Radovan Fuchs

Ochratoxin A in human sera in the area with endemic nephropathy in croatia

Ochratoxin A (OA) is nephrotoxic fungal metabolite (mycotoxin) occurring in foodstuffs. The compound is causally associated with mycotoxin porcine nephropathy, a disease comparable with a human kidney disease called endemic nephropathy (EN). In this paper we presented results obtained over a 10-year period in the hyperendemic village Kaniza, and in control villages where no clinical cases of nephropathy had been found. In the hyperendemic village Kaniza and non-endemic villages the incidence of OA in human blood was up to 4.5% (range 2-50 ng/ml) and up to 2.4% (range 2-10 ng/ml), respectively. Almost all samples of food and feed, collected randomly in the hyperendemic village were found to contain OA. Considering marked exposure to OA in Kaniza, it was assumed that incidence of EN in this population could be related to OA contamination of food and feed.

The occurrence of ochratoxin A in blood in general population of Croatia

The exposure of general population in Croatia to mycotoxin ochratoxin A (OTA) was investigated in five cities: Split, Rijeka, Varazdin, Osijek, and Zagreb. In June 1997, blood donors from each of these cities gave 50 samples of 3 ml plasma each. The mean concentration of OTA, determined using high-pressure liquid chromatography (HPLC), was 0.39 ng/ml of plasma. The highest frequency of OTA-positive samples (>0.2 ng/ml plasma), and the highest number of samples with the concentration exceeding 1.0 ng/ml, were found in Osijek. This difference is probably due to the higher consumption of fresh and dried pork by population of Osijek. The calculated daily intake of OTA, estimated from the mean OTA concentration of all samples in each town (in the range from 0.24 to 0.91 ng/kg b.w. found in Rijeka and Osijek, respectively) is lower than the tolerable daily intake proposed by Joint FAO/WHO Expert Committee on Food Additives (1995) of 16.0 ng OTA/kg b.w.

Ochratoxin A in human kidney diseases

Ochratoxin A (OTA) is an ubiquitous nephrotoxic and carcinogenic mycotoxin considered to be involved in the aetiology of Balkan endemic nephropathy (BEN). The occurrence of this human fatal disease that appears in regions of Bosnia and Herzegovina, Bulgaria, Croatia, Rumania, and Serbia and Monte Negro correlates with very high incidence of otherwise rare urothelial tumours of the renal pelvis and ureters. Although OTA was found more frequently and/or in higher concentration in food and blood of inhabitants in regions with BEN than in other regions, the involvement of OTA in the development of BEN is still open. Patients with chronic renal insufficiency treated with dialysis have higher blood OTA concentrations than healthy persons, and OTA concentration does not decrease with such treatment.

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.

The involvement of mycotoxins in the development of endemic nephropathy

ZusammenfassungDie endemische Nephropathie (EN) ist eine Nierenerkrankung, die noch immer nicht wissenschaftlich erklärt werden kann. Die EN wird von einer hohen Prävalenz von urothelialen Tumoren bei der ländlichen Bevölkerung der endemischen Region begleitet. Man vermutet daher, dass eine natürlich vorkommende nephrotoxische und karzinogene Substanz in die Ätiologie involviert ist. Am meisten wird das Mykotoxin Ochratoxin A (OTA) als schuldige Substanz verdächtigt, da dieses eine gesicherte nephrotoxische und karzinogene Wirkung hat. Die vorliegende Arbeit bringt eine Übersicht über alle relevanten Studien über OTA in der Nahrung beziehungsweise im Blut und im Harn der Bewohner der betroffenen Endemie-Gebiete. Es werden auch Daten über das gleichzeitige Vorkommen von OTA mit anderen Mykotoxinen, wie Zitronin und Fumonisin B1 (FB1) in der Nahrung vorgestellt. Leider existieren keine Studien über das gemeinsame Vorkommen von OTA und anderen Mykotoxinen beim Menschen, und es gibt nur eine Studie über die Exposition mit FB1 in endemischen Gebieten. Diese Übersicht berichtet auch über experimentelle Daten, die in Zellkulturen und Labortieren erhoben wurden, die mit OTA und anderen nephrotoxischen Mykotoxinen (da die meisten Mykotoxin-Kombinationen synergistische Wirkung zeigten) behandelt worden sind. Es wird auch das Vorkommen von OTA- und Aristolochia-Säure-bedingten DNA-Veränderungen diskutiert.SummaryEndemic 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 B1 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 B1 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.

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

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 Kaniža 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 Kaniža (43 % and 18 %, respectively) than in the control village (28 % and 6 %, respectively). OTA concentrations in samples collected in Kaniža were higher in 2000 than in 2005 (p<0.005). Although in both years Sa/So ratio was higher in Kaniža, 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 Kaniža four such samples were collected in 2000 and one in 2005. Okratoksin A i omjer sfinganina i sfingozina u urinu stanovnika s područja endemske nefropatije u Hrvatskoj Najprihvatljivija teorija o etiologiji endemske nefropatije povezuje njezin nastanak s izloženošću nefrotoksičnim mikotoksinima. Dok se izloženost mikotoksinu okratoksinu A (OTA) može dokazati njegovim nalazom u biološkim uzorcima kao što su krv i urin, vrlo kratko zadržavanje fumonizina B1 (FB1) u organizmu to onemogućava. Na pokusnim je životinjama nađeno da je porast omjera koncentracija sfingolipida sfinganina i sfingozina (Sa/So) biološki pokazatelj izloženosti tom mikotoksinu. U ovom istraživanju mjerena je koncentracija OTA i omjer koncentracija Sa/So u urinu 45 stanovnika u endemskom selu Kaniža i 18 stanovnika u kontrolnom selu. Uzorci urina skupljeni su od istih osoba 2000. i 2005. godine. U obje godine učestalost uzoraka koji su sadržavali OTA bila je veća u Kaniži (43 % i 18 %) negoli u kontrolnom selu (28 % i 6 %). Koncentracija OTA također je bila viša u urinima skupljenim u Kaniži negoli u kontrolnom selu. Koncentracija OTA u uzorcima skupljenim u Kaniži 2000. bila je viša nego u uzorcima iz 2005. (p<0.005). Iako je u urinima iz obje godine omjer koncentracija Sa/So bio viši u Kaniži negoli u kontrolnom selu, razlika nije bila statistički značajna. Nije nađen nijedan uzorak skupljen u kontrolnom selu koji bi istodobno sadržavao mjerljivu koncentraciju OTA i omjer Sa/So veći od jedan. Za razliku od uzoraka iz kontrolnog sela, četiri uzorka skupljena u Kaniži u 2000. godini i jedan uzorak u 2005. godini upućivali su na istodobnu izloženost ovim mikotoksinima.

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.

Alleviation of irinotecan toxicity by HI-6 oxime: in vivo study focused on ChE/AChE activity

Acetylcholinesterase (AChE) is an extremely active enzyme necessary for terminating the action of acetylcholine (ACh) in cholinergic synapses. There are many natural and synthetic compounds that inhibit the AChE followed by accumulation of acetylcholine and a generalised cholinergic crisis. Irinotecan is one of frequently used antineoplastic drugs, which has shown remarkable antitumor activity. In spite of beneficial outcomes, the majority of this drug also exhibit side effects that imply the use of special supportive care. The mechanism of action of HI-6 oxime as a therapeutic component with protection/ reactivation of the inhibited AChE is clear at this time. In the present study, we investigated its effect in vivo on the rats when given as pre-treatment and therapy in combination with the same drug. Both HI-6 and irinotecan act as inhibitors of ChE/AChE activity, but show different level of inhibition of ChE in plasma and AChE in liver and brain tissue. Our results indicate that the sequence of administration of HI-6 significantly affected the measured enzyme activity. In rats given HI-6 as therapy the enzyme activity was lower that in those that received HI-6 as pre-treatment. It is possible that when HI-6 given 15 minutes before irinotecan, it had enough time to reach the target and temporarily occupy the enzyme making it unavailable for irinotecan. Hi-6 relieves the cholinergic side effects and possesses acceptable toxicity profile, which might significantly improve the response to therapy. However, further studies are essential.

Therapeutic effect of bis-pyridinium oximes against Tabun poisoning

Organophosphorus compounds are widely used as pesticides and unfortunately as nerve agents in chemical warfare. They are known inhibitors of acetylcholinesterase (AChE, EC 3.1.1.7) an enzyme that hydrolizes the neurotransmitter acetylcholine in the nervous system. The clinical signs of AChE inhibition manifest as hypersalivation, lacrimation, diarrhoea, tremor, respiratory distress, convulsion and seizures. Signs are dose-dependent, leading to severe incapacitation and rapid death. Together with atropine, pyridinium oximes are known to be successfully used to treat poisoning with many organophosphorus compounds. In this paper three new bis-pyridinium compounds: K033 [1, 4-bis(2-hydroxyiminomethylpyridinim)butane dibromide], K027 [1, 4-hydroksyiminomethylpyridinium)-3-(4-carbamoylpyridinium) propane dibromide], K048 [1-(4-hydroxyiminomethylpyridinium)-4-(4-carbamoylpyridinium) butane dibromide] were tested as potential antidotes in tabun poisoned mice. Their antidotal effect was compared with TMB-4 [1, 3-bis(4-hydrxyiminomethylpyridinium) propane dibromide], which is the best-known antidote in tabun poisoning. In all experiments, oxime K033 in doses of 1/4 or 5% of its LD50 was used for the pre-treatment 15 minutes before tabun-intoxication. Also, one or 5 minutes after tabun application experimental animals received oxime K027, K033 or K048 (5% or 1/4 of its LD50) plus atropine sulphate as therapy. The antidotal efficacy of tested compounds was expressed as therapeutic factor (TF) and therapeutic dose (TD). Under same experimental conditions, our experiment selected compound K048 as the most reactivator of tabun inhibited AChE. Namely, this study has shown that the therapeutic regimen consisting of K033 in dose 5% of its LD50 as preatretment and f of LD50 of K048 plus atropine as treatment had the highest TF and TD. The TF was 13.3 LD50 of tabun, TD was 10 LD50 of tabun and insurance survival of all tested animals. In conclusion, treatment with these new bis-pyridinium oximes seems to be a very good alternative for current treatment in tabun poisoning. For this reason, these and other similar compounds require further investigation.

Fumonisin B1: Oxidative status and DNA damage in rats

Fumonisins are mycotoxins that contaminate maize and wheat. The most toxic and the most frequently found fumonisin is fumonisin B1 (FB1) involved in several animal diseases and supposed to be involved in the etiology of some human tumors. FB1 disturbs the metabolism of sphinganine (Sa) and sphingosine (So) increasing the ratio of their concentrations (Sa/So). FB1 was shown to be mutagenic in several studies on cultured cells. Literature data on the studies of FB1 genotoxicity in experimental animals are rather scarce and the mechanism of genotoxicity is not understood. The aim of this study was to elucidate whether DNA lesions in kidney and liver cells of FB1 treated rats are related to changes of oxidative status. Adult male Wister rats were receiving either FB1 dissolved in sterile saline (0.9 % NaCl) (0.5 mg/kg b.w./day) or solvent only (negative control animals) intraperitoneally for two or seven consecutive days and sacrificed 24 h after the last treatment. Ratio of Sa and So concentrations and parameters of oxidative status (activity of catalase, and concentration of protein carbonyls and malondialdehyde – MDA) were measured in plasma, liver and kidney homogenate, while comet assay was performed in liver and kidney. In plasma and homogenates of liver and kidney activity of catalase, concentration of protein carbonyls and MDA were not affected after two days treatment with FB1. At this time point the ratio of Sa and So in all tested samples was increased. After two days treatment tail length and tail intensity measured with comet assay in liver homogenate was not changed, while in kidney they where significantly different from controls. After seven days treatment all measured parameters were significantly different from controls. This study showed that in experimental animals FB1 causes DNA lesions in kidney before affecting catalytic activity of catalase and concentration of protein carbonyls and MDA. In this time point the ratio of Sa and So significantly increases in all tissues. These results taken together confirm that oxidative stress is the consequence and not the cause of DNA damage and that the metabolism of sphingolipids should be involved in the DNA damage caused by FB1 rather than oxidative stress.