Disuccinyl betulin triggers metacaspase-dependent endonuclease G-mediated cell death in unicellular protozoan parasite Leishmania donovani.

The unicellular organism Leishmania undergoes apoptosis-like cell death in response to external stress or exposure to antileishmanial agents. Here, we showed that 3-O,28-O-disuccinyl betulin (DiSB), a potent topoisomerase type IB inhibitor, induced parasitic cell death by generating oxidative stress. The characteristic feature of the death process resembled the programmed cell death (PCD) seen in higher eukaryotes. In the current study, the generation of reactive oxygen species (ROS), followed by the depolarization of mitochondrial membrane potential (ΔΨm), caused a loss in ATP production in Leishmania parasites. This further gave positive feedback to produce a large amount of ROS, which in turn caused oxidative DNA lesions and genomic DNA fragmentation. The treatment of promastigotes with DiSB induced high expression levels of metacaspase protein that led to cell death in this unicellular organism. The PCD was insensitive to benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone (zVAD-fmk), suggesting that the death process was not associated with the activation of caspases. DiSB treatment translocated Leishmania donovani endonuclease G (LdEndoG) from mitochondria to the nucleus, which was responsible for the DNA degradation process. Conditional antisense knockdown of L. donovani metacaspase (LdMC), as well as EndoG, -subverted death of the parasite and rescued cell cycle arrest in G1 phase. The present study on the effector molecules associated with the PCD pathway of the parasite should help to manifest the mechanisms of PCD and also might be exploited in antileishmanial chemotherapy.

Novel betulin derivatives as antileishmanial agents with mode of action targeting type IB DNA topoisomerase.

Toward developing antileishmanial agents with mode of action targeted to DNA topoisomerases of Leishmania donovani, we have synthesized a large number of derivatives of betulin. The compound, a natural triterpene isolated from the cork layer of Betula spp. plants exhibits several pharmacological properties. Three compounds (disuccinyl betulin, diglutaryl dihydrobetulin, and disuccinyl dihydrobetulin) inhibit growth of the parasite as well as relaxation activity of the enzyme type IB topoisomerase [Leishmania donovani topoisomerase I (LdTOP1LS)] of the parasite. Mechanistic studies suggest that these compounds interact with the enzyme in a reversible manner. The stoichiometry of these compounds binding to LdTOP1LS is 1:1 (mole/mole) with a dissociation constant on the order of ∼10(-6) M. Unlike CPT, these compounds do not stabilize the cleavage complex; rather, they abrogate the covalent complex formation. In processive mode of relaxation assay condition, these compounds slow down the strand rotation event, which ultimately affects the relaxation of supercoiled DNA. It is noteworthy that these compounds reduce the intracellular parasite burden in macrophages infected with wild-type L. donovani as well as with sodium antimony gluconate resistant parasite (GE1). Taken together, our data suggest that these betulin derivatives can be exploited as potential drug candidates against threatening drug resistant leishmaniasis.

Comparative antimutagenic and anticancer activity of three fractions of black tea polyphenols thearubigins.

Antimutagenic and anticancer effects of black tea polyphenols theaflavins (TF) and thearubigins (TR) have previously been reported. TR is a complex mixture of polyphenols. In this study, our interest was to fractionate TR and to study the antimutagenic and anticancer activities of the fractions. Three fractions of TR, namely TR-1, TR-2, and TR-3, were isolated by chromatographic processes. Antimutagenic activity of these 3 fractions was carried out on 4 Salmonella strains by Ames assay. Anticancer activity was studied on human leukemic cells U937. Our findings clearly indicated antimutagenic and anticancer activities of the TR-1, TR-2, and TR-3 fractions on Salmonella strains and on U937 cells, respectively. However, all 3 fractions, at or below 100 µg/ml dose, did not show any significant toxic effects on the normal human cells (peripheral blood mononuclear cells). TR-2 was found to be the most active fraction among the 3. Flow cytometric and confocal microscopic studies further indicate that apoptosis induction could be an important mechanism behind the anticancer effects of these fractions. To our knowledge, this study is the first attempt to describe the antimutagenic and anticancer activity of TR fractions, and it also suggests that TR-2 is the most active component of TR.

Role of oxidation-triggered activation of JNK and p38 MAPK in black tea polyphenols induced apoptotic death of A375 cells.

Theaflavins (TF) and thearubigins (TR) are the major polyphenols of black tea. Our previous study revealed that TF- and TR-induced apoptosis of human malignant melanoma cells (A375) is executed via a mitochondria-mediated pathway. In our present study we observed the role of the three most important MAPK (ERK, JNK, and p38) in TF- and TR-induced apoptosis. TF and TR treatment of A375 cells led to sustained activation of JNK and p38 MAPK but not ERK, suggesting that JNK and p38 are the effector molecules in this polyphenol-induced cell death. This idea was further supported by subsequent studies in which JNK and p38 activation was inhibited by specific inhibitors. Significant inhibition was found in TF- and TR-treated A375 cell death pretreated with JNK- or p38-specific inhibitors only. Further, we have found that TF and TR treatment induces a time-dependent increase in intracellular reactive oxygen species generation in A375 cells. Interestingly, treatment with the antioxidant N-acetyl cystein inhibits TF- and TR-induced JNK and p38 activation as well as induction of cell death in A375 cells. We also provide evidence demonstrating the critical role of apoptosis signal-regulating kinase 1 in TF- and TR-induced apoptosis in A375 cells. Taken together our results strongly suggest that TF and TR induce apoptotic death of A375 cells through apoptosis signal-regulating kinase 1, MAPK kinase, and the JNK-p38 cascade, which is triggered by N-acetyl cystein intracellular oxidative stress.

Molecular mechanism of black tea polyphenols induced apoptosis in human skin cancer cells: involvement of Bax translocation and mitochondria mediated death cascade.

Theaflavins (TF) and thearubigins (TR) are the most exclusive polyphenols of black tea. Even though few previous reports showed the anticancer effects of TF through apoptosis, the potential effect of TR has not been appraised. This study investigated the induction of apoptosis in human skin cancer cells after treatment of TF and TR. We report that both TF and TR could exert inhibition of A431 (human epidermoid carcinoma) and A375 (human malignant melanoma) cell proliferation without adversely affecting normal human epidermal keratinocyte cells. Growth inhibition of A375 cells occurred through apoptosis, as evident from cell cycle arrest at G(0)/G(1) phase, increase in early apoptotic cells, externalization of phosphatidylserine and DNA fragmentation. In our pursuit to dissect the molecular mechanism of TF- and TR-induced apoptosis in A375 cells, we investigated whether cell death is being mediated by mitochondria. In our system, Bax translocation to mitochondria persuaded depolarization of mitochondrial membrane potential, cytochrome c release in cytosol and induced activation of caspase-9, caspase-3 and poly (ADP-ribose) polymerase cleavage. Our intricate investigations on apoptosis also explained that TF and TR augmented Bax:Bcl2 ratio, up-regulated the expression of p53 as well as p21 and inhibited phosphorylation of the cell survival protein Akt. Furthermore, TF and TR elicited intracellular reactive oxygen species generation in A375 cells. These observations raise speculations that TF as well as TR might exert chemopreventive effect through cell cycle arrest and induction of apoptogenic signals via mitochondrial death cascade in human skin cancer cells.

Quercetin interferes with iron metabolism in Leishmania donovani and targets ribonucleotide reductase to exert leishmanicidal activity.

OBJECTIVES: The possibility of developing antileishmanial drugs was evaluated by intervention in the parasite's iron metabolism, utilizing quercetin (Qr) under in vivo conditions, and identifying the target of this lipophilic metal chelator against Leishmania donovani. METHODS: Interaction between Qr and serum albumin (SA) was studied by using the intrinsic fluorescence of Qr as a probe. The effect of treatment with Qr and SA on the proliferation of amastigotes was determined by evaluating splenic parasite load. Disintegration of parasites in response to combination treatment was assessed from ultrastructural analysis using a transmission electron microscope. Quenching of the tyrosyl radical of ribonucleotide reductase (RR) in treated amastigotes was detected by an electron paramagnetic resonance study. RESULTS: Treatment with a combination of Qr and SA increased bioavailability of the flavonoid and proved to be of major advantage in promoting the effectiveness of Qr towards the repression of splenic parasite load from 75%, P < 0.01 to 95%, P < 0.002. Qr-mediated down-regulation of RR (P < 0.05), catalysing the rate-limiting step of DNA synthesis in the pathogens, could be related to the deprivation of the enzyme of iron which in turn destabilized the critical tyrosyl radical required for its catalysing activity. CONCLUSIONS: Results have implications for improved leishmanicidal action of Qr in combination with SA targeting RR and suggest future drug design based on interference with the parasite's iron metabolism under in vivo conditions.

Phytochemical investigations and evaluation of antimutagenic activity of the alcoholic extract of Glycosmis pentaphylla and Tabernaemontana coronaria by Ames test.

Chemical investigation of root bark of Glycosmis pentaphylla and stem bark of Tabernaemontana coronaria led to the isolation of three carbazole alkaloids glycozoline, glycozolidine and methyl carbazole 3-carboxylate, two furoquinoline alkaloids skimmianine and dictamine, an acridone alkaloid arborinine, three monomeric indole alkaloids coronaridine, 10-methoxy coronaridine and tabernaemontanine, and two dimeric indole alkaloids voacamine and tabernaelegantine B. Their structures were established by detailed spectral analysis. Mutagenic and antimutagenic potential of methanol extract of both plant materials were evaluated by Ames test against known positive mutagens 2-aminofluorine, 4-nitro-O-phenylenediamine and sodium azide using Salmonella typhimurium TA 98 and TA 100 bacterial strains both in the presence and absence of S9. Both the extracts were non-mutagenic in nature. Both the extracts of G. pentaphylla and T. coronaria exhibited significant antimutagenic activity against NPD and sodium azide for S. typhimurium TA98 and TA100 strains. The results indicated that the extracts could counteract the mutagenicity induced by different genotoxic compounds.

Voacamine alters Leishmania ultrastructure and kills parasite by poisoning unusual bi-subunit topoisomerase IB.

Indole alkaloids possess a large spectrum of biological activities including anti-protozoal action. Here we report for the first time that voacamine, isolated from the plant Tabernaemontana coronaria, is an antiprotozoal agent effective against a large array of trypanosomatid parasites including Indian strain of Leishmania donovani and Brazilian strains of Leishmania amazonensis and Trypanosoma cruzi. It inhibits the relaxation activity of topoisomerase IB of L. donovani (LdTop1B) and stabilizes the cleavable complex. Voacamine is probably the first LdTop1B-specific poison to act uncompetitively. It has no impact on human topoisomerase I and II up to 200µM concentrations. The study also provides a thorough insight into ultrastructural alterations induced in three kinetoplastid parasites by a specific inhibitor of LdTop1B. Voacamine is also effective against intracellular amastigotes of different drug unresponsive field isolates of Leishmania donovani obtained from endemic zones of India severely affected with visceral leishmaniasis. Most importantly, this is the first report demonstrating the efficacy of a compound to reduce the burden of drug resistant parasites, unresponsive to SAG, amphotericin B and miltefosine, in experimental BALB/c mice model of visceral leishmaniasis. The findings cumulatively provide a strong evidence that voacamine can be a promising drug candidate against trypanosomatid infections.

Anticlastogenic effects of black tea polyphenols theaflavins and thearubigins in human lymphocytes in vitro.

Black tea accounts for nearly 80% of total World tea production. It contains dimeric flavanols and polymeric polyphenols known as theaflavins (TF) and thearubigins (TR). TR is exclusively present in black tea. On the basis of our previous potent antimutagenic and anticlastogenic effects of TF and TR in vitro in bacterial system and in vivo in mouse bone marrow cells, we have decided to extend our study in human cells in vitro. This study investigated the anticlastogenic effects of black tea polyphenols TF and TR as measured by chromosomal aberrations (CA) and micronuclei formation (MN) against two known mutagens/carcinogens i.e. benzo[a]pyerne (B[a]P) and aflatoxin B1(AFB1) with S9 activation. A significant decrease in both CA and MN were observed in the human lymphocyte cultures treated with either TF or TR pretreated with either B[a]P or AFB1 (250, 500, 1000 microg/ml) when compared with B[a]P or AFB1 treated cultures alone. TF shows more protective effects than TR in this in vitro system. These results indicate that both TF and TR have significant anticlastogenic effects in vitro in human lymphocytes.

A new bisbenzylisoquinoline alkaloid isolated from Thalictrum foliolosum, as a potent inhibitor of DNA topoisomerase IB of Leishmania donovani.

Chemical investigation of the stem of Thalictrum foliolosum resulted in the isolation of two new bisbenzylisoquinoline alkaloids (1 and 2) along with known protoberberine group of isoquinoline alkaloids thalifendine (3) and berberine (4). The structures of the new compounds were established by detailed 2D NMR spectral analysis with their configurations determined from their optical rotation values and confirmed using circular dichroism. Inhibitory activities of these four compounds against DNA topoisomerase IB of Leishmania donovani were evaluated. Compound 2 exhibited almost complete inhibition of the enzyme activity at 50 µM concentration and it was found to be effective in killing both wild type as well as SAG resistant promastigotes of the parasite.

Therapeutic use of quercetin in the control of infection and anemia associated with visceral leishmaniasis.

Flavonoids are a broad class of plant phenolics that are known to possess a well-established protective effect against membrane lipoperoxidative damages. Oxidative damage of erythrocytes has been implicated in the reduced survival of erythrocytes during leishmanial infection. This study reveals the efficacy of five naturally occurring flavonoids in arresting the development of anemia during the postinfection period. Among the compounds studied, quercetin was most successful in inhibiting the oxidation of proteins and lipids on the red cell membranes of infected animals. Apart from its antianemic property, quercetin also seemed to be highly potent in lowering the parasite load in the spleen. Combination therapy of quercetin with the antileishmanial drug stibanate produced a better decay of .OH in the erythrocytes of the infected animals compared to that induced by quercetin or drug treatment alone. Similar results were obtained in successful prevention of proteolytic degradation resulting in an aversion to early lysis of red cells after simultaneous treatment with quercetin and stibanate. Subsequent studies demonstrated the therapeutic efficacy of the combination treatment in the abatement of both anemia and parasitemia under the diseased condition.

Inhibition of benzo[a]pyrene induced mutagenicity and genotoxicity by black tea polyphenols theaflavins and thearubigins in multiple test systems.

This study investigated the antimutagenic and anticlastogenic effects of black tea polyphenols, theaflavins (TF) and thearubigins (TR) in Salmonella assay in vitro and in vivo in bone marrow cells of mice as measured by chromosomal aberrations (CA) and sister chromatid exchange (SCE) against a known carcinogen, benzo[a]pyrene (B[a]P). A significant decrease in mutagenicity in Salmonella assay and both CA and SCE were observed in all the different concentrations of TF and TR plus B[a]P treated series when compared with B[a]P treated group alone. These results indicate that both TF and TR have significant antimutagenic and anticlastogenic effects.

Anthocephaline, a new indole alkaloid and cadambine, a potent inhibitor of DNA topoisomerase IB of Leishmania donovani (LdTOP1LS), isolated from Anthocephalus cadamba.

Chemical investigation of the stem bark of Anthocephalus cadamba has resulted in the isolation of anthocephaline (1), a new indole alkaloid, along with strictosamide (2), vincosamide (3) and cadambine (4). The structures of the isolated alkaloids (1-4) were established by detailed 2D NMR spectral analysis. Cadambine (4) exhibited potent DNA topoisomerase IB inhibitory activity.

Harmine: evaluation of its antileishmanial properties in various vesicular delivery systems.

Harmine, a beta-carboline amine alkaloid isolated from Peganum harmala, was tested for its antileishmanial properties both in vitro and in vivo. In vitro antileishmanial activity of harmine was encouraging and prompted us to confirm the activity in vivo in hamster models. Harmine was tested both in free form and in different vesicular forms viz. liposomes, niosomes and nanoparticles. The different vesicles were prepared by the published protocols. The percent intercalation of harmine in liposomes, niosomes and nanoparticles was found to be 65, 60 and 20, respectively, when determined at 325 nm (epsilon(M) =2.33 x 10 M(-1) cm(-1)). At an equivalent dose of 1.5 mg/kg body weight, injected subcutaneously (SC) for a total of six doses in 15 days, harmine was found to reduce spleen parasite load by approximately 40, 60, 70 and 80%, respectively in free, liposomal, niosomal and nanoparticular forms. An inverse relationship could be established between the efficacy in the lowering of spleen parasite load and the size of the vesicles. Specific biochemical tests related to normal liver and kidney functions revealed that the toxicity of the drug was reduced in the vesicular forms in the same order as their efficacy and the same was confirmed by the histopathological studies of splenic sections. Cell cycle analysis studies using flow cytometry suggested that although harmine interferes in the cell division stage, it does not induce apoptosis in Leishmania donovani promastigotes. The results using Confocal Microscopy supported that the cell death could be attributed to necrosis due to non-specific membrane damage. Even then, because of its appreciable efficacy in destroying intracellular parasites as well as non-hepatotoxic and non-nephrotoxic nature, harmine, in the vesicular forms, may be considered for clinical application in humans.

Targeting of liposomal flavonoid to liver in combating hepatocellular oxidative damage.

The efficacy of mannosylated liposome formulations with Quercetin (QC, a flavonoid antioxidant isolated from indigenous origin) has been tested in vivo against carbon tetrachloride(CCl(4))-induced liver oxidative damage in rats. Single subcutaneous injection of CCl(4) (40% v/v in olive oil; 1 ml/kg) induces the generation of toxic oxygen radicals and results in hepatocellular damage. The increased serum enzyme levels (glutamate pyruvate transaminase, alkaline phosphatase) and hepatocellular conjugated diene levels by CCl(4) induction were significantly lowered due to pretreatment with mannosylated liposomal QC (MLQ) (0.5 ml liposomal suspension containing 0.27 mg QC), whereas the same amount of free QC was found to be ineffective. In addition, the effectiveness of MLQ on CCl(4)-induced acute liver damage also was evaluated by tissue histopathological examination. Damage produced by CCl(4) in liver reverted to normal with pretreatment of MLQ.

Withaferin A modulates the Spindle assembly checkpoint by degradation of Mad2-Cdc20 complex in colorectal cancer cell lines.

Withania somnifera L. Dunal (Ashwagandha) is used over centuries in the ayurvedic medicines in India. Withaferin A, a withanolide, is the major compound present in leaf extract of the plant which shows anticancer activity against leukemia, breast cancer and colorectal cancer. It arrests the ovarian cancer cells in the G2/M phase in dose dependent manner. In the current study we show the effect of Withaferin A on cell cycle regulation of colorectal cancer cell lines HCT116 and SW480 and its effect on cell fate. Treatment of these cells with this compound leads to apoptosis in a dose dependent manner. It causes the G2/M arrest in both the cell lines. We show that Withaferin A (WA) causes mitotic delay by blocking Spindle assembly checkpoint (SAC) function. Apoptosis induced by Withaferin A is associated with proteasomal degradation of Mad2 and Cdc20, an important constituent of the Spindle Checkpoint Complex. Further overexpression of Mad2 partially rescues the deleterious effect of WA by restoring proper anaphase initiation and keeping more number of cells viable. We hypothesize that Withaferin A kills cancer cells by delaying the mitotic exit followed by inducing chromosome instability.

14-Deoxyandrographolide alleviates ethanol-induced hepatosteatosis through stimulation of AMP-activated protein kinase activity in rats.

Andrographis paniculata (AP) is a traditional medicinal plant of Ayurveda. It grows widely in Asia and is prescribed in the treatment of liver diseases. Here we have investigated the beneficial role of 14-deoxyandrographolide (14-DAG), a bioactive diterpenoid from AP, against alcoholic steatosis in rats. 14-DAG was extracted from aerial parts (leaves and stems) of AP. Rats were fed with ethanol for 8 weeks. Animals were treated with 14-DAG during the last 4 weeks of ethanol treatment. In vitro studies were undertaken in a human hepatocellular liver carcinoma cell line culture. Hepatosteatosis was assessed from histopathological studies of liver sections. Acetyl-CoA, malonyl-CoA, and triglyceride contents were determined using commercially available kits. Fatty acid synthesis was evaluated from incorporation of 1-(14)C acetate. Regulation of fatty acid oxidation and lipogenesis were monitored with immunoblotting and immunoprecipitation studies. Ethanol exposure led to hepatotoxicity, as evident from the marked enhancement in the levels of AST and ALT. The values decreased almost to control levels in response to 14-DAG treatment. Results showed that ethanol feeding induced deactivation of AMP-activated protein kinase (AMPK) that led to enhanced lipid synthesis and decreased fatty acid oxidation, culminating in hepatic fat accumulation. Treatment with 14-DAG activated AMPK through induction of cyclic AMP-protein kinase A pathway. Activation of AMPK was followed by down-regulation of sterol regulatory element binding protein-1c, acetyl-CoA carboxylase, and fatty acid synthase, leading to suppression of lipogenesis. This was associated with up-regulation of sirtuin 1 and depletion of malonyl-CoA, in favor of increased fatty acid oxidation. 14-DAG controlled ethanol-induced hepatosteatosis by interfering with dysregulation of lipid metabolism. In conclusion, our results indicated that 14-DAG was capable of preventing the development of fatty liver through AMPK-mediated regulation of lipid metabolism. This finding supported the hepatoprotective role of 14-DAG, which might serve as a therapeutic option to alleviate hepatosteatosis in chronic alcoholism.

A new ellagic acid glycoside and DNA topoisomerase IB inhibitory activity of saponins from Putranjiva roxburghii.

Chemical investigation of the stem bark and leaves of Putranjiva roxburghii has resulted in the isolation of a new ellagic acid glycoside (5) along with four saponins (1-4). The structures of the isolated compounds were established by detailed spectral analysis. Incidentally putranoside-A methyl ester (4) has been isolated for the first time from this species and the saponins (1-4) exhibited potent DNA topoisomerase IB inhibitory activity.

The lignan glycosides lyoniside and saracoside poison the unusual type IB topoisomerase of Leishmania donovani and kill the parasite both in vitro and in vivo.

Lignans are diphenyl propanoids with vast range of biological activities. The present study provides an important insight into the anti-leishmanial activities of two lignan glycosides, viz. lyoniside and saracoside. These compounds inhibit catalytic activities of topoisomerase IB (LdTopIB) of Leishmania donovani in non-competitive manner and stabilize the LdTopIB mediated cleavage complex formation both in vitro and in Leishmania promastigotes and subsequently inhibit the religation of cleaved strand. These two compounds not only poison LdTopIB but also can interact with the free enzyme LdTopIB. We have also shown that lyoniside and saracoside are cytotoxic to promastigotes and intracellular amastigotes. The protein-DNA complex formation leads to double strand breaks in DNA which ultimately triggers apoptosis-like cell death in the parasite. Along with their cytotoxicity towards sodium antimony gluconate (SAG) sensitive AG83 strain, their ability to kill SAG resistant GE1 strain makes these two compounds potential anti-leishmanial candidates. Not only they effectively kill L. donovani amastigotes inside macrophages in vitro, lyoniside and saracoside demonstrated strong anti-leishmanial efficacies in BALB/c mice model of leishmaniasis. Treatment with these lignan glycosides produce nitric oxide and reactive oxygen species which result in almost complete clearance of the liver and splenic parasite burden. These compounds do not inhibit human topoisomerase IB upto 200µM concentrations and had poor cytotoxic effect on uninfected cultured murine peritoneal macrophages upto 100µM concentrations. Taken together it can be concluded that these compounds can be developed into excellent therapeutic agent against deadly disease leishmaniasis.

14-Deoxyandrographolide targets adenylate cyclase and prevents ethanol-induced liver injury through constitutive NOS dependent reduced redox signaling in rats.

Chronic alcoholism is one of the most common causes of liver diseases worldwide. Nitric oxide (NO) has been proposed to have potential for clinical application against chronic hepatocellular injuries. However, mechanisms underlying hepatoprotective functions of NO in ethanol-induced apoptosis are largely unknown. Sprauge-Dawley rats were exposed to ethanol for 8 weeks. Half of the ethanol-fed animals received 14-deoxyandrographolide (14-DAG) treatment for the last 4 weeks of study. Preventive effect of 14-DAG against ethanol-induced hepatotoxicity involved constitutive nitric oxide synthase (cNOS) activation followed by up-regulation of γ-glutamylcysteine synthetase activity and reduced oxidative stress. Enhanced interaction of cNOS with caveolin-1 caused down-regulation of enzyme activity and led to depletion of NO in the hepatocytes of ethanol-fed animals. 14-DAG acted as activator of adenylate cyclase and modulated cyclic AMP (cAMP) mediated expression of caveolin-1 and calmodulin. This eventually favored activation of cNOS through inhibition of cNOS-caveolin-1 interaction. Our results suggest that, protective effect of 14-DAG against ethanol-induced hepatic injury is based on its ability to reduce oxidative stress through cNOS dependent improvement of redox status. 14-DAG mediated activation of adenylate cyclase-cAMP signaling leading to up-regulation of cNOS may provide a promising approach in the prevention of liver diseases during chronic alcoholism.