Allyl alcohol toxicity in isolated renal epithelial cells: protective effects of low molecular weight thiols

The toxicity of allyl alcohol was studied in freshly isolated renal epithelial cells prepared from male and female rats. Cells from female rats demonstrated a greater susceptibility to allyl alcohol toxicity as assessed by glutathione depletion and loss of cell viability. The sensitivity of female rat renal cells appears to relate to the higher activity of alcohol dehydrogenase found in the female rat kidney, which metabolizes allyl alcohol to the highly reactive aldehyde, acrolein. Pyrazole, an inhibitor of alcohol dehydrogenase, abolished the cytotoxic effects of allyl alcohol whereas inhibition of aldehyde dehydrogenase by disulfiram treatment was found to increase the sensitivity of renal cells to the effects of allyl alcohol. The toxicity of allyl alcohol was decreased by a number of treatments which resulted in increased levels of glutathione or other low molecular weight thiols. These results indicate that acrolein is the toxic metabolite responsible for the renal cell injury following exposure to allyl alcohol, and unless immediately inactivated acrolein interacts with critical nucleophilic sites of the cell and initiates cell injury. These studies demonstrate that freshly isolated kidney cells represent a convenient model system for studies of thiol-mediated protective mechanisms against toxic renal cell injury.     

Extensive protein carbonylation precedes acrolein-mediated cell death in mouse hepatocytes

Allyl alcohol hepatotoxicity is mediated by an alcohol dehydrogenase-derived biotranformation product, acrolein. This highly reactive alpha,beta-unsaturated aldehyde readily alkylates model proteins in vitro, forming, among other products, Michael addition adducts that possess a free carbonyl group. Whether such damage accompanies acrolein-mediated toxicity in cells is unknown. In this work we established that allyl alcohol toxicity in mouse hepatocytes involves extensive carbonylation of a wide range of proteins, and that the severity of such damage to a subset of 18-50 kDa proteins closely correlated with the degree of cell death. In addition to abolishing cytotoxicity and glutathione depletion, the alcohol dehydrogenase inhibitor 4-methyl pyrazole strongly attenuated protein carbonylation. Conversely, cyanamide, an aldehyde dehydrogenase inhibitor, enhanced cytotoxicity and protein carbonylation. Since protein carbonylation clearly preceded the loss of membrane integrity, it may be associated with the toxic process leading to cell death.     

Cytochrome P450 2E1 metabolically activates propargyl alcohol: propiolaldehyde-induced hepatocyte cytotoxicity

Pargyline, an antihypertensive agent and monoamine oxidase inhibitor, induces hepatic GSH depletion and hepatotoxicity in vivo in rats [E.G. De Master, H.W. Sumner, E. Kaplan, F. N. Shirota, H.T. Nagasawa, Toxicol. Appl. Pharmacol. 65 (1982) 390-401]. Propargyl alcohol (2-propyn-1-ol), because of its structural similarity to allyl alcohol, was thought to be activated by alcohol dehydrogenase. However, it is a poor substrate compared to allyl alcohol and it was therefore proposed that propargyl alcohol-induced liver injury involved metabolic activation by catalase/H(2)O(2) [E.G. De Master, T. Dahlseid, B. Redfern, Chem. Res. Toxicol. 7 (1994) 414-419]. In the following we showed that; (1) propargyl alcohol-induced cytotoxicity was markedly enhanced in CYP 2E1-induced hepatocytes and prevented by various CYP 2E1 inhibitors but was only slightly affected when alcohol dehydrogenase was inhibited with methylpyrazole/DMSO or when catalase was inactivated with azide or aminotriazole, (2) hepatocyte GSH depletion preceded cytotoxicity and was inhibited by cytochrome P450 inhibitors but not by catalase/alcohol dehydrogenase inhibitors. GSH conjugate formation during propargyl alcohol metabolism by microsomal mixed function oxidase in the presence of GSH was also prevented by anti-rat CYP 2E1 or CYP 2E1 inhibitors, (3) cytotoxicity was prevented when lipid peroxidation was inhibited with antioxidants, desferoxamine (ferric chelator) or dithiothreitol. Propargyl alcohol-induced cytotoxicity and reactive oxygen species formation were markedly increased in GSH-depleted hepatocytes. All of this evidence suggests that propargyl alcohol-induced cytotoxicity involves metabolic activation by CYP 2E1 to form propiolaldehyde that causes hepatocyte lysis as a result of GSH depletion and lipid peroxidation.     

Cytochrome P450 2E1 metabolically activates propargyl alcohol: propiolaldehyde-induced hepatocyte cytotoxicity

Pargyline, an antihypertensive agent and monoamine oxidase inhibitor, induces hepatic GSH depletion and hepatotoxicity in vivo in rats [E.G. De Master, H.W. Sumner, E. Kaplan, F. N. Shirota, H.T. Nagasawa, Toxicol. Appl. Pharmacol. 65 (1982) 390–401]. Propargyl alcohol (2-propyn-1-ol), because of its structural similarity to allyl alcohol, was thought to be activated by alcohol dehydrogenase. However, it is a poor substrate compared to allyl alcohol and it was therefore proposed that propargyl alcohol-induced liver injury involved metabolic activation by catalase/H₂O₂ [E.G. De Master, T. Dahlseid, B. Redfern, Chem. Res. Toxicol. 7 (1994) 414–419]. In the following we showed that; (1) propargyl alcohol-induced cytotoxicity was markedly enhanced in CYP 2E1-induced hepatocytes and prevented by various CYP 2E1 inhibitors but was only slightly affected when alcohol dehydrogenase was inhibited with methylpyrazole/DMSO or when catalase was inactivated with azide or aminotriazole, (2) hepatocyte GSH depletion preceded cytotoxicity and was inhibited by cytochrome P450 inhibitors but not by catalase/alcohol dehydrogenase inhibitors. GSH conjugate formation during propargyl alcohol metabolism by microsomal mixed function oxidase in the presence of GSH was also prevented by anti-rat CYP 2E1 or CYP 2E1 inhibitors, (3) cytotoxicity was prevented when lipid peroxidation was inhibited with antioxidants, desferoxamine (ferric chelator) or dithiothreitol. Propargyl alcohol-induced cytotoxicity and reactive oxygen species formation were markedly increased in GSH-depleted hepatocytes. All of this evidence suggests that propargyl alcohol-induced cytotoxicity involves metabolic activation by CYP 2E1 to form propiolaldehyde that causes hepatocyte lysis as a result of GSH depletion and lipid peroxidation.     

The metabolic activation of cyanamide to an inhibitor of aldehyde dehydrogenase is catalyzed by catalase

The inhibition of aldehyde dehydrogenase by cyanamide is dependent on an enzyme catalyzed conversion of the latter to an active metabolite. The following results suggest that catalase is the enzyme responsible for this bioactivation. The elevation of blood acetaldehyde elicited by cyanamide after ethanol administration to rats was attenuated more than 90 percent by pretreatment with the catalase inhibitor, 3-amino-1,2,4-triazole. This attenuation was dose dependent and was accompanied by a reduction in total hepatic catalase activity. Although hepatic catalase was also inhibited by cyanamide, a positive correlation between blood acetaldehyde and hepatic catalase activity was observed. In vitro, the activation inhibitor, 3-amino-1,2,4-triazole. This attenuation was dose dependent and was accompanied by a reduction in total hepatic catalase activity. Although hepatic catalase was also inhibited by cyanamide, a positive correlation between blood acetaldehyde and hepatic catalase activity was observed. In vitro, the activation of cyanamide was catalyzed by a) the rat liver mitochondrial subcellular fraction, b) the 50-65% ammonium sulfate mitochondrial fraction and c) purified bovine liver catalase. Cyanamide activation was inhibited by sodium azide. Since much of the hepatic catalase is localized in the peroxisomes and since peroxisomes and mitochondria cosediment, the cyanamide activating enzyme, catalase, is likely of peroxisomal and mitochondrial origin.     

Gene-environmental interactions between alcohol-drinking behavior and ALDH2 and CYP2E1 polymorphisms and their impact on micronuclei frequency in human lymphocytes

Ethanol is converted to acetaldehyde by alcohol dehydrogenase (ADH), cytochrome p4502E1 (CYP2E1) and catalase. This metabolite is then detoxified by aldehyde dehydrogenase 2 (ALDH2), a key enzyme in the elimination of acetaldehyde, via further oxidation to acetic acid. The toxic effects of acetaldehyde are well documented and may be partially mediated by genotoxic damage. In the present study, we investigated the effects of alcohol-drinking behavior and genetic polymorphisms in two different genes (ALDH2 and CYP2E1) on the micronuclei (MN) frequency in 248 healthy Japanese men. Genotyping was performed by PCR-RFLP analysis. The ALDH2 variant (deficient type) was significantly associated with an increased MN frequency in subjects drinking more than three times/wk, while habitual drinkers with wild-type CYP2E1 also had a significantly increased MN frequency. Furthermore, when the subjects were divided into eight groups according to their drinking frequency and genotypes of ALDH2 and CYP2E1, we found that habitual drinkers with homozygous CYP2E1*1/*1 and heterozygous ALDH2*1/*2 or homozygous ALDH2*2/*2 showed the highest mean MN frequency. In the present study, we found clear associations among ALDH2 and CYP2E1 gene polymorphisms, alcohol-drinking behavior and genotoxic effects in a healthy Japanese population. Therefore, analysis of the polymorphisms of alcohol-metabolizing enzymes may lead to elucidation of the mechanism(s) for individual susceptibilities to the toxicity of ethanol metabolites.     

Increased toxicity by transforming growth factor-beta 1 in liver cells overexpressing CYP2E1

Ethanol treatment causes an increase in expression of TGF-beta1 and CYP2E1 in the centrilobular area. Alcoholic liver disease is usually initiated in the centrilobular region of the liver. We hypothesized that the combination of TGF-beta1 and CYP2E1 produces increased oxidative stress and liver cell toxicity. To test this possibility, we studied the effects of TGF-beta1 on the viability of HepG2 E47 cells that express human CYP2E1, and C34 HepG2 cells, which do not express CYP2E1. E47 cells underwent greater growth inhibition and enhanced apoptosis after TGF-beta1 treatment, as compared to the C34 cells. There was an enhanced production of reactive oxygen species (ROS) and a decline in reduced glutathione (GSH) levels in the TGF-beta1-treated E47 cells and the enhanced cell death could be prevented by antioxidants. The CYP2E1 inhibitor diallyl sulfide prevented the potentiated cell death in E47 cells validating the role of CYP2E1. Mitochondrial membrane potential declined in the TGF-beta1-treated E47 cells, prior to developing toxicity, and cell death could be prevented by trifluoperazine, an inhibitor of the mitochondrial membrane permeability transition. TGF-beta1 also produced a loss of cell viability in hepatocytes from pyrazole-treated rats with elevated levels of CYP2E1, compared to control hepatocytes. In conclusion, increased toxic interactions by TGF-beta1 plus CYP2E1 can occur by a mechanism involving increased production of intracellular ROS and depletion of GSH, resulting in mitochondrial membrane damage and loss of membrane potential, followed by apoptosis. Potentiation of TGF-beta1-induced cell death by CYP2E1 may contribute to mechanisms of alcohol-induced liver disease.     

Inhibition of CYP2E1 leads to decreased malondialdehyde–acetaldehyde adduct formation in VL-17A cells under chronic alcohol exposure

AimEthanol metabolism leads to the formation of acetaldehyde and malondialdehyde. Acetaldehyde and malondialdehyde can together form malondialdehyde–acetaldehyde (MAA) adducts. The role of alcohol dehydrogenase (ADH) and cytochrome P4502E1 (CYP2E1) in the formation of MAA-adducts in liver cells has been investigated.Main methodsChronic ethanol treated VL-17A cells over-expressing ADH and CYP2E1 were pretreated with the specific CYP2E1 inhibitor — diallyl sulfide or ADH inhibitor — pyrazole or ADH and CYP2E1 inhibitor — 4-methyl pyrazole. Malondialdehyde, acetaldehyde or MAA-adduct formation was measured along with assays for viability, oxidative stress and apoptosis.Key findingsInhibition of CYP2E1 with 10 μM diallyl sulfide or ADH with 2 mM pyrazole or ADH and CYP2E1 with 5 mM 4-methyl pyrazole led to decreased oxidative stress and toxicity in chronic ethanol (100 mM) treated VL-17A cells. In vitro incubation of VL-17A cell lysates with acetaldehyde and malondialdehyde generated through ethanol led to increased acetaldehyde (AA)-, malondialdehyde (MDA)-, and MAA-adduct formation. Specific inhibition of CYP2E1 or ADH and the combined inhibition of ADH and CYP2E1 greatly decreased the formation of the protein aldehyde adducts. Specific inhibition of CYP2E1 led to the greatest decrease in oxidative stress, toxicity and protein aldehyde adduct formation, implicating that CYP2E1 accelerates the formation of protein aldehyde adducts which can be an important mechanism for alcohol mediated liver injury.SignificanceCYP2E1-mediated metabolism of ethanol leads to increased AA-, MDA-, and MAA-adduct formation in liver cells which may aggravate liver injury.     

Depletion of S-adenosyl-l-methionine with cycloleucine potentiates cytochrome P450 2E1 toxicity in primary rat hepatocytes

S-Adenosyl-l-methionine (SAM) is the principal biological methyl donor. Methionine adenosyltransferase (MAT) catalyzes the only reaction that generates SAM. Hepatocytes were treated with cycloleucine, an inhibitor of MAT, to evaluate whether hepatocytes enriched in cytochrome P450 2E1 (CYP2E1) were more sensitive to a decline in SAM. Cycloleucine decreased SAM and glutathione (GSH) levels and induced cytotoxicity in hepatocytes from pyrazole-treated rats (with an increased content of CYP2E1) to a greater extent as compared to hepatocytes from saline-treated rats. Apoptosis caused by cycloleucine in pyrazole hepatocytes appeared earlier and was more pronounced than control hepatocytes and could be prevented by incubation with SAM, glutathione reduced ethyl ester and antioxidants. The cytotoxicity was prevented by treating rats with chlormethiazole, a specific inhibitor of CYP2E1. Cycloleucine induced greater production of reactive oxygen species (ROS) in pyrazole hepatocytes than in control hepatocytes, and treatment with SAM, Trolox, and chlormethiazole lowered ROS formation. In conclusion, lowering of hepatic SAM levels produced greater toxicity and apoptosis in hepatocytes enriched in CYP2E1. This is due to elevated ROS production by CYP2E1 coupled to lower levels of hepatoprotective SAM and GSH. We speculate that such interactions e.g. induction of CYP2E1, decline in SAM and GSH may contribute to alcohol liver toxicity.     

Effect of ethanol on glutathione concentration in isolated hepatocytes.

1. Ethanol induces a decrease in GSH (reduced glutathione) concentration is isolated hepatocytes. Maximal effects appear at 20 mM-ethanol. The concentration-dependence of this decrease is paralleled by the concentration-dependence of the activity of alcohol dehydrogenase. 2. Pyrazole, a specific inhibitor of alcohol dehydrogenase, prevents the ethanol-induced GSH depletion. 3. Acetaldehyde, above 0.05 mM, also promotes a decrease in GSH concentration in hepatocytes. 4. Disulfiram (0.05 mM), an inhibitor of aldehyde dehydrogenase, potentiates the fall in GSH concentration caused by acetaldehyde. 5. The findings support the hypothesis that acetaldehyde is responsible for the depletion of GSH induced by ethanol. 6. Methionine prevents the effect of alcohol or acetaldehyde on GSH concentration in hepatocytes.     

CYP2E1 potentiation of LPS and TNFα-induced hepatotoxicity by mechanisms involving enhanced oxidative and nitrosative stress, activation of MAP kinases, and mitochondrial dysfunction

The mechanisms by which alcohol causes cell injury are not clear. A major mechanism that is the focus of considerable research is the role of lipid peroxidation and oxidative stress in alcohol toxicity. Many pathways have been suggested to play a role in how alcohol induces oxidative stress. Considerable attention has been given to alcohol-elevated production of lipopolysaccharide (LPS) and TNFα and to alcohol induction of CYP2E1. These two pathways are not exclusive of each other, however, associations and interactions between them, especially in vivo, have not been extensively evaluated. We have shown that increased oxidative stress from induction of CYP2E1 in vivo sensitizes hepatocytes to LPS and TNF toxicity and that oxidants, such as peroxynitrite, activation of p38 and JNK MAP kinases, inactivation of NF-kB protective pathways and mitochondrial dysfunction are downstream mediators of this CYP2E1-LPS/TNF potentiated hepatotoxicity. This review will summarize studies showing potentiated interactions between these two risk factors in promoting liver injury and the mechanisms involved.     

The metabolism of ethanol-derived acetaldehyde by alcohol dehydrogenase (EC 1.1.1.1) and aldehyde dehydrogenase (EC 1.2.1.3) in Drosophila melanogaster larvae.

Both aldehyde dehydrogenase (ALDH, EC 1.2.1.3) and the aldehyde dehydrogenase activity of alcohol dehydrogenase (ADH, EC 1.1.1.1) were found to coexist in Drosophila melanogaster larvae. The enzymes, however, showed different inhibition patterns with respect to pyrazole, cyanamide and disulphiram. ALDH-1 and ALDH-2 isoenzymes were detected in larvae by electrophoretic methods. Nonetheless, in tracer studies in vivo, more than 75% of the acetaldehyde converted to acetate by the ADH ethanol-degrading pathway appeared to be also catalysed by the ADH enzyme. The larval fat body probably was the major site of this pathway.     

Alcohol and acetaldehyde in rat's milk following ethanol administration

Alcohol and acetaldehyde were measured in milk and peripheral blood in chronic alcoholic rats, at 5 and 15 days of lactation. Ethanol in blood increased throughout lactation and the levels of acetaldehyde were much higher than in nonlactating alcoholic rats. The concentration of acetaldehyde in milk was always ca. 50% of that in blood, whereas that of ethanol varied within the range of 44-80% of the blood levels. Blood alcohol levels in the corresponding sucking pups were much lower than in maternal blood and increased throughout lactation. The time course of ethanol and acetaldehyde concentration in blood and milk were determined in normal lactating rats after cyanamide (40 mg/kg) and ethanol administration (2 or 4 g/kg). Milk alcohol reached higher concentrations than in blood within the first hour of ethanol administration, decreasing and remaining constant thereafter at ca. 65% of those in blood. Acetaldehyde levels in milk were always 35-45% lower than in blood. No alcohol dehydrogenase activity was found in homogenates of mammary tissue; however there was some aldehyde dehydrogenase activity. A significant decrease in mammary tissue aldehyde dehydrogenase was found in chronic alcoholic rats. The role of this enzyme is discussed.     

Role of intracellular calcium and phospholipase A2 in arachidonic acid-induced toxicity in liver cells overexpressing CYP2E1

Liver cells (HepG2 and primary hepatocytes) overexpressing CYP2E1 and exposed to arachidonic acid (AA) were previously shown to lose viability together with enhanced lipid peroxidation. These events were blocked in cells pre-incubated with antioxidants (alpha-tocopherol, glutathione ethyl ester), or in HepG2 cells not expressing CYP2E1. The goal of the current study was to evaluate the role of calcium and calcium-activated hydrolases in these CYP2E1-AA interactions. CYP2E1-expressing HepG2 cells treated with AA showed an early increase in cytosolic calcium and partial depletion of ionomycin-sensitive calcium stores. These changes in calcium were blocked by alpha-tocopherol. AA activated phospholipase A2 (PLA2) in CYP2E1-expressing liver cells, and this was inhibited by PLA2 inhibitors or alpha-tocopherol. PLA2 inhibitors prevented the cell death caused by AA, without affecting CYP2E1 activity or lipid peroxidation. AA toxicity and PLA2 activation were inhibited in calcium-depleted cells, but not by removal of extracellular calcium alone. Removal of extracellular calcium inhibited the early increase in cytosolic calcium caused by AA. CYP2E1 overexpressing HepG2 cells exposed to AA showed a decrease in mitochondrial membrane potential, which was prevented by the PLA2 inhibitors. These results suggest that AA-induced toxicity to CYPE1-expressing cells: (i) is associated with release of Ca2+ from intracellular stores that depends mainly on oxidative membrane damage; (ii) is associated with activation of PLA2 that depends on intracellular calcium and lipid peroxidation; (iii) does not depend on increased influx of extracellular calcium, and (iv) depends on the effect of converging events (lipid peroxidation, intracellular calcium, activation of PLA2) on mitochondria to induce bioenergetic failure and necrosis. These interactions may play a role in alcohol liver toxicity, which requires polyunsaturated fatty acids, and involves induction of CYP2E1.     

International Commission for Protection against Environmental Mutagens and Carcinogens. ICPEMC Working Paper No. 15/5. Acute aldehyde syndrome and chronic aldehydism

Different types of alcohol dehydrogenase and of aldehyde dehydrogenase lead to different blood acetaldehyde levels. With respect to acetaldehyde levels in human blood 3 types can be distinguished: (1) the normal range, (2) the acute aldehyde syndrome, and (3) the chronic aldehydism. Acetaldehyde is electrophilic and reacts with nucleophilic groups of various macromolecules including DNA. Acetyldehyde inhibits synthetic and metabolic pathways, it interferes with the polymerization of tubulin and stimulates collagen synthesis. By depletion of cellular glutathione levels, acetaldehyde leads to lipid peroxidation and to the formation of malonaldehyde. There are indications that acetaldehyde may play a role in positively reinforcing mood changes induced by alcohol in humans.     

Depletion of cytosolic or mitochondrial thioredoxin increases CYP2E1-induced oxidative stress via an ASK-1-JNK1 pathway in HepG2 cells

Thioredoxin is an important reducing molecule in biological systems. Increasing CYP2E1 activity induces oxidative stress and cell toxicity. However, whether thioredoxin protects cells against CYP2E1-induced oxidative stress and toxicity is unknown. SiRNA were used to knockdown either cytosolic (TRX-1) or mitochondrial thioredoxin (TRX-2) in HepG2 cells expressing CYP2E1 (E47 cells) or without expressing CYP2E1 (C34 cells). Cell viability decreased 40-60% in E47 but not C34 cells with 80-90% knockdown of either TRX-1 or TRX-2. Depletion of either thioredoxin also potentiated the toxicity produced either by a glutathione synthesis inhibitor or by TNFα in E47 cells. Generation of reactive oxygen species and 4-HNE protein adducts increased in E47 but not C34 cells with either thioredoxin knockdown. GSH was decreased and adding GSH completely blocked E47 cell death induced by either thioredoxin knockdown. Lowering TRX-1 or TRX-2 in E47 cells caused an early activation of ASK-1, followed by phosphorylation of JNK1 after 48 h of siRNA treatment. A JNK inhibitor caused a partial recovery of E47 cell viability after thioredoxin knockdown. In conclusion, knockdown of TRX-1 or TRX-2 sensitizes cells to CYP2E1-induced oxidant stress partially via ASK-1 and JNK1 signaling pathways. Both TRX-1 and TRX-2 are important for defense against CYP2E1-induced oxidative stress.     

Toxicity of endosulfan on kidney of male rats in relation to drug metabolizing enzymes and microsomal lipid peroxidation

Endosulfan administration (po, 15 and 30 days at 7.5 and 10 mg/kg body wt respectively) inhibited the activity of microsomal mixed function oxidases in kidney tissue of male rats. Microsomal and cytosolic protein contents of kidney were significantly increased following 30 days endosulfan exposures. Profound induction in the activity profiles of alcohol dehydrogenase and cytosolic glutathione s-transferase was noticed, however, no such change was apparent in the activity of aldehyde dehydrogenase. Microsomal preparations from treated animals showed a dose and duration dependent increase in spontaneous lipid peroxidation. The observed biochemical changes persisted even after 7 days normalcy allowance provided after the endosulfan (10 mg/kg body wt) withdrawl. The results suggest a substantial renal toxicity of endosulfan to male rats in relation to microsomal mixed function oxidases and associated functions which possibly resulted from lipid peroxidative damage of microsomal membrane in treated animals.