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PubMed1990

Acrolein stimulates eicosanoid release from bovine airway epithelial cells.

Injury to the airway mucosa after exposure to environmental irritants is associated with pulmonary inflammation and bronchial hyperresponsiveness. To better understand the relationships between mediator release and airway epithelial cell injury during irritant exposures, we studied the effects of acrolein, a low-molecular-weight aldehyde found in cigarette smoke, on arachidonic acid metabolism in cultured bovine tracheal epithelial cells. Confluent airway epithelial cell monolayers, prelabeled with [3H]arachidonic acid, released significant levels of 3H activity when exposed (20 min) to 100 microM acrolein. [3H]arachidonic acid products were resolved using reverse-phase high-performance liquid chromatography. Under control conditions the released 3H activity coeluted predominantly with the cyclooxygenase product, prostaglandin (PG) E2. After exposure to acrolein, significant "peaks" in 3H activity coeluted with the lipoxygenase products 12-hydroxyeicosatetraenoic acid (HETE) and 15-HETE, as well as with PGE2, PGF2 alpha, and 6-keto-PGF1 alpha. Dose-response relationships for acrolein-induced release of immunoreactive PGF2 alpha and PGE2 from unlabeled epithelial monolayers demonstrated 30 microM acrolein as the threshold dose, with 100 microM acrolein inducing nearly a fivefold increase in both PGF2 alpha and PGE2. Cellular viability after exposure to 100 microM acrolein, determined by released lactate dehydrogenase activity, was not affected until exposure periods were greater than or equal to 2 h. These results implicate the airway epithelial cell as a possible source of eicosanoids after exposure to acrolein.

Acrolein↗
PubMed1993

Acrolein increases airway sensitivity to substance P and decreases NEP activity in guinea pigs.

The effects of acrolein exposure on airway responses to intravenous substance P were determined in guinea pigs exposed to vehicle or 1.6 ppm acrolein for 7.5 h on 2 consecutive days and examined 1, 4, 8, 15, and 28 days after exposure by use of pulmonary mechanics and bronchoalveolar lavage (BAL). Lung, trachea, liver, and BAL fluid were also assayed for neutral endopeptidase (NEP) activity 1, 7, and 28 days after exposure. Pulmonary inflammation and epithelial damage were prominent 1 day after acrolein exposure. NEP activity was decreased in the lungs, trachea, and liver 1 and 7 days after acrolein. Twenty-eight days after exposure, NEP activity in the lungs and liver was not significantly different in vehicle- and acrolein-exposed guinea pigs but was still reduced in tracheal tissue. The BAL NEP activity in acrolein-exposed guinea pigs was approximately twice that of vehicle control guinea pigs at all three time points. Acrolein caused a prolonged increase in airway sensitivity to substance P. Experiments performed in the presence of thiorphan suggested that the acrolein-induced reduction in NEP may contribute to increased airway sensitivity to aerosolized substance P, but the increase in airway sensitivity to intravenous substance P may occur by additional mechanisms.

Acrolein↗
PubMed1988

Pathobiological effects of acrolein in cultured human bronchial epithelial cells.

The ability of the highly reactive aldehyde acrolein to affect growth, membrane integrity, differentiation, and thiol status and to cause DNA damage has been studied at serum- and thiol-free conditions using cultured human bronchial epithelial cells. Acrolein markedly decreases colony survival at 3 microM whereas about 10-fold higher concentrations are required to increase membrane permeability, measured as uptake of trypan blue dye. Acrolein at micromolar concentrations also causes epithelial cells to undergo squamous differentiation as indicated by decreased clonal growth rate, dose-dependent increased formation of cross-linked envelopes, and increased cell planar surface area. Acrolein causes a marked and dose-dependent cellular depletion of total and specific free low-molecular-weight thiols as well as protein thiols. Exposure to acrolein did not cause oxidation of glutathione indicating that thiol depletion occurred by direct conjugation of reduced glutathione to acrolein without concomitant generation of active oxygen species. Furthermore, acrolein is genotoxic and causes both DNA single strand breaks and DNA protein cross-links in human bronchial epithelial cells. The results indicate that acrolein causes several cytopathic effects that relate to multistage carcinogenesis in the human bronchial epithelium.

Acrolein↗
PubMed1984

The acute effects of single and repeated injections of acrolein and other aldehydes.

The irritating aldehyde acrolein was injected intraperitoneally into mice. A single injection at 4 mg/kg gave rise to a 5-fold increase in plasma total lactate dehydrogenase (LDH) activity, with the peak after approximately 10 h. The pattern of LDH isoenzymes was not altered. Repeated injections (daily or weekly) caused a progressively less pronounced effect on the LDH activity. Experiments with formaldehyde and crotonaldehyde gave essentially the same results. The LD50 for acrolein i.p. in mice was increased from a level of 7 mg/kg to a level of 12 mg/kg by pretreatment with sublethal doses of 4 mg/kg/day for 5 days. Thus, the response to repeated acrolein injections, in terms of LDH and LD50, indicates an acquired tolerance against the irritant. Likewise, pretreatment with formaldehyde or crotonaldehyde could induce tolerance, in terms of LDH activity, towards a subsequent injection of acrolein. Histopathological examination revealed that spleen, adrenals and thymus were affected. The thymus markedly decreased in size after repeated injections of acrolein, crotonaldehyde or formaldehyde. Adrenalectomized mice given acrolein showed no thymus atrophy. A single injection of aldehyde caused an increased level of the adrenal hormone corticosterone in blood plasma. Adrenalectomized mice still showed a certain tolerance, in terms of LDH activity, after repeated injections of acrolein, but the increase in plasma LDH activity was smaller than for normal animals. Treatment with acrolein for six days did not change the level of reduced glutathione or the glutathione S-transferase activity in liver cytosol, but the rate of glutathione synthesis was increased. It is concluded that adrenalectomy does not completely prevent the development of tolerance in mice. It is possible that an increased metabolism can partially explain the acquired tolerance.

Acrolein↗
PubMed1994

Aldose reductase-catalyzed reduction of acrolein: implications in cyclophosphamide toxicity.

Acrolein, a highly cytotoxic aldehyde, is a metabolic by-product of the antineoplastic agent cyclophosphamide and is responsible for the development of hemorrhagic cystitis, a serious side effect of cyclophosphamide therapy. Aldose reductase (EC 1.1.1.21), a member of the aldo-keto reductase superfamily, catalyzes the NADPH-dependent reduction of acrolein to allyl alcohol (Km = 80 microM, kcat = 87 min-1). Aldose reductase is expressed at different levels in individuals. This suggests that individual differences in the reductive metabolism of acrolein may be a determinant of acrolein toxicity. In addition to being a substrate, acrolein also produces a time-dependent 7-20-fold increase in the activity of aldose reductase toward a variety of substrates. This involves initial binding of acrolein to a second site (Ks = 58 microM). Acrolein activation of aldose reductase results not only in higher kcat values for all substrates but also in higher Km values and decreased catalytic efficiencies. Acrolein activation of aldose reductase reduces its affinity for aldose reductase inhibitors.

Acrolein↗
PubMed1989

Metabolism of the glutathione-acrolein adduct, S-(2-aldehydo-ethyl)glutathione, by rat liver alcohol and aldehyde dehydrogenase.

The oxidative and reductive metabolism of the acrolein-glutathione adduct, S-(2,aldehydo-ethyl)glutathione, by rat liver aldehyde dehydrogenase (ALDH) and alcohol dehydrogenase (ADH) was characterized. The glutathione-acrolein adduct is oxidized to the respective acid by two different forms of ALDH contained in rat liver cytosol which are distinct from two forms of ALDH present in the mitochondria also capable of oxidizing the aldehyde moiety of the adduct. Extensive kinetic characterization (Km, Vmax and V/K parameters) of the ALDH enzymes suggest that the glutathione-acrolein adduct is oxidized most efficiently by one form of mitochondrial ALDH which is 3.5 to 175 times more active (based on V/K comparisons) than the other forms of mitochondrial and cytosolic ALDH evaluated. The glutathione-acrolein adduct is also subject to reductive metabolism by rat liver ALH. However, the Km value (877 microM) for reduction of the adduct suggests that this would be a minor pathway of metabolism. Collectively, these results indicate that the glutathione-acrolein adduct formed after exposure to acrolein, or as a result of allyl alcohol oxidation and cyclophosphamide metabolism, can be oxidized by hepatic ALDH or ADH, respectively. However, the kinetic parameters for these pathways suggest that micromolar concentrations of this adduct may accumulate before these enzyme systems mediate significant oxidative or reductive pathways of detoxification. The proposition that the glutathione-acrolein adduct may play a role in acrolein-mediated hepatotoxicity is discussed.

Alcohol Dehydrogenase↗
PubMed1986

The embryolethality and teratogenicity of acrolein in cultured rat embryos.

Acrolein, a three-carbon unsaturated aldehyde, is teratogenic to rats in vivo following intraamniotic administration but has been reported not to be teratogenic in vitro in the rat whole embryo culture system. In this study the effects of acrolein on rat embryos cultured in the standard medium consisting of rat serum were assessed over a narrow-concentration range. Additionally, a comparison was done of the effects of culture in a serum medium vs. a serum-free medium. In the serum medium acrolein caused 100% embryolethality at 140 microM and was found to be teratogenic in the concentration range of 80-120 microM. In the serum-free medium acrolein was 100% embryolethal at 20 microM and was teratogenic in the range of 5-15 microM. The EC50 for malformations in the serum medium was 137 microM, whereas that for embryolethality was 115 microM; the EC50s for malformations and embryolethality in the serum-free medium were 2.8 microM and 8.3 microM, respectively. Malformations were observed in the brain, facial area, and heart in addition to blebs and twisted or kinked bodies. Decreases in yolk sac diameter, crown-rump length, head length, number of somites, morphological score, and protein content were observed within the teratogenic ranges in each type of medium. Thus acrolein is teratogenic and embryolethal in vitro as well as in vivo. Dissociation between embryolethality and teratogenicity was seen in the serum-free medium. The slope of the acrolein log concentration-response curve in the serum-free medium was twice that in the serum medium, indicating that acrolein may have a different mechanism of action in this medium.

Abnormalities, Drug-Induced↗
PubMed1989

Effects of phosphoramide mustard and acrolein, cytotoxic metabolites of cyclophosphamide, on mouse limb development in vitro.

Phosphoramide mustard and acrolein are toxic and reactive metabolites of the widely used anticancer drug and known teratogen cyclophosphamide. To study the mechanism(s) involved and to determine which of the active metabolites of cyclophosphamide is responsible for the production of limb malformations, the effects of exposure of cultured limb buds to phosphoramide mustard and acrolein were investigated. Fore- and hindlimbs were excised from ICR mice on day 12 of gestation and cultured in roller bottles for 6 days. Limbs were exposed to either phosphoramide mustard or acrolein (10 or 50 micrograms/ml) for the first 20 hours of the culture period. Exposure to phosphoramide mustard produced limb reduction malformations in both the fore- and hindlimbs; total limb bone area was greatly reduced, while the relative contribution of the paw to this area in forelimbs was increased. There was a fourfold reduction in both DNA and RNA; protein content was reduced only by one-half. Alkaline phosphatase activity was significantly decreased in fore- and hindlimbs exposed to phosphoramide mustard, whereas creatine phosphokinase activity was only reduced in hindlimbs in the limbs exposed to the higher concentration of phosphoramide mustard. Exposure to acrolein also produced malformed limbs with a mangled appearance; however, total limb bone area and the relative contribution of the long bones versus paw structures were not altered. Acrolein exposure had little effect on growth parameters such as DNA (decreased only in hindlimbs exposed to 50 micrograms/ml), RNA (increased in hindlimbs exposed to 50 micrograms/ml), or protein content. Alkaline phosphatase and creatine phosphokinase activities were not altered in acrolein-exposed fore- or hindlimbs. Thus, phosphoramide mustard and acrolein have dramatically different effects on developing limbs in vitro; this observation may indicate that they have different targets and/or mechanisms of action as teratogens in the limb. The effects of phosphoramide mustard are very similar to those of "activated" cyclophosphamide (4-hydroperoxycyclophosphamide).

Acrolein↗
PubMed1995

Acrolein-induced smooth muscle hyperresponsiveness and eicosanoid release in excised ferret tracheae.

Acrolein is a ubiquitous toxic air pollutant that can have adverse lung effects. To understand the mechanism governing airway reactivity in relation to acrolein uptake, in vitro experiments were conducted in which excised tracheae from ferrets were exposed for 1 hr to a unidirectional constant flow (100 ml/min) of an acrolein-in-air mixture at several concentrations (0-12.5 ppm). During exposure, acrolein uptake into the trachea was determined by a chromatographic analysis of gas samples taken at the entrance and at the exit of the trachea. Smooth muscle contractility in response to carbachol (CCh), acetylcholine (ACh), and potassium chloride (KCl) was measured following exposure, and eicosanoids released in the perfusate baths were assayed. The results indicate that the fractional uptake into an excised ferret trachea was strongly dependent on inlet concentration, implying that diffusion and reaction processes of acrolein in airway tissue are not linear. Only the low concentration of acrolein caused an increase of eicosanoid release from the exposed tracheae in the perfusate bath; it is possible that, at higher exposure concentration, the epithelium was sloughed off and most of the eicosanoids were lost. Although acrolein did not alter smooth muscle response to KCl, it did increase the contractile responses to CCh and ACh, suggesting an alteration in the pharmacomechanical but not the electromechanical coupling of ferret tracheal smooth muscle; therefore, it is more likely that this hyperresponsiveness occurs primarily by a mobilization of intracellular Ca2+ stores rather than by an increased influx of extracellular Ca2+ through voltage-dependent channels.

Acrolein↗
PubMed1997

Acrolein-induced cell death in human alveolar macrophages.

Acrolein is an environmental air pollutant that is known to suppress respiratory host defense against infections. The mechanism of the decrease in host defense is not yet clear. In this study, the effects of acrolein on human alveolar macrophages and their function were examined. Acrolein caused dose-dependent cytotoxicity to alveolar macrophages as demonstrated by the induction of apoptosis and necrosis. In addition, at lower doses, acrolein caused induction of heme oxygenase 1 protein; however, stress protein 72 (SP72) was not induced. These findings demonstrated that acrolein caused a dose-dependent selective induction of a stress response, apoptosis, and necrosis in human alveolar macrophages. Macrophage function was assessed by release of cytokines in response to endotoxin stimulation. Acrolein caused a dose-dependent inhibition of release of IL-1beta, TNF-alpha, and IL-12. The inhibition of cytokine release and cytotoxicity to alveolar macrophages may in part be responsible for acrolein-induced immunosuppression of the lung.

Acrolein↗
PubMed2002

Measurement of 4-hydroxylation of ifosfamide in human liver microsomes using the estimation of free and protein-bound acrolein and codetermination of keto- and carboxyifosfamide.

PURPOSE: The aim of the present study was to determine the turnover (4-hydroxylation and N-dechloroethylation) of ifosfamide in a total of 25 human liver microsomal preparations in which the codetermination of keto- and carboxyifosfamide as well as the calculation of free and protein-bound acrolein was carried out for the first time. METHODS: The 4-hydroxylation of ifosfamide was estimated by using acrolein (free and protein-bound) and a newly developed procedure involving the codetermination of keto- and carboxyifosfamide (LC/MS). The ifosfamide N-dechloroethylation was determined as the sum of 2- and 3-dechloroethylifosfamide (LC/MS). RESULTS: Using the usual estimation of liberated free acrolein in 25 human liver microsomal preparations, the 4-hydroxylation of ifosfamide amounted to 0.28+/-0.16 nmol/min. nmol(P450). However, after calculating the 4-hydroxylation as the sum of free and protein-bound acrolein and keto- and carboxyifosfamide, a ninefold higher activity (2.40+/-0.73 nmol/min. nmol(P450)) was found. The percentage of the inactive metabolites keto- (25/25) and carboxyifosfamide (5/25) in the 4-hydroxylation amounted to only 0.79-5.25% (mean 2.90%). The ifosfamide N-dechloroethylation (mean 0.21+/-0.11 nmol/min. nmol(P450)) determined as the sum of 2- and 3-dechloroethylifosfamide was estimated as 8.3+/-4.3% of the total ifosfamide turnover. The application of the relative substrate-activity factor (RSF)-approach and the calculation of the contribution of various isoforms in the ifosfamide 4-hydroxylation yielded the following results: CYP 3A4: 58+/-31%, CYP 2A6: 25+/-15%, and CYP 2C9: 5+/-2% of the total measured 4-hydroxylation. A correlation between 4-hydroxylation and the N-dechloroethylation rates of ifosfamide and the activities of isoenzymes indicates the involvement of both CYP 3A4 ( P=0.026) and CYP 2C9 ( P=0.012) in the 4-hydroxylation reaction and of CYP 3A4 ( P<0.01) in the N-dechloroethylation reaction. CONCLUSIONS: The estimation of protein-bound acrolein should be included in the calculation of the ifosfamide 4-hydroxylation besides liberated free acrolein. Because of the small amounts of the inactive metabolites keto- and carboxyifosfamide, the exclusive determination of acrolein only (free and protein-bound) seems to suffice for the calculation of total ifosfamide hydroxylation. Using this method the hepatic in vitro turnover of ifosfamide was estimated as 92% for 4-hydroxylation (CYP 3A4 and CYP 2A6 mediated) and 8% for N-dechloroethylation (CYP 3A4 mediated), and in this way, a relative overestimation of the N-dechloroethylation of ifosfamide on the whole metabolism is avoided.

Acrolein↗
PubMed1984

Selected responses of hypertension-sensitive and resistant rats to inhaled acrolein.

The Dahl selected rat lines, one susceptible to salt-induced hypertension (DS) and the other resistant to salt-induced hypertension (DR), were exposed to filtered air, 0.4, 1.4, or 4.0 ppm acrolein for 6 h/day, 5 days/week for 62 days. All of the DS rats exposed to 4.0 ppm acrolein died within the first 11 days, while 60% of the DR animals survived the duration of the study. Neither dose dependent blood pressure changes nor altered behavioral characteristics were evident in either rat strain following acrolein exposure. Exposure to 4.0 ppm acrolein increased the level of several serum enzymes in the DR rats which survived. This concentration of acrolein also led to pulmonary edema and a significant increase in lung connective tissue in these animals. There was a marked difference in the pulmonary pathology observed in DS and DR rats exposed to 4.0 ppm acrolein. The lungs of moribund DS rats exhibited severe airway epithelial necrosis with edema and hemorrhage, while surviving DR rats primarily showed a proliferative change. Following exposure to 0.4 and 1.4 ppm acrolein, both rat lines displayed similar pathologic changes. Epithelial hyperplasia and/or clusters of macrophages were usually found near terminal bronchiolar areas. These findings suggest that further investigation of the physiopathologic sensitivity of the DS rat line may elucidate a model for investigating the underlying characteristics of stress susceptible populations.

Acrolein↗
PubMed2004

Acrolein induces axolemmal disruption, oxidative stress, and mitochondrial impairment in spinal cord tissue.

Acrolein, a byproduct of oxidative stress and lipid peroxidation, has been implicated in neurodegenerative disorders such as Alzheimer's disease, but not in spinal cord trauma, as a possible key factor in neuronal degeneration. Using an isolated guinea pig spinal cord model, we have found that acrolein, in a dose- and time-dependent manner, inflicts severe membrane disruption, a factor thought to be critical in triggering axonal deterioration and cell death. The concentration threshold of such detrimental effect is shown to be around 1 microM when acrolein was exposed for 4 h. The membrane damage is likely mediated in part by reactive oxygen species and lipid peroxidation, which were elevated in response to acrolein exposure. Antioxidants were able to significantly reduce acrolein-mediated membrane disruption which further supports the role of reactive oxygen species in the loss of membrane integrity. Mitochondrial function was also impaired after acrolein exposure which not only implicates but emphasizes the role of this organelle in reactive oxygen species generation. In summary, our data strongly suggest that at a clinically relevant concentration, acrolein can severely compromise membrane integrity and may further serve as an initiating toxin triggering secondary injury cascades following the initial physical insult to the spinal cord.

Acrolein↗
PubMed2004

Detoxication of the environmental pollutant acrolein by a rat liver aldo-keto reductase.

Acrolein is a highly reactive hazardous air pollutant of human health concern, particularly as it is a component of cigarette smoke. It can be metabolized by enzymes including the aldo-keto reductase (AKR) family of enzymes. AKR7A1 is a member of the AKR7 sub-family and can catalyse the reduction of toxic aldehydes, including alpha-unsaturated carbonyl compounds, to alcohols [Biochem. J. 312 (1995) 535]. In this study, the role of AKR7A1 in protecting against acrolein toxicity has been assessed by stably-expressing a cDNA encoding AKR7A1 in Chinese hamster V79 cells. Cells expressing AKR7A1 showed over 2-fold increased resistance to acrolein compared to V79 cells alone, as measured by 3-[4,4-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assays. IC50 increased from 45 microM in control V79-pCI-neo cells to 125microM for V79-AKR7A1 cells. Cells expressing AKR7A1 were also found to be less susceptible to DNA damage, showing a decrease in mutation rate in the presence of acrolein as measured by hypoxanthine guanine phosphoribosyl transferase (HGPRT) mutagenicity assays. The mutation rate for acrolein-exposed control cells was 20-fold higher than for acrolein-exposed AKR7A1-expressing cells. These results indicate that AKR7A1 has the potential to protect against acrolein-induced damage in vivo.

Acrolein↗
PubMed2003

Induction of cellular glutathione and glutathione S-transferase by 3H-1,2-dithiole-3-thione in rat aortic smooth muscle A10 cells: protection against acrolein-induced toxicity.

There is increasing evidence that aldehydes, including acrolein generated endogenously during the degradation process of biological molecules or the metabolism of foreign chemicals may be involved in the pathogenesis of cardiovascular diseases, such as atherosclerosis. Because glutathione (GSH) and GSH S-transferase (GST) are a major cellular defense against the toxic effects of reactive aldehydes, in this study we have characterized the inducibility of GSH and GST by the unique chemoprotective agent, 3H-1,2-dithiole-3-thione (D3T) and their protective effects against acrolein-induced toxicity in rat aortic smooth muscle A10 cells. Incubation of rat aortic A10 cells with micromolar concentrations of D3T resulted in a concentration- and time-dependent induction of both GSH and GST. Treatment of A10 cells with D3T also led to induction of gamma-glutamylcysteine synthetase, the key enzyme involved in GSH biosynthesis. Notably, the levels of GSH and GST remained higher than basal levels 72 h after removal of D3T from the culture media. To examine the protective effects of D3T-induced GSH and GST against reactive aldehyde-mediated toxicity, A10 cells were pretreated with D3T and then exposed to acrolein. Pretreatment of A10 cells with D3T resulted in a marked decrease of acrolein-induced toxicity as determined by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide reduction assay and morphological changes. To further demonstrate the involvement of GSH and GST in protecting against acrolein-induced toxicity, buthionine sulfoximine (BSO) and sulfasalazine were used to inhibit cellular GSH biosynthesis and GST activity, respectively. Either depletion of cellular GSH by BSO or inhibition of cellular GST by sulfasalazine led to a marked potentiation of acrolein-induced toxicity in A10 cells. Furthermore, co-treatment of cells with BSO was found to greatly abolish the protective effects of D3T on acrolein-induced toxicity. Taken together, our results demonstrate for the first time that both GSH and GST in aortic smooth muscle cells can be induced by D3T, and that this increased cellular defense affords great protection against reactive aldehyde-induced cardiovascular cell injury.

Acrolein↗
PubMed2002

Acrolein-induced cytotoxicity in cultured human bronchial epithelial cells. Modulation by alpha-tocopherol and ascorbic acid.

Acrolein is a highly reactive unsaturated hazardous air pollutant of human health concern, particularly as a component of cigarette smoke. In this study, the mechanisms of acrolein-induced cytotoxicity in human bronchial epithelial cells (HBE1) and the modulating effects of antioxidants were examined. Our results show that acrolein induces a cell death pathway in human bronchial epithelial cells, which retain key features of apoptosis, as indicated by phosphatidylserine (PS) externalization and DNA fragmentation. Acrolein-induced apoptosis was associated with depletion of cellular GSH and intracellular generation of oxidants. Supplementation of cells with either alpha-tocopherol or ascorbic acid was found to strongly inhibit acrolein-induced apoptosis and to prevent the increase in the generation of intracellular oxidants, although GSH depletion was unaffected. Moreover, recovery of cellular GSH levels after acrolein exposure was enhanced following either alpha-tocopherol or ascorbic acid supplementation. The intracellular generation of oxidants following acrolein exposure seems to be an important event triggering the apoptotic response in this model system.

Acrolein↗
PubMedDate not supplied

Identification of 3,N4-propanodeoxycytidine 5'-monophosphate formed by the reaction of acrolein with deoxycytidine 5'-monophosphate.

Acrolein reacts with deoxycytidine 5'-monophosphate at physiological pH to form one major adduct. A second minor adduct can be detected when a 3-fold excess of acrolein is present in the reaction mixture. The products were separated by ion-pair HPLC on two reverse-phase columns connected in series using triethylammonium formate as ion-pair reagent. The major adduct was characterized as 3-(5'-monophospho-2'-deoxyribosyl)-7,8, 9-trihydro-7-hydroxy-pyrimido [3,4-c]pyrimidin-2-one (3,N4-propanodeoxcytidine 5'-monophosphate). This mixture of diastereomers was formed by addition of C1 of acrolein to the exocyclic amino group at the 4-position of cytidine, followed by ring closure between C3 of acrolein and N3 of the heterocyclic ring. In order to address the utility of 32P postlabeling for the detection of this exocyclic adduct in acrolein-modified nucleic acids, an acrolein-deoxycytidine 3'-monophosphate reaction mixture was subjected to 32P postlabeling. 3-Dephosphorylation with nuclease P1 and the 3'-phosphatase activity of T4 polynucleotide kinase yields a nucleotide 5'-[32P] monophosphate which cochromatographs with 3,N4-propanodeoxycytidine 5'-monophosphate. These data indicate that 32P postlabeling and 3'-dephosphorylation can be used in conjunction with ion-pair HPLC for the detection and quantitation of acrolein-modified nucleotides.

Acrolein↗
PubMed2005

Differences in lysine adduction by acrolein and methyl vinyl ketone: implications for cytotoxicity in cultured hepatocytes.

Acrolein is a highly toxic environmental pollutant that readily alkylates the epsilon-amino group of lysine residues in proteins. In model systems, such chemistry involves sequential addition of two acrolein molecules to a given nitrogen, forming bis-Michael-adducted species that undergo aldol condensation and dehydration to form Nepsilon-(3-formyl-3,4-dehydropiperidino)lysine. Whether this ability to form cyclic adducts participates in the toxicity of acrolein is unknown. To address this issue, we compared the chemistry of protein adduction by acrolein to that of its close structural analogue methyl vinyl ketone, expecting that the alpha-methyl group would hinder the intramolecular cyclization of any bis-adducted species formed by methyl vinyl ketone. Both acrolein and methyl vinyl ketone displayed comparable protein carbonylating activity during in vitro studies with the model protein bovine serum albumin, confirming the alpha,beta,-unsaturated bond of both compounds is an efficient Michael acceptor for protein nucleophiles. However, differences in adduction chemistry became apparent during the use of electrospray ionization-MS to monitor reaction products in a lysine-containing peptide after modification by each compound. For example, although a Schiff base adduct was detected following reaction of the peptide with acrolein, an analogous species was not formed by methyl vinyl ketone. Furthermore, while ions corresponding to mono- and bis-Michael adducts were detected at the N-terminus and lysine residues following peptide modification by both carbonyls, only acrolein modification generated ions attributable to cyclic adducts. Despite these differences in adduction chemistry, in mouse hepatocytes, the two compounds exhibited very comparable abilities to induce rapid, concentration-dependent cell death as well as protein carbonylation. These findings suggest that the acute toxicity of short-chain alpha,beta-unsaturated carbonyl compounds involves their ability to form acyclic Michael addition adducts rather than Schiff conjugates or heterocyclic adducts.

Acrolein↗