein (1.5 microM) also resulted in significant loss of plasma membrane-specific Na+/K(+)-ATPase as well as plasma membrane P-SH content (p < 0.05 for both). When cells were treated with ouabain, reductions in amino acid uptake were observed, and this appeared to mimic the effect of acrolein exposure. When isolated plasma membranes were exposed to a known SH-alkylating agent, N-ethylmaleimide, losses of Na+/K(+)-ATPase and P-SH content were observed and were similar to the effects following exposure to acrolein. These results demonstrate that acrolein exposure results in alterations of plasma membrane-dependent transport in pulmonary artery endothelial cells, leading to reduced availability of precursor amino acids used in GSH and protein synthesis. This plasma membrane injury is accompanied by reductions in the GSH and P-SH contents of these cells. Loss of the plasma membrane P-SH appears to be associated with specific inactivation of Na+/K(+)-ATPase.

Acrolein↗