Low doses of ochratoxin A upregulate the protein expression of organic anion transporters Oat1, Oat2, Oat3 and Oat5 in rat kidney cortex.

Mycotoxin ochratoxin A (OTA) is nephrotoxic in various animal species. In rodents, OTA intoxication impairs various proximal tubule (PT) functions, including secretion of p-aminohippurate (PAH), possibly via affecting the renal organic anion (OA) transporters (Oat). However, an effect of OTA on the activity/expression of specific Oats in the mammalian kidney has not been reported. In this work, male rats were gavaged various doses of OTA every 2nd day for 10 days, and in their kidneys we studied: tubule integrity by microscopy, abundance of basolateral (rOat1, rOat3) and brush-border (rOat2, rOat5) rOat proteins by immunochemical methods, and expression of rOats mRNA by RT-PCR. The OTA treatment caused: a) dose-dependent damage of the cells in S3 segments of medullary rays, b) dual effect upon rOats in PT: low doses (50-250 microg OTA/kg b.m.) upregulated the abundance of all rOats, while a high dose (500 microg OTA/kg b.m.) downregulated the abundance of rOat1, and c) unchanged mRNA expression for all rOats at low OTA doses, and its downregulation at high OTA dose. Changes in the expression of renal Oats were associated with enhanced OTA accumulation in tissue and excretion in urine, whereas the indicators of oxidative stress either remained unchanged (malondialdehyde, glutathione, 8-hydroxydeoxyguanosine) or became deranged (microtubules). While OTA accumulation and downregulation of rOats in the kidney are consistent with the previously reported impaired renal PAH secretion in rodents intoxicated with high OTA doses, the post-transcriptional upregulation of Oats at low OTA doses may contribute to OTA accumulation and development of nephrotoxicity.

Optimisation of a gas chromatography-mass spectrometry method for the simultaneous determination of tetrahydrocannabinol and its metabolites in rat urine.

In order to evaluate the effect of irinotecan (IRI) on urinary elimination of delta-9-tetrahydrocannabinol (THC) in a rat experimental model, we developed an analytical method for the determination of the mass concentration of THC and its metabolites [11-hydroxy-delta-9-tetrahydrocannabinol (THC-OH) and 11-nor-9-carboxy-delta-9-tetrahydrocannabinol (THC-COOH)] in the urine of rats treated only with THC and treated simultaneously with THC and irinotecan. For this purpose, hydrolysis and solid phase extraction conditions of the investigated analytes were optimised and a gas chromatography-mass spectrometry (GC-MS) method was developed to determine all three analytes in rat urine. The most effective hydrolysis method for THC, THC-OH, and THC-COOH conjugates was so-called tandem hydrolysis by the ß-glucuronidase enzyme from Escherichia coli at 50 °C for 2 hours and followed by alkaline hydrolysis. The proposed method was then applied for determining concentrations of analytes in 24-hour rat urine. THC was not detected in either sample, THC-OH was detected in 50 % of samples, and THC-COOH in all of the samples. Enhanced urinary THC-COOH excretion was noted in rats administered combined treatment compared to single THC treatment. The method described herein was suitable for determining the mass concentration of THC metabolites in the rat urine due to its sensitivity (detection limits: 0.8-1.0 µg/L), accuracy (>96 %), and precision (RSD <6 %).

The involvement of mycotoxins in the development of endemic nephropathy.

Endemic nephropathy is a human kidney disease that still escapes scientific explanation. It is accompanied by a high incidence of urothelial tumors in rural populations in endemic areas, which suggests that a natural nephrotoxic and carcinogenic compound may be involved in the etiology. The most imputed causative agent of endemic nephropathy is the mycotoxin ochratoxin A (OTA), because of its confirmed nephrotoxic and carcinogenic action. This paper presents a review of studies of OTA in food collected in the endemic areas and in blood and urine of their residents. Data on the co-occurrence of OTA and other nephrotoxic and carcinogenic mycotoxins such as citrinin and fumonisin B(1) in food are also presented. Unfortunately, there is no study on the co-occurrence of OTA and other mycotoxins in humans and there is only one study on fumonisin B(1) exposure in endemic areas. The paper also presents experimental data on cultured cells and laboratory animals treated with combinations of OTA and other nephrotoxic mycotoxins, because most such combinations show a synergistic effect. The occurrence of OTA- and aristolochic acid-DNA adducts is also presented.

The involvement of oxidative stress in ochratoxin A and fumonisin B1 toxicity in rats.

The aim of this study was to find out whether very low doses of nephrotoxic and hepatotoxic mycotoxins ochratoxin A (OTA) and fumonisin B1 (FB1) induce oxidative stress in rat kidney and liver and whether their effect is synergistic. Rats were treated orally with OTA (5 ng/kg b.w. and 50 microg/kg b.w.) and FB1 (200 ng/kg b.w. and 50 microg/kg b.w.), or their combinations. Malondialdehyde (MDA) and protein carbonyls (PCs) concentration in kidney was affected with lower dose of OTA than in liver (p<0.05). FB1 did not affect MDA and PCs concentrations in the liver, while in the kidney both FB1 doses increased MDA concentration (p<0.05). The combination of the lower doses of OTA+FB1 increased the MDA and PCs concentration both in the liver and the kidney, compared to controls and animals treated with respective doses of mycotoxins (p<0.05). The combinations of mycotoxins reduced the catalase activity only in the kidney when compared to controls (p<0.05). In contrast to the increased kidney concentrations of MDA and PCs even with very low doses of OTA and FB1, the activity of catalase and SOD does not change. Combinations of OTA+FB1 affected almost all parameters, which indicates their potential to produce oxidative damage.

Butyrylcholinesterase activity and plasma lipids in dexamethasone treated rats.

The paper describes the effect of glucocorticoid dexamethasone (DM) given intraperitoneally on the catalytic activity of butyrylcholinesterase (BuChE) measured in plasma, liver and white adipose tissue of rats of both sexes. Effects of DM on the concentration of plasma lipids and lipoproteins were also tested. Rats were given multiple (2 and 4) pharmacological doses (0.4 and 3.0 mg kg(-1) body mass) of DM. All animals were sacrificed 48 hours after the last dose. Administration of DM significantly decreased the catalytic activity of BuChE in plasma and liver of all treated groups regardless of sex. BuChE catalytic activity in white adipose tissue differed depending on the dose and frequency of administration. In contrast to liver where both doses caused significant BuChE inhibition, the lower DM dose did not inhibit BuChE activity in adipose tissue, and the inhibition achieved by the higher dose was not as strong as in liver. This result corroborates an earlier hypothesis that BuChE is also synthesized in the adipose tissue. DM significantly increased plasma concentrations of triglycerides, total cholesterol and high-density lipoprotein (HDL) cholesterol and decreased the low-density lipoprotein (LDL) cholesterol concentration. Neither positive correlation between BuChE and triglycerides nor negative correlation between BuChE and HDL was found. Changes in lipid profile during DM treatment were not sex- and time-dependent.

Two different clean-up procedures for liquid chromatographic determination of ochratoxin A in urine.

This paper describes two different procedures for extraction of ochratoxin A (OTA) from urine samples: one using acidic chloroform-methanol mixture, followed by solid-phase extraction (SPE) clean-up and the other using commercial Chem Elut columns and a chloroform-formic acid mixture. The recovery of OTA using the procedure with silica gel columns was 82% with a R.S.D. < 8.4% and the detection and quantitation limits were 0.5 and 1.5 ng OTA/ml, respectively. The recovery of OTA in the second procedure with urine samples purified only on commercial Chem Elut columns was 95% with R.S.D. < 4.0%, and detection and quantitation limits 0.3 and 0.9 ng/ml, respectively. Both procedures of OTA extraction effectively eliminate interfering substances and give reliable and repeatable results. However, the procedure with Chem Elut columns gave higher recovery and lower detection and quantitation limits. It was successfully applied in determining OTA in human urine samples.

Ochratoxin A-induced apoptosis in rat kidney tissue.

The aim of our study was to find whether ochratoxin A (OTA) induces the apoptosis and/or necrosis of kidney tissue in rats. In the first experiment, the highest number of apoptotic cells was found in rats sacrificed one day after OTA administration (1.00 mg/kg b.w., i.p.). The number of apoptotic cells reduced gradually and they were not seen nine days after OTA administration. A possible dose-dependence of histological changes was checked in kidney tissue of rats given 0.25, 0.50 or 1.00 mg of OTA/kg b.w., i.p. three times a week for four weeks. The number of apoptotic cells showed a clear dose-dependence, but necrosis was absent even at the highest doses. The time-dependent appearance of lesions related to OTA administration was checked by administering 0.50 mg OTA/kg body weight to rats, and sacrificing them one day after 1, 3, 6, and 9 doses/administrations, or 6 and 21 day after 12 doses/administrations. Long-term administration is associated with continued and increased apoptosis without necrosis, suggestive of OTA's role in the pathogenesis of progressive renal atrophy.

The effect of dichlorvos treatment on butyrylcholinesterase activity and lipid metabolism in rats.

This paper describes the effects of dichlorvos (DDVP) on butyrylcholinesterase (BuChE) activity with possible consequences for lipid and lipoprotein metabolism in rats. The rats of both sexes were given a single and multiple doses of DDVP (8.0 mg/kg body weight) with two-day intervals between administrations, ensuring the continuous inhibition of BuChE activity without lethal outcome. BuChE activity was measured in plasma, liver, and white and brown adipose tissue. The recovery of BuChE activity was observed only in white adipose tissue of female rats 10 days after treatment. Our results show that DDVP significantly decreases BuChE activity in female and male rat plasma (40-60%; P < 0.05), and significantly increases triglycerides (60-600%; P < 0.05) and total cholesterol (35-75%; P < 0.05). In contrast to the increased HDL-cholesterol (20-30%; P < 0.05), LDL-cholesterol decreased (30-40%; P < 0.05). The decrease of BuChE activity and the changes in concentrations of lipids and lipoproteins were observed throughout the experiment. Our results contribute to the hypothesis that BuChE may play a role in lipid and lipoprotein metabolism.

The effect of cycloheximide on butyrylcholinesterase activity in vivo.

The paper describes the catalytic activity of butyrylcholinesterase (BuChE) measured in plasma, liver, white adipose tissue, heart, and brain of rats intraperitoneally administered a single non-lethal dose of cycloheximide (2.0 mg/kg body weight; CHM). The BuChE assay was performed on rats of both sexes either administered CHM or saline (controls), and killed 2, 3, 4, 5, 10 days later. A significant decrease of BuChE catalytic activity was observed in all tested tissues except plasma. In animals of both sexes, the lowest BuChE catalytic activity was found in the liver (2-6%), while it was higher in white adipose tissue, heart, and brain. However, the respective values remained significantly different from controls (33-67%, 49-62%, and 14-71% in males, and 24-82%, 72-86%, and 33-67% in females). Since there was no effect of CHM on BuChE catalytic activity in plasma, the data suggest that CHM inhibits the synthesis of BuChE rather than its active site.

Evaluation of the cyto/genotoxicity profile of oxime K048 using human peripheral blood lymphocytes: an introductory study.

This study investigates the effects of oxime K048 (730, 200, and 7.3nM) on the viability and chromosome stability of human peripheral blood lymphocytes (PBLs) after a 30min exposure in vitro. Cytotoxicity was tested by a viability assay with ethidium bromide and acridine orange. For the evaluation of the genotoxic potential, we used comet assays, cytokinesis-blocked micronucleus (CBMN) assay, and chromosome aberration (CA) analysis. We found acceptable cytotoxicity for K048 (9.7±2.1% non-viable PBL at highest concentration vs. 7.3±2.5% in control; apoptosis dominated over necrosis). Overall primary DNA damage was low and not significantly different from controls. The hOGG1-comet assay showed a slight increase in the level of oxidative DNA damage. In oxime treated PBLs, we found 13-19 MN compared to 15 MN in control cultures. The frequencies and types of CA in oxime-treated PBLs did not significantly differ from controls. K048 showed acceptable biocompatibility at the level of cell viability and chromatin/chromosome integrity. Since no increase in secondary genome damage was detected, the primary DNA lesions may have resulted from treatment-induced cell stress, subsequently becoming repaired and not fixed as chromosome aberrations. The toxicity profile of K048 should be further studied and compared with other clinically relevant oximes.

Urine ochratoxin a and sphinganine/sphingosine ratio in residents of the endemic nephropathy area in Croatia.

The most plausible theory of the aetiology of endemic nephropathy links it with exposure to nephrotoxic mycotoxin ochratoxin A (OTA). In this study, the concentration of OTA and sphinganine/sphingosine (Sa/So) ratio, the biomarker of another nephrotoxic mycotoxin fumonisin B1 exposure, were analysed in 45 human urine samples collected in the endemic village of Kaniza in Croatia and in 18 samples from control village. Samples were collected twice from the same persons in 2000 and 2005. In both years the frequency of OTA-positive samples was higher in Kaniza (43 % and 18 %, respectively) than in the control village (28 % and 6 %, respectively). OTA concentrations in samples collected in Kaniza were higher in 2000 than in 2005 (p<0.005). Although in both years Sa/So ratio was higher in Kaniza, the difference from the control group was not statistically significant. No control sample contained OTA and had the Sa/So ratio >1 at the same time, while in Kaniza four such samples were collected in 2000 and one in 2005.