Antiproliferative and uncoupling effects of delocalized, lipophilic, cationic gallic acid derivatives on cancer cell lines. Validation in vivo in singenic mice.

Tumor cells principally exhibit increased mitochondrial transmembrane potential (ΔΨ(m)) and altered metabolic pathways. The therapeutic targeting and delivery of anticancer drugs to the mitochondria might improve treatment efficacy. Gallic acid exhibits a variety of biological activities, and its ester derivatives can induce mitochondrial dysfunction. Four alkyl gallate triphenylphosphonium lipophilic cations were synthesized, each differing in the size of the linker chain at the cationic moiety. These derivatives were selectively cytotoxic toward tumor cells. The better compound (TPP(+)C10) contained 10 carbon atoms within the linker chain and exhibited an IC50 value of approximately 0.4-1.6 µM for tumor cells and a selectivity index of approximately 17-fold for tumor compared with normal cells. Consequently, its antiproliferative effect was also assessed in vivo. The oxygen consumption rate and NAD(P)H oxidation levels increased in the tumor cell lines (uncoupling effect), resulting in a ΔΨ(m) decrease and a consequent decrease in intracellular ATP levels. Moreover, TPP(+)C10 significantly inhibited the growth of TA3/Ha tumors in mice. According to these results, the antineoplastic activity and safety of TPP(+)C10 warrant further comprehensive evaluation.

Microsomal oxidative damage promoted by acetaminophen metabolism.

Adverse reactions of acetaminophen have been associated to oxidative stress, which may be elicited by reactive oxygen species (ROS) and/or production of the metabolite NAPQI. Both phenomena would arise through the activity of liver cytochrome P450 (CYP450) system, but their contribution to this oxidative stress is yet to be clarified. A NADPH oxidase activity has been proposed in rat liver microsomes. This activity may be due to the presence of NAD(P)H oxidase (NOX) isoforms in liver endoplasmic reticulum. Both NOX and the CYP450 system activities can catalyze ROS generation using NADPH as a cofactor. Therefore, acetaminophen biotransformation, which requires NADPH, may promote ROS generation through either activity or both. To discriminate between these possibilities, rat liver microsomes were incubated with acetaminophen and NADPH in the presence or absence of specific inhibitors. Incubation with NADPH and acetaminophen elicited lipid peroxidation and decreased thiol content and glutathione-S-transferase (GST) activity. The NOX inhibitors apocynin and plumbagin prevented all these phenomena but the decrease in thiol content. In contrast, this decrease was completely prevented by the specific CYP450 system inhibitor SKF-525A. These data suggest that ROS generation following incubation of microsomes with acetaminophen and NADPH appears to be mainly caused by a NOX activity. In light of these data, toxicity of acetaminophen is discussed.

Mechanisms underlying iron and copper ions toxicity in biological systems: Pro-oxidant activity and protein-binding effects.

Iron and copper ions, in their unbound form, may lead to the generation of reactive oxygen species via Haber-Weiss and/or Fenton reactions. In addition, it has been shown that copper ions can irreversibly and non-specifically bind to thiol groups in proteins. This non-specific binding property has not been fully addressed for iron ions. Thus, the present study compares both the pro-oxidant and the non-specific binding properties of Fe(3+) and Cu(2+), using rat liver cytosol and microsomes as biological systems. Our data show that, in the absence of proteins, Cu(2+)/ascorbate elicited more oxygen consumption than Fe(3+)/ascorbate under identical conditions. Presence of cytosolic and microsomal protein, however, differentially altered oxygen consumption patterns. In addition, Cu(2+)/ascorbate increased microsomal lipid peroxidation and decreased cytosolic and microsomal content of thiol groups more efficiently than Fe(3+)/ascorbate. Finally, Fe(3+)/ascorbate and Cu(2+)/ascorbate inhibited in different ways cytosolic and microsomal glutathione S-transferase (GST) activities, which are differentially sensitive to oxidants. Moreover, in the absence of ascorbate, only Cu(2+) decreased the content of cytosolic and microsomal thiol groups and inhibited cytosolic and microsomal GST activities. Catechin partially prevented the damage to thiol groups elicited by Fe(3+)/ascorbate and Cu(2+)/ascorbate but not by Cu(2+) alone. N-Acetylcysteine completely prevented the damage elicited by Cu(2+)/ascorbate, Fe(3+)/ascorbate and Cu(2+) alone. N-Acetylcysteine also completely reversed the damage to thiol groups elicited by Fe(3+)/ascorbate, partially reversed that of Cu(2+)/ascorbate but failed to reverse the damage promoted by Cu(2+) alone. Our data are discussed in terms to the potential damage that the accumulation of iron and copper ions can promote in biological systems.

Antioxidant properties of a dry product from Vitis vinifera seeds (Leucoselect) in rat liver endoplasmic reticulum / Propiedades antioxidantes de un producto seco de semillas de Vitis vinifera (Leucoselect) en retículo endoplásmico de hígado de rata

Leucoselect is a commercial dry product obtained from grape seeds and enriched in procyanidins, which display antioxidant activity in virtue to their ability to scavenge oxygen free radicals and to chelate transition metal ions. The hypoxanthine/xanthine oxidase and Cu2+/ascorbate systems are capable of generating reactive oxygen species; the latter system can also promote non-specific binding of copper ions to proteins. Therefore, we assessed the ability of Leucoselect to inhibit oxidative phenomena elicited by both oxidative systems on rat liver microsomes: lipid peroxidation, oxidation of protein thiols, and inhibition of the cytochrome P450 system. The antioxidant activity of Leucoselect was a reflection of its ability to scavenge oxygen free radicals, chelate copper ions, and protect microsomal membranes through direct interaction. These mechanisms were displayed in a dependent manner with the type of biomolecule studied and also with the oxidative system employed, which is an interesting phenomenon to consider when evaluating the antioxidant activity of herbal products.

Leucoselect es un producto comercial seco obtenido de semillas de uva y enriquecido en procianidinas, las cuales presentan actividad antioxidante debido a su capacidad para atrapar radicales libres y quelar metales de transición. Los sistemas hipoxantina/xantina oxidasa y Cu2+/ascorbato generan especies reactivas del oxígeno; este último sistema también promueve la unión inespecífica de iones cobre a proteínas. Por lo tanto, evaluamos la capacidad de Leucoselect para inhibir los fenómenos oxidativos producidos por ambos sistemas oxidantes en microsomas hepáticos de rata: lipoperoxidación, oxidación de tioles proteicos e inhibición de la actividad del sistema citocromo P450. La actividad antioxidante de Leucoselect fue un reflejo de su capacidad de atrapar radicales libres del oxígeno, quelar iones cobre y proteger membranas microsómicas por interacción directa. Dichos mecanismos se manifestaron en forma dependiente del tipo de biomolécula estudiada y del sistema oxidante empleado, fenómeno interesante de considerar al evaluar la actividad antioxidante de preparados herbales.

Melatonin protects the cytochrome P450 system through a novel antioxidant mechanism.

Melatonin, an endogenous hormone, is used as an antioxidant drug in doses quite higher than the endogenous circulating levels of this hormone. Hepatic endoplasmic reticulum contains the cytochrome P450 (CYP450) system, which catalyzes one biotransformation pathway of melatonin; this organelle is also one of the main sources of reactive oxygen species in cells. Therefore, we proposed that the antioxidant activity of this hormone may have a biological relevance in the organelle where it is biotransformed. To evaluate this postulate, we used Fe(3+)/ascorbate, an oxygen free radical generating system that leads to lipid peroxidation, loss of protein-thiol content, and activation of UDP-glucuronyltransferase in rat liver microsomes. We found that mM concentrations of melatonin prevented all these oxidative phenomena. We also found that Fe(3+)/ascorbate leads to structural alterations in the CYP450 monooxygenase, the enzyme that binds the substrate in the CYP450 system catalytic cycle, probably through direct oxidation of the protein, and also inhibited p-nitroanisole O-demethylation, a reaction catalyzed by the CYP450 system. Notably, melatonin prevented both phenomena at microM concentrations. We provide evidence suggesting that melatonin may be oxidized by oxygen free radicals. Thus, we postulate that melatonin may be acting as an oxygen free radical scavenger, and Fe(3+)/ascorbate-modified melatonin would be directly protecting the CYP450 system through an additional specific mechanism. Pharmacological relevance of this phenomenon is discussed.

Comparative effects of superoxide anion and hydrogen peroxide on microsomal and cytosolic glutathione S-transferase activities of rat liver.

Glutathione S-transferases (GSTs) are isoenzymes occurring in the cytoplasm and as integral membrane proteins. In addition to their role in drug metabolism by conjugating electrophilic and lipophilic compounds with glutathione (GSH), these enzymes display multiple functions in cells, including antioxidant action. It has been generalized that reactive oxygen species (ROS) inhibit cytosolic GSTs and activate microsomal GSTs; some evidence shows, however, that different ROS-generating systems can inhibit microsomal GST activity. We therefore tested the effect of Fe3+/ascorbate, another ROS-generating system, on cytosolic and microsomal GST activities from rat liver cytosol and microsomes, respectively, and compared it to that of hydrogen peroxide (H2O2). We found that, while both agents displayed similar inhibitory effects on cytosolic GST activity, they promoted opposite effects on microsomal GST activity. Using specific antioxidant enzymes, we corroborated that the effect of Fe3+/ascorbate involves generation of O2(.-) without dismutation into H2O2. Since these ROS have physicochemical properties and redox potentials that are very distinct, their reactivity is different, and their oxidative action is likely to have different targets. We discuss how these properties are related with the oxidative potency of ROS, especially those of O2(.-) and H2O2.

DPPH and oxygen free radicals as pro-oxidant of biomolecules.

Numerous investigations exist about the alterations that oxygen free radicals can provoke on biomolecules; these modifications can be prevented and/or reversed by different antioxidants agents. On the other hand, 2,2-diphenyl-1-picrylhydrazyl radical (DPPH), a stable nitrogen synthetic radical, is used to evaluate the antioxidant capacity of medicinal herbal products; however, the structural changes that this radical provoke on the herbal active principles are not clear yet. In this work, we compared the redox reactivity of oxygen free radicals and DPPH radical on phospholipids and protein thiol groups present in rat liver microsomes. Cu2+/ascorbate was used as generator system of oxygen free radical and as antioxidant, an extract of Buddleja globosa's leaves. Cu2+/ascorbate provoked microsomal lipid peroxidation, microsomal thiols oxidation and oxygen consumption; all of these phenomena were inhibited by B. globosa extract. On the other hand, DPPH was bleached in different extension by the herbal extract and phosphatidyl choline; beside, DPPH decreased microsomal thiols content, but this phenomenon were not prevented by the herbal extract. Furthermore, DPPH did not induce oxygen consumption and neither modified the oxygen consumption induced by Cu2+/ascorbate. Distinct redox mechanisms may explain the differences between the reactivity of DPPH and oxygen free radicals on biomolecules, which is discussed.

Nitroaryl-1,4-dihydropyridines as antioxidants against rat liver microsomes oxidation induced by iron/ascorbate, nitrofurantoin and naphthalene.

1,4-Dihydropyridines (DHPs) used in the treatment of cardiovascular diseases, are calcium channel antagonists and also antioxidant agents. These drugs are metabolized through cytochrome P(450) oxidative system, majority localized in the hepatic endoplasmic reticulum. Several lipophilic drugs generate oxidative stress to be metabolized by this cellular system. Thus, DHP antioxidant properties may prevent the oxidative stress associated with hepatic biotransformation of drugs. In this work, we tested the antioxidant capacity of several synthetic nitro-phenyl-DHPs. These compounds (I-IV) inhibited the microsomal lipid peroxidation, UDPGT oxidative activation and microsomal thiols oxidation; all phenomena induced by Fe(3+)/ascorbate, a generator system of oxygen free radicals. As the same manner, these compounds inhibited the oxygen consumption induced by Cu(2+)/ascorbate in the absence of microsomes. Furthermore, compound III (2,6-dimethyl-4-(4-nitrophenyl)-1,4-dihydropyridin-3,5-ethyl-dicarboxylate) and compound V (N-ethyl-2,6-dimethyl-4-(4-nitrophenyl)-1,4-dihydropyridin-3,5-methyl-dicarboxylate) inhibited the microsomal lipid peroxidation induced by Nitrofurantoin and naphthalene in the presence of NADPH. Oxidative stress induced on endoplasmic reticulum may alter the biotransformation of drugs, so, modifying their plasmatic concentrations and therapeutic effects. When drugs which are activated by biotransformation are administered together with antioxidant drugs, such as DHPs, oxidative stress induced in situ may be prevented.

Copper modifies liver microsomal UDP-glucuronyltransferase activity through different and opposite mechanisms.

Treatment of hepatic microsomes with Fe(3+)/ascorbate activates UDP-glucuronyltransferase (UGT), a phenomenon totally prevented and reversed by reducing agents. At microM concentrations, iron and copper ions catalyze the formation of ROS through Fenton and/or Haber-Weiss reactions. Unlike iron ions, indiscriminate binding of copper ions to thiol groups of proteins different from the specialized copper-binding proteins may occur. Thus, we hypothesize that incubation of hepatic microsomes with the Cu(2+)/ascorbate system will lead to both UGT oxidative activation and Cu(2+)-binding induced inhibition, simultaneously. We studied the effects of Cu(2+) alone and in the presence of ascorbate on rat liver microsomal UGT activity. Our results show that the effects of both copper alone and in the presence of ascorbate were copper ion concentration- and incubation time-dependent. At very low Cu(2+) (25nM), this ion did not modify UGT activity. In the presence of ascorbate, however, UGT activity was increased. At higher copper concentrations (10 and 50microM), this ion led to UGT activity inhibition. In the presence of ascorbate, 10microM Cu(2+) activated UGT at short incubation periods but inhibited this enzyme at longer incubation times; 50microM Cu(2+) only inhibited UGT activity. Thiol reducing agent 2,4-dithiothreitol prevented and reversed UGT activation while EDTA prevented both, UGT activation and inhibition. Our results are consistent with a model in which Cu(2+)-induced oxidation of UGT leads to the activation of the enzyme, while Cu(2+)-binding leads to its inhibition. We discuss physiological and pathological implications of these findings.

Topical anti-inflammatory activity of 2alpha-hydroxy pentacyclic triterpene acids from the leaves of Ugni molinae.

Leaf extracts of Ugni molinae Turcz. are used in the Chilean cosmetic industry on the assumption that they have decongestant, regenerative, and anti-aging properties. A bioassay-guided fractionation of this plant material showed that some extracts have potent anti-inflammatory activities. Further fractionation led to the isolation and identification of betulinic acid, a mixture of ursolic and oleanolic acids, and the 2alpha-hydroxy derivatives alphitolic, asiatic, and corosolic acids. The latter three were evaluated in vivo in the mouse ear assay for their topical anti-inflammatory activity, inducing inflammation with either arachidonic acid (AA) or 12-O-tetradecanoylphorbol-13 acetate (TPA). Only corosolic acid was active in the AA assay, with similar potency to nimesulide, but all three triterpene acids inhibited TPA-induced inflammation with potencies comparable to that of indomethacin.

Microsomal UDP-glucuronyltransferase in rat liver: oxidative activation.

Activation of microsomal UDP-glucuronyltransferase (UDPGT) activity by treatment of hepatic microsomes with either detergents or Fe(3+)/ascorbate pro-oxidant system has been reported; however, definite mechanisms underlying these effects have not been clarified. In this work, we characterize Fe(3+)/ascorbate-induced activation of UDPGT activity prior to solubilization with Triton X-100 and after the oxidation process provoked the solubilization of the enzyme. We observed a time-dependent increase in UDPGT activity up to 20 min. incubation of the microsomes with Fe(3+)/ascorbate (3-times); after 20 min. incubation, however, we observed a time-dependent decrease in this activity to basal levels after 4 hr incubation. Treatment of microsomes with 0.1% Triton X-100 (5 min.) lead to a similar increase in UDPGT activity; higher detergent concentrations produced a dose-dependent decrease in this activity to basal levels with 1% Triton X-100. Interestingly, UDPGT activity was susceptible to activation only when associated to microsomal membranes and the loss of activation correlated with the solubilization of this activity. UDPGT activation by either Fe(3+)/ascorbate or Triton X-100 was correlated with an increase in p-nitrophenol apparent K(m) and V(max) values. This activation was prevented or reversed by the reducing agents glutathione, cysteine or dithiothreitol when it was induced by the Fe(3+)/ascorbate. Furthermore, the latter provoked a significant decrease in microsomal thiol content, effect not observed after treatment with Triton X-100. Our results suggest that the main mechanism responsible for Fe(3+)/ascorbate-induced UDPGT activation is likely to be the promotion of protein sulfhydryl oxidation; this mechanism appears to be different from detergent-induced UDPGT activation.

Possible mechanisms underlying copper-induced damage in biological membranes leading to cellular toxicity.

It is generally accepted that copper toxicity is a consequence of the generation of reactive oxygen species (ROS) by copper ions via Fenton or Haber-Weiss reactions. Copper ions display high affinity for thiol and amino groups occurring in proteins. Thus, specialized proteins containing clusters of these groups transport and store copper ions, hampering their potential toxicity. This mechanism, however, may be overwhelmed under copper overloading conditions, in which copper ions may bind to thiol groups occurring in proteins non-related to copper metabolism. In this study, we propose that indiscriminate copper binding may lead to damaging consequences to protein structure, modifying their biological functions. Therefore, we treated liver subcellular membrane fractions, including microsomes, with Cu2+ ions either alone or in the presence of ascorbate (Cu2+/ascorbate); we then assayed both copper-binding to membranes, and microsomal cytochrome P450 oxidative system and GSH-transferase activities. All assayed sub-cellular membrane fractions treated with Cu2+ alone displayed Cu2+-binding, which was significantly increased in the presence of Zn2+, Hg2+, Cd2+, Ag+1 and As3+. Treatment of microsomes with Cu2+ in the microM range decreased the microsomal thiol content; in the presence of ascorbate, Cu2+ added in the nM concentrations range induced a significant microsomal lipoperoxidation; noteworthy, increasing Cu2+ concentration to > or =50 microM led to non-detectable lipoperoxidation levels. On the other hand, microM Cu2+ led to the inhibition of the enzymatic activities tested to the same extent in either presence or absence of ascorbate. We discuss the possible significance of indiscriminate copper binding to thiol proteins as a possible mechanism underlying copper-induced toxicity.

Bases bioquímicas de la acción de drogas antichagásicas / Biochemical bases of the action of anti-chagasic

El avance en el conocimiento de la bioquímica de parásitos, que ha ocurrido en los años recientes, ha conducido al desarrollo de nuevas drogas y ha permitido entender el modo de acción de muchas de ellas. La acción de algunas drogas tripanosomicidas se debería a la generación de metabolitos que son radicales libres, incluyendo productos de reducción parcial del oxígeno. El T. cruzi es muy susceptible al daño celular producido por estos metabolitos , ya que las enzimas que destruyen las especies activadas de oxigeno en mamíferos tienen muy baja actividad o no existen en el parásito. Drogas en uso, como son el nifurtimox, el benznidazol y el cristal violeta actuarían generando radicales libres. Otro posible sitio de ataque quimioterapéutico en estudio es la biosíntesis del glutatión, el cual participa en la eliminación de radicales libres y en la conjugación y detoxicación de numerosas drogas. También se estudia la manera de interferir con la reducción del glutatión oxidado que en el T. cruzi, a diferencia del huésped, requiere del cofactor tripanotión. Drogas experimentales como el alopurinol y análogos de purinas basan su modo de acción en la incapacidad del parásito de sintetizar purinas de novo y en una relativa baja especificidad de la enzima succino-AMP sintetasa. La cadena respiratoria del parásito también presenta importantes diferencias con la del huésped