Cytoprotection by almond skin extracts or catechins of hepatocyte cytotoxicity induced by hydroperoxide (oxidative stress model) versus glyoxal or methylglyoxal (carbonylation model).

Oxidative and carbonyl stress are detrimental in the pathogenesis of diabetic complications, as well as in other chronic diseases. However, this process may be decreased by dietary bioactive compounds. Almond skin is an abundant source of bioactive compounds and antioxidants, including polyphenolic flavonoids, which may contribute to the decrease in oxidative and carbonyl stress. In this study, four Almond Skin Extracts (ASEI, ASEII, ASEIII, and ASEIV) were prepared by different methods and evaluated for their antioxidant activity. The order of the polyphenol content (total muM gallic acid equivalents) of the four extracts was found to be, in decreasing order of effectiveness: ASEI>ASEIII>ASEIV>ASEII. The order of Ferric-reducing antioxidant power (FRAP, microM FeSO(4)/g) value, in decreasing order was ASEI (216)>ASEIII (176)>ASEIV (89)>ASEII (85). The order of ASE effectiveness for decreasing protein carbonyation induced by the copper Fenton reaction was ASEI>ASEIV>ASEII>ASEIII. The order of antioxidant effectiveness for inhibiting tertiary-butyl hydroperoxide (TBH) induced microsomal lipid peroxidation was ASEI>ASEIV>ASEII, ASEIII. Also, the order of ASE effectiveness for inhibiting TBH induced hepatocyte cell death was: ASEIII, ASEIV>ASEI, ASEII. Catechin also protected hepatocytes from TBH induced hepatocyte, lipid peroxidation and cytotoxicity. In a cell free model, equimolar concentrations of catechin or epicatechin rescued serum albumin from protein carbonylation induced by methylglyoxal (MGO). Catechin, epicatechin and ASEI all decreased gloxal induced hepatocyte cell death and reactive oxygen species (ROS) formation in GSH-depleted hepatocytes. Catechin and epicatechin protected against GO or MGO induced hepatocyte cell death, protein carbonylation and ROS formation. Catechin was more effective than epicatechin. Our results suggest that (a) bioactive almond skin constituents in the non-lipophilic polyphenol extract were the most effective at protecting hepatocytes against hydroperoxide induced hepatocyte oxidative stress and in protecting against dicarbonyl induced cytotoxicity; (b) catechins, the major polyphenol in the extract, were also effective at preventing GO or MGO cytotoxicity likely by trapping GO and MGO and/or rescuing hepatocytes from protein carbonylation.

Interactions between herbal remedies and medicinal drugs--considerations about Cuba.

The use of herbal products to treat a wide range of conditions is rapidly leading to increased intake of phytochemicals. This is one of the main reasons for reinforcing the surveillance of the safety, efficacy and quality control of traditional and complementary medicines. Herbal preparations can interact with a drug at pharmacokinetic, pharmacodynamic and pharmacogenetic levels. In this article interactions between herbal products and conventional medicines are reviewed. Reports about side effects of traditional medicines and main interactions between herbal medicines and conventional drugs in Cuba are also included. Herbal products are currently not subject to the rigorous testing indispensable for conventional drugs. However, if potential drug interactions are to be predicted, it is essential that the ability of herbal products to interfere with drug-metabolizing enzyme systems is fully established.

Hepatocyte cytotoxicity induced by hydroperoxide (oxidative stress model) or glyoxal (carbonylation model): prevention by bioactive nut extracts or catechins.

Carbonyl and oxidative stress play important roles in the development of diabetic complications and have been shown to be augmented by various natural compounds and pharmacological agents. Nuts are a rich source of bioactive compounds and antioxidants and various beneficial health effects of nuts have been reported. This study was conducted to evaluate the cytoprotectiveness of various nut extracts and bioactive compounds found in nuts for decreasing cytotoxicity, lipid peroxidation and protein carbonylation in cell toxicity models of diabetes-related carbonyl (glyoxal) and oxidative stress (hydroperoxide). Methanol, ethyl acetate or water were used to prepare crude hazelnut and walnut extracts, which were then used to screen for in vitro cytoprotection of freshly isolated rat hepatocytes against these toxins. The order of protection by nut extracts against hydroperoxide induced cell death was: walnut methanolic extract>walnut aqueous extract>lipophilic walnut extract>hazelnut aqueous extract>hazelnut methanolic extract whereas the lipophilic hazelnut extract did not protect against cell death. The order of protection against lipid peroxidation was the same except for the hazelnut methanolic extract, which prevented lipid peroxidation better than the hazelnut aqueous extract. Catechin, epicatechin and epigallocatechin gallate (EGCG) were investigated for possible protective effects against carbonyl stress cell death and protein carbonylation in hepatocytes. Catechin protected against glyoxal induced cell death and protein carbonylation, and even elicited protection when added to hepatocytes 30 min after the addition of glyoxal. When catechin and epicatechin were compared for protectiveness against glyoxal induced carbonyl stress in hepatocytes, epicatechin protected more effectively than catechin against cell death and protein carbonylation at 120 min. Both compounds also elicited better protection when premixed with glyoxal before addition to hepatocytes, compared to not premixing with glyoxal. Our results suggest (a) that bioactive nut constituents in the non-lipophilic extracts were more effective than lipophilic extracts for cytoprotection against hydroperoxide induced oxidative stress, (b) catechin compounds under physiological conditions were likely effective at preventing glyoxal cytotoxicity by trapping glyoxal or reversing early stage carbonylation (Schiff base formation).

Hepatocyte susceptibility to glyoxal is dependent on cell thiamin content.

Glyoxal, a reactive dicarbonyl, is detoxified primarily by the glyoxalase system utilizing glutathione (GSH) and by the aldo-keto reductase enzymes which utilizes NAD[P]H as the co-factor. Thiamin (Vitamin B(1)) is an essential coenzyme for transketolase (TK) that is part of the pentose phosphate pathway which helps maintain cellular NADPH levels. NADPH plays an intracellular role in regenerating glutathione (GSH) from oxidized GSH (GSSG), thereby increasing the antioxidant defenses of the cell. In this study we have focused on the prevention of glyoxal toxicity by supplementation with thiamin (3mM). Thiamin was cytoprotective and restored NADPH levels, glyoxal detoxification and mitochondrial membrane potential. Hepatocyte reactive oxygen species (ROS) formation, lipid peroxidation and GSH oxidation were decreased. Furthermore, hepatocytes were made thiamin deficient with oxythiamin (3mM) as measured by the decreased hepatocyte TK activity. Under thiamin deficient conditions a non-toxic dose of glyoxal (2mM) became cytotoxic and glyoxal metabolism decreased; while ROS formation, lipid peroxidation and GSH oxidation was increased.

Mitochondrial function and toxicity: role of B vitamins on the one-carbon transfer pathways.

The B vitamins are water-soluble vitamins that are required as coenzymes for reactions essential for cellular function. This review focuses on the essential role of vitamins in maintaining the one-carbon transfer cycles. Folate and choline are believed to be central methyl donors required for mitochondrial protein and nucleic acid synthesis through their active forms, 5-methyltetrahydrofolate and betaine, respectively. Cobalamin (B12) may assist methyltetrahydrofolate in the synthesis of methionine, a cysteine source for glutathione biosynthesis. Pyridoxal, pyridoxine and pyridoxamine (B6) seem to be involved in the regeneration of tetrahydrofolate into the active methyl-bearing form and in glutathione biosynthesis from homocysteine. Other roles of these vitamins that are relevant to mitochondrial functions will also be discussed. However these roles for B vitamins in cell function are mostly theoretically based and still require verification at the cellular level. For instance it is still not known what B vitamins are depleted by xenobiotic toxins or which cellular targets, metabolic pathways or molecular toxic mechanisms are prevented by B vitamins. This review covers the current state of knowledge and suggests where this research field is heading so as to better understand the role vitamin Bs play in cellular function and intermediary metabolism as well as molecular, cellular and clinical consequences of vitamin deficiency. The current experimental and clinical evidence that supplementation alleviates deficiency symptoms as well as the effectiveness of vitamins as antioxidants will also be reviewed.

Cellular and in vivo hepatotoxicity caused by green tea phenolic acids and catechins.

Tea phenolic acids and catechins containing gallic acid moieties are most abundant in green tea, and various medical benefits have been proposed from their consumption. In the following, the cytotoxicities of these major tea phenolics toward isolated rat hepatocytes have been ranked and the mechanisms of cytotoxicity evaluated. The order of cytotoxic effectiveness found was epigallocatechin-3-gallate>propyl gallate>epicatechin-3-gallate>gallic acid, epigallocatechin>epicatechin. Using gallic acid as a model tea phenolic and comparing it with the tea catechins and gallic acid-derivative food supplements, the major cytotoxic mechanism found with hepatocytes was mitochondrial membrane potential collapse and ROS formation. Epigallocatechin-3-gallate was also the most effective at collapsing the mitochondrial membrane potential and inducing ROS formation. Liver injury was also observed in vivo when these tea phenolics were administered ip to mice, as plasma alanine aminotransferase levels were significantly increased. In contrast, GSH conjugation, methylation, metabolism by NAD(P)H:quinone oxidoreductase 1, and formation of an iron complex were important in detoxifying the gallic acid. In addition, for the first time, the GSH conjugates of gallic acid and epigallocatechin-3-gallate have been identified using mass spectrometry. These results add insight into the cytotoxic and cytoprotective mechanisms of the simple tea phenolic acids and the more complex tea catechins.

Herbal monoterpene alcohols inhibit propofol metabolism and prolong anesthesia time.

2,6-Diisopropylphenol (Propofol) is a short-acting intravenous anesthetic that is rapidly metabolized by glucuronidation and ring hydroxylation catalyzed by cytochrome P450. The goal of this research was to determine whether dietary monoterpene alcohols (MAs) could be used to prolong the anesthetic effect of propofol by inhibiting propofol metabolism in animals. Mice were injected intraperitoneally (i.p.) with MAs (100-200) mg/kg followed by the administration of 100 mg/kg propofol 40 min later via an i.p. injection. The time of the anesthesia of each mouse was recorded. It was found that (+/-)-borneol, (-)-carveol, trans-sobrerol, and menthol significantly extended the anesthetic effect of propofol (>3 times). The concentration of propofol in the mouse blood over time (up to 180 min) also increased in mice pre-treated with (-)-borneol, (-)-carveol, and trans-sobrerol. The volume of distribution of propofol decreased in the (-)-borneol (p<0.05), pre-treated group as compared to the propofol control group. Moreover, the maximum blood concentration of propofol and the concentration of propofol in the blood as indicated by the area under the curve were significantly increased in (-)-borneol and (-)-carveol pre-treated groups. Additional evidence using rat hepatocytes showed that (-)-borneol inhibited propofol glucuronidation whereas trans-sobrerol and (-)-carveol inhibited cytochrome P450 dependent microsomal aminopyrine N-demethylation. These results suggest that (-)-borneol extends propofol-induced anesthesia by inhibiting its glucuronidation in the mouse whereas trans-sobrerol (-)-carveol extends propofol-induced anesthesia by inhibiting P450 catalyzed propofol metabolism.

Preventing hepatocyte oxidative stress cytotoxicity with Mangifera indica L. extract (Vimang).

Vimang is an aqueous extract of Mangifera indica used in Cuba to improve the quality of life in patients suffering from inflammatory diseases. In the present study we evaluated the effects of Vimang at preventing reactive oxygen species (ROS) formation and lipid peroxidation in intact isolated rat hepatocytes. Vimang at 20, 50 and 100 microg/ml inhibited hepatocyte ROS formation induced by glucose-glucose oxidase. Hepatocyte cytotoxicity and lipid peroxidation induced by cumene hydroperoxide was also inhibited by Vimang in a dose and time dependent manner at the same concentration. Vimang also inhibited superoxide radical formation by xanthine oxidase and hypoxanthine. The superoxide radical scavenging and antioxidant activity of the Vimang extract was likely related to its gallates, catechins and mangiferin content. To our knowledge, this is the first report of cytoprotective antioxidant effects of Vimang in cellular oxidative stress models.

Oxidative stress mediated idiosyncratic drug toxicity.

The following describes a novel screening method for "new chemical entities" (NCEs), suitable for ADMET studies, that measures ability to form prooxidant radicals on metabolism and their ability to induce oxidative stress in intact cells. The accelerated molecular cytotoxic mechanism screening (ACMS) techniques used with isolated rat hepatocytes showed that cytotoxicity is usually initiated as a result of macromolecular covalent binding or macromolecular oxidative stress. While P450 is likely responsible for drug metabolic activation in the liver, intestine, lung, and in other nonhepatic tissues, where P450 levels are low, peroxidases including prostaglandin synthetase peroxidase can catalyze xenobiotic one-electron oxidation to form prooxidant free radicals that may cause toxicity or carcinogenesis. Inflammation markedly activates H2O2, generating NADPH oxidase and peroxidase of certain immune cells when they infiltrate tissues including the liver. Myeloperoxidase and NADPH oxidase in the Kupffer cells (resident macrophages of the liver) also become activated during inflammation. The addition of noncytotoxic concentrations of peroxidase/H2O2 to the hepatocyte incubate markedly increased drug cytotoxicity and prooxidant radical formation as shown by glutathione or lipid oxidation. Many drugs that have hepato- or gastrointestinal (GI) toxicity problems or were withdrawn from the market for safety problems, e.g., troglitazone, tolcapone, mefenamic acid, diclofenac, and phenylbutazone, were markedly more toxic and prooxidant in this inflammation model system, whereas other drugs, e.g., entacapone, were not toxic in this inflammation model. Some of the idiosyncratic hepatotoxicity responsible for recent drug withdrawals may therefore result from commonplace sporadic inflammatory episodes during drug therapy.

Applications of cytotoxicity assays and pre-lethal mechanistic assays for assessment of human hepatotoxicity potential.

While drug toxicity (especially hepatotoxicity) is the most frequent reason cited for withdrawal of an approved drug, no simple solution exists to adequately predict such adverse events. Simple cytotoxicity assays in HepG2 cells are relatively insensitive to human hepatotoxic drugs in a retrospective analysis of marketed pharmaceuticals. In comparison, a panel of pre-lethal mechanistic cellular assays hold the promise to deliver a more sensitive approach to detect endpoint-specific drug toxicities. The panel of assays covered by this review includes steatosis, cholestasis, phospholipidosis, reactive intermediates, mitochondria membrane function, oxidative stress, and drug interactions. In addition, the use of metabolically competent cells or the introduction of major human hepatocytes in these in vitro studies allow a more complete picture of potential drug side effect. Since inter-individual therapeutic index (TI) may differ from patient to patient, the rational use of one or more of these cellular assay and targeted in vivo exposure data may allow pharmaceutical scientists to select drug candidates with a higher TI potential in the drug discovery phase.

Potential toxicity of flavonoids and other dietary phenolics: significance for their chemopreventive and anticancer properties.

Flavonoids, including isoflavones, are natural components in our diet and, with the burgeoning interest in alternative medicine, are increasingly being ingested by the general population. Plant phenolics, which form moieties on flavonoid rings, such as gallic acid, are also widely consumed. Several beneficial properties have been attributed to these dietary compounds, including antioxidant, anti-inflammatory, and anticarcinogenic effects. Flavonoid preparations are marketed as herbal medicines or dietary supplements for a variety of alleged nontoxic therapeutic effects. However, they have yet to pass controlled clinical trials for efficacy, and their potential for toxicity is an understudied field of research. This review summarizes the current knowledge regarding potential dietary flavonoid/phenolic-induced toxicity concerns, including their pro-oxidant activity, mitochondrial toxicity (potential apoptosis-inducing properties), and interactions with drug-metabolizing enzymes. Their chemopreventive activity in animal in vivo experiments may result from their ability to inhibit phase I and induce phase II carcinogen metabolizing enzymes that initiate carcinogenesis. They also inhibit the promotion stage of carcinogenesis by inhibiting oxygen radical-forming enzymes or enzymes that contribute to DNA synthesis or act as ATP mimics and inhibit protein kinases that contribute to proliferative signal transduction. Finally, they may prevent tumor development by inducing tumor cell apoptosis by inhibiting DNA topoisomerase II and p53 downregulation or by causing mitochondrial toxicity, which initiates mitochondrial apoptosis. While most flavonoids/phenolics are considered safe, flavonoid/phenolic therapy or chemopreventive use needs to be assessed as there have been reports of toxic flavonoid-drug interactions, liver failure, contact dermatitis, hemolytic anemia, and estrogenic-related concerns such as male reproductive health and breast cancer associated with dietary flavonoid/phenolic consumption or exposures.

Biomechanical evaluation of proximal humeral fracture fixation supplemented with calcium phosphate cement.

BACKGROUND: Proximal humeral fractures are common injuries, and numerous surgical methods have been described for their treatment. The biomechanical characteristics of various internal fixation devices that are used to treat these fractures have not been extensively studied, nor has the potential beneficial effect of calcium phosphate cement supplementation. METHODS: We used a cadaveric three-part proximal humeral osteotomy model to perform a biomechanical evaluation of three types of internal fixation devices: a cloverleaf plate, an angled blade-plate, and Kirschner wires. The effect of supplementing the fixation with SRS (Skeletal Repair System) calcium phosphate cement was evaluated as well. Eighteen pairs of fresh-frozen humeri were obtained, and the bone-mineral density of each specimen was measured. In each pair, one specimen was secured with internal fixation alone and the contralateral specimen was secured with internal fixation combined with calcium phosphate cement. The specimens were tested cyclically in abduction and in external rotation for 250 cycles to evaluate interfragmentary motion. The specimens were then loaded to failure in external rotation to measure torsional load to failure and torsional stiffness. RESULTS: Overall, there were no significant differences between the specimens treated with the blade and cloverleaf plates, whereas the specimens treated with Kirschner wires demonstrated more interfragmentary motion, less stiffness, and lower torque to failure. In general, supplementation with calcium phosphate cement led to significant improvements in the mechanical performance of all three forms of internal fixation as demonstrated by a significant decrease in interfragmentary motion, a significant increase in torque to failure, and a significant increase in torsional stiffness. The addition of calcium phosphate cement increased the stiffness of even the most osteoporotic specimens to levels that were higher than those of the most osteodense specimens that had been treated with internal fixation alone. CONCLUSION: The initial biomechanical properties of internal fixation as measured with use of a proximal humeral osteotomy model and three methods of fixation were significantly improved by the addition of calcium phosphate cement.