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PubMed2003

Evaluation of genotoxic risk and oxidative DNA damage in mammalian cells exposed to mycotoxins, patulin and citrinin.

Mycotoxins are fungal secondary metabolites with very diversified toxic effects in humans and animals. In the present study, patulin (PAT) and citrinin (CTN), two prevalent mycotoxins, were evaluated for their genotoxic effects and oxidative damage to mammalian cells, including Chinese hamster ovary cells (CHO-K1), human peripheral blood lymphocytes, and human embryonic kidney cells (HEK293). PAT, but not CTN, caused a significant dose-dependent increase in sister chromatid exchange (SCE) frequency in both CHO-K1 and human lymphocytes. PAT also elevated the levels of DNA gap and break in treated CHO-K1. In the single cell gel electrophoresis (SCGE) assay, exposure of HEK293 to concentrations above 15 microM of PAT induced DNA strand breaks; the tail moment values also greatly increased after posttreatment with formamidopyrimidine-DNA glycosylase (Fpg). This suggests that in human cells PAT is a potent clastogen with the ability to cause oxidative damage to DNA. However, no significant change in the tail moment values in CTN-treated cultures was found, suggesting that CTN is not genotoxic to HEK293. Incubation of HEK293 with CTN increased the mRNA level of heat shock protein 70 (HSP70), but not that of human 8-hydroxyguanine DNA glycosylase 1 (hOGG1). PAT treatment did not modulate the expression of either HSP70 or hOGG1 mRNA.

Animals↗
PubMed2005

Evaluation of citrinin occurrence and cytotoxicity in Monascus fermentation products.

Monascus purpureus and its fermentation products have been used in food coloring and meat preservation in Asia for centuries and have also been recently used as dietary supplements because of their cholesterol-lowering ability. However, the presence of the mycotoxin citrinin (CTN), a secondary metabolite of Monascus species, in fermentation products is a potential threat to public health. In the present study, HPLC was used to analyze CTN levels in lipid and aqueous extracts of commercialized Monascus products. CTN was detected in lipid extracts of all examined samples at concentrations varying between 0.28 and 6.29 microg/g, but was not found in aqueous extracts. When human embryonic kidney cells (HEK293) were incubated for 72 h with Monascus extracts, the concentrations causing 50% cell death by all lipid extracts were in the range of 1.8-4.7 mg/mL, whereas aqueous extracts showed a lower cytotoxicity. Incubation of HEK293 cells with 60 microM pure CTN for 72 h caused cell viability to fall to 50% of control levels. In addition, coadministration of pure CTN and lipid extracts from Monascus samples significantly enhanced CTN cytotoxicity for HEK293 cells using the MTT assay. These results provide the first information about the cytotoxic effects of various Monascus samples and CTN-Monascus mixtures on a human cell line.

Cell Death↗
PubMed2006

Ochratoxin A and citrinin loads in stored wheat grains: impact of grain dust and possible prediction using ergosterol measurement.

Crop storage should be carried out under hygienic conditions to ensure safe products, but sometimes grain dust which has settled from previous storage may be left over and incorporated to the following stored grains. This paper describes the results obtained using a lab model developed in order to assess the impact of grain dust incorporation for its direct contribution as a contaminant but also as an inoculum in stored wheat. Settled grain dust (4 samples) released from Belgian grain storages were collected and analysed by HPLC for ergosterol, ochratoxin A (OTA) and citrinin (CIT) content. For OTA and for ergosterol, there was a high degree of variability in concentrations found in the dust samples (from 17.3-318 ng g(-1) and from 39-823 microg g(-1), respectively) whilst for CIT, the range was less significant (from 137-344 ng g(-1)). Incorporation of grain dust into wheat storage contributed to an increase in the concentrations of mycotoxins in the stored grain. Dust acts as a contaminant and as an inoculum. According to these two ways, patterns of mycotoxin generation vary with the nature of the mycotoxin, the mycotoxigenic potential of dust and the water activity of the wheat. OTA and CIT showed a very versatile image when considering the amounts of toxins produced under the selected experimental conditions. The development of a robust tool to forecast the mycotoxigenicity of dust was based on the determination of ergosterol content as a general marker of fungal biomass. Present results suggest that this predictive tool would only be valid for predicting the contamination level of CIT and OTA at reasonable moisture content (14-20%). The potential risk of having highly contaminated batches from stock to stock may thus occur and this paper discusses possible pathways leading to OTA and CIT contamination either under wet or dry storage conditions. We therefore, recommend taking precautionary measures not only by controlling and maintaining moisture at a reasonable level during storage of the raw materials but also by paying more attention to the cleaning of the stores before loading in the new harvests.

Anti-Bacterial Agents↗
PubMed1984

Effect of simultaneous prenatal exposure to ochratoxin A and citrinin in the rat.

Ochratoxin A (OA) and citrinin (CT) are food-borne mycotoxins produced by several fungal species of the genera Aspergillus and Penicillium. Both are teratogenic in the rat. To determine the prenatal effects of simultaneous exposure to these toxins, pregnant Sprague-Dawley rats were injected either with a single individual subthreshold teratogenic dose of OA (1 mg/kg) or CT (30 mg/kg) or with both toxins. Toxins were dissolved in 5% sodium bicarbonate and administered subcutaneously on one of gestation d 5, 6, 7, 8, 10, 11, or 14. Maternal body weight gain of animals in the combination group was similar to other treatment groups and the control. Approximately 22-40% mortality in dams occurred on gestation d 5, 6, 7, and 14. Other than d 7, there was no significant effect on the number of implants. Treatment on d 5 or 7 resulted in increased fetal resorptions. Fetal body weights were not decreased significantly. OA and CT in combination resulted in a significant increase in gross malformations on d 6 and 7, visceral anomalies on d 5, 7, 8, and 10, and skeletal defects on d 5, 6, 7, 8, 10, and 14. When administered individually, OA and CT resulted in very few fetal resorptions. Fetal body weights were not significantly different except on d 8 of gestation following CT treatment. Individual toxin treatment resulted in minimal malformations on all gestation days. These results suggest that OA and CT, when administered concurrently, may interact to enhance prenatal toxicity and teratogenicity, and these results have focussed attention on the public health hazards of contamination of food with these mycotoxins.

Abnormalities, Drug-Induced↗
PubMed1990

Occurrence of some mycotoxins (aflatoxin, ochratoxin A, citrinin, zearalenone and vomitoxin) in various Egyptian feeds.

Ninety five samples of various Egyptian feedstuffs were investigated for the aflatoxin B1, B2, G1, and G2 means thin layer chromatography (TLC). Out of these samples 44.2% were positive (maize, rice crack, rice germ, rice germ cake, rice bran, wheat bran, cotton seed, cotton seed cake, peanut, and mixed feed for broilers, egg production, calf fattening and milk production). High percentage (90.5%) of the positive samples were contaminated with less than 100 ppb total aflatoxins. Peanut from "Ismailia" showed the highest contamination-mean of 400 ppb aflatoxin B1. The contamination relationship between kernels and shell of the same pods of the peanut was 1:7. The lowest contamination-mean was 5 ppb B1 in soya bean samples. All samples of horse bean and fish meal were negative. Aflatoxin B1 was present alone so frequently (in 76.2% of the positive samples). The relationship between the concentrations of aflatoxins B2:G1:B1 was 1:2.3:22.4. 51 different samples of foods and feeds from various Egyptian regions were collected and investigated for the nephrotoxic mycotoxin ochratoxin A means TLC. Twelve samples (23.5%) from them were designated as positive samples. The positive samples belonged to white maize, wheat, wheat bran, beans, rice germ, rice germ cake, broilers feed, egg production feed, and milk production feed; whereas the yellow maize (hybrid), soya beans, wheat soya meal, rice crack, cotton seed, cotton seed cake, and fish meal samples were negative. The contamination range was from 4 ppb to 577 ppb with an average of 58.2 +/- 22.9 ppb. Half of the positive samples was contaminated with 10-100 ppb whereas 41.7% from the positive samples had less than 10 ppb and 8.3% only had more than 100 ppb. Citrinin is existing in Egyptian food and feedstuffs. Out of 52 different samples--from various Egyptian regions-15.4% were positive. These were rice bran, rice germ, maize (white), wheat bran, cotton seed cake and fish meal. The highest contamination was in fish meal (40-70 ppb) whereas the lowest was in wheat bran (3 ppb). Mean of the contamination level was 25.9 +/- 3.4 ppb with a range of 3-70 ppb. For the first time in the Egyptian foods and feeds will be informed about the presence of the mycotoxin zearalenone with a high concentration. From several Egyptian places, 64 samples were collected (4 samples for each food or feed stuff).(ABSTRACT TRUNCATED AT 400 WORDS)

Aflatoxins↗
PubMed1992

The influence of contamination with separate mycotoxins (aflatoxins, ochratoxin A, citrinin, patulin, penicillic acid or sterigmatocystin) on the in vitro dry matter and organic matter digestibilities of some roughages (berseem hay and wheat straw).

In vitro study on berseem hay and wheat straw was undertaken to investigate the the effect of mycotoxin contamination on dry matter and organic matter digestibilities. The data revealed a negative effect of most studied mycotoxins on the materials digestibility. Among the investigated mycotoxins, penicillic acid with its two concentrations (5 and 10 nmol) was the most negative, affecting digestibilities of both feed materials. Wheat straw digestibility was more influenced than berseem hay by the ochratoxin A, citrinin and sterigmatocystin (besides the penicillic acid) particularly with their high level (10 nmol). Yet, some mycotoxins act as antibiotics which may affect only the harmful flora but encourage the rumen microflora resulting in slight improvement of digestibility. The rumen conditions were able to metabolize or deform the used levels of all mycotoxins studied. Thus, there were no detectable residues of these mycotoxins in the digestion media after the in vitro fermentation.

Aflatoxins↗
PubMed1998

Aneuploidogenic and clastogenic potential of the mycotoxins citrinin and patulin.

The aneuploidogenic and clastogenic potentials of the mycotoxins citrinin (CIT) and patulin (PAT) were studied by determining inhibition of microtubule assembly under cell-free conditions and by measuring induction of mitotic arrest and micronuclei in cultured Chinese hamster V79 cells. Both CIT and PAT inhibited cell-free microtubule polymerization in a concentration-dependent manner. PAT, but not CIT, bound covalently to reactive thiol groups of microtubule proteins. At concentrations without gross cytotoxicity, mitotic arrest and CREST-positive micronuclei, i.e. micronuclei containing whole chromosomes/chromatids, were induced by CIT and PAT in V79 cells. The time course of micronucleus induction and positive CREST staining indicate the aneuploidogenic potential of CIT and PAT. CREST-negative micronuclei, i.e. micronuclei containing acentric chromosomal fragments, were induced by PAT but not by CIT, implying a clastogenic potential of PAT. The aneuploidogenic and clastogenic potential of PAT and the aneuploidogenic potential of CIT may well contribute to the putative carcinogenicity of these mycotoxins in long-term animal studies.

Aneuploidy↗
PubMed1977

Produciton and biological activity of patulin and citrinin from Penicillium expansum.

Penicillium expansum isolated from meat and apples produced both patulin and citrinin. Toxin identity was confirmed by spectroscopic and physical methods. The mean lethal dose in chicken embryos was determined for toxins administered both singly and in various ratios. Data from simultaneous administration of mycotoxin combinations plotted as isobolograms showed and additive effect. Both toxins were teratogenic in chicken embryos.

Animals↗
PubMed1986

Citrinin mycotoxicosis in the rabbit: ultrastructural alterations.

Citrinin was given to rabbits as a single oral dose of 120 or 67 mg/kg. Rabbits were killed at 4, 6, 8, 10, and 12 hours post dosing, and the kidneys were fixed by intravascular perfusion. Ultrastructural alterations were evident by 4 hours after treatment. In the proximal tubule, alterations were brush border disruption, cytoplasmic rarefaction, and swelling of interdigitating processes. At higher doses, mitochondria were condensed and distorted. Medullary and straight cortical distal tubules had marked distention of the intercellular spaces and disorganization of interdigitating processes. Changes in cortical and outer medullary collecting ducts were similar but less severe. Renal alterations were suggestive of damage to membrane structure and/or transport functions and interference with cellular bioenergetics. Leukocytic infiltration was associated with damaged tubules indicating a contribution of inflammation to the development of the lesions.

Animals↗
PubMed2004

Structurally related mycotoxins ochratoxin A, ochratoxin B, and citrinin differ in their genotoxic activities and in their mode of action in human-derived liver (HepG2) cells: implications for risk assessment.

To elucidate the effects of three structurally related mycotoxins, namely, ochratoxin A (OTA), ochratoxin B (OTB), and citrinin (CIT), on human health, we investigated their acute toxic, mitogenic, and genotoxic effects in the human-derived liver cell line (HepG2). These compounds are found in moldy foods in endemic areas of nephropathy, which is associated with urinary tract cancers. In agreement with previous experiments, we found that OTA causes a dose-dependent induction of micronuclei (MN) and DNA migration in the single-cell gel electrophoresis (SCGE) assay, which was statistically significant at concentrations of > or =5 microg/ml. In contrast, OTB was devoid of genotoxic activity under identical conditions, but the compound caused pronounced inhibition of cell division even at doses lower than OTA (10 microg/ml). CIT caused an effect similar to that of OTA in MN assays (significant at dose levels of > or =2.5 microg/ml) but was negative in the SCGE test. All compounds failed to induce mutations in Salmonella/microsome assays in strains TA 98 and TA 100 after addition of HepG2-derived enzyme homogenate (S9-mix). By use of DNA-centromeric probes we found that induction of MN by OTA involves chromosome breaking effects (55-60% of the MN were centromere negative), whereas CIT-induced MN were predominantly centromere positive (78-82%). Our findings indicate that OTB is devoid of genotoxic activity in human-derived cells and therefore probably not a genotoxic carcinogen in humans. In contrast, CIT was an equally potent inducer of MN in HepG2 cells as OTA, but this effect is caused by a different mechanism, namely, aneuploidy. Furthermore, our data suggest that combined exposure to structurally related mycotoxins that cause DNA damage via completely different mechanisms may significantly increase the cancer risk of humans consuming moldy foods.

Cell Line, Tumor↗
PubMed2004

Effect of citrinin and in association with aflatoxin B(1) on the infectivity and proliferation of Toxoplasma gondii in vitro.

Macrophages exposed to 10 mug/mL citrinin (CTR) or 0.01 mug CTR mixed with 0.04 mug aflatoxin B1 (AFB1) for a period of 2 h at 37 masculine C, were infected with 10(6) Toxoplasma gondii tachyzoites/muL. The parasites were treated with mycotoxins (2 h at 37 masculine C) before being added to the macrophage culture. The number of tachyzoites was quantified 2, 24, 48, 72 and 96 h after infection. During the first 2 hours, 59% infectivity was observed in the control. After exposure to CTR or the mixture of toxins (CTR-AFB1), macrophages were infected with 77.5% and 75% of the inoculated tachyzoites, respectively. Similarly, 72.3% of the cells were infected when cultured together with previously treated parasites. The treatment with CTR-AFB1 gave rise to 2.9 times more tachyzoites than the control at 72 h. An increased number of parasites was recovered from macrophages exposed to CTR after 96 h, and to CTR-AFB1 after 72 h of culture; The number of tachyzoites recovered from the supernatant was 1.94 and 2.06 times higher, respectively, than in the control (5 x 10(5) +/- 0.054 /mL).

Aflatoxin B1↗
PubMed1976

Effects of dietary citrinin and aflatoxin on leg problems of broilers growtn on wire floors.

Dietary levels of citrinin of 62.5, 125 and 500 mug./g. diet or aflatoxin at a dietary level of 2.5 mug./g. of diet fed from 1-21 days of age did not inccrease the incidence of leg problems compared to control groups receiving no dietary nycotoxins. These data indicate that dietary contamination with these toxins does not account for the incidence of occasionally observed leg probelms in younger broiler chicks.

Aflatoxins↗
PubMed1980

Isolation and identification of citrinin produced by Penicillium lanosum.

Penicillium lanosum, when grown on corn, produces a metabolite which increases water intake and excretion by chicks. During a 5-hr test period, chicks fed inoculated corn as the only feed began excreting water within 2 hr and excreted as much as 36 ml, whereas chicks fed untreated corn did not excrete measurable amounts. Chicks fed the inoculated corn drank more water than those fed untreated corn. The metabolite could not be extracted from oven-dried corn but was removed from air-dried corn which had been moistened with acid and extracted with chloroform and then with methanol. It was soluble in 1% sodium bicarbonate and precipitated as yellow cystals when the solution was acidified to pH 1.5. The precipitate was identified as citrinin based on the results of thin layer chromatography, ultraviolet, infrared, mass spectrometer, fluorescence excitation and emission, and nuclear magnetic resonance spectra.

Animals↗
PubMed1977

Inhibition of sterol synthesis by citrinin in a cell-free system from rat liver and yeast.

Citrinin, a fungal metabolite known as an antibiotic, strongly inhibited the labeled acetate incorporation into nonsaponifiable lipids by a cell-free system from rat liver but not the labeled mevalonate incorporation. Of the enzymes involved in cholesterol synthesis, two enzymes, acetoacetyl-CoA thiolase (EC 2.3.1.9) and 3-hydroxy-3-methylglutaryl-CoA reductase (EC 1.1.1.34), were specifically inhibited by the antibiotic. The concentration required for 50% inhibition was 0.2 mM for the former enzyme and 0.5 mM for the latter. Essentially the same results were obtained with a cell-free system from yeast although higher concentrations of the antibiotic were required for inhibition.

Acetyl-CoA C-Acetyltransferase↗
PubMed2005

Serial analysis of gene expression in Monascus aurantiacus producing citrinin.

OBJECTIVE: To construct a tag expression library of Monascus aurantiacus that could produce citrinin maximally on the thirteenth (0.966 mg/mL) day in the submerged culture. METHODS: Total RNA was extracted from the mycelium, cDNA was synthesized using the SuperScript choice system, and then, a SAGE library was successfully constructed according to the MicroSAGE method. RESULTS: Five hundred and ninety eight clones were obtained in SAGE library, and 120 clones were picked out randomly for identification and sequencing purpose. Eighty nine clones had positive inserts, 26 clones had no inserts and the remaining 5 clones had no site of NlaIII enzyme in inserts. There were seven repeated clones. CONCLUSION: With the aid of SAGE2000 software, 901 tags were obtained from 89 clones, representing 686 unique transcripts. Six unique tags of them belong to highly expressed genes (Number of tags > or = 10) and 143 unique tags to moderately expressed genes (repeat tags > or = 2).

Anti-Bacterial Agents↗
PubMedDate not supplied

Synchronous analysis method for detection of citrinin and the lactone and acid forms of monacolin K in red mold rice.

The Monascus product known as red mold rice (RMR) has been found to contain the cholesterol-lowering agent monacolin K (MK), including the lactone form (MKL) and the acid form (MKA) and mycotoxin citrinin (CT). In current studies, CT and MK are usually detected by different analysis methods, which have a high level of error, and are inconvenient, expensive, and time-consuming. The goal of this study is to establish a rapid synchronous analysis method for the detection of CT, MKL, and MKA levels in RMR. In this study, CT, MKL, and MKA are extracted by the same extraction method and are then separated in a reversed-phase high-performance liquid chromatography (HPLC) C18 column. The elution from the C18 column is then passed through an ultraviolet detector and introduced directly into the fluorescence detector. The results show that higher recovery rates of CT, MKL, and MLK are yielded from RMR powder by extracting with 95% ethanol (10 mL) at 60 degrees C for 30 min. Regarding the optimal conditions of HPLC, the peaks of CT, MKL, and MKA can be clearly separated from any noise peaks by isocratic elution with optimum mobile phase, acetonitrile-water-trifluoroacetate (55 + 45 + 0.05, v/v).

Acetonitriles↗
PubMed2006

[The method of the simultaneous determination of the citrinin and ochratoxin A content in cereals and cereal products].

The simplifyed method of simultaneous determination of the citrinin (CT) and ochratoxin A (OTA) in cereals is described. The extraction of mycotoxins was carried out by small volumes of solvents (water/aceton/hexane) without additional sample clean up and concentration. The extracts were analysed by HPLC using mixture of methanol/ethylacetate/phosphoric acid, pH 2.2 as a mobile phase and fluorescence detection (lamda ex 330 nm, lamda em 495 nm). The reliability and reproducibility of results were improved by usage of internal standard--methyl-derivative of ochratoxin A. Average recovery of CT and OTA was 70%, relative standard deviation 12% and 7%, respectively, limit of detection 0.003 mg/kg.

Chromatography, High Pressure Liquid↗