María Eugenia Letelier

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 effects of 1,4-dihydropyridine and nitroso aryl derivatives on the Fe+3/ascorbate-stimulated lipid peroxidation in rat brain slices

1. Lipid peroxidation in rat brain slices was induced by Fe+3/ascorbate. 2. Brain lipid peroxidation, as measured by malondialdehyde formation, was inhibited by all the tested nitro aryl 1,4-dihydropyridine derivatives over a wide range of concentrations. The time-course antioxidant effects of the most representative agents were assessed. On the basis of both time-course and IC50 experiments the tentative order of antioxidant activity on rat brain slices could be: nicardipine>nisoldipine> (R,S/S,R)-furnidipine > (R,R/S,S)-furnidipine>nitrendipine>nimodipine> nifedipine. 3. 1,4-Dihydropyridine derivatives that lack of a nitro group in the molecule (isradipine, amlodipine) also inhibited lipid peroxidation in rat brain slices but at higher concentrations than that of nitro-substituted derivatives. 4. All the tested nitroso aryl derivatives [2,6-dimethyl-4-(2-nitrosophenyl)-3,5-pyridinedicar. boxylic acid dimethyl ester (NTP), nitrosotoluene, nitrosobenzene] were more potent inhibitors of lipid peroxidation than were the parent nitro compounds. In conclusion, on the basis of the IC50 values determined, the rank order of antioxidant potency for these derivatives can be established as: ortho-nitrosotoluene>NTP>nitrosobenzene.

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.

t-Butyl-4-hydroxyanisole, a novel respiratory chain inhibitor: Effects on Trypanosoma cruzi epimastigotes

t-Butyl-4-hydroxyanisole, an antioxidant food additive, inhibited the growth of Trypanosoma cruzi by almost 100% at 0.5 mM concentration. This compound inhibited 70% of oxygen consumption of epimastigotes. The redox level of NAD(P) was shifted to a more reduced state and inversely the redox level ofcytochrome b changed to a more oxidized state. This hydroxyanisole thus is a new electron transport chain inhibitor. This compound and related ones, or the respiratory chain of T. cruzi, may be important in the design of antichagasic drugs.t‐Butyl‐4‐hydroxyanisole, an antioxidant food additive, inhibited the growth of Trypanosoma cruzi by almost 100% at 0.5 mM concentration. This compound inhibited 70% of oxygen consumption of epimastigotes. The redox level of NAD(P) was shifted to a more reduced state and inversely the redox level ofcytochrome b changed to a more oxidized state. This hydroxyanisole thus is a new electron transport chain inhibitor. This compound and related ones, or the respiratory chain of T. cruzi, may be important in the design of antichagasic drugs.

Some properties of the microsomal system metabolizing DDT in Triatoma infestans

Abstract 1. 1. Microsomes prepared from Triatoma infestans fifth-instar larvae metabolize DDT to a compound similar to Kelthane (metabolite No. 3), and to a derivative of apparently a phenolic nature (metabolite No. 2). 2. 2. The production of both metabolites requires either NADPH or NADH, but only the formation of metabolite No. 3 is nicotinamide—and magnesium— dependent. 3. 3. Optimum pH for the formation of metabolite No. 3 is 8·5, while it is 9·0 for metabolite No. 2. The K m values for each activity were also slightly different. 4. 4. The activity for each metabolite was increased by pretreatment with phenobarbital, but the induction kinetics was different in each case. 5. 5. These observations suggest that two rather than one microsomal enzyme are involved in DDT metabolism in T. infestans .

Role of glutathione in the susceptibility of Trypanosoma cruzi to drugs

1. Glutathione (G-SH) concentration, gamma-glutamyltranspeptidase and glutathione S-transferase activities were studied in several strains of T. cruzi epimastigotes. GSH varied from 1.04 mM for the LQ strain to 0.61 mM for the Tulahuen strain. 2. Cultures of the LQ strain presented more resistance to drugs than those of the Tulahuen. It was necessary a concentration of nifurtimox 4 times higher and one of benznidazole 10 times higher in order to inhibit approximately to 50% the growth of LQ strain cultures when compared with the Tulahuen strain. 3. Buthionine sulfoximine decreased the concentration of glutathione to about 50% in the LQ and Tulahuen strains and potentiated the toxicity of nifurtimox and benznidazole in T. cruzi epimastigote cultures. These results suggest that glutathione is an important factor in the resistance of T. cruzi to nifurtimox and benznidazole.

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.

Redox behaviour of nifuroxazide: generation of the one-electron reduction product.

The electrochemical properties of nifuroxazide have been investigated in aqueous and aqueous-DMF mixed solvents. In aqueous media, a single, irreversible four-electron reduction occurs to give the hydroxylamine derivative. In mixed media, a reversible one-electron reduction to form a nitro radical anion takes place. Cyclic voltammetric studies show that the anion radical product is stable, although the nitro radical anion intermediate shows a tendency to undergo further chemical reactions. A comparison with the voltammetric behaviour of other nitrofurans such as nifurtimox, nitrofurazone and furazolidone is made. The electrochemically-obtained parameters are correlated with the in vivo studies of oxygen consumption on Trypanosoma cruzi cell suspensions.

Acid and alkaline phosphatase activity in trypanosoma cruzi epimastigotes

Phosphatase activity in intact Trypanosoma cruzi epimastigotes has been demonstrated. After subcellular fractionation three activities were characterized: (a) a membrane-bound microsomal acid activity with an optimum pH of 4.0 and a Km of 1.2 mM, strongly inhibited by tartrate and fluoride; (b) a soluble cytosolic acid activity with an optimum pH of 5.5 and a Km of 0.95 mM, strongly inhibited by p-hydroxymercuribenzoate, EDTA and copper ions and activated by cyanide, manganese and magnesium ions; and (c) a soluble cytosolic alkaline activity with an optimum pH of 8.0 and a Km of 3.8 mM, inhibited by p-hydroxymercuribenzoate, fluoride, EDTA, and copper, calcium and zinc ions. This activity was increased by magnesium and manganese ions.

Mechanisms underlying the inhibition of the cytochrome P450 system by copper ions.

Copper toxicity has been associated to the capacity of free copper ions to catalyze the production of superoxide anion and hydroxyl radical, reactive species that modify the structure and/or function of biomolecules. In addition, nonspecific Cu2+‐binding to thiol enzymes, which modifies their catalytic activities, has been reported. Cytochrome P450 (CYP450) monooxygenase is a thiol protein that binds substrates in the first and limiting step of CYP450 system catalytic cycle, necessary for the metabolism of lipophilic xenobiotics. Therefore, copper ions have the potential to oxidize and bind to cysteinyl residues of this monooxygenase, altering the CYP450 system activity. To test this postulate, we studied the effect of Cu2+ alone and Cu2+/ascorbate in rat liver microsomes, to independently evaluate its nonspecific binding and its pro‐oxidant effects, respectively. We assessed these effects on the absorbance spectrum of the monooxygenase, as a measure of structural damage, and p‐nitroanisole O‐demethylating activity of CYP450 system, as a marker of functional impairment. Data obtained indicate that Cu2+ could both oxidize and bind to some amino acid residues of the CYP450 monooxygenase but not to its heme group. The differences observed between the effects of Cu2+ and Cu2+/ascorbate show that both mechanisms are involved in the catalytic activity inhibition of CYP450 system by copper ions. The significance of these findings on the pharmacokinetics and pharmacodynamics of drugs is discussed. Copyright