Oxidative stress as a mechanism of combined OTA and CTN toxicity in rat plasma, liver and kidney.

Ochratoxin A (OTA) and citrinin (CTN) commonly coexist in grains. Aiming to evaluate oxidative stress in OTA + CTN toxicity, male Wistar rats were orally treated with two doses of OTA (0.125 and 0.250 mg kg-1 of body weight (b.w.)), CTN (2 mg kg-1 of b.w.) and resveratrol (RSV; 20 mg kg-1 of b.w.) and combined daily during 3 weeks. Protein carbonyl concentrations were measured in kidneys and liver; catalytic activity of glutathione peroxidase (GPx), superoxide dismutase (SOD), catalase (CAT) and glutathione (GSH) level in plasma, kidneys and liver, while malondialdehyde (MDA) concentration was measured in plasma, kidneys, liver and urine. Mycotoxin treatment significantly increased MDA concentration in plasma and kidney and decreased SOD activity in the liver. Rats treated with CTN and OTA125 + CTN had lower plasma GPx activity. Concentration of GSH in the kidney and protein carbonyls in the kidney and liver as well as GPx activity in the kidney and liver, SOD activity in the kidney and CAT activity in the liver were not affected. Protective effect of RSV was observed on GSH in the kidney and plasma and MDA in the kidney, plasma and urine. Oxidative stress is involved in OTA + CTN toxicity in vivo because such treatment affects parameters of oxidative stress, particularly in plasma. RSV can reduce but not overcome oxidative stress induced by combined OTA and CTN treatment.

Early toxic effects of fumonisin B1 in rat liver.

Mycotoxin fumonisin B(1) (FB(1)) is hepatotoxic and carcinogenic in experimental animals. It is known that long-term exposure of experimental animals to FB(1) causes apoptosis and lipid peroxidation. In this study, male adult Wistar rats were treated with single FB(1) doses (5, 50, and 500 microg/kg b.w.) and sacrificed 4, 24, and 48 hours after treatment. Parameters of oxidative stress, histopathological changes, and DNA damage were monitored in the liver of treated and control animals. Parameters of oxidative stress were not affected by such treatment. A significant increase in apoptotic cells appeared in animals when 5 microg/kg b.w. dose was given and sacrificed after 24 hours with further increase at higher doses. In contrast to the number of mitotic figures and karyomegaly seen mostly at lower FB(1) doses, necrosis was the prominent feature at higher doses. Significant increase in liver cells DNA mobility was observed 48 hours following treatment with 50 and 500 microg/kg b.w. as compared to control (tail length 15.2 +/- 0.3, 16.4 +/- 0.5, and 13.5 +/- 0.1 mum, respectively). Tail intensity appeared to be more sensitive parameter for detecting DNA damage even at 5 microg/kg b.w. after 48 hours (1.69 +/- 0.27% DNA; control 0.59 +/- 0.11% DNA). This study proved that FB(1)-induced DNA damage is time- and dose-dependent, and that it could be caused in Wistar rats by a single dose.

DNA damage by ochratoxin A in rat kidney assessed by the alkaline comet assay.

There are few studies of ochratoxin A (OTA) genotoxicity in experimental animals and the results obtained with cell cultures are inconsistent, although the carcinogenic potential of OTA for the kidney of experimental animals has been well established. We studied the genotoxic potential of OTA in the kidney of adult female Wistar rats (5 in each group) treated intraperitoneally with OTA (0.5 mg kg body weight-1 day-1 for 7, 14, and 21 days) measuring DNA mobility on agarose gel stained with ethidium-bromide using standard alkaline single-cell gel electrophoresis (comet assay). Negative control animals were treated with solvent (Tris buffer, 1.0 mg/kg) and positive control animals were treated with methyl methanesulfonate (40 mg/kg) according to the same schedule. OTA concentrations in plasma and kidney homogenates in 7-, 14-, and 21-day treated animals were 4.86 +/- 0.53, 7.52 +/- 3.32, 7.85 +/- 2.24 microg/mL, and 0.87 +/- 0.09, 0.99 +/- 0.06, 1.09 +/- 0.15 microg/g, respectively. In all OTA-treated groups, the tail length, tail intensity, and tail moment in kidney tissue were significantly higher than in controls (P < 0.05). The tail length and tail moment were higher after 14 days than after 7 days of treatment (P < 0.05), and still higher after 21 days (P < 0.05). The highest tail intensity was observed in animals treated for 21 days, and it differed significantly from animals treated for 7 and 14 days (P < 0.05). OTA concentrations in plasma and kidney tissue increased steadily and OTA concentration in kidney tissue strongly correlated with tail intensity and tail moment values. These results confirm the genotoxic potential of OTA, and show that the severity of DNA lesions in kidney correlates with OTA concentration.

Seed-borne fungi and ochratoxin A contamination of dry beans (Phaseolus vulgaris L.) in the Republic of Croatia.

The study was designed to identify seed-borne fungi on bean (Phaseolus vulgaris L.) crops grown in 13 counties of the Republic of Croatia and their association with ochratoxin A (OTA) production. Bean samples (N=45) were collected in Croatia in 2001 shortly after the harvest and were stored at -20 degrees C for mycological and mycotoxin analyses. The most common fungi isolated were Cladosporium spp. (98%) Alternaria spp. (75%), Aspergillus spp. (73%), Rhizopus spp. (73%), Penicillium spp. (69%), Fusarium spp. (38%), Botrytis spp. (27%), Trichothecium spp. (24%), and Chaetomium spp. (18%). OTA was found only in samples contaminated with Penicillium and Aspergillus spp. Using HPLC (detection limit 0.25 microg/kg), OTA was found in 17 out of 45 samples (38%), and the mean concentration in positive samples was 0.41+/-0.21 microg OTA/kg. Beans from south Croatia (Adriatic coast) were OTA-free and the least mould-infected, while the mean OTA concentration and mould infection of samples from other regions were similar. The OTA contamination of beans in our country is low. Although beans are not severely contaminated with OTA, their consumption may contribute to the exposure to OTA from other commodities.

Organophosphate polyneuropathy in chicks.

Young animals are resistant to organophosphate-induced delayed neuropathy (OPIDP), although biochemical changes on Neuropathy Target Esterase (NTE) caused by neuropathic organophosphorus esters (OP) are similar to those observed in the sensitive hen. We report here that the resistance of chicks to single doses of neuropathic OPs is not absolute because ataxia was produced in 40-day-old chicks by 2,2-dichlorovinyl dibutyl phosphate (DBDCVP, 5.0 or 10.0 mg/kg s.c.) and by diisopropyl phosphorofluoridate (DFP, 2.0 mg/kg s.c.). However, the clinical picture was different from that usually seen in hens; spasticity and complete recovery being the main features. alpha-Tolyl sulphonyl fluoride (PMSF, 300 mg/kg s.c.) promoted both DBDCVP neuropathy (5.0 or 10.0 mg/kg s.c.) and non-neuropathic doses of DFP (1.5 mg/kg s.c.) or DBDCVP (1.0 mg/kg s.c.). The lowest promoting dose of PMSF given 24 hr after 1.5 mg/kg of DFP was 30 mg/kg. Higher doses had a more severe effect but no further increase of OPIDP severity was obtained with doses ranging from 90 to 300 mg/kg. PMSF (30 mg/kg) protected 40-day-old chicks from subsequent doses of neuropathic OPs even when a promoting dose of PMSF followed. At 60 days of age, chicks' resistance to OPIDP decreased because lower doses of neuropathic OPs became effective and, similarly to hens, PMSF did not fully protect from subsequent promotion. In 40-day-old chicks the threshold of NTE inhibition for OPIDP development was 95-97% (DBDCVP 5.0 mg/kg). When promotion followed initiation, the minimal effective inhibition of NTE for initiation by neuropathic OPs was about 90%. In 36-day-old chicks, PMSF (300 mg/kg) promoted OPIDP when given up to 5 days after DFP (1.5 mg/kg) when residual NTE inhibition in brain and sciatic nerve was about 40%. We conclude that chicks' resistance to OPIDP might reflect either a less effective initiation by phosphorylated NTE or a more efficient repair mechanism or both, and also that promotion is likely to involve a target other than NTE.

Age sensitivity to organophosphate-induced delayed polyneuropathy. Biochemical and toxicological studies in developing chicks.

Young animals are resistant to organophosphate-induced delayed polyneuropathy (OPIDP). The putative target protein in the nervous system for initiation of OPIDP in the adult hen is an enzyme called Neuropathy Target Esterase (NTE), which is dissected by selective inhibitors among nervous tissue esterases hydrolysing phenyl valerate (PV). We report here that the pool of PV-esterases sensitive to paraoxon was different in peripheral nerves of chicks as compared to that of hens while that of brain and spinal cord was not. NTE activity decreased with age in brain, spinal cord and peripheral nerve, but its sensitivity to several inhibitors remained unchanged. In the adult hen more than 70% inhibition of peripheral nerve NTE by neuropathic OPs is followed by deficit of retrograde axonal transport, axonal degeneration and paralysis. Similar NTE inhibition in 40-day-old or younger chicks however is not followed by changes in retrograde axonal transport nor by OPIDP. Chicks aged 60 to 80 days are only marginally sensitive to a single dose of DFP otherwise clearly neuropathic to hens. In vitro and in vivo phosphorylation by DFP and subsequent aging of brain NTE is similar both in chicks and in hens. The recovery of NTE activity monitored in vivo after inhibition by DFP is faster (half-life of about 3 days) in chick peripheral nerves as compared to chick brain, hen brain and hen peripheral nerve (half-life of about 5 days). It is concluded that the reduced sensitivity to OPIDP in chicks is not due to differences in OP-NTE interactions. The resistance might be explained by a more efficient repair mechanism, as suggested by the faster recovery of peripheral nerve NTE activity.

Phenylmethanesulfonyl fluoride delays the recovery from crush of peripheral nerves in hens.

Several esterase inhibitors (carbamates, phosphinates and sulfonyl halides) have been shown to promote organophosphate-induced delayed polyneuropathy (OPIDP). The mechanism of promotion is not understood, but indirect evidence suggests impairments of peripheral nerve repair. Also, other toxic neuropathies, such as those caused by 2,5-hexanedione in hens and bromophenylacetylurea in rats, have been reported to be promoted by phenylmethanesulfonyl fluoride (PMSF). Hen sciatic nerve was crushed at the bifurcation. Either mild or heavy pressure was applied by forceps obtaining a mild and rapidly recovering lesion (possibly myelinic) or a more severe, long-lasting lesion (possibly axonal), respectively. Hens were then treated with PMSF (120 mg/kg s.c. or 200 mg/kg s.c. x 2, 24 h apart) either before (5-48 h) crush or afterwards (5-48 h). Controls received vehicle only. Animals were observed for reappearance of digit movements, and standing and walking ability. PMSF treatment did not change the clinical outcome when animals received a mild crush. In hens receiving the more severe crush the reappearance of digit movements and the complete clinical recovery were observed after 43 +/- 14 and 63 +/- 9 days, respectively. In animals treated with PMSF there was a significant delay in both reappearance of digit movements (56 +/- 11 days when PMSF was given 24 and 48 h before crush, and 55 +/- 10 days, when given 24 and 48 h after crush) and in clinical recovery (75 +/- 15 and 80 +/- 18 days, respectively). It is concluded that traumatic axonopathy as well as toxic neuropathies can be promoted by PMSF. Moreover, it appears that PMSF promotion involves a target and a mechanism which are present in healthy axons and do not need to be activated by the insult to the axon.

Selective promotion by phenylmethanesulfonyl fluoride of peripheral and spinal cord neuropathies initiated by diisopropyl phosphorofluoridate in the hen.

This paper reports studies in hens showing that diisopropyl phosphorofluoridate (DFP) neuropathy is promoted by PMSF when initiated either in central (spinal cord) or peripheral nervous system. Moreover, the critical site for promotion is in peripheral nerve axons rather than in their cell bodies. Selective promotion in peripheral nerves was achieved by giving PMSF into sciatic artery monolaterally (7 mg/kg) to birds where neuropathy was initiated by DFP, either systematically (0.3 mg/kg s.c.) or intra-arterially (0.04 mg/kg in the same artery). Birds developed monolateral neuropathy in the leg where PMSF was delivered. Promotion of spinal cord neuropathy was achieved by giving PMSF (120 mg/kg s.c.) to birds where neuropathy was initiated selectively in spinal cord. This was obtained by protecting peripheral axons with intra-arterial bilateral injections of PMSF (0.55 x 2 mg/kg) followed by DFP (0.3, 0.4 or 0.7 mg/kg s.c.). The resulting syndrome was characterized by spastic ataxia.

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.

The ability of fungal isolates from human lung aspergilloma to produce mycotoxins.

This study included 11 adult patients (seven men and four women) who had been surgically treated for pulmonary aspergilloma in the Republic of Croatia within two years. Mycological analysis was positive for Aspergillus genus in five samples of surgically removed tissue. A. fumigatus was isolated in three and A. versicolor in two samples. Their mycotoxigenic potency was determined by thin layer chromatography. A. fumigatus strains were found to produce aflatoxin B1 (AFB), and two of them aflatoxin G1. A. versicolor strains produced AFB1 and sterigmatocystin. Neither isolated Aspergillus strain produced aflatoxin G2 or ochratoxin A. Fungal growth and production of mycotoxins are the consequences of interaction of fungus, host and environment. One has also to take into consideration that the production of mycotoxins in vitro does not reflect what these fungi may produce in human organisms.

Changes in plasma lipids after a non-lethal dose of cycloheximide in rats.

This paper describes a study of the effect of a single intraperitoneal non-lethal dose of cycloheximide (CHM; 2.0 mg/kg body weight) on the concentration of plasma lipids and lipoproteins in male rats killed one, two, three, four and nine days after receiving the dose. The concentration of triglycerides, total cholesterol, high-density lipoproteins (HDL)-cholesterol and low-density lipoproteins (LDL)-cholesterol was measured in treated and control animals. The effect of CHM on the concentration of triglycerides, total cholesterol, HDL-cholesterol, and LDL-cholesterol was visible in rat plasma throughout the study. Total cholesterol and HDL-cholesterol concentrations showed the same pattern of changes, probably due to the reversible inhibition of apolipoprotein apo A-I synthesis by CHM. The concentration of triglycerides decreased after a lag period of three days when the reserves of apolipoprotein apo B, the main apolipoprotein of very low-density lipoproteins (VLDL)-cholesterols produced in the liver, were consumed.

Experimental mycotoxicosis in chickens induced by ochratoxin A and penicillic acid and intervention with natural plant extracts.

The combined toxic effect of ochratoxin A (OTA) and penicillic acid (PA) on the body mass, the weight and pathomorphology of some internal organs was studied in 85 broiler chickens fed a mouldy diet containing 130, 300 or 800 ppb OTA and 1000-2000 ppb PA. The main pathomorphological changes were cloudy swelling and granular degeneration in the epithelium and mononuclear cell proliferation and activation of capillary endothelium in the kidney and liver; degenerative changes and depletion of lymphoid cells in lymphoid organs (bursa of Fabricius, thymus and spleen) were also seen. Protective effects of 5% total water extract of artichoke and a new natural phytosubstance Rosallsat against these pathomorphological changes were observed. A significant decrease in body mass and relative weight of lymphoid organs was found after 6 weeks of exposure and a greater decrease after 10 weeks of exposure to OTA and PA, and a protective effect of artichoke extract and a slight effect of Rosallsat against that decrease was observed. A significant increase in relative weight of liver and kidneys was also observed as well as a protective effect of artichoke extract against that increase. The quantity of OTA and the percentage of positive samples were significantly lower in tissues of chickens treated with artichoke extract or Rosallsat in addition to OTA than in those treated with only OTA.

Animal studies related to Balkan endemic nephropathy.

Basic field studies related to the animal population were performed in the region of Slavonski Brod, Republic of Croatia, where Balkan endemic nephropathy is an endemic disease. Pathological changes in several animal species from the locality were examined. The pig population in the area is numerous. Morphologically and physiologically pigs make an excellent animal model for studies of human diseases. Their use in studies should be encouraged, especially because there is a possibility that pigs and humans suffer from the same type of intoxication with a specific xenobiotic of natural origin. According to the mycotoxin theory about the aetiology of Balkan endemic nephropathy, pork meat might be one of the possible hazards for humans. Experiments on laboratory animals provide an excellent possibility to monitor several aspects of pathogenesis and all stages of pathomorphological changes which might then be correlated with Balkan endemic nephropathy. However, the experimental species should be critically chosen because some spontaneous, species-specific lesions of the kidneys are easily mistaken for changes induced experimentally.

Contamination of food with mycotoxins and human health.

Mycotoxins are natural contaminants of cereals and other commodities throughout the world. They are produced by various strains of moulds, particularly in tropical countries. Due to significant trade of cereals, humans in temperate countries can also be exposed to mycotoxins. The most common route of exposure to mycotoxins is ingestion, but it may also involve dermal, respiratory, and parenteral routes, the last being associated with drug abuse. Apart from acute and chronic toxic effects on human health called mycotoxicosis, some mycotoxins are proved or suspected human carcinogens. This paper describes various human diseases caused by ergot, afflatoxin, ochratoxin A, 3-nitropropionic acid, trichothecene, zearalenone, and fumonisin. It also gives a quick review of human carcinogenicity evaluations of the international Agency for Research on Cancer and of regulatory limits of mycotoxin concentrations in various commodities.

Ochratoxin A in blood of healthy population in Zagreb.

Healthy blood donors from the city of Zagreb were checked for the presence of a nephrotoxic mycotoxin ochratoxin A (OTA) in the plasma. Samples of blood were collected in June, September, and December 1997, and March 1998, totalling 200 or 50 in each round. The concentrations of OTA were measured using high pressure liquid chromatography (HPLC) method (detection limit 0.2 ng OTA/ml of plasma). The frequency of OTA-positive samples (> 0.2 ng/ml of plasma) showed significant seasonal variation (P < 0.001). The frequency of OTA-positive samples was the highest in March (65%) and it gradually decreased towards December (12%). The high frequency of positive samples coincided with seasons favouring growth of moulds and production of toxins. The daily intake of OTA by healthy persons in Zagreb was estimated from the mean concentration of OTA in samples collected during the whole year (0.19 ng OTA/ml plasma). The estimated daily intake was 0.26 ng/kg b.w., that is, substantially below the tolerable daily intake proposed by World Health Organization (16.0 ng/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.

Toxic effects of mycotoxins in humans.

Mycotoxicoses are diseases caused by mycotoxins, i.e. secondary metabolites of moulds. Although they occur more frequently in areas with a hot and humid climate, favourable for the growth of moulds, they can also be found in temperate zones. Exposure to mycotoxins is mostly by ingestion, but also occurs by the dermal and inhalation routes. Mycotoxicoses often remain unrecognized by medical professionals, except when large numbers of people are involved. The present article reviews outbreaks of mycotoxicoses where the mycotoxic etiology of the disease is supported by mycotoxin analysis or identification of mycotoxin-producing fungi. Epidemiological, clinical and histological findings (when available) in outbreaks of mycotoxicoses resulting from exposure to aflatoxins, ergot, trichothecenes, ochratoxins, 3-nitropropionic acid, zearalenone and fumonisins are discussed.

Interactions between neuropathy target esterase and its inhibitors and the development of polyneuropathy.

This paper combines new and old data in order to offer a modified perspective of the mechanism of organophosphate-induced delayed polyneuropathy. Neuropathy target esterase (NTE) is though to be the molecular target and neuropathy to be initiated with a two-step mechanism: progressive inhibition of NTE and aging of the phosphorylated enzyme. When neuropathic organophosphates modify more than 70% of NTE in this way, neuropathy develops 2 weeks later. Other chemicals producing an inhibited NTE, which is incapable of aging, were thought to be not neuropathic. When given before a challenging dose of a neuropathic organophosphate they protect animals from neuropathy. However, recent evidence indicates that aging may not always be essential in causing neuropathy. In fact, mipafox and methamidophos as well as certain classic protective inhibitors such as carbamate and sulfonyl fluoride form an inhibited NTE which apparently does not age and yet produces neuropathy. We propose that all NTE inhibitors may have the potential to cause neuropathy. In analogy with pharmacological models of drug-receptor interactions, NTE inhibitors might have variable intrinsic activities to initiate neuropathy once attached to the protein. Strong neuropathic chemicals require about 70% inhibition of NTE, others 80-90%, and the least potent almost 100%. These differences have been amplified by means of promotion. Different levels of NTE inhibition as caused by different compounds were promoted by the same dose of phenylmethanesulfonyl fluoride to similar degrees of ataxia. Conversely nearly complete NTE inhibitions obtained in chicks with different chemicals were promoted to varying severities of ataxia. Protection from delayed polyneuropathy by the least neuropathic inhibitors can be explained by their weak intrinsic activity: occupying NTE, they prevent the binding of more neuropathic compounds. Methamidophos represents a particular example because it is protective at lower doses and neuropathic at high doses. Moreover, the levels of NTE inhibited by methamidophos which can be promoted to neuropathy are lower than those required for classic protective chemicals and higher than those of classic neuropathic OPs. This suggests that methamidophos has an intermediate position between the most and the least neuropathic NTE inhibitors.

Antidotal efficacy of quinuclidinium oximes against soman poisoning.

The efficiency of newly synthesized oxime derivatives of quinuclidinium were tested in vitro on soman inhibited acetylcholinesterase (AChE) of human erythrocytes and in vivo using soman poisoned mice. For this purpose, the inhibitory power of oximes (IC50), acute toxicity (LD50) as well as reactivating and protective capacities with respect to soman-inhibited AChE were determined for each of the oximes. All oximes tested were ineffective in vitro but protected mice very efficiently (BM-1 protects against 4LD50 of soman). The results indicate that the in vivo effectiveness of quinuclidinium oximes against soman poisoning may not be related to reactivation or protection of AChE but rather to some other mechanism of the cholinergic system.