Prevention of benznidazole-induced prolonging effect on the pentobarbital sleeping time of rats using different thiol-containing compounds.

Benznidazole (BZ) is a nitroimidazolic chemotherapeutic agent employed against the acute and indeterminate phase of Chagas' disease, a tropical sickness afflicting more than twenty million people in Latin America. BZ has serious toxic side effects forcing people to stop treatment. These effects were attributed to the nitroreductive metabolic activation of BZ to a hydronitroxide radical or the hydroxylamine, which would covalently bind to cellular components. One of these deleterious effects is the prolongation on the pentobarbital sleeping time of rats. This results from the covalent binding of BZ reactive metabolites, arisen during its nitroreductive metabolism, to the phospholipid component of the mixed function oxidase which biotransform the barbiturate. In this study, the potential ability of different thiol containing drugs to trap BZ reactive metabolites and to prevent BZ effect on the pentobarbital sleeping time was tested. Our HPLC studies evidenced that cysteine, N-acetylcysteine, penicillamine and glutathione were able to trap BZ reactive metabolites in vitro to produce one or two adducts. Reduced lipoic acid instead, decreased the intensity of the nitroreductive process without leading to detectable adducts. The in vivo administration of the thiol drugs, at dosage regimes available in literature, was able to markedly prevent the BZ prolongation effect on the sleeping time. Whether these thiols might prevent other BZ toxic effects without harming its chemotherapeutic actions remains to be established.

Trypanocidal effect of SKF525A, proadifen, on different developmental forms of Trypanosoma cruzi.

SKF525A, an inhibitor and inducer of cytochrome P450, was tested on different developmental stages of Trypanosoma cruzi. Growth, motility and structure of epimastigotes, motility and infectivity of trypomastigotes, and infectivity of trypomastigotes to Vero cells in culture were abolished by the drug at 10-100 microM concentrations. When blood from infected mice was treated with the drug, and used to infect 8 day-old mice, no parasites were observed at 0.6-1 mM, and all animals survived. Blood cell morphology was well preserved, and the sleeping time of pentobarbital-treated mice inoculated with the same amount of drug was not increased. The present results suggest that SKF525A or other related inhibitors of cytochrome P450 coned be tested as an additive for blood sterilization in blood banks.

Late protective effects of the anticalmodulin drug fluphenazine on carbon tetrachloride-induced liver necrosis.

Fluphenazine (FP) treatment (50 mg/kg bw, ip in saline) 30 min before or 6 or 10 h after CCl4 administration (1 ml/kg ip in olive oil) significantly prevented the liver necrosis produced by the hepatotoxin at 24 h. FP had enhancing effects on the covalent binding of CCl4 reactive metabolites to cellular constituents and on CCl4 induced lipid peroxidation. FP lowered body temperature of the CCl4-poisoned animals during the 24 h observation period. The obtained results are compatible but do not prove the hypothesis that calmodulin (CaM) had participation in late occurring events preceding necrosis. FP lowering action on body temperature, however, might also play a role in the effects of this drug on the onset of CCl4 induced liver necrosis. FP levels in liver tissue as determined by gas chromatography-mass spectrometry evidenced the presence of the drug in amounts sufficient to inhibit CaM and that suggests that not all preventive effects of FP are due to its indirect actions on the central nervous system via decreased body temperature.

Trifluopromazine late preventive effects on carbon tetrachloride-induced liver necrosis.

Trifluopromazine (TFPro) administration to rats (50 mg/kg, ip) 30 min before or 6 or 10 hr after CCl4 treatment (1 ml/kg ip in olive oil) partially prevented necrogenic effects of this compound at 24 hr. TFPro has only minor effects on the covalent binding (CB) of CCl4-reactive metabolites to cellular constituents and even an enhancing action on CCl4-promoted lipid peroxidation (LP). Determination of TFPro levels in liver 1 and 3 hr after administration by gas chromatography/mass spectrometry showed its presence in that tissue at concentrations well above those needed for calmodulin (CaM) inhibitory effects of this drug. TFPro lowered body temperature in CCl4-treated animals during the 24-hr observation period. Protective effects of TFPro at 6 or 10 hr, when most of the CB and all of the LP has already occurred, suggest but do not prove a role for CaM in late stages of CCl4-induced necrogenic effects. Decreases in the body temperature of CCl4-poisoned animals provoked by TFPro might also play a role in the preventive actions of this drug.

Nicotinamide late protective effects against carbon tetrachloride-induced liver necrosis.

Nicotinamide (NIC) is known to increase the synthesis of pyridine nucleotides and also to inhibit the hydrolysis of them to ADP-ribose, which in turn is involved in Ca2+ release from mitochondria via the ADP ribosylation of crucial mitochondrial proteins. In this work, we test the potential ability of NIC to be a late protective agent against CCl4-induced liver necrosis. We observed that 1 g/kg po NIC, 30 min before or 6 or 10 hr after CCl4 (1 ml/kg), given ip as a 20% (v/v) solution in olive oil, was able to significantly prevent the necrogenic effect of the hepatotoxin at 24 hr as evidenced by determination of isocitric dehydrogenase activity in plasma or by histological observation. NIC administration 6 hr after CCl4 prevented fatty liver induced by hepatotoxin at 24 hr. NIC did not modify CCl4-induced lipid peroxidation process at 1 hr after CCl4 and decreased the covalent binding of 14CCl4 to lipids. NIC decreased the levels of 14CCl4 reaching the liver when given 30 min before hepatotoxin but not when given 6 hr after it. NIC lowered body temperature of rats at 1, 3, and 6 hr and augmented it at 24 hr after CCl4. NIC concentrations in liver as determined by GC/MS/SIM analysis were 21 micrograms/g liver 1 hr after administration and 53 micrograms/g at 3 hr. Late preventive effects of NIC against CCl4 induced liver necrosis when given at 6 or 10 hr after CCl4 are compatible with the hypothesis that NIC restores mitochondrial ability for Ca2+ uptake. This hypothesis remains to be proved and is being further challenged in our laboratory.

Prevention of CCl4-induced liver necrosis by the calcium chelator arsenazo III.

Arsenazo III (AIII) (100 mg/kg ip in saline) administration to Sprague-Dawley male rats 30 min before or 6 or 10 hr after CCl4 [1 ml/kg ip as a 20% (v/v) solution in olive oil] significantly prevented liver necrosis but not fatty liver caused by the hepatotoxin at 24 hr as demonstrated either by histology or by determination of isocitric acid dehydrogenase in plasma. AIII did not modify the CCl4 concentrations reaching the liver, the intensity of the covalent binding of CCl4-reactive metabolites to hepatic microsomal lipids, or the CCl4-promoted lipid peroxidation process at either 1 or 3 hr of poisoning. AIII administration enhanced glutathione (GSH) levels in liver and significantly prevented the CCl4-induced minor decreases in GSH content and the CCl4-induced increases in calcium content at 24 hr of intoxication. AIII treatment further enhanced the CCl4-induced decreases in body temperature of the poisoned rats. Results suggest that AIII's preventive effects might be related to its very well-known calcium-chelating properties, but that additional factors related to AIII's ability to increase GSH content in liver or to decrease body temperature of CCl4-intoxicated animals may also play a role.

Ultrastructural alterations in testes from rats treated with cysteine.

Cysteine administration in relatively large doses has been repetitively employed as preventive agent against chemically induced cell injury or as radioprotector. In this work we report that administration of a dose standard for those purposes (1.9 gr/kg, po in water) causes significant ultrastructurally evident alterations in testes at 24h. Damage involves Sertoli cells and spermatids. Alterations found in the former include dilatation of nuclear membrane and of the smooth (SER) and rough endoplasmic reticulum (RER) and detachment of ribosomes from RER. Cytoplasm appeared more sparse and electron lucent than in controls and contained more lipid droplets and lysosomes. Mitochondria exhibited alterations in shape and size. Damage in spermatids consisted of the appearance of irregular shape of the nucleus and alterations in their acrosomal caps. There was no histochemical evidence for either calcium accumulation or lipid peroxidation occurrence in testes of cysteine-treated animals. Results indicate that the large doses of cysteine employed in prevention of radiation or chemical effects is able to cause injury to Sertoli cells of the testes. Damage observed does not reach irreversible stages but may be sufficient to lead to production of abnormal spermatids.

Prevention of carbon tetrachloride-induced liver necrosis by the chelator alizarin sodium sulfonate.

The administration of the calcium chelator alizarin sodium sulfonate (ASR) (100 mg/kg ip in saline) 30 min before or 6 or 10 hr after CCl4 (1 ml/kg ip as a 20% v/v solution in olive oil) partially prevents the necrogenic effect of the hepatotoxin at 24 hr, but prevention of CCl4 fat accumulation was not observed. Protective action cannot be attributed to potential decreasing effects of ASR on CCl4 levels reaching the liver, on the covalent binding of CCl4-reactive metabolites to cellular components, or on CCl4-induced lipid peroxidation because ASR does not modify these parameters significantly. ASR administration increases GSH levels in livers of both control and CCl4-poisoned animals and decreases the calcium content of intoxicated animals at 24 hr of poisoning. ASR significantly lowers the body temperature of CCl4-treated animals at different times of the intoxication process. Present and previous results from our laboratory on the preventive effects of another very specific calcium chelator, calcion, and several anticalmodulins suggest that the beneficial effects of ASR might be associated with its calcium chelating ability. Other protective effects of ASR, such as lowering body temperature or increasing GSH content in liver, cannot be excluded.

Reductive biotransformation of xenobiotics by the sheep ruminal content.

1. Sheep ruminal content was able to reduce nitro groups from nitrobenzene and azo groups from dimethylamino-azobenzene. 2. Results might be of interest in relation to ruminants exposed to environmental chemicals via oral route. 3. Biotransformation of xenobiotics in rumen might give to deleterious products appearing later in meat and/or milk or harming the ruminant itself.

Disruption of mitochondrial function as the basis of the trypanocidal effect of trifluoperazine on Trypanosoma cruzi.

The tricyclic anti-calmodulin drug trifluoperazine (TFP) inhibited growth and motility of epimastigotes of Trypanosoma cruzi, at concentrations lower than 100 microM, and motility and infectivity of the bloodstream trypomastigote form at 200 microM. Electron microscopy of TFP-treated epimastigotes showed that the major effect was at the mitochondrial level, with gross swelling and disorganization. The oligomycin-sensitive, mitochondrial ATPase was completely inhibited by 20 microM TFP, and the same drug concentration caused a 60% decrease in intracellular ATP content. The results suggest that the trypanocidal effect of TFP may be related more to mitochondrial damage than to the well-known anticalmodulin effect of the drug.

Ultrastructural alterations in ovaries from nifurtimox or benznidazole-treated rats: their relation to ovarian nitroreductive biotransformation of both drugs.

Chagas' disease is a parasitic chronic condition affecting several million people in Latin America. Two drugs are used in the chemotherapy of Chagas' disease: nifurtimox (Nfx) and benznidazole (Bz). Both are nitroderivatives whose deleterious effects are related to their reductive biotransformation. In this work we report that rat ovaries exhibited Bz and Nfx nitroreductase activity. The Bz nitroreductase was only found in the mitochondrial fraction and was partially inhibited by CO. The Nfx nitroreductase activity was maximal in ovarian mitochondria but was also present in microsomes and in the cytosol. The microsomal enzyme was completely inhibited by CO while that in mitochondria was only partially inhibited by CO. The cytosolic activity only proceeded using hypoxanthine as substrate and was inhibited by allopurinol. The cytosolic activity was able to proceed in part under oxygen. All the other Bz or Nfx nitroreductases were completely inhibited by atmospheric oxygen. The potential participation of cytochrome P450, flavoenzymes, iron-sulfur-protein, and xanthinooxidase in both nitroreductive processes is discussed. The administration of either Nfx or Bz to female rats produced ultrastructural degenerative effects in the different cell types of ovaries. Specific alterations such as swelling, disruption, disorganization, and loss of matrix components were observed in ovarian mitochondria. These alterations occurred irrespectively of the ovarian cycle stage. The potential reproductive toxicological consequences of Bz or Nfx administration are analyzed.

Further studies on the mechanism of the late protective effects of phenylmethylsulfonyl fluoride on carbon tetrachloride-induced liver necrosis.

We previously reported that phenylmethylsulfonyl fluoride (PMSF) administration to rats (100 mg/kg, ip in olive oil) as late as 6 or 10 hr after CCl4 (1 ml/kg, ip as a 20% v/v solution in olive oil) can partially prevent the necrogenic response to the hepatotoxin at 24 hr. Here we confirm that observation by electron microscopy and provide further evidence that only in these circumstances were nuclear clumping of chromatin, slight dilatation of the endoplasmic reticulum, myelin figures and lipid droplets in the cytoplasm, large numbers of lysosomes and peroxisomes, glycogen, and slightly swollen mitochondria observable in the protected animals. A very minor part of the late protective effects of PMSF might be due to the effects of this drug on decreasing the intensity of covalent binding of CCl4-reactive metabolites or the intensity of CCl4-induced lipid peroxidation still occurring 6 or 10 hr after CCl4. PMSF administration did not prevent CCl4-induced decreases in cytochrome P450 content or glucose-6-phosphatase activity but partially prevented CCl4-induced calcium accumulation in liver. PMSF treatment increased glutathione and glycogen content in CCl4-poisoned animals, but did not markedly modify protein/phospholipid synthesis or degradation processes. Results suggest that the late protective effects of PMSF administration in CCl4-induced liver necrosis might be due to a favorable modulation of the calcium-calmodulin system similar to that previously described for other drugs.

Carbon tetrachloride-induced liver cell injury in the Mongolian gerbil (Meriones unguiculatus).

1. Male Mongolian gerbils (Meriones unguiculatus) liver activates CCl4 to free radicals that bind covalently to cellular components (CB) and stimulate a lipid peroxidation (LP) process to a larger extent than the rat liver. 2. CCl4 administration results in a less intense necrogenic effect in gerbils than in rats and does not cause fatty liver. 3. CCl4 causes less intense effects on liver ultrastructure or calcium metabolism but more marked depression of glucose 6 phosphatase activity (G6P-ase) in gerbils than in rats. 4. Results suggest that a better ability of gerbil liver to keep calcium homeostasis than rat liver might be the cause of their relative resistance to necrosis. Higher intensity of CB and LP in gerbils than in rats might explain more intense effects on G6P-ase.

Late preventive effects against carbon tetrachloride-induced liver necrosis of the calcium chelating agent Calcion.

In agreement with the hypothesis that changes in calcium homeostasis might be significant in late stages of chemically-induced liver cell injury, a calcium chelating agent, Calcion, was able to partially prevent CCl4-induced liver necrosis observed at 24 h, when treatment was given as late as 6 or 10 h after the hepatotoxin. Calcion had minor or no effects on covalent binding of reactive metabolites to cellular components, or on lipid peroxidation or on CCl4 levels reaching the liver. Calcion treatment of CCl4-poisoned animals decreased CCl4-induced calcium increases in liver and increased glutathione levels decreased by hepatotoxin at 24 h. Calcion treatment was not able to prevent CCl4-induced fatty liver. Calcion protective effects were body temperature dependent but they were cancelled when Calcion-treated poisoned animals were kept normothermic. Results suggest that Calcion protective effects might be linked to calcium chelation or alternatively that they might derive from decreases in body temperature.

Further studies on the late preventive effects of the anticalmodulin trifluoperazine on carbon tetrachloride-induced liver necrosis.

Trifluoperazine (TFP) (50 mg/kg ip) administration to rats 6 or 10 hr after CCl4 (1 ml/kg ip in olive oil) significantly prevented liver necrosis but not fatty liver caused by the hepatotoxin at 24 hr as evidenced by either histology or electron microscopy. TFP given 6 hr after CCl4 significantly decreased the CCl4-induced increases in liver calcium content. TFP raised four to five times the liver glycogen content in control rats but was unable to modify decreased glycogen content of CCl4 poisoned animals. TFP administration increased phospholipid and protein synthesis as evidenced by studies on 32P incorporation into microsomal phospholipid and by experiments on [14C]leucine incorporation in microsomal protein fractions from control rat livers. No significant changes were observed in microsomal phospholipid degradation as studied by decay of label from 32P-prelabeled microsomal lipids or in increased protein degradation as evidenced by decay of label from [14C-guanidino]arginine-prelabeled microsomal proteins found in livers of control rats after TFP treatment. Electron microscopy observations of liver from control animals treated with TFP evidenced accumulation of glycogen in areas close to smooth endoplasmic reticulum (SER); large Golgi areas with an abundant number of lysosomes, and minor dilatation effects on the rough endoplasmic reticulum (RER) and nuclear membrane. Results suggest that TFP preventive effects might be due to the anticalmodulin actions of this drug.

Glutathione (GSH) content in livers from control and carbon tetrachloride poisoned rats treated with the calmodulin inhibitors thioridazine, imipramine or chlorpromazine.

Administration to rats of the calmodulin (CaM) inhibitors Thioridazine (TDZ) or Imipramine (IMP) (50 mg/kg ip) or Chlorpromazine (CPZ) at a dose of 25 mg/kg, ip 6 hr after olive oil or CCl4 (1 ml/kg, ip as a 20% olive oil solution) significantly increased the GSH content in liver of CCl4 poisoned animals but not in controls. The analysis of present observations and past results with Trifluoperazine, suggest that increases in GSH content in CCl4 poisoned animals treated with protective anticalmodulins are a consequence of prevention and not the cause of their preventive effects.

Effects of carbon tetrachloride on the liver of chickens. Early biochemical and ultrastructural alterations in the absence of detectable lipid peroxidation.

Administration of CCl4 i.p. to Leghorn chickens did not promote lipid peroxidation of liver microsomal lipids, as evidenced by either increased diene conjugation or by decreased arachidonic acid content. The hepatotoxin did not produce liver necrosis 24 h after dosing, but decreased the cytochrome P-450 content, and aminopyrine N-demethylase and glucose 6 phosphatase activities at 1, 3, 6 and 24 h. CCl4 administration produced dilation of the rough endoplasmic reticulum and detachment of ribosomes from their membranes. These observations suggest that lipid peroxidation is not the key event in the production of these biochemical and ultrastructural alterations, elicited by CCl4.

Effects of nifurtimox or benznidazole administration on rat testes: ultrastructural observations and biochemical studies.

Ultrastructural and biochemical alterations in testes of Sprague-Dawley rats receiving either nifurtimox (Nfx) or benznidazole (Bz) (both 100 mg/kg po) were studied. Nfx produced intense deleterious effects on Steroli cells consisting of dilatation of endoplasmic reticulum and perinuclear membrane, alterations in shape and size of mitochondria, increased lysosomal activity, detachment of ribosomes, and alterations in shape and configuration of spermatids and mature spermatozoa. Bz induced alterations were similar in nature but far less intense and observable only in some cells or preparations or animals but not in others. Testicular Nfx but not Bz nitroreductase activity was detected in microsomal and cytosolic fractions. Microsomal Nfx nitroreductase activity was not inhibited by CO. The cytosolic activity in the presence of hypoxanthine was inhibited by allopurinol and that in the presence of N-methylnicotinamide was inhibited by menadione. All these enzymatic activities were inhibitable by oxygen except the cytosolic one in the presence of N-methylnicotinamide. No evidence for lipid peroxidation was found in testes from Nfx treated animals. Covalent binding of Bz reductive metabolites to testicular proteins and phospholipids was found. Toxicological and pharmacological implications for patients suffering Chagas' disease and receiving these drugs were analyzed.

Increased glutathione (GSH) content in livers of control and carbon tetrachloride poisoned rats treated with the anticalmodulin drug trifluoperazine (TFP).

TFP administration to rats (50 mg/kg, ip) 6 hr after olive oil or CCl4 (1 mg/kg, ip as a 20% olive oil solution) significantly increases the GSH content in liver at 24 hr. The role of GSH increases in the late preventive effects against CCl4 hepatotoxicity is discussed. A link between the calcium/calmodulin (CaM) system and the GSH status in liver is postulated.

Carbon tetrachloride activation by highly purified liver mitochondrial preparations.

Highly purified rat liver mitochondrial preparations were found to be able to activate CCl4 to reactive metabolites that bind covalently to lipids. Part of the process is of an enzymatic nature, but most of it is non-enzymatic. The enzymatic mitochondrial CCl4 activation operates more efficiently under anaerobic conditions; it requires NADPH, is CO sensitive, is inducible by phenobarbital pretreatment and is only weakly inhibited by high concentrations of cyanide or azide. The non-enzymatic mitochondrial CCl4 activation is not inhibited by CO and proceeds equally well under air or nitrogen.