PRESENCE OF CITRININ IN GRAINS AND ITS POSSIBLE HEALTH EFFECTS.

BACKGROUND: Citrinin is a mycotoxin produced by several species of the genera Aspergillus, Penicillium and Monascus and it occurs mainly in stored grain. Citrinin is generally formed after harvest and occurs mainly in stored grains, it also occurs in other plant products. Often, the co-occurrence with other mycotoxins is observed, especially ochratoxin A, which is usually associated with endemic nephropathy. At the European Union level, systematic monitoring of Citrinin in grains began with the aim of determining its highest permissible amount in food. Thus, far the systematic monitoring of the above mentioned mycotoxin in Croatia is yet to begin. MATERIALS AND METHODS: The main goal of this study was to determine the presence of Citrinin in grains sampled in the area of Medimurje, Osijek-Baranja, Vukovar-Srijem and Brod-Posavina County. For the purpose of identification and quantification of citrinin, high performance liquid chromatograph (HPLC) with fluorescence was used (Calibration curve k > 0.999; Intra assay CV = 2.1%; Inter assay CV = 4.3%; LOQ < 1 µg/kg). RESULTS: From the area of Medimurje County, 10 samples of corn and 10 samples of wheat were analyzed. None of the samples contained Citrinin (<1 µg/kg). From the area of Osijek-Baranja and Vukovar-Srijem County, 15 samples from each County were analyzed. The mean value for the samples of Osijek-Baranja County was 19.63 µg/kg (median=15.8 µg/kg), while for Vukovar-Srijem County the mean value of citrinin was 14,6 µg/kg (median=1.23 µg/kg). From 5 analyzed samples from Brod-Posavina County, one of the samples contained citrinin in the amount of 23.8 µg/kg, while the registered amounts in the other samples were <1 µg/kg. CONCLUSION: The results show that grains from several Counties contain certain amounts of Citrinin possibly indicating a significant intake of citrinin in humans. It must be stated that grains and grain-based products are the basis of everyday diet of all age groups, especially small children, where higher intake of citrinin can occur. Consequently, we emphasize the need for systematic analysis of larger amount of samples, from both large grains and small grains, especially in the area of Brod-Posavina County, in order to obtain more realistic notion of citrinin contamination of grains and to asses the health risk in humans.

Metals of Deep Ocean Water Increase the Anti-Adipogenesis Effect of Monascus-Fermented Product via Modulating the Monascin and Ankaflavin Production.

Deep ocean water (DOW) obtained from a depth of more than 200 m includes abundant nutrients and minerals. DOW was proven to positively increase monascin (MS) and ankaflavin (AK) production and the anti-adipogenesis effect of Monascus-fermented red mold dioscorea (RMD). However, the influences that the major metals in DOW have on Monascus secondary metabolite biosynthesis and anti-adipogenesis remain unknown. Therefore, the major metals in DOW were used as the culture water to produce RMD. The secondary metabolites production and anti-adipogenesis effect of RMD cultured with various individual metal waters were investigated. In the results, the addition of water with Mg, Ca, Zn, and Fe increased MS and AK production and inhibited mycotoxin citrinin (CT). However, the positive influence may be contributed to the regulation of pigment biosynthesis. Furthermore, in the results of cell testing, higher lipogenesis inhibition was seen in the treatments of various ethanol extracts of RMD cultured with water containing Mg, K, Zn, and Fe than in those of RMD cultured with ultra-pure water. In conclusion, various individual metals resulted in different effects on MS and AK productions as well as the anti-adipogenesis effect of RMD, but the specific metals contained in DOW may cause synergistic or comprehensive effects that increase the significantly positive influence.

Comparison of extraction methods for analysis of citrinin in red fermented rice.

There is no standard method for the extraction and analysis of citrinin in red fermented rice (RFR). In the study, five extraction methods were compared for their efficiency to analyse citrinin in RFR by HPLC-FLD (reversed phase high performance liquid chromatography with fluorescence detection), including, (1) ultrasonic extraction with EW solution (ethanol:water, 7:3, v/v); (2) ultrasonic extraction with TEF solvent mixtures (toluene:ethyl acetate:formic acid, 7:3:1, v/v); (3) shaking extraction with EW; (4) shaking extraction with EF solvent mixtures (ethyl acetate:formic acid, 1:1, v/v); (5) shaking combined with ultrasonic extraction in EW. Comparison of chromatograms of citrinin by HPLC-FLD with different extraction methods revealed that EW was the best extraction solvent. It was also found that shaking combined with ultrasonic extraction in EW was the most efficient extraction method to extract citrinin from RFR for qualitative and quantitative analysis.

Effect of feeding graded doses of Citrinin on clinical and teratology in female Wistar rats.

Citrinin is the one of the well-known mycotoxins, which is possibly spread all over the world. The graded doses of citrinin (1, 3 and 5 ppm CIT in feed) in female Wistar rats 10 weeks prior to mating, during mating and during organogenesis resulted in resorptions and post implantation losses, decreased fetal body weights and crown-rump lengths in fetuses of all groups. Various developmental anomalies recorded in fetuses of treated rats included gross (wrist drop, curled tail, stretched forelimb, subcutaneous haematoma), skeletal (incomplete ossification of skull bones, incomplete fusion of vertebral bodies, complete and partial agenesis of sternaebrae, metacarpals, metatarsals and phalanges, fused ribs and swing out ribs) and visceral (internal and external hydrocephalus, cerebellar hypoplasia, microphthalmia, roundening of heart, contracted kidneys, dilated renal pelvis and cryptorchid testes). The results suggest that CIT has adverse effects on fetal development which may be due to the longer bioavailability of citrinin in the animals.

Citrinin induces apoptosis in HL-60 cells via activation of the mitochondrial pathway.

The mycotoxin citrinin (CTN), a frequent natural contaminants of certain food and feeds, is known to be cytotoxic and genotoxic to various mammalian cells. To investigate the death mode of cells exposed to CTN, human promyelocytic leukemia (HL-60) cells were chosen to identify the apoptotic process induced by CTN. Morphological evidence of apoptosis, including nuclei fragmentation and DNA laddering formation, was clearly observed 24h after exposure to CTN. Flow cytometry analysis revealed that apoptotic cells in the hypodiploid region dramatically increased in cultures treated with CTN at concentrations above 50muM. Results of Western blotting showed that CTN induced the formation of processed caspase-3, -6, -7, -9, but not caspase-8, in a dose-dependent manner; CTN also induced a time-dependent increase in caspase-3 catalytic activity. The apoptosis triggered by CTN in HL-60 was accompanied by the cytochrome c release from mitochondria to cytoplasm. The presence of antioxidants in cultures did not effectively suppress CTN-induced cytotoxicity and caspase-3 activity. These findings suggest that CTN induces apoptosis in HL-60 cells by stimulating cytochrome c release followed by activation of multiple caspases, but oxidative stress may not play a role in the apoptotic process.

Supressão da resposta imunitária humoral causada pela citrinina / Humoral immunosupression caused by citrinin

Avaliou-se o efeito imunotóxico causado por exposição a baixas doses de citrinina (2,5mg kg-1) em camundongos albinos expostos à micotoxina antes (n=15), durante (n=15) e após (n=15) a imunização com antígeno inerte, representado por eritrócitos de carneiro - sheep red blood cells (SRBC). Quinze camundongos foram usados como controle (não intoxicados). Sete dias após o tratamento, os animais foram sangrados e os títulos de anticorpos anti-SRBC e de complemento foram determinados. A citrinina diminuiu os títulos de anticorpos primários em todos os grupos intoxicados. A intoxicação antes e após a imunização provocou diminuição em 87,5 por cento nos títulos médios de anticorpos específicos. A exposição simultânea à imunização gerou diminuição de 75 por cento. Houve acentuada redução nos níveis de complemento circulante, detectada nos animais previamente intoxicados (93,8 por cento), ou intoxicados juntamente com a imunização (87,5 por cento).

Citrinin production and stability in cheese.

Citrinin is a nephrotoxic fungal metabolite that has been demonstrated to be mutagenic in hepatocytes. It can be produced by several fungal species that belong mainly to the genus Penicillium and has been isolated from many feeds and human foods. Cheese is a very sensitive product because it can be naturally contaminated by citrinin-producing molds. The purpose of this study was to determine whether citrinin can be produced in cheeses and whether it is stable in these products. Both toxigenic strains of Penicillium citrinum and Penicillium expansum used were able to produce citrinin in cheese at 20 degrees C, but not at 4 degrees C. Up to 600 mg of citrinin per kg of cheese was obtained after 10 days of incubation. Interestingly, fresh goat cheese appeared to be a more favorable substrate for toxigenesis than did yeast extract-sucrose medium. Although contamination was mainly superficial, 33% of the toxin remained in cheese after trimming. Moreover, citrinin appeared to be very stable in some of the tested cheeses (goat cheese, Saint Marcellin, Soignon). For all cheeses tested, more than 50% of the initial content of citrinin was still present after 8 days of storage. Taken together, these results suggest that the contamination of cheeses by wild strains of Penicillium must be avoided.

Balkan endemic nephropathy and associated urinary tract tumours: a review on aetiological causes and the potential role of mycotoxins.

A series of publications in the 1950s described a kidney disease in Bulgaria, the former Yugoslavia and Romania that became known as Balkan endemic nephropathy (BEN). The disease was qualified by World Health Organisation (WHO) experts as 'progressive and very gradually developing renal failure with insidious onset.... The last stage shows marked fibrosis...'. BEN is characterized by tubular degeneration, interstitial fibrosis and hyalinization of glomeruli accompanied by enzymuria and impaired renal function without nephrotic syndrome. Later, an association between BEN and tumours of the kidney pelvis and ureter was recognized, so that the problem of BEN became not only nephrological, but also oncological. There may also be an association with increased urinary bladder cancer incidence, although many confounding factors may interfere in the analysis of data for this organ. In view of the very intimate association between BEN and the urinary tract tumours (UTT), the term 'endemic uropathy' has been proposed. Several hypothesis concerning the aetiology of these diseases has been investigated, which include: predisposing genes factors, environmental factors (heavy metals, minerals, bacteria, leptospira, viruses, fungal toxins and, most recently, pliocene lignites). This paper reviews the different hypotheses about the aetiology of endemic uropathy and pays particular attention to the role of fungal toxins.

Mechanism of citrinin-induced dysfunction of mitochondria. I. Effects on respiration, enzyme activities and membrane potential of renal cortical mitochondria.

Citrinin depresses the phosphorylation efficiency of rat renal cortical mitochondria, as inferred from the decrease of the respiratory control coefficient (RC) and ADP/O ratios. The transmembrane potential (delta psi) developed by energized mitochondria and the depolarization upon ADP addition are also decreased. Citrinin (1.0 mM) inhibits almost all enzymes linked to the respiratory chain and increases the activity of succinate cytochrome c reductase and succinate oxidase (coupled). Malate and glutamate dehydrogenases are also inhibited. The inhibitory action of citrinin on phosphorylation efficiency could be related to the following findings: the effect on complex I; the action on the ATP synthetase complex; the partial inhibition of the transmembrane potential.

Citrinin as a possible cause of the pruritus, pyrexia, haemorrhagic syndrome in cattle.

An outbreak of the pruritus, pyrexia, haemorrhagic syndrome affected eight of a herd of 175 cows which was divided into two groups of 115 and 60 according to yield. There was no difference in management between them but citrus pulp pellets were fed only to the larger group in which the eight cows were affected. Silage, which had been made without the use of additives, was also fed to both groups. The citrus pulp was visibly mouldy and contained 30 to 40 parts per billion of citrinin. Signs of the syndrome occurred within three days of the cows starting to ingest the citrus pulp, which was fed for 21 days, and the last case occurred six days after the feeding of citrus pulp ceased. Five calves whose dams had been fed citrus pulp were subsequently born with superior prognathism. In contrast to the eight cows that developed the syndrome only one out of 68 heifers which were fed larger quantities of citrus pulp for 10 days developed mild signs of the syndrome and then recovered, suggesting that older animals may be more susceptible. The clinical signs, gross pathology and histopathology are described and compared with those of previous outbreaks. Mycotoxins, particularly citrinin, were strongly implicated as the cause of this outbreak.

Citrinin mycotoxicosis in the rabbit: clinicopathological alterations.

Citrinin, a nephrotoxic mycotoxin, was dissolved in 0.5 N-NaOH neutralized with HCl and given in a single oral dose of 120 mg/kg (Trial I) or 80 or 100 mg/kg (Trial II) to male New Zealand white rabbits weighing 2.0-2.7 kg. In Trial I, sequential measurements of clinicopathological parameters were made over a 24-hr period. Azotaemia and metabolic acidosis with haemoconcentration and hypokalaemia developed within 4-12 hr. In Trial II, clinicopathological and urinary parameters were measured daily for 7 days. Increased blood urea nitrogen and serum-creatine levels and decreased creatinine clearance indicated renal failure; these values were most abnormal on days 2-4, returning to normal or near normal by day 7 in rabbits that survived. Urine analysis indicated tubular dysfunction and necrosis with glucosuria, isosthenuria and cylindruria; most urinary parameters were normal by day 7.