Selective protection by stably transfected human ALDH3A1 (but not human ALDH1A1) against toxicity of aliphatic aldehydes in V79 cells

Toxic medium chain length alkanals, alkenals, and 4-hydroxyalkenals that are generated during lipid peroxidation are potential substrates for aldehyde dehydrogenase (ALDH) isoforms. We have developed transgenic cell lines to examine the potential for either human ALDH1A1 or ALDH3A1 to protect against damage mediated by these toxic aldehydes. Using crude cytosols from stably transfected cell lines, these aldehydes were confirmed to be excellent substrates for ALDH3A1, but were poorly oxidized by ALDH1A1. Expression of ALDH3A1 by stable transfection in V79 cells conferred a high level of protection against growth inhibition by the medium-chain length aldehyde substrates with highest substrate activity, including hexanal, trans-2-hexenal, trans-2-octenal, trans-2-nonenal, and 4-hydroxy-2-nonenal (HNE). This was reflected in a parallel ability of ALDH3A1 to prevent depletion of glutathione by these aldehydes. Expression of hALDH3 completely blocked the potent induction of apoptosis by HNE in both V79 cells and in a RAW 264.7 murine macrophage cell line, consistent with the observed total prevention of HNE-protein adduct formation. Structure-activity studies indicated that the rank order of potency for the contributions of HNE functional groups to toxicity was aldehyde >/=C2=C3 double bond>C4-hydroxyl group. Oxidation of the aldehyde moiety of HNE to a carboxyl by ALDH3A1 expressed in stably transfected cell lines drastically reduced its potency for growth inhibition and apoptosis induction. In contrast, ALDH1A1 expression provided only moderate protection against trans-2-nonenal (t2NE), and none against the other six-nine carbon aldehydes. Neither ALDH1A1 nor ALDH3A1 conferred any protection against acrolein, acetaldehyde, or chloroacetaldehyde. A small degree of protection against malondialdehyde was afforded by ALDH1A1, but not ALDH3A1. Paradoxically, cells expressing ALDH3A1 were 1.5-fold more sensitive to benzaldehyde toxicity than control V79 cells. These studies demonstrate that expression of class 3 ALDH, but not class 1 ALDH, can be an important determinant of cellular resistance to toxicity mediated by aldehydes of intermediate chain length that are produced during lipid peroxidation.     

Aldehyde dehydrogenase 7A1 (ALDH7A1) attenuates reactive aldehyde and oxidative stress induced cytotoxicity

Mammalian aldehyde dehydrogenase 7A1 (ALDH7A1) is homologous to plant ALDH7B1 which protects against various forms of stress such as increased salinity, dehydration and treatment with oxidants or pesticides. Deleterious mutations in human ALDH7A1 are responsible for pyridoxine-dependent and folinic acid-responsive seizures. In previous studies, we have shown that human ALDH7A1 protects against hyperosmotic stress presumably through the generation of betaine, an important cellular osmolyte, formed from betaine aldehyde. Hyperosmotic stress is coupled to an increase in oxidative stress and lipid peroxidation (LPO). In this study, cell viability assays revealed that stable expression of mitochondrial ALDH7A1 in Chinese hamster ovary (CHO) cells provides significant protection against treatment with the LPO-derived aldehydes hexanal and 4-hydroxy-2-nonenal (4HNE) implicating a protective function for the enzyme during oxidative stress. A significant increase in cell survival was also observed in CHO cells expressing either mitochondrial or cytosolic ALDH7A1 treated with increasing concentrations of hydrogen peroxide (H(2)O(2)) or 4HNE, providing further evidence for anti-oxidant activity. In vitro enzyme activity assays indicate that human ALDH7A1 is sensitive to oxidation and that efficiency can be at least partially restored by incubating recombinant protein with the thiol reducing agent β-mercaptoethanol (BME). We also show that after reactivation with BME, recombinant ALDH7A1 is capable of metabolizing the reactive aldehyde 4HNE. In conclusion, ALDH7A1 mechanistically appears to provide cells protection through multiple pathways including the removal of toxic LPO-derived aldehydes in addition to osmolyte generation.     

Oxidation of 4-hydroxy-2-nonenal by succinic semialdehyde dehydrogenase (ALDH5A)

Elevated levels of 4-hydroxy-trans-2-nonenal (HNE) are implicated in the pathogenesis of numerous neurodegenerative disorders. Although well-characterized in the periphery, the mechanisms of detoxification of HNE in the CNS are unclear. HNE is oxidized to a non-toxic metabolite in the rat cerebral cortex by mitochondrial aldehyde dehydrogenases (ALDHs). Two possible ALDH enzymes which might oxidize HNE in CNS mitochondria are ALDH2 and succinic semialdehyde dehydrogenase (SSADH/ALDH5A). It was previously established that hepatic ALDH2 can oxidize HNE. In this work, we tested the hypothesis that SSADH oxidizes HNE. SSADH is critical in the detoxification of the GABA metabolite, succinic semialdehyde (SSA). Recombinant rat SSADH oxidized HNE and other alpha,beta-unsaturated aldehydes. Inhibition and competition studies in rat brain mitochondria showed that SSADH was the predominant oxidizing enzyme for HNE but only contributed a portion of the total oxidizing activity in liver mitochondria. In vivo administration of diethyldithiocarbamate (DEDC) effectively inhibited (86%) ALDH2 activity but not HNE oxidation in liver mitochondria. The data suggest that a relationship between the detoxification of SSA and the neurotoxic aldehyde HNE exists in the CNS. Furthermore, these studies show that multiple hepatic aldehyde dehydrogenases are able to oxidize HNE.     

Mechanisms involved in the protection of UV-induced protein inactivation by the corneal crystallin ALDH3A1

Various lines of evidence have shown that ALDH3A1 (aldehyde dehydrogenase 3A1) plays a critical and multifaceted role in protecting the cornea from UV-induced oxidative stress. ALDH3A1 is a corneal crystallin, which is defined as a protein recruited into the cornea for structural purposes without losing its primary function (i.e. metabolism). Although the primary role of ALDH3A1 in the metabolism of toxic aldehydes has been clearly demonstrated, including the detoxification of aldehydes produced during UV-induced lipid peroxidation, the structural role of ALDH3A1 in the cornea remains elusive. We therefore examined the potential contribution of ALDH3A1 in maintaining the optical integrity of the cornea by suppressing the aggregation and/or inactivation of other proteins through chaperone-like activity and other protective mechanisms. We found that ALDH3A1 underwent a structural transition near physiological temperatures to form a partially unfolded conformation that is suggestive of chaperone activity. Although this structural transition alone did not correlate with any protection, ALDH3A1 substantially reduced the inactivation of glucose-6-phosphate dehydrogenase by 4-hydroxy-2-nonenal and malondialdehyde when co-incubated with NADP(+), reinforcing the importance of the metabolic function of this corneal enzyme in the detoxification of toxic aldehydes. A large excess of ALDH3A1 also protected glucose-6-phosphate dehydrogenase from inactivation because of direct exposure to UVB light, which suggests that ALDH3A1 may shield other proteins from damaging UV rays. Collectively, these data demonstrate that ALDH3A1 can reduce protein inactivation and/or aggregation not only by detoxification of reactive aldehydes but also by directly absorbing UV energy. This study provides for the first time mechanistic evidence supporting the structural role of the corneal crystallin ALDH3A1 as a UV-absorbing constituent of the cornea.     

Molecular mechanisms of ALDH3A1-mediated cellular protection against 4-hydroxy-2-nonenal

Evidence suggests that aldehydic molecules generated during lipid peroxidation (LPO) are causally involved in most pathophysiological processes associated with oxidative stress. 4-Hydroxy-2-nonenal (4-HNE), the LPO-derived product, is believed to be responsible for much of the cytotoxicity. To counteract the adverse effects of this aldehyde, many tissues have evolved cellular defense mechanisms, which include the aldehyde dehydrogenases (ALDHs). Our laboratory has previously characterized the tissue distribution and metabolic functions of ALDHs, including ALDH3A1, and demonstrated that these enzymes may play a significant role in protecting cells against 4-HNE. To further characterize the role of ALDH3A1 in the oxidative stress response, a rabbit corneal keratocyte cell line (TRK43) was stably transfected to overexpress human ALDH3A1. These cells were studied after treatment with 4-HNE to determine their abilities to: (a) maintain cell viability, (b) metabolize 4-HNE and its glutathione conjugate, (c) prevent 4-HNE-protein adduct formation, (d) prevent apoptosis, (e) maintain glutathione homeostasis, and (f) preserve proteasome function. The results demonstrated a protective role for ALDH3A1 against 4-HNE. Cell viability assays, morphological evaluations, and Western blot analyses of 4-HNE-adducted proteins revealed that ALDH3A1 expression protected cells from the adverse effects of 4-HNE. Based on the present results, it is apparent that ALDH3A1 provides exceptional protection from the adverse effects of pathophysiological concentrations of 4-HNE such as may occur during periods of oxidative stress.     

Arachidonic acid suppresses growth of human lung tumor A549 cells through down-regulation of ALDH3A1 expression

Expression of aldehyde dehydrogenase 3A1 (ALDH3A1) in certain normal and tumor cells is associated with protection against the growth inhibitory effect of reactive aldehydes generated during membrane lipid peroxidation. We found that human lung tumor (A549) cells, which express high levels of ALDH3A1 protein, were significantly less susceptible to the antiproliferative effects of 4-hydroxynonenal compared to human hepatoma HepG2 or SK-HEP-1 cells that lack ALDH3A1 expression. However, A549 cells became susceptible to lipid peroxidation products when they were treated with arachidonic acid. The growth suppression of A549 cells induced by arachidonic acid was associated with increased levels of lipid peroxidation and with reduced ALDH3A1 enzymatic activity, protein, and mRNA levels. Furthermore, arachidonic acid treatment of the A549 cells resulted in an increased expression of peroxisome proliferator-activated receptor gamma (PPARgamma), whereas NF-kappaB binding activity was inhibited. Blocking PPARgamma using a selective antagonist, GW9662, prevented the arachidonic acid-mediated reduction of ALDH3A1 expression as well as the growth inhibition of A549 cells, suggesting the central role of PPARgamma in these phenomena. The increase in PPARgamma and the reduction in ALDH3A1 were also prevented by exposing cells to vitamin E concomitant with arachidonic acid treatment. In conclusion, our data show that the arachidonic acid-induced suppression of A549 cell growth is associated with increased lipid peroxidation and decreased ALDH3A1 expression, which may be due to activation of PPARgamma.     

Expression and initial characterization of human ALDH3B1

Aldehyde dehydrogenases (ALDHs) are critical enzymes in the metabolism of endogenous and exogenous aldehydes. The human genome contains 19 putatively functional ALDH genes; ALDH3B1 belongs to the ALDH3 family. While recent studies have linked the ALDH3B1 locus to schizophrenia, nothing was known, until now, about the properties and significance of the ALDH3B1 protein. The aim of this study was to characterize the ALDH3B1 protein. Human ALDH3B1 was baculovirus-expressed and found to be catalytically active towards medium- and long-chain aliphatic aldehydes and the aromatic aldehyde benzaldehyde. Western blot analyses indicate that ALDH3B1 is highly expressed in kidney and liver and moderately expressed in various brain regions. ALDH3B1-transfected HEK293 cells were significantly protected against cytotoxicity induced by the lipid peroxidation product octanal when compared to vector-transfected cells. This study shows for the first time the functionality, expression and protective role of ALDH3B1 and indicates a potential physiological role of ALDH3B1 against oxidative stress.     

Omeprazole as a potent activator of human cytosolic aldehyde dehydrogenase ALDH1A1

BackgroundAccumulation of lipid aldehydes plays a key role in the etiology of human diseases where high levels of oxidative stress are generated. In this regard, activation of aldehyde dehydrogenases (ALDHs) prevents oxidative tissue damage during ischemia-reperfusion processes. Although omeprazole is used to reduce stomach gastric acid production, in the present work this drug is described as the most potent activator of human ALDH1A1 reported yet.MethodsDocking analysis was performed to predict the interactions of omeprazole with the enzyme. Recombinant human ALDH1A1 was used to assess the effect of omeprazole on the kinetic properties. Temperature treatment and mass spectrometry were conducted to address the nature of binding of the activator to the enzyme. Finally, the effect of omeprazole was evaluated in an in vivo model of oxidative stress, using E. coli cells expressing the human ALDH1A1.ResultsOmeprazole interacted with the aldehyde binding site, increasing 4–6 fold the activity of human ALDH1A1, modified the kinetic properties, altering the order of binding of substrates and release of products, and protected the enzyme from inactivation by lipid aldehydes. Furthermore, omeprazole protected E. coli cells over-expressing ALDH1A1 from the effects of oxidative stress generated by H₂O₂ exposure, reducing the levels of lipid aldehydes and preserving ALDH activity.ConclusionOmeprazole can be repositioned as a potent activator of human ALDH1A1 and may be proposed for its use in therapeutic strategies, to attenuate the damage generated during oxidative stress events occurring in different human pathologies.     

Detoxification of promutagenic aldehydes derived from methylpyrenes by human aldehyde dehydrogenases ALDH2 and ALDH3A1

Methylated polycyclic aromatic hydrocarbons can be metabolically activated via benzylic hydroxylation and sulpho conjugation to reactive esters, which can induce mutations and tumours. Yet, further oxidation of the alcohol may compete with this toxification. We previously demonstrated that several human alcohol dehydrogenases (ADH1C, 2, 3 and 4) oxidise various benzylic alcohols (derived from alkylated pyrenes) to their aldehydes with high catalytic efficiency. However, all these ADHs also catalysed the reverse reaction, the reduction of the aldehydes to the alcohols, with comparable or higher efficiency. Thus, final detoxification requires elimination of the aldehydes by further biotransformation. We have expressed two human aldehyde dehydrogenases (ALDH2 and 3A1) in bacteria. All pyrene aldehydes studied (1-, 2- and 4-formylpyrene, 1-formyl-6-methylpyrene and 1-formyl-8-methylpyrene) were high-affinity substrates for ALDH2 (K_m = 0.027–0.9 μM) as well as ALDH3A1 (K_m = 0.78–11 μM). Catalytic efficiencies (k_cat/K_m) were higher for ALDH2 than ALDH3A1 by a moderate to a very large margin depending on the substrate. Most important, they were also substantially higher than the catalytic efficiencies of the various ADHs for the reduction the aldehydes to the alcohols. These kinetic properties ensure that ALDHs, and particularly ALDH2, can complete the ADH-mediated detoxification.     

Antioxidant function of corneal ALDH3A1 in cultured stromal fibroblasts

Aldehyde dehydrogenase 3A1 (ALDH3A1) is highly expressed in epithelial cells and stromal keratocytes of mammalian cornea and is believed to play an important role in cellular defense. To explore a potential protective role against oxidative damage, a rabbit corneal fibroblastic cell line (TRK43) was stably transfected with the human ALDH3A1 and subjected to oxidative stress induced by H(2)O(2), mitomycin C (MMC), or etoposide (VP-16). ALDH3A1-transfected cells were more resistant to H(2)O(2,) MMC, and VP-16 compared to the vector-transfected cells. All treatments induced apoptosis only in vector-transfected cells, which was associated with increased levels of 4-hydroxy-2-nonenal (4-HNE)-adducted proteins. Treatment with H(2)O(2) resulted in a rise in reduced glutathione (GSH) levels in all groups but was more pronounced in the ALDH3A1-expressing cells. Treatment with the DNA-damaging agents led to GSH depletion in control groups, although the depletion was significantly less in ALDH3A1-expressing cells. Increased carbonylation of ALDH3A1 but not significant decline in enzymatic activity was observed after all treatments. In conclusion, our results suggest that ALDH3A1 may act to protect corneal cells against cellular oxidative damage by metabolizing toxic lipid peroxidation products (e.g., 4-HNE), maintaining cellular GSH levels and redox balance, and operating as an antioxidant.     

Trans-4-oxo-2-nonenal potently alters mitochondrial function

Alzheimer disease elevates lipid peroxidation in the brain and data indicate that the resulting lipid-aldehydes are pathological effectors of lipid peroxidation. The disposition of 4-substituted nonenals derived from arachidonate (20:4, n-6) and linoleate (18:2, n-6) oxidation is modulated by their protein adduction targets, their metabolism, and the nature of the 4-substitutent. Trans-4-oxo-2-nonenal (4-ONE) has a higher toxicity in some systems than the more commonly studied trans-4-hydroxy-2-nonenal (HNE). In this work, we performed a structure-function analysis of 4-hydroxy/oxoalkenal upon mitochondrial endpoints. We tested the hypotheses that 4-ONE, owing to a highly reactive nature, is more toxic than HNE and that HNE toxicity is enantioselective. We chose to study freshly isolated brain mitochondria because of the role of mitochondrial dysfunction in neurodegenerative disorders. Whereas there was little effect related to HNE chirality, our data indicate that in the mitochondrial environment, the order of toxic potency under most conditions was 4-ONE>HNE. 4-ONE uncoupled mitochondrial respiration at a concentration of 5μM and inhibited aldehyde dehydrogenase 2 (ALDH2) activity with an IC(50) of approximately 0.5μM. The efficacy of altering mitochondrial endpoints was ALDH2 inhibition>respiration=mitochondrial swelling=ALDH5A inhibition>GSH depletion. Thiol-based alkenal scavengers, but not amine-based scavengers, were effective in blocking the effects of 4-ONE upon respiration. Quantum mechanical calculations provided insights into the basis for the elevated reactivity of 4-ONE>HNE. Our data demonstrate that 4-ONE is a potent effector of lipid peroxidation in the mitochondrial environment.     

The enzymatic activity of human aldehyde dehydrogenases 1A2 and 2 (ALDH1A2 and ALDH2) is detected by Aldefluor, inhibited by diethylaminobenzaldehyde and has significant effects on cell proliferation and drug resistance

There has been a new interest in using aldehyde dehydrogenase (ALDH) activity as one marker for stem cells since the Aldefluor flow cytometry-based assay has become available. Diethylaminobenzaldehyde (DEAB), used in the Aldeflour assay, has been considered a specific inhibitor for ALDH1A1 isoform. In this study, we explore the effects of human ALDH isoenzymes, ALDH1A2 and ALDH2, on drug resistance and proliferation, and the specificity of DEAB as an inhibitor. We also screened for the expression of 19 ALDH isoenzymes in K562 cells using TaqMan Low Density Array (TLDA). We used lentiviral vectors containing the full cDNA length of either ALDH2 or ALDH1A2 to over express the enzymes in K562 leukemia and H1299 lung cancer cell lines. Successful expression was measured by activity assay, Western blot, RT-PCR, and Aldefluor assay. Both cell lines, with either ALDH1A2 or ALDH2, exhibited higher cell proliferation rates, higher clonal efficiency, and increased drug resistance to 4-hydroperoxycyclophosphamide and doxorubicin. In order to study the specificity of known ALDH activity inhibitors, DEAB and disulfiram, we incubated each cell line with either inhibitor and measured the remaining ALDH enzymatic activity. Both inhibitors reduced ALDH activity of both isoenzymes by 65-90%. Furthermore, our TLDA results revealed that ALDH1, ALDH7, ALDH3 and ALDH8 are expressed in K562 cells. We conclude that DEAB is not a specific inhibitor for ALDH1A1 and that Aldefluor assay is not specific for ALDH1A1 activity. In addition, other ALDH isoenzymes seem to play a major role in the biology and drug resistance of various malignant cells.     

Alda-1, an ALDH2 activator,protects against hepatic ischemia/reperfusion injury in rats via inhibition of oxidative stress

Previous studies have proved that activation of aldehyde dehydrogenase two (ALDH2) can attenuate oxidative stress through clearance of cytotoxic aldehydes, and can protect against cardiac, cerebral, and lung ischemia/reperfusion (I/R) injuries. In this study, we investigated the effects of the ALDH2 activator Alda-1 on hepatic I/R injury. Partial warm ischemia was performed in the left and middle hepatic lobes of Sprague-Dawley rats for 1?h, followed by 6?h of reperfusion. Rats received either Alda-1 or vehicle by intravenous injection 30?min before ischemia. Blood and tissue samples of the rats were collected after 6-h reperfusion. Histological injury, proinflammatory cytokines, reactive oxygen species (ROS), cellular apoptosis, ALDH2 expression and activity, 4-hydroxy-trans-2-nonenal (4-HNE) and malondialdehyde (MDA) were measured. BRL-3A hepatocytes were subjected to hypoxia/reoxygenation (H/R). Cell viability, ROS, and mitochondrial membrane potential were determined. Pretreatment with Alda-1 significantly alleviated I/R-induced elevations of alanine aminotransferase and aspartate amino transferase, and significantly blunted the pathological injury of the liver. Moreover, Alda-1 significantly inhibited ROS and proinflammatory cytokines production, 4-HNE and MDA accumulation, and apoptosis. Increased ALDH2 activity was found after Alda-1 administration. No significant changes in ALDH2 expression were observed after I/R. ROS was also higher in H/R cells than in control cells, which was aggravated upon treatment with 4-HNE, and reduced by Alda-1 treatment. Cell viability and mitochondrial membrane potential were inhibited in H/R cells, which was attenuated upon Alda-1 treatment. Activation of ALDH2 by Alda-1 attenuates hepatic I/R injury via clearance of cytotoxic aldehydes.     

ALDH2 protects against stroke by clearing 4-HNE

Aldehyde dehydrogenase 2 (ALDH2) is a mitochondrial enzyme that metabolizes ethanol and toxic aldehydes such as 4-hydroxy-2-nonenal (4-HNE). Using an unbiased proteomic search, we identified ALDH2 deficiency in stroke-prone spontaneously hypertensive rats (SHR-SP) as compared with spontaneously hypertensive rats (SHR). We concluded the causative role of ALDH2 deficiency in neuronal injury as overexpression or activation of ALDH2 conferred neuroprotection by clearing 4-HNE in in vitro studies. Further, ALDH2-knockdown rats revealed the absence of neuroprotective effects of PKCε. Moderate ethanol administration that is known to exert protection against stroke was shown to enhance the detoxification of 4-HNE, and to protect against ischemic cerebral injury through the PKCε-ALDH2 pathway. In SHR-SP, serum 4-HNE level was persistently elevated and correlated inversely with the lifespan. The role of 4-HNE in stroke in humans was also suggested by persistent elevation of its plasma levels for at least 6 months after stroke. Lastly, we observed that 21 of 1 242 subjects followed for 8 years who developed stroke had higher initial plasma 4-HNE levels than those who did not develop stroke. These findings suggest that activation of the ALDH2 pathway may serve as a useful index in the identification of stroke-prone subjects, and the ALDH2 pathway may be a potential target of therapeutic intervention in stroke.     

PPARgamma-mediated ALDH1A3 suppression exerts anti-proliferative effects in lung cancer by inducing lipid peroxidation

Context: The metabolic function of peroxisome proliferator-activated receptor gamma (PPARγ) in lung cancer remains unclear.

Objectives: To determine the relationship of PPARγ on ALDH1A3-induced lipid peroxidation to inhibit lung cancer cell growth.

Materials and methods: In silico analysis using microarray dataset was performed to screen the positive correlation between PPARγ and all ALDH isoforms. NUBIscan software and ChIP assay were used to identify the binding sites (BSs) of PPARγ on ALDH1A3 promoter. The expression of ALDH1A3 under thiazolidinedione (TZD) treatment was evaluated by QPCR and Western Blot in HBEC and H1993 cell lines. Upon treatment of TZD, colony formation assay was used to check cell growth inhibition and 4-hydroxy-2-nonenal (4HNE) production as lipid peroxidation marker was determined by Western Blot in PPARγ positive cell H1993 and PPARγ negative cell H1299.

Results: Compared to other ALDH isoforms, ALDH1A3 showed the highest positive correlation to PPARγ expression. ALDH1A3 upregulated PPARγ expression while PPARγ activation suppressed ALDH1A3. Among 2 potential screened PPARγ response elements, BS 1 and 2 in the promoter of ALDH1A3 gene, PPARγ bound directly to BS2. Ligand activation of PPARγ suppressed mRNA and protein expression of ALDH1A3. Growth inhibition was observed in H1993 (PPARγ positive cell) treated with PPARγ activator and ALDH inhibitor compared to H1299 (PPARγ negative cell). PPARγ activation increased 4HNE which is known to be suppressed by ALDH1A3.

Conclusions: ALDH1A3 suppression could be one of PPARγ tumor suppressive function. This study provides a better understanding of the role of PPARγ in lung cancer.     

Cytotoxicity and mutagenicity of dibenzo[a,l]pyrene and (+/-)-dibenzo[a,l]pyrene-11,12-dihydrodiol in V79MZ cells co-expressing either hCYP1A1 or hCYP1B1 together with human glutathione-S-transferase A1

We have used V79MZ hamster lung fibroblasts stably transfected with human cytochrome P450-1A1 (hCYP1A1; cell line designated V79MZh1A1) or P450-1B1 (hCYP1B1; cell line designated V79MZh1B1) alone, or in combination with human glutathione-S-transferase (GST) alpha-1 (hGSTA1), in order to examine GST protection against cytotoxicity and mutagenicity of dibenzo[a,l]pyrene (DBP) and the intermediate dihydrodiol metabolite (+/-)-DBP-11,12-dihydrodiol (DBPD). At comparable expression levels of hCYP1A1 and hCYP1B1, both DBP and DBPD were more cytotoxic in V79MZ1A1 (IC(50)=2.7 and 0.7nM, respectively) than in V79MZh1B1 (IC(50)=6.0 and 4.8nM, respectively). In contrast, both DBP and DBPD were two- to four-fold more mutagenic in V79MZh1B1 than in V79MZ1A1. Co-expression of hGSTA1 with hCYP1A1 decreased DBP cytotoxicity two-fold compared to V79MZh1A1 with hCYP1A1 alone, and provided a small, yet still statistically significant, 1.3-fold protection against DBPD. Protection against mutagenicity of these compounds was comparable to that for cytotoxicity in cells expressing hCYP1A1. In V79MZh1B1 cells, co-expression of hGSTA1 conferred up to five-fold protection against DBP cytotoxicity, and up to nine-fold protection against the (+/-)-DBP-dihydrodiol cytotoxicity relative to the cells expressing hCYP1B1 alone. Co-expression of hGSTA1 also reduced mutagenicity of DBP or its dihydrodiol to a lesser extent (1.3-1.8-fold) than the protection against cytotoxicity in cells expressing hCYP1B1. These findings demonstrate that the protective efficacy of hGSTA1 against DBP and DBPD toxicity is variable, depending on the compound or metabolite present, the specific cytochrome P450 isozyme expressed, and the specific cellular damage endpoint examined.     

Deficiency in a mitochondrial aldehyde dehydrogenase increases vulnerability to oxidative stress in PC12 cells

Mitochondrial aldehyde dehydrogenase 2 (ALDH2) plays a major role in acetaldehyde detoxification. The alcohol sensitivity is associated with a genetic deficiency of ALDH2. We have previously reported that this deficiency influences the risk for late-onset Alzheimer's disease. However, the biological effects of the deficiency on neuronal cells are poorly understood. Thus, we obtained ALDH2-deficient cell lines by introducing mouse mutant Aldh2 cDNA into PC12 cells. The mutant ALDH2 repressed mitochondrial ALDH activity in a dominant negative fashion, but not cytosolic activity. The resultant ALDH2-deficient transfectants were highly vulnerable to exogenous 4-hydroxy-2-nonenal, an aldehyde derivative generated by the reaction of superoxide with unsaturated fatty acid. In addition, the ALDH2-deficient transfectants were sensitive to oxidative insult induced by antimycin A, accompanied by an accumulation of proteins modified with 4-hydroxy-2-nonenal. Thus, these findings suggest that mitochondrial ALDH2 functions as a protector against oxidative stress.     

Inhibition of succinic semialdehyde dehydrogenase activity by alkenal products of lipid peroxidation

Lipid peroxidation causes the generation of the neurotoxic aldehydes acrolein and 4-hydroxy-trans-2-nonenal (HNE). These products are elevated in neurodegenerative diseases and acute CNS trauma. Previous studies demonstrate that mitochondrial class 2 aldehyde dehydrogenase (ALDH2) is susceptible to inactivation by these alkenals. In the liver and brain another mitochondrial aldehyde dehydrogenase, succinic semialdehyde dehydrogenase (SSADH/ALDH5A1), is present. In this study, we tested the hypothesis that aldehyde products of lipid peroxidation inhibit SSADH activity using the endogenous substrate, succinic semialdehyde (SSA, 50 microM). Acrolein potently inhibited SSADH activity (IC(50)=15 microM) in rat brain mitochondrial preparations. This inhibition was of an irreversible and noncompetitive nature. HNE inhibited activity with an IC(50) of 110 microM. Trans-2-hexenal (HEX) and crotonaldehyde (100 microM each) did not inhibit activity. These data suggest that acrolein and HNE disrupt SSA metabolism and may have subsequent effects on CNS neurochemistry.     

Aldehyde dehydrogenase 1B1 (ALDH1B1) is a potential biomarker for human colon cancer

Aldehyde dehydrogenases (ALDHs) belong to a superfamily of NAD(P)⁺-dependent enzymes, which catalyze the oxidation of endogenous and exogenous aldehydes to their corresponding acids. Increased expression and/or activity of ALDHs, particularly ALDH1A1, have been reported to occur in human cancers. It is proposed that the metabolic function of ALDH1A1 confers the “stemness” properties to normal and cancer stem cells. Nevertheless, the identity of ALDH isozymes that contribute to the enhanced ALDH activity in specific types of human cancers remains to be elucidated. ALDH1B1 is a mitochondrial ALDH that metabolizes a wide range of aldehyde substrates including acetaldehyde and products of lipid peroxidation (LPO). In this study, we immunohistochemically examined the expression profile of ALDH1A1 and ALDH1B1 in human adenocarcinomas of colon (N = 40), lung (N = 30), breast (N = 33) and ovary (N = 33) using an NIH tissue array. The immunohistochemical expression of ALDH1A1 or ALDH1B1 in tumor tissues was scored by their intensity (scale = 1–3) and extensiveness (% of total cancer cells). Herein we report a 5.6-fold higher expression score for ALDH1B1 in cancerous tissues than that for ALDH1A1. Remarkably, 39 out of 40 colonic cancer specimens were positive for ALDH1B1 with a staining intensity of 2.8 ± 0.5. Our study demonstrates that ALDH1B1 is more profoundly expressed in the adenocarcinomas examined in this study relative to ALDH1A1 and that ALDH1B1 is dramatically upregulated in human colonic adenocarcinoma, making it a potential biomarker for human colon cancer.