Chagas disease: Present status of pathogenic mechanisms and chemotherapy.

There are approximately 7.8 million people in Latin America, including Chile, who suffer from Chagas disease and another 28 million who are at risk of contracting it. Chagas is caused by the flagellate protozoan Trypanosoma cruzi. It is a chronic disease, where 20%-30% of infected individuals develop severe cardiopathy, with heart failure and potentially fatal arrhythmias. Currently, Chagas disease treatment is more effective in the acute phase, but does not always produce complete parasite eradication during indeterminate and chronic phases. At present, only nifurtimox or benznidazole have been proven to be superior to new drugs being tested. Therefore, it is necessary to find alternative approaches to treatment of chronic Chagas. The current treatment may be rendered more effective by increasing the activity of anti-Chagasic drugs or by modifying the host's immune response. We have previously shown that glutathione synthesis inhibition increases nifurtimox and benznidazole activity. In addition, there is increasing evidence that cyclooxygenase inhibitors present an important effect on T. cruzi infection. Therefore, we found that aspirin reduced the intracellular infection in RAW 264.7 cells and, decreased myocarditis extension and mortality rates in mice. However, the long-term benefit of prostaglandin inhibition for Chagasic patients is still unknown.

Trypanosoma cruzi: In vitro effect of aspirin with nifurtimox and benznidazole.

Nifurtimox and benznidazole are the only active drugs against Trypanosoma cruzi; however, they have limited efficacy and severe side effects. During primoinfection, T. cruzi infected macrophages mount an antiparasitic response, which the parasite evades through an increase of tumor growth factor beta and PGE(2) activation as well as decreased iNOS activity. Thus, prostaglandin synthesis inhibition with aspirin might increase macrophage antiparasitic activity and increase nifurtimox and benznidazole effect. Aspirin alone demonstrated a low effect upon macrophage antiparasitic activity. However, isobolographic analysis of the combined effects of aspirin, nifurtimox and benznidazole indicated a synergistic effect on T. cruzi infection of RAW cells, with combinatory indexes of 0.71 and 0.61, respectively. The observed effect of aspirin upon T. cruzi infection was not related with the PGE(2) synthesis inhibition. Nevertheless, NO() levels were restored by aspirin in T. cruzi-infected RAW cells, contributing to macrophage antiparasitic activity improvement. Thus, the synergy of aspirin with nifurtimox and benznidazole is due to the capability of aspirin to increase antiparasitic activity of macrophages.

Chagas disease: Present status of pathogenic mechanisms and chemotherapy

There are approximately 7.8 million people in Latin America, including Chile, who suffer from Chagas disease and another 28 million who are at risk of contracting it. Chagas is caused by the flagellate protozoan Trypanosoma cruzi. It is a chronic disease, where 20 percent-30 percent of infected individuals develop severe cardiopathy, with heart failure and potentially fatal arrhythmias. Currently, Chagas disease treatment is more effective in the acute phase, but does not always produce complete parasite eradication during indeterminate and chronic phases. At present, only nifurtimox or benznidazole have been proven to be superior to new drugs being tested. Therefore, it is necessary to find alternative approaches to treatment of chronic Chagas. The current treatment may be rendered more effective by increasing the activity of anti-Chagasic drugs or by modifying the host's immune response. We have previously shown that glutathione synthesis inhibition increases nifurtimox and benznidazole activity. In addition, there is increasing evidence that cyclooxygenase inhibitors present an important effect on T. cruzi infection. Therefore, we found that aspirin reduced the intracellular infection in RAW 264.7 cells and, decreased myocarditis extension and mortality rates in mice. However, the long-term benefit of prostaglandin inhibition for Chagasic patients is still unknown.

Study of cytochrome P450 2E1 and its allele variants in liver injury of nondiabetic, nonalcoholic steatohepatitis obese women.

CYP2E1 enzyme is related to nonalcoholic steatohepatitis (NASH) due to its ability for reactive oxygen species production, which can be influenced by polymorphisms in the gene. The aim of this study was to investigate hepatic levels, activity, and polymorphisms of the CYP2E1 gene to correlate it with clinical and histological features in 48 female obese NASH patients. Subjects were divided into three groups: (i) normal; (ii) steatosis; and (iii) steatohepatitis. CYP2E1 protein level was assayed in microsomes from liver biopsies, and in vivo chlorzoxazone hydroxylation was determined by HPLC. Genomic DNA was isolated for genotype analysis through PCR. The results showed that liver CYP2E1 content was significantly higher in the steatohepatitis (45%; p=0.024) and steatosis (22%; p=0.032) group compared with normal group. Chlorzoxazone hydroxylase activity showed significant enhancement in the steatohepatitis group (15%, p=0.027) compared with the normal group. c2 rare allele of RsallPstl polymorphisms but no C allele of Dral polymorphism was positively associated with CHZ hydroxylation, which in turn is correlated with liver CYP2E1 content (r=0.59; p=0.026). In conclusion, c2 allele is positively associated with liver injury in NASH. This allele may determine a higher transcriptional activity of the gene, with consequent enhancement in pro-oxidant activity of CYP2E1 thus affording liver toxicity.

Buthionine sulfoximine has anti-Trypanosoma cruzi activity in a murine model of acute Chagas' disease and enhances the efficacy of nifurtimox.

L-buthionine (S,R)-sulfoximine (BSO) at a dose of 220 mg/kg of body weight/day showed an anti-Trypanosoma cruzi effect in infected mice, increasing their survival rate and decreasing the parasitemias and parasite burden in the hearts. Treatment with BSO plus nifurtimox caused an increase in the survival rate in comparison to the rates with treatment with each drug alone.

Trypanosoma cruzi: activities of lapachol and alpha- and beta-lapachone derivatives against epimastigote and trypomastigote forms.

Derivatives of natural quinones with biological activities, such as lapachol, alpha- and beta-lapachones, have been synthesized and their trypanocidal activity evaluated in vitro in Trypanosoma cruzi cells. All tested compounds inhibited epimastigote growth and trypomastigote viability. Several compounds showed similar or higher activity as compared with current trypanocidal drugs, nifurtimox and benznidazole. The results presented here show that the anti-T. cruzi activity of the alpha-lapachone derivatives can be increased by the replacement of the benzene ring by a pyridine moiety. Free radical production and consequently oxidative stress through redox cycling or production of electrophilic metabolites are the potential biological mechanism of action for these synthetic quinones.

Study of Cytochrome P450 2E1 and its allele Variants in Liver Injury of Nondiabetic, Nonalcoholic Steatohepatitis Obese Women

CYP2E1 enzyme is related to nonalcoholic steatohepatitis (NASH) due to its ability for reactive oxygen species production, which can be influenced by polymorphisms in the gene. The aim of this study was to investigate hepatic levels, activity, and polymorphisms of the CYP2E1 gene to correlate it with clinical and histological features in 48 female obese NASH patients. Subjects were divided into three groups: (i) normal; (ii) steatosis; and (iii) steatohepatitis. CYP2E1 protein level was assayed in microsomes from liver biopsies, and in vivo chlorzoxazone hydroxylation was determined by HPLC. Genomic DNA was isolated for genotype analysis through PCR. The results showed that liver CYP2E1 content was significantly higher in the steatohepatitis (45 percent; p=0.024) and steatosis (22 percent; p=0.032) group compared with normal group. Chlorzoxazone hydroxylase activity showed significant enhancement in the steatohepatitis group (15 percent, p=0.027) compared with the normal group. c2 rare allele of RsallPstl polymorphisms but no C allele of Dral polymorphism was positively associated with CHZ hydroxylation, which in turn is correlated with liver CYP2E1 content (r=0.59; p=0.026). In conclusion, c2 allele is positively associated with liver injury in NASH. This allele may determine a higher transcriptional activity of the gene, with consequent enhancement in pro-oxidant activity of CYP2E1 thus affording liver toxicity.

Relationship between in vivo chlorzoxazone hydroxylation, hepatic cytochrome P450 2E1 content and liver injury in obese non-alcoholic fatty liver disease patients.

The aim of the present study was to test the hypothesis that induction of cytochrome P450 2E1 (CYP2E1) in the liver of patients with non-alcoholic fatty liver disease (NAFLD) is correlated both with the in vivo activity of the cytochrome and with the development of liver injury. For this purpose, the liver content of CYP2E1 was determined by Western blot and the CYP2E1 activity by the in vivo hydroxylation of chlorzoxazone (CLZ). The study groups were obese women with an average body mass index (BMI) of 40.3kg/m(2), who underwent therapeutic gastroplasty or gastrectomy with a gastro-jejunal anastomosis. Further, the hepatic histology was determined to establish the pathological score grouping the subjects into three categories: control, steatosis and steatohepatitis. The liver CYP2E1 content and the CLZ hydroxylation of obese patients with steatosis and, particularly, with steatohepatitis were significantly higher than controls and correlated positively with both the severity of the liver damage. These data provide evidence that CYP2E1 would be involved in the mechanism of liver injury found in obese NAFLD patients. Also, the correlation between liver CYP2E1 content and in vivo CLZ hydroxylation would validate the latter as a reliable indicator of liver injury in NAFLD, thus providing a simple and not invasive method to study these patients.

Redox up-regulated expression of rat liver manganese superoxide dismutase and Bcl-2 by thyroid hormone is associated with inhibitor of kappaB-alpha phosphorylation and nuclear factor-kappaB activation.

Recently, we demonstrated that 3,3',5-triiodothyronine (T3) induces oxidative stress in rat liver, with enhancement in the DNA binding of nuclear factor-kappaB (NF-kappaB) and the NF-kappaB-dependent expression of tumor necrosis factor-alpha (TNF-alpha). In this study, we show that T3 administration (daily doses of 0.1 mg/kg i.p. for three consecutive days) elicited a calorigenic response and higher liver O2 consumption rates, with increased serum levels of TNF-alpha (ELISA), liver inhibitor of kappaB (IkappaB-alpha) phosphorylation (Western blot analysis), and hepatic NF-kappaB DNA binding (EMSA) at 56-72 h after treatment. Within this time interval, liver manganese superoxide dismutase (MnSOD) activity and the protein expression of MnSOD and Bcl-2 are enhanced. These changes are abrogated by the administration of alpha-tocopherol (100 mg/kg i.p.) prior to T3. It is concluded that T3 treatment leads to the redox upregulation of MnSOD and Bcl-2 in rat liver, in association with TNF-alpha release and activation of the IkappaB-alpha kinase/NF-kappaB cascade, which may constitute a protective mechanism against free radical toxicity involving cell death signaling.

[Cytochrome P450 activity and its alteration in different diseases]. / Actividad del citocromo P450 y su alteración en diversas patologías.

Cytochrome P450 (CYP) enzymes participate in the metabolism of a variety of naturally occurring and foreign compounds by reactions requiring NADPH and O2. The diversity of reactions catalyzed and its extensive substrate specificity render CYP enzymes as one of the most versatile known catalysts. Individual members of the CYP superfamily are expressed in almost every cell type in the body. As compared to hepatic enzymes, the regulation of human extrahepatic CYPs has not been so well studied. In general, the levels of some hepatic CYP enzymes are depressed by diseases, causing potential and documented impairment of drug clearence and clinical drug toxicity. However, modulation of CYPs is enzyme selective and this selectivity differs in different diseases. This article reviews some basic concepts about CYP and its regulation in some disease states such as hypertension, diabetes, obesity and hepatic, infectious and inflammatory diseases.

Increase in long-chain polyunsaturated fatty acid n - 6/n - 3 ratio in relation to hepatic steatosis in patients with non-alcoholic fatty liver disease.

Hepatic steatosis is a major feature associated with NAFLD (non-alcoholic fatty liver disease). The aims of the present study were to assess the levels of PUFA (polyunsaturated fatty acids) in liver total lipids, triacylglycerols (triglycerides) and phospholipids of NAFLD patients in relation to those in adipose tissue and hepatic indexes related to oxidative stress as factors contributing to hepatic steatosis. Eleven control subjects and 19 patients with NAFLD were studied. Analysis of liver and abdominal adipose tissue fatty acids was carried out by GLC. The liver content of protein carbonyl groups and malondialdehyde were taken as indexes related to oxidative stress. NAFLD patients had a depletion in LCPUFA (long-chain PUFA) of the n -6 and n -3 series in liver triacylglycerols, with decreased 20:4, n -6/18:2, n -6 and (20:5, n -3+22:6, n -3)/18:3, n -3 ratios, whereas liver phospholipids contained higher n -6 and lower n -3 LCPUFA. These findings were accompanied by an enhancement of (i) n -6/ n -3 ratio in liver and adipose tissue, (ii) 18:1, n -9 trans levels in adipose tissue, and (iii) hepatic lipid peroxidation and protein oxidation indexes. It is concluded that a marked enhancement in LCPUFA n -6/ n -3 ratio occurs in the liver of NAFLD patients, a condition that may favour lipid synthesis over oxidation and secretion, thereby leading to steatosis. Depletion of hepatic LCPUFA may result from both defective desaturation of PUFA, due to inadequate intake of precursors, such as 18:3, n -3, and higher intake of the 18:1, n -9 trans isomer leading to desaturase inhibition, and from an increased peroxidation of LCPUFA due to oxidative stress.

Oxidative stress-related parameters in the liver of non-alcoholic fatty liver disease patients.

Oxidative stress is implicated in the pathogenesis of non-alcoholic fatty liver disease (NAFLD). In the present study, hepatic and plasma oxidative stress-related parameters were measured and correlated with clinical and histological findings in 31 NAFLD patients showing increased body mass index. Liver protein carbonyl content was enhanced by 403% in patients with steatosis (n=15) compared with control values (n=12), whereas glutathione content, superoxide dismutase (SOD) activity and the ferric reducing ability of plasma (FRAP) were decreased by 57%, 48% and 21% (P<0.05) respectively. No changes in microsomal p-nitrophenol hydroxylation and the total content of cytochrome P450 (CYP) or CYP2E1 were observed. Patients with steatohepatitis (n=16) exhibited protein carbonyl content comparable with that of controls, whereas glutathione content, SOD and catalase activities were decreased by 27%, 64% and 48% (P<0.05). In addition, FRAP values in patients with steatohepatitis were reduced by 33% and 15% (P<0.05) when compared with controls and patients with steatosis respectively, whereas p-nitrophenol hydroxylation (52%) and CYP2E1 content (142%) were significantly increased (P<0.05) compared with controls. It is concluded that oxidative stress is developed in the liver of NAFLD patients with steatosis and is exacerbated further in patients with steatohepatitis, which is associated with CYP2E1 induction. Substantial protein oxidation is followed by proteolysis of the modified proteins, which may explain the co-existence of a diminished antioxidant capacity and protein oxidation in the liver of patients with steatohepatitis.

Homocysteine and essential hypertension.

The authors examine the available clinical and experimental data supporting the view that homocysteine, an alternative risk factor of cardiovascular disease, may play a role in the pathogenesis of essential hypertension. The mechanism of this disease has not been elucidated, but it may be related to impairment of vascular endothelial and smooth muscle cell function. Therefore, the occurrence of endothelial dysfunction could contribute to alterations of the endothelium-dependent vasomotor regulation. Elevated homocysteinemia diminishes the vasodilation by nitric oxide, increases oxidative stress, stimulates the proliferation of vascular smooth muscle cells, and alters the elastic properties of the vascular wall. Thus, homocysteine contributes to elevate the blood pressure. Also it is known that elevated plasma levels of homocysteine could lead to oxidant injury to the endothelium. The correction of elevated homocysteinemia by administration of vitamins B12 and B6 plus folic acid, could be a useful adjuvant therapy of hypertension. However, further controlled randomized trials are necessary to establish the efficacy and tolerability of these potentially therapeutic agents.

Implications of oxidative stress and homocysteine in the pathophysiology of essential hypertension.

The present review examines the clinical and experimental data to support the view that homocysteine and oxidative stress, two alternative risk factors of vascular disease, may play a role in the pathogenesis of primary or essential hypertension. Although the precise mechanism of this disease has not been elucidated, it may be related to impairment of vascular endothelial and smooth muscle cell function. Thus, the occurrence of endothelial dysfunction could contribute to alterations of the endothelium-dependent vasomotor regulation. Hyperhomocysteinemia limits the bioavailability of nitric oxide, increases oxidative stress, stimulates the proliferation of vascular smooth muscle cells, and alters the elastic properties of the vascular wall. The link between oxidative stress and hyperhomocysteinemia is also biologically plausible, because homocysteine promotes oxidant injury to the endothelium. Cumulated evidence suggests that the diminution of oxidative stress with antioxidants or the correction of hyperhomocysteinemia with vitamins-B plus folic acid, could be useful as an adjuvant therapy for essential hypertension. Further studies involving long-term trials could help to assess the tolerability and efficacy of the use of these therapeutic agents.

Effects of red wine consumption on kidney FA composition.

We studied the effects of red wine consumption on the FA composition of rat kidney. Four groups of adult male rats were fed a balanced diet for 10 wk. The drinking fluid was water (control), red wine, alcohol-free red wine, or ethanol (12.5%, vol/vol). FA composition, lipid peroxidation, and cytochrome P450 content were determined in the kidney. The antioxidant capacity of plasma was also measured. Ethanol decreased the content of long-chain PUFA, whereas red wine maintained the levels of arachidonic (20:4n-6) and eicosapentaenoic (20:5n-3) acids and alcohol-free red wine significantly increased the levels of 20:4n-6. Lipid peroxidation in the red wine and alcohol-free red wine groups was significantly lower than that of both the control and ethanol groups. The diminished renal lipid peroxidation was associated with an increased antioxidant capacity of plasma. Renal cytochrome P450 was elevated by 50% in the ethanol group and diminished by 20% in the alcohol-free red wine group. These data suggest that moderate red wine consumption could contribute to the preservation of the contents of n-3 and n-6 PUFA, particularly 20:4n-6, in rat kidney. Although ethanol increased the content of cytochrome P450 in the kidney, this effect was eliminated by the nonalcoholic components of red wine.

Rat kidney antioxidant response to long-term exposure to flavonol rich red wine.

This study evaluated the antioxidant defense system of the rat kidney following chronic exposure to red wine rich in flavonols. Both ethanol and antioxidant non-alcoholic wine components, mainly polyphenols, could contribute to the antioxidant status of kidney. Adult rats were given separately, water, ethanol (12.5%), red wine or alcohol-free red wine. After ten weeks of treatment, blood samples were obtained to determine plasma antioxidant capacity (FRAP, ferric reducing ability of plasma), uric acid and ethanol levels. Kidney tissues (cortex and papilla) were separated to perform measurements of reduced glutathione (GSH), glutathione disulfide (GSSG), lipid peroxidation (malondialdehyde, MDA) and the antioxidant enzymes catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px). The activity of (Na + K)-ATPase, a membrane-bound enzyme, was also assessed. Red wine in plasma, elevated the FRAP without changing the concentration of uric acid; in kidney, it diminished the MDA production and elevated the GSH/GSSG ratio and the activity of CAT and GSH-Px. The activity of SOD did not change. Despite the finding that renal (Na + K)-ATPase activity was upregulated by ethanol, it was not altered by either red wine or alcohol-free red wine. The effects on the antioxidant enzymes could be attributed to ethanol, but the increase in the FRAP and GSH/GSSG ratio is attributed to the non-alcoholic components of red wine. These data suggest that there is an enhancement of the antioxidant defense potential in kidney and plasma, after chronic red wine consumption. Both ethanol and the non-alcoholic antioxidant constituents of red wine could be responsible for these effects.

Modulation of cytochrome P450 activity in the kidney of rats following long-term red wine exposure.

Cytochrome P450 (CYP)-dependent oxidation of lauric acid, p-nitrophenol and ethanol by microsomal fractions of kidney were studied in control rats and in animals given either ethanol, red wine, or alcohol-free red wine for 10 weeks. Ethanol increased the total CYP content and specifically CYP 2E1, as well as p-nitrophenol and ethanol oxidation. The effects of ethanol treatment on the content and activity of CYP 2E1 were attenuated when red wine was administered, while the alcohol-free red wine values were similar to those of the control group. Although lauric acid hydroxylation was decreased by red wine treatment, the content of CYP 4A1 was not influenced by drinking fluids. We conclude that red wine administration attenuates the ethanol-induced enhancement of microsomal activities dependent on CYP 2E1 of rat kidney. Our results suggest that the non-alcoholic constituents of red wine could account for this modulation.

Modulation of rat liver cytochrome P450 activity by prolonged red wine consumption.

Cytochrome P450-dependent oxidation of lauric acid, p-nitrophenol and ethanol by liver microsomal fractions were studied in control rats and in animals given either ethanol, red wine, or alcohol-free red wine for 10 weeks. Ethanol increased the total cytochrome P450 and the isoenzyme 2E1 content, as well as the p-nitrophenol hydroxylation and ethanol oxidation. These effects of ethanol treatment were attenuated by red wine administration. Red wine increased the total antioxidant capacity of plasma, whereas the alcohol-free red wine decreased the cytochrome P450 content and decreased the oxidation of lauric acid, p-nitrophenol and ethanol to values lower than control. It is concluded that red wine administration attenuates the ethanol-induced enhancement in liver microsomal parameters dependent on cytochrome P450 2E1 activity, an affect that seems to be accomplished by the non-alcoholic constituents of red wine known to have antioxidant properties.

Changes in (Na + K)-adenosine triphosphatase activity and ultrastructure of lung and kidney associated with oxidative stress induced by acute ethanol intoxication.

UNLABELLED: STUDY AND OBJECTIVES: (Na + K)-adenosine triphosphatase (ATPase) activity, oxidative stress parameters, and morphologic characteristics of the lung and kidney of rats under acute ethanol intoxication were assessed to investigate the pathogenic mechanism of tissue damage. DESIGN AND INTERVENTIONS: Adult rats were given ethanol (5.5 g/kg) 3 h before performing the biochemical and morphologic studies. Oxidative stress was assessed by measuring the levels of reduced glutathione (GSH) and glutathione disulfide (GSSG), the activities of key antioxidant enzymes (ie, catalase [CAT], superoxide dismutase [SOD], and glutathione peroxidase [GSH-Px]) and malondialdehyde production. (Na + K)-ATPase, a membrane-bound enzyme, also was assayed. RESULTS: In the lung, ethanol increased MDA production by 60%, decreased GSH levels by 33%, decreased SOD and GSH-Px activity by 10%, and decreased (Na + K)-ATPase activity by 55%, whereas CAT activity was unaltered. Impaired surfactant secretion and cell adhesion of lung epithelial cells were found. In the kidney, ethanol did not influence the activity of (Na + K)-ATPase or lipid peroxidation, despite the reduction of both GSH and the GSH/GSSG ratio. Focally thickened glomerular basement membrane, apoptosis of foot processes, and tubulointerstitial fibrosis were found. CONCLUSIONS: These data suggest that oxidative stress plays a role in mediating the ethanol-induced down-regulation of lung (Na + K)-ATPase. GSH depletion seems to be a major determinant of this effect. Independent mechanisms seem to account for the morphologic alterations of these organs.

Renal effects of experimental obstructive jaundice: morphological and functional assessment

Background. The pathophysiology of renal impairments occurring in obstructive jaundice has been extensively studied, but underlying mechanism of these derangements remains unclear. The aim of the present study was to investigate the time-related morphological and functional changes occurring in the kidneys of rats undergoing obstructive jaundice. Methods. Histological examination, renal function assessment and determination of (Na + K)-ATPase activity were performed in the kidneys of rats 7, 14, and 21 days following bile duct ligation (BDL) or sham operation (sham). Results. Glomerular filtration rate was unaffected by BDL throughout the period of the study. Tubular effects occurred at days 7 ant 14, being more marked at day 7, and consisted of an increase of about twice in the fractional excretion of sodium and chloride, paralleled by a decreased proximal and distal tubular reabsorption of sodium of about 50 and 40 percent, respectively. Natriuresis was consistent with augmentation of osmolar clearance but it was not associated with changes in the acivity of renal (Na+ + K+)-ATPase. The ability to dilute urine was imparied at days 14 and 21 after BDL. Additionally, these effects were accompanied by decreased tubulointerstitial fibrosis and vasodilation of inner medullary capillaries. At day 21, the parameters of tubular function in BDL and sham groups were not significantly different. Conclusions. These data support the view that rasied natriuresis taking place in the initial 2 weeks following BDL is due mainly to tubular effects. The contribution of hemodynamic, paracrine and humoral mediators is discussed