Reduction of the ochratoxin A-induced cytotoxicity in Vero cells by aspartame

Ochratoxin A (OTA) is a mycotoxin produced by Aspergillus ochraceus as well as other moulds. This mycotoxin contaminates animal feed and human food and is nephrotoxic for all animal species studied so far. OTA is immunosuppressive, genotoxic, teratogenic and carcinogenic. Recently lipid peroxidation induced by OTA has been reported. OTA, a structural analogue of phenylalanine, inhibits protein synthesis by competition with phenylalanine in the phenylalanine-tRNA aminoacylation reaction, constituting the main mechanism of OTA-induced cytotoxicity. Since it seems impossible to avoid contamination of foodstuffs by toxigenic fungi, investigation is required for preventing the toxicity of OTA. An attempt to prevent its toxic effect, mainly the inhibition of protein synthesis, has been made using aspartame (L-aspartyl-L-phenylalanine methyl ester) a structural analogue of both OTA and phenylalanine. Protein synthesis was assayed in monkey kidney cells (Vero cells) treated by increasing concentrations of OTA (10-100 microM). After 24 h incubation, protein synthesis was inhibited by OTA in a concentration dependent manner (the 50% inhibitory concentration, IC50, was c. 14.5 microM). Aspartame (A19), at tenfold higher concentrations than OTA (100-1000 microM), was found to partially protect against the OTA-induced inhibition of protein synthesis in Vero cells, and more efficiently when added 24 h prior to the toxin (IC50 34 microM) than together (IC50 22 microM). As expected A19(250 microM) prevented the OTA-induced leakage of certain enzymes, including lactate dehydrogenase, gamma-glutamyl transferase, alkaline phosphatase, into the culture medium, and the concomitant decrease of their intracellular activity in OTA (25 microM)-treated cells. In order to investigate the effect of aspartame (A19) on OTA-protein binding as explanation of the above results, the mycotoxin time- and concentration-dependent binding to human samples was studied in static diffusion cells with two compartments separated by a dialysis membrane. When A19 (34 microM) was added to the upper compartment containing plasma before installing OTA (50, 250, 1240 microM) in the lower one. OTA binding was largely prevented (95-98%). When A19 (34 microM) was added to the lower compartment simultaneously with the toxin (50, 250, 1240 microM), for the lowest concentration of OTA, the same efficiency was shown in preventing OTA binding, but at the two high concentrations A19 seemed less efficient.     

Pentoxifylline reduces nephrotoxicity associated with cyclosporine in the rat by its rheological properties

BACKGROUND: The goals of this study were to evaluate whether administration of pentoxifylline (POF) reduces the nephrotoxicity associated with cyclosporine (CsA) in the rat, and whether the effect of POF is related to its rheological properties. METHODS: Mean arterial pressure was measured by an intraarterial catheter. Glomerular filtration rate and renal plasma flow were determined by measuring inulin and para-aminohippurate clearances, after double-blind coadministration for 10 days of CsA (25 mg/kg/day) with either vehicle or POF (45 mg/kg every 12 hr). These results were compared with those obtained in control rats. Blood viscosity and erythrocyte deformability were also evaluated after treatment using a cone plate viscometer and a filtration method, respectively. RESULTS: No changes were observed in mean arterial pressure in both groups compared with controls. Glomerular filtration rate was significantly lower in CsA-treated rats (0.3+/-0.1 ml/min/100 g) than in control animals (0.6+/-0.1 ml/min/100 g, P<0.02). The coadministration of CsA with POF normalized the glomerular filtration rate (0.6+/-0.1 ml/min/100 g). A parallel decrease in renal plasma flow was observed in CsA-treated rats compared with controls (CsA+vehicle: 1.5+/-0.2 vs. control: 2.2+/-0.1 ml/min/100 g, P<0.02), this effect completely reversed by cotreatment with POF (3.1+/-0.2 ml/min/100 g). Blood viscosity was significantly higher in CsA-treated rats than in the control group (CsA+vehicle: 5.6+/-0.7 vs. control: 5.0+/-0.4 m x Pa x s, P<0.05). This effect was associated with a lower erythrocyte deformability (CsA+vehicle: 1.2+/-0.2 vs. control: 1.5+/-0.3 ml/min, P<0.05). These rheological abnormalities were normalized by coadministration with POF (blood viscosity: 4.9+/-0.7 m x Pa x s and erythrocyte deformability: 1.9+/-0.4 ml/min, P<0.05). CONCLUSIONS: Our results show that administration of POF prevents the nephrotoxicity associated with CsA. This beneficial effect could be related to its rheological properties.     

Dietary stearic acid reduces plasma and hepatic cholesterol concentrations without increasing bile acid excretion in cholesterol-fed hamsters

Although there is general agreement that saturated fatty acids elevate plasma cholesterol concentrations, the relative effects of individual fatty acids on cholesterol and bile acid metabolism are less clear. In this study, cholesterol and bile acid responses to diets enriched in different saturated fatty acids were investigated in hamsters. The six diets examined were as follows: 5% fat (g/100 g) enriched in palmitic acid (16:0) with no cholesterol, 5% fat 16:0-enriched, 0.05% cholesterol (wt/wt), and four diets containing 0.05% cholesterol and 15% fat with each diet enriched in lauric (12:0), myristic (14:0), palmitic (16:0), or stearic acid (18:0). Total plasma cholesterol concentration was significantly greater in hamsters fed the 14:0-enriched diet relative to those fed the 18:0-enriched diet (P < 0.05). Both plasma and liver cholesterol concentrations of hamsters fed 18:0 did not differ from those of the group fed no dietary cholesterol. In all instances, differences in total plasma cholesterol were accounted for within the HDL fraction; no significant treatment differences in VLDL or LDL cholesterol were found. Total daily fecal bile acid excretion was higher in hamsters fed the 15% fat 16:0 diet compared with those fed no dietary cholesterol (P < 0.05), but not significantly different from other treatment groups. There was greater deoxycholic acid excretion (P < 0.05) from hamsters fed the 14:0 and 16:0 diets compared with those fed the 18:0-enriched diet. Small intestinal + gallbladder bile acids, an index of pool size, did not differ significantly among the groups. The observed relative hypocholesterolemic effect of stearic acid was not mediated by increased bile acid excretion.     

Dietary saturated fatty acids increase cholesterol synthesis and fecal steroid excretion in healthy men and women

In a strictly controlled 6-week trial with 47 healthy volunteers we have determined the effect of replacement of polyunsaturated by saturated fatty acids on the fecal steroid excretion and on the rate of whole body cholesterol synthesis, as measured both by the sterol balance method and by the concentration of the cholesterol precursor lathosterol in serum. Subjects were fed mixed natural diets, of which the total fat content was kept constant at 45% energy. Consumption of polyunsaturated fatty acids, mainly linoleic acid, was 21% energy for the first 3-week period (P:S ratio 1.9), and 5% of energy (P:S ratio 0.2) for the next 3-week period, or vice versa. Cholesterol intake as determined by analysis of duplicate diets was 41 mg MJ-1 (about 500 mg day-1) during both periods. Feces were collected for 5 days at the end of both periods. The steroid composition of the feces was not affected by the change of diets. The fecal excretion of neutral steroids was significantly higher on the low P:S high-saturated-fat (2.25 +/- 0.68 mmol day-1) than on the high P:S high-linoleic-acid diet (2.00 +/- 0.69 mmol day-1; P < 0.01). The excretion of bile acids was similar (0.77 +/- 0.40 and 0.79 +/- 0.41 mmol day-1, respectively). The cholesterol balance and the rate of cholesterol synthesis were higher during the low P:S (1.86 +/- 0.83 mmol day-1) than during the high P:S period (1.55 +/- 0.85 mmol day-1; P < 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)     

Dietary conjugated linoleic acid reduces plasma lipoproteins and early aortic atherosclerosis in hypercholesterolemic hamsters

Conjugated linoleic acid is a collective term used to designate a mixture of positional and geometric isomers of linoleic acid in which the double bonds are conjugated. Unlike linoleic acid, there is a paucity of information regarding the effect of dietary conjugated linoleic acid on plasma lipoproteins and aortic atherosclerosis. Therefore, fifty hamsters were divided into five groups of ten and fed 0 (Control), 0.06 (LOW), 0.11 (MEDIUM), and 1.1 (HIGH) en% conjugated linoleic acid or 1.1 en% linoleic acid. Blood samples were taken at 4, 8 and 11 weeks for plasma lipid analyses and for plasma tocopherol assay at sacrifice. Animals fed the conjugated linoleic acid-containing diets collectively had significantly reduced levels of plasma total cholesterol, non-high density lipoprotein cholesterol, (combined very low and low density lipoprotein) and triglycerides with no effect on high density lipoprotein cholesterol, as compared to CONTROLs. Linoleic acid-fed animals relative to CONTROLs also had reduced plasma total cholesterol, non-high density lipoprotein cholesterol and triglycerides, but only the latter was statistically significant. Compared to the CONTROL group, plasma tocopherol/total cholesterol ratios determined from plasma pools for the LOW, MEDIUM and HIGH conjugated linoleic acid and linoleic acid groups were increased by 48%, 48%, 86% and 29%, respectively, suggesting a tocopherol-sparing effect, at least for the conjugated linoleic acid treatment. Morphometric analysis of aortas revealed less early atherosclerosis in the conjugated linoleic acid and linoleic acid-fed hamsters compared to the CONTROL group.     

Plasma and hepatic cholesterol levels and fecal neutral sterol excretion are altered in hamsters fed straw mushroom diets

The effect of the fruiting body and mycelium of Volvariella volvacea (straw mushroom) on the concentrations of plasma lipids, liver cholesterol, fecal neutral sterol and bile acid excretions was investigated in male Golden Syrian hamsters. The hamsters were fed a purified hypercholesterolemic diet (0.1% cholesterol, 10% fat) for 4 wk to elevate plasma lipid concentrations. Twelve hamsters with elevated plasma total cholesterol were randomly assigned to each treatment group: control (5% cellulose), mushroom fruiting body (5%) and mushroom mycelium (5%). After 4 wk of mushroom diet consumption, the plasma total cholesterol, HDL cholesterol, and combined VLDL + LDL cholesterol concentrations (mmol/L) were significantly lower than control in the group fed the fruiting body-diet (40, 38 and 43%, respectively) (P < 0.05). The liver cholesterol levels were significantly lower in both the mushroom fruiting body- and the mycelium-fed groups (28 and 21% in terms of concentration; 39 and 30% in terms of total content, respectively) (P < 0.05) than that in the control group. Fecal neutral sterol excretion in the mushroom fruiting body- and mycelium-fed groups was significantly higher (81 and 74%, respectively) (P < 0.05) than that in the control group. Although no significant differences (P > 0.05) in the excretion of fecal bile acids were observed among groups fed the mushroom diets and the control diet, the mushroom fruiting body diet-fed hamsters apparently had less bacterial degradation of cholic acid as indicated by a significantly greater proportion (P < 0.05) of fecal cholic acid than in controls. They also had a significantly lower proportion of fecal deoxycholic acid (P < 0.05). This study suggests that the fruiting body of the straw mushroom lowers elevated plasma cholesterol in hypercholesterolemic hamsters, whereas the mycelium does not.     

Sex- and strain-specific expression of cytochrome P450s in ochratoxin A-induced genotoxicity and carcinogenicity in rats

There is an urgent need for drugs capable of inhibiting renal calcifications, nephrocalcinosis and stones included, in humans. Current anticalcification medication is based mainly on alkalinization of the metabolism using potassium-containing citrate alone, despite the fact that calcium stone patients suffer marginally from both magnesium and potassium deficiency. We investigated the anticalcification efficacy of oral potassium citrate versus the combined administration of this drug and magnesium citrate in the magnesium-deficient rat developing corticomedullary nephrocalcinosis and luminal microliths in the long term. Among other things we employed specific stains for calcium and oxalate, light microscopy and element analysis for renal tissue and calcifications, respectively. In addition, minerals in renal tissue, urine and plasma were determined, as well as the state of extracellular calcium homeostasis. Magnesium deficiency caused pure calcium phosphate tissue deposits, containing no magnesium, but no deposition of calcium oxalate in the tubular lumen; tissue magnesium, calcium and phosphorus were increased, and there was marked potassium wastage via urine; despite mild hypercalcemia other signs of hyperparathyroidism were not found. Alkalinization with the two kinds of medication evoked an increase in urinary pH, citrate, and potassium; however, potassium citrate alone tended to aggravate renal concretions, whereas the combination of this drug with magnesium citrate completely prevented concretions. It was concluded that: (1) magnesium deficiency-induced calcifications are oxalate-free and are not sensitive to mobilization by alkalinization with potassium citrate, which might explain the failure of the drug to prevent stone recurrence in clinical stone patients, and (2) the combination of potassium citrate and magnesium citrate, which shows enormous anticalcification efficacy, deserves high priority in clinical trials aimed at evaluating strategies for the prevention of stones.     

Therapeutic trials for promotion of faecal excretion of PCDFs by the administration of rice bran fiber and cholestyramine in Yusho patients

Two girls with Swyer syndrome (SS) were described. Diagnosis was established according to clinical data and ultrasound, laparoscope, histopathological, hormonal and cytogenetical examinations. One presents diagnostic possibilities followed advanced methods in genetics. The GTG and RBG high resolution bounding technique and replication analysis of short arms (Xp and Yp) were employed. Normal structure of end segments of X and Y chromosomes was mentioned. Molecular DNA analysis by polymerase chain reaction (PCR) did not find any mutation in SRY gene. Normal structure of this gene does not exclude possibility of SS existence. Our data implicates on the other mechanism of these disturbances.     

Increased plasma levels of substance P and disturbed water excretion in patients with liver cirrhosis

We describe a surgical technique for replacing the hip joint capsule using synthetic mesh after oncological resections of the proximal femur that resulted in gross intraoperative instability of the prosthetic reconstruction. The results of its use in 13 patients, 6 of whom also had pelvic resections, are described. These patients were selected from a total group of 88 patients undergoing proximal femoral replacement, 75 of whom did not require capsular replacement (none of these 75 patients have experienced dislocation). In the group requiring capsular reconstruction, 1 of 4 patients with bipolar hemiarthroplasty and 4 of 9 patients with total hip replacements experienced dislocation after operation. Of the dislocated total hip replacements, 1 remains chronically dislocated, and 3 were successfully stabilized by open reduction with further capsular augmentation. Given that the resections involved removal of most of the soft tissues stabilizing the hip joint, we believe that the technique of capsular reconstruction is useful in this difficult group of patients.     

Dietary magnesium deficiency increases Gi alpha levels in the rat heart after myocardial infarction

OBJECTIVE: Magnesium (Mg) is crucial for the function of G proteins which play important roles in mediating the inotropic effects of beta adrenergic agonists in the heart and are altered in heart failure. This study was performed to determine whether or not dietary Mg deficiency alters functional activity and levels of the two major ventricular G proteins, Gi alpha and Gs alpha in the heart after myocardial infarction (MI). METHODS: Six week old rats were fed an Mg adequate or deficient diet for 6 weeks. At the end of week 3, MI was induced by coronary artery ligation. A sham operation was performed as control. After surgery, surviving animals were maintained on their assigned diets for another 3 weeks. Then, cardiac function was measured, plasma and tissue were collected. RESULTS: Severe hypomagnesemia and increased plasma catecholamine level were observed in all animals fed the Mg deficient diet. A significant reduction of myocardial Mg concentration accompanied by elevated plasma and myocardial calcium concentrations was observed in MI animals with existing Mg deficiency vs. animals fed the Mg adequate diet. Cardiac function was impaired in MI rats and further reduced in MI rats with existing Mg deficiency. Gi alpha level was not altered by either Mg deficiency or MI alone, but was dramatically elevated in animals with combined Mg deficiency and MI (9.9 +/- 0.7 arbitrary unit.mg-1 protein) as compared to MI alone (5.8 +/- 0.6, P < 0.05) and Mg deficiency alone (6.1 +/- 0.8, P < 0.05). Gs alpha level did not differ between groups. GppNHp-, but not fluoride-stimulated adenylyl cyclase activity was slightly reduced in MI animals with existing Mg deficiency. CONCLUSION: The findings suggest that dietary Mg deficiency increases the expression of Gi alpha in the heart after MI, while levels and function of Gs alpha are not compromised during dietary Mg deficiency either with or without MI.     

Effect of Tityus serrulatus scorpion toxin on serum gastrin levels in anaesthetized rat

Scorpion toxin induces gastric secretion of acid and pepsin in rats. These effects seem to be mediated by the release of acetylcholine and histamine. However, the role of gastrin in the scorpion-toxin-induced gastric secretion is unknown. We describe the effects of the T1 fraction purified from Tityus serrulatus scorpion venom on serum and on antral tissue gastrin levels in anaesthetized rats. Gastrin levels in serum and in the antral mucosa were measured before and at intervals 5, 15, 30, 60, 90 up to 120 min after the intravenous injection of saline or the T1 fraction of scorpion venom (0.25 mg/kg) into anaesthetized rats. Antral G-cells were submitted to immunocytochemistry and electron microscopy. The data on gastrin were correlated with the gastric juice volume, and the acid and pepsin output increases induced by toxin. Scorpion toxin induced a significant increase in volume, acid output and pepsin output of gastric juice and gastrin serum levels 15-60 min after injection. Simultaneous measurements of antral gastrin levels did not show significant effects. The number of dense, intermediate and empty granules per microm(2) in the cytoplasm of antral G-cells was not significantly changed 60 min after saline or toxin injection. Scorpion toxin significantly increased serum gastrin; levels in rats.     

Correlation of plasma corticosterone levels with running activity in the blinded rat

Blinded female reats were placed in running-wheel cages to monitor the phase of their activity cycle. After approximately a month of adaptation to the wheels, jugular vein blood samples were withdrawn at the beginning and end of the running phase of activity and analyzed for plasma corticosterone. By the time blood samples were obtained, the blined rats' activity cycles had desynchronized from the lighting cycle of the room in an apparently free-running fashion, and were out of phase with each other. The corticosterone levels were high at the beginning of the running phase and low at the end: begin run corticosterone was 34.5 +/- 13, end run corticosteronewas 14.2 +/- 10 (mean +/-SD in mug/100 ml of plasma; t = 7.93, df = 82, P less than 0.001). It was concluded that blinded rats do have an adrenocortical rhythm and that it is in phase with the activity cycle of each individual rat.     

The marine toxin okadaic acid reduces O2- generation and tyrosine phosphorylation in LPS-primed rat neutrophils

Contrasting effects of okadaic acid (OKA) on neutrophil (PMN) superoxide anion (O2-) generation have been reported. In this study, we examined the effect of OKA on phorbol myristate acetate (PMA)-stimulated O2- generation in rat PMNs primed with LPS in vivo (LPS-PMN) and saline-treated rat PMNs (SAL-PMN). The following results were observed: (1) OKA, but neither genistein nor vanadate, markedly reduced O2- generation in a dose and time-dependent manner; (2) genistein, a tyrosine kinase inhibitor, as well as OKA, reduced tyrosine phosphorylation; (3) sodium orthovanadate, a tyrosine phosphatase inhibitor, potently enhanced tyrosine phosphorylation. Our studies suggest that OKA might reduce tyrosine phosphorylation by affecting the activity of tyrosine phosphatases regulated by serine-threonine phosphorylation.     

Renal urate excretion at various plasma concentrations in the rat: a free-flow micropuncture study

Free-flow micropuncture experiments were performed in male Sprague-Dawley rats undergoing moderate mannitol diuresis and infused with urate-containing solutions. The resulting plasma urate concentrations ranged from 37.5 +/- 2.4 to 601.2 +/- 23.8 muM. With urate loading, the fraction of filtered urate excreted in pelvic urine increased from 0.32 +/- 0.02 to 0.92 +/- 0.05 mu M, but net secretion was not observed. At normal urate levels net reabsorption occurred along superficial proximal tubules, whereas net secretion could be demonstrated at the highest plasma urate levels. Net movements of urate did not appear to occur across the walls of the lower segments of nephrons.     

Leukocyte adhesion molecules in the liver and plasma cytokine levels in endotoxin-induced rat liver injury

BACKGROUND: The interactions between polymorphonuclear neutrophils (PMNs) and sinusoidal endothelial cells (SECs) have been known to be involved in the pathogenesis of acute liver injury. It has been also reported that tumor necrosis factor-alpha (TNF-alpha) up-regulates ICAM-1 expression on SECs and that interleukin-8 (IL-8) provokes rapid activation of CD11/CD18 on PMNs. These findings expand into the relationship between the expression of leukocyte adhesion molecules (ICAM-1, CD11a/CD18 and CD11b/CD18) in liver tissues and plasma TNF and IL-8 levels after lipopolysaccharide (LPS)-induced liver injury in rats. METHODS: Male Wistar rats weighing 200-250 g were treated with 2 mg LPS/kg intravenously in a 0.2- to 0.25-ml volume. Liver and blood samples were obtained at 1, 3, 8, and 12 h after LPS exposure. Plasma TNF and IL-8 levels were measured using bioassay and specific enzyme-linked immunosorbent assay, respectively. Liver samples were fixed and studied by immunohistochemistry using specific monoclonal antibodies against ICAM-1, CD11a, and CD11b. RESULTS: The TNF level showed a peak at 1 h (23.3 +/- 11.4 IU/ml), and the IL-8 level showed a peak at 3 h (343.1 +/- 110.5 ng/ml) after LPS exposure. An increase in the number of PMNs in the liver was observed as early as 1 h and continued until 12 h after LPS exposure. PMNs adhered to degenerated SECs and hepatocytes. ICAM-1 on SECs was diffusely and strongly expressed at 8 h, and PMNs adhered to SECs expressed both CD11a and CD11b. ICAM-1 was also observed on hepatocytes. CONCLUSION: These data suggest that PMN-SEC and PMN-hepatocyte interactions via leukocyte adhesion molecules, related to inflammatory cytokines such as TNF and IL-8, exist and play an important role in the pathogenesis of acute liver injury.     

Effects of choline-free plasma induced by choline oxidase on regional levels of choline and acetylcholine in rat brain

Choline-free plasma (CFP) was induced in rats by intravenous (IV) injection of 56.0 x 10(2) units kg-1 of choline oxidase (ChO) which completely metabolized the free Ch circulating in the plasma for at least 15.0 h and caused subsequent significant decrease in the concentration of free Ch in the three brain regions examined, the striatum, hippocampus, and cortex. However, the treatment did not affect concentrations of acetylcholine (ACh) in these regions. By contrast, intraperitoneal (IP) injection of 1.0 mmol kg-1 Ch chloride resulted in a maximum concentration of free Ch in plasma in 5 min, after which tissue Ch in all regions examined increased (p < 0.001). Concomitant increases were observed in cortical and hippocampal ACh (p < 0.05) 20 min after the injection. It is thus suggested that the brain may possess compensative mechanisms to prevent the supply of free Ch from circulating to the brain during synthesis of ACh in the brain. It is also suggested that the CFP rat would be a useful and readily available animal model for future study.     

Effects of fosfomycin and imipenem/cilastatin on nephrotoxicity and renal excretion of vancomycin in rats

PURPOSE: The effects of fosfomycin and imipenem/cilastatin on the nephrotoxicity of vancomycin were studied in rats, and those on the renal handling of vancomycin were also investigated in perfused kidneys. METHODS: The protective effects of fosfomycin and imipenem/cilastatin on vancomycin nephrotoxicity were evaluated by increases in plasma concentration of creatinine and urea nitrogen in rats. The urinary excretion of vancomycin was measured and analyzed kinetically in the perfused rat kidney. RESULTS: The nephrotoxicity induced by vancomycin (500 mg/kg, i.v.) was inhibited almost completely by co-administration of fosfomycin or imipenem/cilastatin. In the perfused rat kidney, the excretion ratio of vancomycin was less than those of p-aminohippurate and cimetidine, and greater than that of arbekacin, suggesting the secretion and reabsorption of vancomycin in renal tubules. The tissue/perfusate ratios of unbound vancomycin were not significantly changed by co-treatment with fosfomycin or imipenem/cilastatin. Imipenem/cilastatin significantly decreased the excretion ratio of vancomycin. Fosfomycin also decreased vancomycin excretion ratio, although this effect was not significant. CONCLUSIONS: The renal handling of vancomycin was different from those of organic anions and cations and an aminoglycoside antibiotic. The protective effects of fosfomycin and imipenem/cilastatin against the nephrotoxicity of vancomycin might be partly due to the change in renal handling of vancomycin, probably in its tubular secretion/ reabsorption, in rats.