Ca(2+)-calmodulin antagonist chlorpromazine and poly(ADP-ribose) polymerase modulators 4-aminobenzamide and nicotinamide influence hepatic expression of BCL-XL and P53 and protect against acetaminophen-induced programmed and unprogrammed cell death in mice.

Acetaminophen (AAP), the analgesic hepatotoxicant, is a powerful inducer of oxidative stress, DNA fragmentation, and apoptosis. The anti-apoptotic oncogene bcl-XL, and the pro-apoptotic oncogene p53 are two key regulators of cell cycle progression and/or apoptosis subsequent to DNA damage in vitro and in vivo. This study investigated the effect of AAP on the expression of these oncogenes and whether agents that modulate DNA fragmentation (chlorpromazine, CPZ) and DNA repair through poly(ADP-Ribose) polymerase (PARP) activity (4-AB: 4-aminobenzamide) can protect against AAP-induced hepatotoxicity by inhibiting oxidative stress, DNA fragmentation, and/or by altering the expression of bcl-XL and p53. In addition, the protective effect of supplemental nicotinamide (NICO), known to be depleted in cells with high PARP activity during DNA repair, is similarly evaluated. Male ICR mice (3 months old) were administered vehicle alone; nontoxic doses of 4-AB (400 mg/kg, ip), NICO (250 mg/kg, ip) or CPZ (25 mg/kg, ip), hepatotoxic dose of AAP alone (500 mg/kg, ip), or AAP plus one of the protective agents 1 h later. All animals were sacrificed 24 h following AAP administration. Serum alanine aminotransferase activity (ALT), hepatic histopathology and lipid peroxidation, DNA damage, and expression of bcl-XL and p53 (western blot analysis) were compared in various groups. All of the three agents significantly prevented AAP-induced liver injury, lipid peroxidation, DNA damage, and associated apoptotic and necrotic cell deaths, 4-AB being the most effective and NICO the least. Compared to control, there was a considerable decrease in bcl-XL expression, and an increase in p53 expression in AAP-exposed livers. The effect of AAP on bcl-XL was antagonized and that on p53 was synergized by the PARP-modulator 4-AB as well as NICO, whereas the endonuclease inhibitor CPZ was without effect on either bcl-XL or p53 expression. These results suggest that the hepatotoxic effect of AAP involves multiple mechanisms including oxidative stress, upregulation of endonuclease (or caspase-activated DNAse) and alteration of pro- and anti-apoptotic oncogenes. The observed antagonism of AAP-induced hepatocellular apoptosis and/or necrosis by modulators of multiple processes including DNA repair suggests the likelihood that a more effective therapy against AAP intoxication should involve a combination of antidotes.

Smokeless tobacco, oxidative stress, apoptosis, and antioxidants in human oral keratinocytes.

We have investigated the effects of a smokeless tobacco extract (STE) on lipid peroxidation, cytochrome c reduction, DNA fragmentation and apoptotic cell death in normal human oral keratinocyte cells, and assessed the protective abilities of selected antioxidants. The cells, isolated and cultured from human oral tissues, were treated with STE (0-300 microl;g/ml) for 24 h. Superoxide anion production was determined by cytochrome c reductase. Oxidative tissue damage was determined by lipid peroxidation and DNA fragmentation, whereas apoptotic cell death was assessed by flow cytometry. STE-induced fragmentation of genomic DNA was also determined by gel electrophoresis. The comparative protective abilities of vitamin C (75 microM), vitamin E (75 microM), a combination of vitamins C & E (75 microM each), and a novel grape seed proanthocyanidin (IH636) extract (GSPE) (100 microg/ml) against STE induced oxidative stress and tissue damage were also determined. Following treatment of the cells with 300 microg STE/ml 1.5-7.6-fold increases in lipid peroxidation, cytochrome c reduction and DNA fragmentation were observed. The addition of the antioxidants to cells treated with STE provided 10-54% decreases in these parameters. Approximately 9, 29, and 35% increases in apoptotic cell death were observed following treatment with 100, 200, and 300 microg STE/ml, respectively, and 51-85% decreases in apoptotic cell death were observed with the antioxidants. The results demonstrate that STE produces oxidative tissue damage and apoptosis, which can be attenuated by antioxidants including vitamin C, vitamin E, a combination of vitamins C plus E and GSPE. GSPE exhibited better protection against STE than vitamins C and E, singly and in combination.

Diclofenac induced in vivo nephrotoxicity may involve oxidative stress-mediated massive genomic DNA fragmentation and apoptotic cell death.

Diclofenac (DCLF) is a nonsteroidal anti-inflammatory drug that is widely used for the treatment of osteoarthritis, rheumatoid arthritis, ankylosing spondylitis, and acute muscle pain conditions. Toxic doses of DCLF can cause nephrotoxicity in humans and experimental animals. However, whether this DCLF-induced nephrotoxicity involves apoptotic cell death in addition to necrosis is unknown. The goals of this investigation were to determine whether DCLF-induced nephrotoxicity involves oxidative stress and apoptotic type genomic DNA fragmentation, and if so, whether DCLF-induced oxidative stress and DNA fragmentation cause apoptotic cell death in mouse kidneys. Male ICR mice (CD-1; 25-45 g), fed ad libitum, were administered nephrotoxic doses of DCLF (100, 200, 300 mg/Kg, po) and sacrificed 24 h later. Blood was collected to evaluate renal injury (BUN), lipid peroxidation (MDA: malondialdehyde levels), and superoxide dismutase (SOD) activity (a marker of oxidative stress). Kidney tissues were analyzed both quantitatively and qualitatively to determine the degree and type of DNA damage, and evaluated histopathologically for the presence of apoptotic characteristics in the nucleus of diverse types of kidney cells. Results show that diclofenac is a powerful nephrotoxicant (at 100, 200, and 300 mg/kg: 4.7-, 4.9-, and 5.0-fold increases in BUN compared to the control, respectively) and a strong inducer of oxidative stress (significant increase in MDA levels). Oxidative stress induced by DCLF was also coupled with massive kidney DNA fragmentation (100, 200, and 300 mg/kg: 3-, 8-, and 10-fold increases compared to control, respectively). A dose-dependent increase in MDA levels and SOD activity was also observed, which indicated a link between oxidative stress and nephrotoxicity. Qualitative analysis of DNA fragmentation by gel electrophoresis showed a DNA ladder indicative of Ca2+-Mg2+-endonuclease activation. Histopathological examination of kidney sections revealed numerous apoptotic nuclei across proximal and distal tubular cell linings. Collectively, these data for the first time suggest that DCLF-induced nephrotoxicity may involve production of reactive oxygen species leading to oxidative stress and massive genomic DNA fragmentation, and these two free radical mediated events may ultimately translate into apoptotic cell death of kidney cells in vivo, and reveal a DNA-active role for DCLF.

Induction of P4502E1 by acetone in isolated rabbit hepatocytes. Role of increased protein and mRNA synthesis.

The molecular mechanism(s) underlying induction of the hepatic microsomal cytochrome P4502E1 (2E1) by xenobiotics (e.g. ethanol and acetone) is controversial. Proposed mechanisms include increased rates of enzyme synthesis due to elevated 2E1 mRNA levels, enhanced translation of pre-existing mRNA, or stabilization of 2E1 protein. To further assess which, if any, of these events predominates during the initial stages of 2E1 protein induction, we investigated the effects of acetone treatment on 2E1 content in cultured rabbit hepatocytes, an in vitro system that allows for precise control of the cellular mileau. Hepatocytes harvested from female rabbits and plated on plastic dishes with serum-supplemented medium were 90-100% viable for at least 48 hr in culture. Analysis of immunoreactive 2E1 content and aniline hydroxylase activity in microsomes isolated from hepatocytes cultured for up to 24 hr revealed that 2E1 expression was equal to that of microsomes from unplated cells and by 48 hr of culture, 2E1 levels decreased by only 35%. Moreover, microsomes isolated from cells exposed to 17 mM acetone for 24 hr exhibited a 53 and 62% increase in aniline hydroxylase activity and 2E1 content, respectively, compared to untreated cells. To explain these increases, the rate of 2E1 protein synthesis was determined in untreated cells or in cells treated with 17 mM acetone by first exposing hepatocytes to medium supplemented with 35S-labeled methionine and cysteine ([35S]Met/Cys) and subsequently assessing radiolabel incorporation into 2E1 protein. While no difference was found between untreated and acetone-treated cells in the incorporation of [35S]Met/Cys into trichloracetic acid-precipitable microsomal proteins, immunoaffinity purification of 2E1 revealed that incorporation of 35S-labeled amino acids specifically into 2E1 was elevated by acetone to 200% of control values. Treatment of hepatocytes with the transcriptional inhibitor, alpha-amanitin, markedly inhibited this acetone-mediated increase in [35S]Met/Cys incorporation into 2E1. Analysis of hepatocyte RNA revealed that acetone increased 2E1 mRNA to 130 and 160% of control levels at 6 and 24 hr, respectively, and that these increases were prevented by pretreatment with alpha-amanitin. Our results indicate that acetone increases 2E1 protein levels in cultured rabbit hepatocytes by stimulating its rate of de novo synthesis. Since this increase in 2E1 synthesis stems, at least in part, from the acetone-mediated enhancement of hepatocyte 2E1 mRNA content and is inhibitable by alpha-amanitin, transcriptional activation of the rabbit CYP2E1 gene is apparently involved in the induction of 2E1 protein by acetone.

Free radicals and grape seed proanthocyanidin extract: importance in human health and disease prevention.

Free radicals have been implicated in over a hundred disease conditions in humans, including arthritis, hemorrhagic shock, atherosclerosis, advancing age, ischemia and reperfusion injury of many organs, Alzheimer and Parkinson's disease, gastrointestinal dysfunctions, tumor promotion and carcinogenesis, and AIDS. Antioxidants are potent scavengers of free radicals and serve as inhibitors of neoplastic processes. A large number of synthetic and natural antioxidants have been demonstrated to induce beneficial effects on human health and disease prevention. However, the structure-activity relationship, bioavailability and therapeutic efficacy of the antioxidants differ extensively. Oligomeric proanthocyanidins, naturally occurring antioxidants widely available in fruits, vegetables, nuts, seeds, flowers and bark, have been reported to possess a broad spectrum of biological, pharmacological and therapeutic activities against free radicals and oxidative stress. We have assessed the concentration- or dose-dependent free radical scavenging ability of a novel IH636 grape seed proanthocyanidin extract (GSPE) both in vitro and in vivo models, and compared the free radical scavenging ability of GSPE with vitamins C, E and beta-carotene. These experiments demonstrated that GSPE is highly bioavailable and provides significantly greater protection against free radicals and free radical-induced lipid peroxidation and DNA damage than vitamins C, E and beta-carotene. GSPE was also shown to demonstrate cytotoxicity towards human breast, lung and gastric adenocarcinoma cells, while enhancing the growth and viability of normal human gastric mucosal cells. The comparative protective effects of GSPE, vitamins C and E were examined on tobacco-induced oxidative stress and apoptotic cell death in human oral keratinocytes. Oxidative tissue damage was determined by lipid peroxidation and DNA fragmentation, while apoptotic cell death was assessed by flow cytometry. GSPE provided significantly better protection as compared to vitamins C and E, singly and in combination. GSPE also demonstrated excellent protection against acetaminophen overdose-induced liver and kidney damage by regulating bcl-X(L) gene, DNA damage and presumably by reducing oxidative stress. GSPE demonstrated excellent protection against myocardial ischemia-reperfusion injury and myocardial infarction in rats. GSPE was also shown to upregulate bcl(2) gene and downregulate the oncogene c-myc. Topical application of GSPE enhances sun protection factor in human volunteers, as well as supplementation of GSPE ameliorates chronic pancreatitis in humans. These results demonstrate that GSPE provides excellent protection against oxidative stress and free radical-mediated tissue injury.

Cholesteryl hemisuccinate treatment protects rodents from the toxic effects of acetaminophen, adriamycin, carbon tetrachloride, chloroform and galactosamine.

In addition to its use as a stabilizer/rigidifier of membranes, cholesteryl hemisuccinate, tris salt (CS) administration has also been shown to protect rats from the hepatotoxic effects of carbon tetrachloride (CCl4). To further our understanding of the mechanism of CS cytoprotection, we examined in rats and mice the protective abilities of CS and the non-hydrolyzable ether form of CS, gamma-cholesteryloxybutyric acid, tris salt (CSE) against acetaminophen-, adriamycin-, carbon tetrachloride-, chloroform- and galactosamine-induced toxicity. The results of these studies demonstrated that CS-mediated protection is not selective for a particular species, organ system or toxic chemical. A 24-h pretreatment of both rats and mice with a single dose of CS (100mg/kg, i.p.), resulted in significant protection against the hepatotoxic effects of CCl4, CHCl3, acetaminophen and galactosamine and against the lethal (and presumably cardiotoxic) effect of adriamycin administration. Maximal CS-mediated protection was observed in experimental animals pretreated 24 h prior to the toxic insult. These data suggest that CS intervenes in a critical cellular event that is an important common pathway to toxic cell death. The mechanism of CS protection does not appear to be dependent on the inhibition of chemical bioactivation to a toxic reactive intermediate (in light of the protection observed against galactosamine hepatotoxicity). However, based on the data presented, we can not exclude the possibility that CS administration inhibits chemical bioactivation. Our findings do suggest that CS-mediated protection is dependent on the action of the intact anionic CS molecule (non-hydrolyzable CSE was as protective as CS), whose mechanism has yet to be defined.

Inhibition of cell division in hepatoma cell cultures by chlordecone and carbon tetrachloride combination.

The propensity of chlordecone (CD) to potentiate the hepatotoxic and lethal effects of CCl(4) is well established. Mirex (M), a close structural analogue of CD, or phenobarbital (PB), both powerful inducers of hepatic microsomal drug metabolizing enzymes, are much weaker potentiators of CCl(4) toxicity. Considerable evidence has accumulated to suggest that this increase in CCl(4) toxicity caused by CD is due to the failure of the hepatocellular regeneration, tissue repair and hepatolobular restoration mechanisms. This interaction occurs at concentrations of CD and CCl(4) that are individually non-toxic and do not interfere with hepatocellular division. To test this unique interaction at cellular level, we employed a rapidly dividing Reuber hepatoma cell line in vitro. Cells were pretreated with a non-toxic dose of either CD, M or PB and exposed to a single addition of CCl(4) in the concentration range 5 to 40 mm 16 days later. The results indicate that CD + CCl(4) combination specifically arrested hepatocellular division. The inhibition of cell division occurred at individually non-toxic concentrations of CD and CCl(4). M + CCl(4) or PB + CCl(4) failed to manifest similar effects. At higher concentrations, these combinations caused cellular toxicity, resulting in cell death. Suppression of cell division might play an important role in the progression of chemical-induced toxicities in the liver. This unique observation opens up new avenues to investigate biochemical molecular mechanisms underlying the interference with hepatocellular division.

Extensive alteration of genomic DNA and rise in nuclear Ca2+ in vivo early after hepatotoxic acetaminophen overdose in mice.

Hepatotoxic doses of acetaminophen cause early impairment of Ca2+ homeostasis. In this in vivo study, 600 mg/kg acetaminophen caused total nuclear Ca2+ and % fragmented DNA to rise in parallel from 2-6 hr, followed by large later increases mirroring frank liver injury. Agarose gel electrophoresis revealed substantial loss of large genomic DNA from 2 hours onward, with accumulation of DNA fragments in a ladder-like pattern resembling apoptosis. Extensive late cleavage of DNA probably resulted from cell death, whereas degradative loss of large genomic DNA at 2 hours arose at an early enough point to contribute to acetaminophen-induced liver necrosis in mice.

Oxidative stress is the master operator of drug and chemically-induced programmed and unprogrammed cell death: Implications of natural antioxidants in vivo.

ROS, RNS, BRIs and ROS-RNS hybrids are produced during drug or chemical metabolism in vivo. These reactive species are instrumental to the culmination of cellular oxidative stress (OS). OS, once turned on, does not spare any vital intracellular macromolecule, such as glutathione, DNA, RNA, proteins, enzymes, lipids and ATP. Since concentration gradients of such components are very delicately balanced for normal cellular functioning, a gross perturbation leads to cell injury and cell death. Abundant evidence now suggests that intracellular antioxidants keep OS in check and maintain homeostasis. Our laboratory has focused on the role of OS in orchestrating various forms of cell death during drug and chemically-induced target organ toxicity and their counteraction by various natural or synthetic antioxidants in in vivo models. Despite complexity of the in vivo models, results show that metabolism of xenobiotics are invariably associated with different degrees of OS and natural antioxidants such as grape seed extract, bitter melon extract (Momordica charantia) and N-acetylcysteine (NAC) which were very effective in counteracting organ toxicities by minimizing events linked to OS (lipid peroxidation and total glutathione), and CAD-mediated DNA fragmentation. Phytoextract exposure rescued cells from toxic assaults, protected genomic integrity, and minimized apoptotic, necrotic and apocrotic (oncotic necrosis) cell deaths. Pre-exposure mode was more effective than post-exposure route. Overall scenario suggests that OS may have been the prime modulator of death and/or survival programs, whereas, antioxidants may have imparted a dual role in either erasing death signals or reviving survival signals, and a combination of antioxidants may be more beneficial than a single entity to influence a number of intracellular events operating simultaneously to neutralize chaotic toxicological consequences.

Protection against drug- and chemical-induced multiorgan toxicity by a novel IH636 grape seed proanthocyanidin extract.

Grape seed proanthocyanidins have been demonstrated to exhibit a broad spectrum of pharmacological, therapeutic and chemoprotective properties. In our previous studies, IH636 grape seed proanthocyanidin extract (GSPE, commercially known as ActiVin) demonstrated excellent concentration- and dose-dependent free radical scavenging abilities in both in vitro and in vivo models and provided significantly better protection than vitamins C, E and beta-carotene. GSPE demonstrated significant cytotoxicity towards human breast, lung and gastric adenocarcinoma cells, while enhancing the growth and viability of normal human gastric mucosal cells and macrophage J774A.1 cells. In this study, the bioavailability and protective ability of GSPE were examined against acetaminophen-induced hepatoxicity, amiodarone-induced pulmonary toxicity, doxorubicin-induced cardiotoxicity, cadmium chloride-induced nephrotoxicity, dimethylnitrosamine-induced spleenotoxicity and O-ethyl-S,S-dipropyl phosphorodithioate (MOCAP)-induced neurotoxicity in mice. In each experiment, half of the test animals were orally fed GSPE for 7-10 days prior to drug/chemical exposure, while the other half received no GSPE. Parameters of analysis included changes in serum chemistry [alanine amino-transferase (ALT), blood urea nitrogen and creatine kinase], histopathology and integrity of genomic DNA. The results indicated that GSPE preexposure prior to the drugs/chemicals such as acetaminophen, amiodarone, doxorubicin, cadmium chloride or dimethylnitrosamine treatment, provided near complete protection in terms of serum chemistry changes (ALT, blood urea nitrogen and creatine kinase) and inhibition of both forms of cell death, e.g., apoptosis and necrosis. DNA damage in various tissues triggered by these agents was significantly reduced. Histopathological examination of the organs evaluated reflected similar patterns to those of the serum chemistry and DNA results. MOCAP exposure showed symptoms of severe neurotoxicity coupled with serum chemistry changes in the absence of any significant genomic change or brain pathology. GSPE afforded only partial protection in the brain tissue. These results suggest that GSPE exposure is bioavailable and provides significant multiorgan protection against drug- and chemical-induced toxic assaults.

The role of the nucleus and other compartments in toxic cell death produced by alkylating hepatotoxicants.

Hepatocellular necrosis occurs under a wide range of pathological conditions. In most cases, toxic cell death takes place over a finite span of time, delayed from the point of initial injury and accompanied by homeostatic counterresponses that are varied and complex. The present strategies for discovering critical steps in cell death recognize that (1) different toxins produce similar morphologic changes that precede killing in widely varied cell types, and that (2) lethal events are likely to involve one or more compartmentalized functions that are common to most cells. Investigations of the plasma membrane, endoplasmic reticulum, cytoplasm, mitochondrion, and nucleus have greatly advanced our understanding of acute hepatocellular necrosis. This report examines each compartment but emphasizes molecular changes in the nucleus which may explain cell death caused by alkylating hepatotoxicants. Accumulating knowledge about two distinct modes of cell death, necrosis and apoptosis, indicates that loss of Ca2+ regulation and subsequent damage to DNA may be critical steps in lethal damage to liver cells by toxic chemicals.

A hepatotoxic dose of acetaminophen modulates expression of BCL-2, BCL-X(L), and BCL-X(S) during apoptotic and necrotic death of mouse liver cells in vivo.

The protein BCL-X(L) and protein product of proto-oncogene bcl-2 act as apoptosis antagonists, and BCL-X(S) serve as a dominant death promoter, including apoptosis following exposure to chemotherapeutic drugs. This investigation examined whether some aspects of the highly integrated process of acetaminophen (AAP)-induced hepatotoxicity involve down-regulation or upregulation of expression of BCL-2, BCL-X(L) and BCL-X(S) in mouse liver in vivo. Male ICR mice (CD-1; 35-45 g) were treated ip with a hepatotoxic dose of AAP (500 mg/kg) and sacrificed 0, 6, and 18 h later. Blood was collected upon sacrifice for determination of serum alanine aminotransferase (ALT) activity and the liver was sectioned for histopathological diagnosis of necrosis/apoptosis. Portions of liver tissues were also used for DNA extraction (for gel electrophoresis) and Western blot analysis. This study demonstrates that administration of a hepatotoxic dose of AAP to ICR mice results in severe liver injury (ALT leakage >200-fold at 6 h and >600-fold at 18 h) leading to massive cell death by apoptosis (diagnosed by nuclear ultrastructure, histopathology, and DNA ladder), in addition to necrosis coupled with spectacular changes in the BCL-X(L) expression (6 and 18 h after AAP administration). Western blot analysis of the liver proteins revealed that mouse liver expresses two proteins, BCL-X(L) and BCL-X(S), and does not express BCL-2. As the toxicity progressed, during 6 and 18 h post-AAP administration, the BCL-X(L) protein band shifted to a slower mobility band which might represent a phosphorylated form of BCL-X(L). Appearance of this higher molecular weight BCL-X(L) protein band correlated with massive apoptotic death of liver cells along with ladder-like DNA fragmentation. In the same time period, death inhibitory gene bcl-2 remained unexpressed, and the level of expression of BCL-X(S) remained unaltered. Whether the consistent level of expression of BCL-X(S) reflected inability of AAP to influence its expression remains unknown. Unaltered expression of BCL-X(S) in the near total absence of BCL-2 expression raises questions regarding the death promoting role of BCL-X(S) in vivo. The precise role of modified form of BCL-X(L) remains elusive. However, this study may have demonstrated for the first time drug-induced changes in the expression of anti-apoptotic gene BCL-X(L), and a positive link between AAP-induced apoptotic death and modification of BCL-X(L) protein in vivo.

Potentiation of CCl4 and CHCl3 hepatotoxicity and lethality by various alcohols.

Various aliphatic alcohols potentiate the toxicity of a wide range of xenobiotics including several haloalkanes. The present series of experiments were designed to test: (i) whether a single subtoxic dose of alcohol can potentiate CCl4 and CHCl3 hepatoxicity, and (ii) whether this potentiation leads to greater animal lethality. Selected members of a homologous series of straight chain alcohols were chosen for this study. Methanol, ethanol, isopropanol, t-butanol, pentanol, hexanol, octanol, decanol, and eicosanol at equimolar doses (10 mmol/kg) were tested in the present investigation. Each alcohol was administered orally to male Sprague-Dawley rats (175-250 g) 18 hr prior to a single oral administration of CCl4 or CHCl3. Liver injury was assessed by plasma transaminases (alanine aminotransferase, ALT; aspartate aminotransferase, AST) and histopathological examination of liver sections 24 hr after the halomethane treatment. None of these alcohols alone increased plasma ALT or AST significantly, whereas CCl4 or CHCl3 administration to alcohol-treated animals resulted in significant elevation of plasma transaminases. Eicosanol (20-carbon alcohol) did not potentiate the toxicity of either halomethane. Methanol, ethanol, isopropanol, and decanol in combination with CCl4 caused massive liver damage but failed to augment CCl4 lethality. t-Butanol, pentanol, hexanol, and octanol significantly decreased the LD50 of CCl4. The hepatotoxic effects of CHCl3 were potentiated by all of the alcohols and the LD50s were also decreased significantly. On a comparative basis, alcohol-potentiated CHCl3 toxicity was greater than the toxicity of CCl4. These findings indicate that even though halomethane liver injury might be potentiated by alcohols, the underlying mechanisms differ among alcohols since not all alcohols potentiate the lethal effects of these halomethanes.

Role of cellular energy status in tocopheryl hemisuccinate cytoprotection against ethyl methanesulfonate-induced toxicity.

Previous studies from our laboratory have demonstrated that the administration of alpha-tocopheryl hemisuccinate (TS), but not unesterified alpha-tocopherol (T), protects hepatocytes from a variety of toxic insults including chemicals, drugs, metals, and oxidative stress. One possible mechanism for this unique cytoprotection is that succinate released from cellular TS is used as a supplemental energy source during a toxic challenge. To test this hypothesis, we examined the effect of TS (25 microM) administration on cell viability, lipid peroxidation, and several cellular energy-related processes such as mitochondrial membrane potential (MMP, psi delta), lactate formation, and ATP and K+ concentrations in isolated hepatocyte suspensions during a toxic challenge with the alkylating agent, ethyl methanesulfonate (EMS). Data from these studies demonstrate that EMS treatment results in rapid cell death and lipid peroxidation following 2 h of incubation. Preceding EMS-induced cell death was a rapid loss of MMP, intracellular ATP and K+ levels, and mitochondrial ultrastructure as well as a transient increase in cellular lactate production. Pretreatment of hepatocytes with TS prior to EMS exposure prevented the loss of MMP and mitochondrial ultrastructural changes as well as lipid peroxidation and cell death. Cellular ATP levels and lactate production did not reflect the protection afforded to TS-treated hepatocytes. Protection against EMS-induced toxicity was not observed when hepatocytes were: (i) pretreated with TS and esterase inhibitors (preventing T and succinate release from TS); (ii) pretreated with other lipophilic succinate derivatives (cholesteryl hemisuccinate, monomethyl and dimethyl succinate); or (iii) pretreated with T and sodium succinate. Unlike monomethyl succinate, cytoprotective TS pretreatment did not stimulate gluconeogenesis or glycolysis. Hepatocytes isolated from rats pretreated for 24 h with T were not protected from the toxic effects of EMS, unlike TS-pretreated rats. In conclusion, TS cytoprotection against the mitochondrial toxicant EMS appears to be related to the hepatocellular accumulation of TS and the maintenance of mitochondrial function (MMP). Based on our earlier findings and the present observations, we propose that a unique subcellular disposition for TS and the subsequent release of T and succinate at a critical mitochondrial site is responsible for the observed cytoprotection.

In vivo protection of dna damage associated apoptotic and necrotic cell deaths during acetaminophen-induced nephrotoxicity, amiodarone-induced lung toxicity and doxorubicin-induced cardiotoxicity by a novel IH636 grape seed proanthocyanidin extract.

Grape seed extract, primarily a mixture of proanthocyanidins, has been shown to modulate a wide-range of biological, pharmacological and toxicological effects which are mainly cytoprotective. This study assessed the ability of IH636 grape seed proanthocyanidin extract (GSPE) to prevent acetaminophen (AAP)-induced nephrotoxicity, amiodarone (AMI)-induced lung toxicity, and doxorubicin (DOX)-induced cardiotoxicity in mice. Experimental design consisted of four groups: control (vehicle alone), GSPE alone, drug alone and GSPE+drug. For the cytoprotection study, animals were orally gavaged 100 mg/Kg GSPE for 7-10 days followed by i.p. injections of organ specific three drugs (AAP: 500 mg/Kg for 24 h; AMI: 50 mg/Kg/day for four days; DOX: 20 mg/Kg for 48 h). Parameters of study included analysis of serum chemistry (ALT, BUN and CPK), and orderly fragmentation of genomic DNA (both endonuclease-dependent and independent) in addition to microscopic evaluation of damage and/or protection in corresponding PAS stained tissues. Results indicate that GSPE preexposure prior to AAP, AMI and DOX, provided near complete protection in terms of serum chemistry changes (ALT, BUN and CPK), and significantly reduced DNA fragmentation. Histopathological examination of kidney, heart and lung sections revealed moderate to massive tissue damage with a variety of morphological aberrations by all the three drugs in the absence of GSPE preexposure than in its presence. GSPE+drug exposed tissues exhibited minor residual damage or near total recovery. Additionally, histopathological alterations mirrored both serum chemistry changes and the pattern of DNA fragmentation. Interestingly, all the drugs, such as, AAP, AMI and DOX induced apoptotic death in addition to necrosis in the respective organs which was very effectively blocked by GSPE. Since AAP, AMI and DOX undergo biotransformation and are known to produce damaging radicals in vivo, the protection by GSPE may be linked to both inhibition of metabolism and/or detoxification of cytotoxic radicals. In addition, its' presumed contribution to DNA repair may be another important attribute, which played a role in the chemoprevention process. Additionally, this may have been the first report on AMI-induced apoptotic death in the lung tissue. Taken together, these events undoubtedly establish GSPE's abundant bioavailability, and the power to defend multiple target organs from toxic assaults induced by structurally diverse and functionally different entities in vivo.

A novel proanthocyanidin IH636 grape seed extract increases in vivo Bcl-XL expression and prevents acetaminophen-induced programmed and unprogrammed cell death in mouse liver.

Several molecular events in the apoptotic or necrotic death of hepatocytes induced by acetaminophen (AAP) now appear to be well defined. Recent studies also indicate that select expression of bcl-Xl is possibly modified during AAP-induced liver injury. The purpose of this study was several-fold: (i) to examine the hepatoprotective ability of short-term (3-day) and long-term (7-day) exposures of a grape seed proanthocyanidin extract (GSPE) on AAP-induced liver injury and animal lethality; (ii) to monitor effects of GSPE on one of the prime targets of AAP, i.e., hepatocellular genomic DNA and associated apoptotic and necrotic death; and (iii) to unravel changes in the pattern of expression of an antiapoptotic gene, bcl-Xl in the liver. In order to investigate these events, male ICR mice (30-40 g) were administered nontoxic doses of GSPE (3 or 7 days, 100 mg/kg, po), followed by hepatotoxic doses of AAP (400 and 500 mg/kg, ip), and sacrificed 24 h later. Serum was analyzed for alanine aminotransferase activity (ALT) and the liver for histopathological diagnosis of apoptosis/necrosis. The ability of AAP to promote apoptotic DNA fragmentation and its counteraction by GSPE in the liver was also evaluated quantitatively (by a sedimentation assay) and qualitatively (by agarose gel electrophoresis). Portions of livers were also subjected to Western blot analysis (27,000g fraction of liver homogenates) to examine the pattern of expression of cell death inhibitory gene bcl-Xl. Results indicate that 7-day GSPE preexposure induced dramatic protection and markedly decreased liver injury and animal lethality culminated by AAP, when compared to a short-term 3-day exposure. Abrogation of toxicity was also mirrored in DNA fragmentation. Histopathological evaluation of liver sections showed remarkable counteraction of AAP-toxicity by this novel GSPE and substantial inhibition of both apoptotic and necrotic liver cell death. Agarose gel electrophoresis revealed that 7-day GSPE preexposure prior to AAP administration completely blocked Ca(2+)/Mg(2+)-Ca(2+)/Mg(2+)-dependent-endonuclease-mediated ladder-like fragmentation of genomic DNA and significantly altered the bcl-Xl expression. The most dramatic changes observed in this study were: (i) substantial increase in the expression of bcl-Xl in the liver by 7-day GSPE exposure alone; (ii) significant modification bcl-Xl expression by AAP alone; and (iii) dramatic inhibition of AAP-induced modification of bcl-Xl (phosphorylation?) expression by GSPE. In summary, these observations demonstrate that GSPE preexposure may significantly attenuate AAP-induced hepatic DNA damage, apoptotic and necrotic cell death of liver cells, and, most remarkably, antagonize the influence of AAP-induced changes in bcl-Xl expression in vivo.

Unique organoprotective properties of a novel IH636 grape seed proanthocyanidin extract on cadmium chloride-induced nephrotoxicity, dimethylnitrosamine (DMN)-induced splenotoxicity and mocap-induced neurotoxicity in mice.

Several observations, both in humans and laboratory animals, have suggested that proanthocyanidins exhibit a broad spectrum of pharmacological, therapeutic and chemoprotective properties. Specifically, some of our earlier studies have shown that IH636 grape seed proanthocyanidin extract (GSPE, commercially known as ActiVin) provides excellent concentration- and dose-dependent protection against toxicities induced by diverse agents, such as acetaminophen, hydrogen peroxide, 12-O-tetradecanoylphorbol-13-acetate (TPA), smokeless-tobacco extract, idarubicin and 4-hydroxyperoxycyclophosphamide in both in vitro and in vivo models. In some models, GSPE proved to be a better cytoprotectant than vitamins C, E and beta-carotene. The purpose of this investigation was three fold: (i) to indirectly assess the bioavailability of GSPE in multiple target organs, (ii) quantify GSPE's capacity to avert cadmium chloride (CdCl2)-induced nephrotoxicity, dimethylnitrosamine (DMN)-induced splenotoxicity and O-ethyl-S,S-dipropyl phosphorodithioate (MOCAP)-induced neurotoxicity, and lastly (iii) to evaluate possible mechanisms of protection in mice. In order to determine all these, three separate experiments were designed and each experiment consisted of four groups, such as vehicle control, GSPE alone, toxicant alone and GSPE + toxicant. GSPE was administered orally (100 mg/Kg) for 7-8 days prior to the toxicant exposure. Parameters of the analyses included evaluation of serum chemistry changes (ALT, BUN and CK), histopathology and integrity of genomic DNA, both quantitatively and qualitatively. Results indicate that GSPE preexposure prior to cadmium chloride and DMN provided near complete protection in terms of serum chemistry changes (ALT, BUN and CK) and inhibition of both forms of cell death. e.g., apoptosis and necrosis. DNA damage, a common denominator usually associated with both apoptosis and necrosis was significantly reduced by GSPE treatment. Histopathological examination of organs correlated strongly with the changes in serum chemistry and the DNA modification data. Surprisingly, MOCAP exposure showed symptoms of neurotoxicity coupled with serum chemistry changes in the absence of any significant genomic DNA damage or brain pathology. Although, GSPE appeared to partially protect the neural tissue, it powerfully antagonized MOCAP-induced mortality. Taken together, this study suggests that in vivo GSPE-preexposure may protect multiple target organs from a variety of toxic assaults induced by diverse chemical entities.

Acetaminophen-induced cytotoxicity in cultured mouse hepatocytes: correlation of nuclear Ca2+ accumulation and early DNA fragmentation with cell death.

Hepatotoxic doses of acetaminophen cause widespread alkylation of liver and early loss of cytosolic Ca2+ regulation. Although the precise location and target of lethal alkylation are not known, Ca2+ accumulation is viewed as a possible link between cell alkylation and cell death. We have recently shown that Ca2+ accumulates in the nucleus and that DNA fragments in vivo before the development of acetaminophen-induced necrosis in mice. The present study examined cultured hepatocytes for nuclear damage and its association with cell death in vitro. Positive results would argue for two key points. (1) Nonparenchymal cell damage does not explain DNA fragmentation induced by acetaminophen in vivo. (2) A chemical that causes necrosis can produce DNA damage considered characteristic of apoptosis. Hepatocytes from NIH Swiss mice were isolated by collagenase perfusion, cultured in Williams' E medium for 24 hr, and exposed to acetaminophen. Cytotoxicity was assessed by lactate dehydrogenase leakage and release of [3H]adenine from a prelabeled nucleotide pool. Genomic DNA fragmentation was assessed quantitatively by colorimetric analysis and qualitatively by agarose gel electrophoresis. Acetaminophen caused DNA damage from 1-4 hr onward and produced significant release of lactate dehydrogenase and [3H]adenine nucleotides at later times. Agarose gel electrophoresis revealed a "ladder" of DNA fragments characteristic of Ca(2+)-mediated endonuclease activation. Cytotoxicity correlated with nuclear Ca2+ accumulation (r greater than 0.895, p less than 0.05) and with percentage DNA fragmentation (r greater than 0.835, p less than 0.05). Nuclear changes in vitro generally reproduced those observed in vivo. Collectively, these findings demonstrate that nuclear Ca2+ accumulation and DNA fragmentation appear as early events that correlate directly with later cytotoxicity. These changes may contribute to acetaminophen-induced injury leading to cell death in vitro and necrosis in vivo.

DMBA induces programmed cell death (apoptosis) in the A20.1 murine B cell lymphoma.

The mechanism by which 7,12-dimethylbenz[a]anthracene (DMBA) produces cytotoxicity in lymphocytes was investigated in these studies using the murine A20.1 B cell lymphoma. Results show that in vitro exposure of these cells to 10-30 microM DMBA for 4 hr produced an increase in intracellular Ca2+, DNA fragmentation, and subsequent cell death. Elevation of Ca2+ and DNA fragmentation induced by DMBA were greatly pronounced when the A20.1 cells were exposed at high cell density (10(7) cells/ml). DMBA-induced DNA fragmentation and cell death were inhibited by coexposure of A20.1 cells to a calcium chelator (EDTA), a general nuclease and polymerase inhibitor (aurintricarboxylic acid), and a protein synthesis inhibitor (cycloheximide). These agents have been previously shown to inhibit apoptosis in lymphocytes and other cells exposed to chemical agents. We also found that cyclosporin A, an inhibitor of Ca(2+)-dependent pathways of T and B cell activation, prevented apoptosis in the A20.1 cell line. These results demonstrate that DMBA induces programmed cell death (apoptosis) in the A20.1 murine B cell lymphoma by Ca(2+)-dependent pathways. The increased sensitivity of A20.1 at high cell density to Ca2+ elevation and DNA fragmentation suggests that cell to cell interactions may also be important in this process.