Action of citrinin on bacterial chromosomal and plasmid DNA in vivo and in vitro

Citrinin, a mycotoxin of Penicillium citrinum and other species of the genera Penicillium and Aspergillus, caused the following effects at different concentrations in Escherichia coli. In vivo at 100 micrograms/ml single-strand breaks were caused in the chromosomal DNA. In the presence of 100 micrograms/ml, UV (254 nm)-induced DNA damage was repaired in the bacterial cells without need for a complete growth medium. At 300 micrograms/ml lambda ts prophage was induced in a lysogenic E. coli strain. In an E. coli strain carrying a F' lac plasmid, 4.7% of the cells displayed the Lac- phenotype after treatment with 200 micrograms of citrinin per ml, suggesting elimination of the F' factor. In vitro, DNA repair synthesis was observed at 5 micrograms of citrinin per ml in permeabilized cells, and replicative DNA synthesis was inhibited at 200 micrograms/ml. In these systems synthesis of stable RNAs was slightly diminished at 300 micrograms/ml, and protein synthesis was not affected at concentrations up to 450 micrograms/ml. Lambda and ColE1 plasmid DNA were cleaved in vitro when small amounts of copper ions were present. This DNA-attacking activity was prevented by NADPH, catalase, and superoxide dismutase and by higher concentrations of hydroxyl radical scavengers, suggesting the involvement of free radicals in the mechanism of action of citrinin on DNA.     

Action of citrinin on bacterial chromosomal and plasmid DNA in vivo and in vitro.

Citrinin, a mycotoxin of Penicillium citrinum and other species of the genera Penicillium and Aspergillus, caused the following effects at different concentrations in Escherichia coli. In vivo at 100 micrograms/ml single-strand breaks were caused in the chromosomal DNA. In the presence of 100 micrograms/ml, UV (254 nm)-induced DNA damage was repaired in the bacterial cells without need for a complete growth medium. At 300 micrograms/ml lambda ts prophage was induced in a lysogenic E. coli strain. In an E. coli strain carrying a F' lac plasmid, 4.7% of the cells displayed the Lac- phenotype after treatment with 200 micrograms of citrinin per ml, suggesting elimination of the F' factor. In vitro, DNA repair synthesis was observed at 5 micrograms of citrinin per ml in permeabilized cells, and replicative DNA synthesis was inhibited at 200 micrograms/ml. In these systems synthesis of stable RNAs was slightly diminished at 300 micrograms/ml, and protein synthesis was not affected at concentrations up to 450 micrograms/ml. Lambda and ColE1 plasmid DNA were cleaved in vitro when small amounts of copper ions were present. This DNA-attacking activity was prevented by NADPH, catalase, and superoxide dismutase and by higher concentrations of hydroxyl radical scavengers, suggesting the involvement of free radicals in the mechanism of action of citrinin on DNA.     

Interaction of copper(II) with the fungal metabolite, citrinin.

Copper(II) reacts with citrinin to form 1:1 and 1:2 chelates. The formation constants of these copper(II) chelates have been determined in the solvent 50% (v/v) dioxane-water. Citrinin in the solid state is a p-quinone methide. It may exist as an equilibrium mixture of the p-quinone and o-quinone methides in solution. The experimental evidence indicates that upon chelate formation it exists predominantly in the o-quinone form.     

Combinatory effects of citrinin and ochratoxin A in immortalized human proximal tubule cells

Ochratoxin A (OTA) and citrinin (CIT) are two mycotoxins often occurring together in grains and cereals. Although both are nephrotoxic and can induce apoptosis, combination effects have not been examined up to now. Therefore, the aim of this study was to take a close look at the interactions of citrinin and OTA in cultured human proximal tubule-derived cells (IHKE cells). The cytotoxicity of both mycotoxins was studied, measuring the metabolic activity and the cell number. Furthermore, caspase 3-activation as a marker for apoptosis was examined for both mycotoxin alone and in combination. The results show that citrinin had an antagonistic effect on ochratoxin A induced caspase 3-activation in concentrations of 2.5 and 5 μmol/l. Higher concentrations (7.5 and 15 μmol/l) lead to additive effects, lower citrinin concentrations (0.25 and 1 μmol/l) did not show any effect at all. The observed decrease in caspase 3-activity was specific for the combination with OTA, since the combination of citrinin with cisplatin did not show any effect. Citrinin did not influence of the OTA-induced apoptosis when added two hours after applying ochratoxin A. Also the combination of both toxins decreased the uptake of OTA into the cells which might be an explanation for the antagonistic effect of citrinin in certain concentrations. However, the transport into cells can not be the only explanation. so further examinations are necessary. Presented at the 27^th Mykotoxin-Workshop. Dortmund, Germany, June 13–15, 2005.     

The effect of culture preservation techniques on patulin and citrinin production by Penicillium expansum Link

AIMS: To study the influence of culture preservation methods and culture conditions on the production of the mycotoxins patulin and citrinin by Penicillium expansum. METHODS AND RESULTS: Ten strains of Penicillium expansum were preserved using subculture and maintenance at 4 degrees C, mineral oil, drying on silica gel and freeze-drying. Patulin and citrinin production was assessed on yeast extract sucrose agar (YES) and grape juice agar (GJ), using TLC before and after 0.5, 2-3, 6 and 12 months preservation. Citrinin was detected in all cultures for all preservation techniques on YES. The patulin profiles obtained differed with strain and culture media used. CONCLUSIONS: Citrinin production seems to be a stable character for the tested strains. There is a tendency for patulin detection with time apparently more consistent for silica gel storage and freeze-drying, especially when the strains are grown on GJ. SIGNIFICANCE AND IMPACT OF THE STUDY: Variability in the profiles of the mycotoxins tested seems to be more strain-specific than dependent on the preservation technique used.     

Production of lipase free of citrinin by Penicillium citrinum

Lipase (Glycerol ester hydrolase E.G. 3.1.1.3) from a Brazilian strain of Penicillium citrinum free of the mycotoxin citrinin has been investigated. Citrinin production was inhibited by using culture medium containing olive oil, soybean oil and corn oil as carbon sources. Potassium concentration and pH play an important role in citrinin production. Potassium concentration lower than 30 mM and pH below 4.5 inhibited the mycotoxin production. P. citrinum produced lipase free of extraneous proteins and citrinin when cultured using, as nitrogen source, ammonium sulphate (lipase activity of 7.88 U/mg) and yeast extract (lipase activity of 4.95 U/mg) with olive oil as carbon source. This data is relevant to the larger scale production of lipases for food technology applications, from Penicillium citrinum.     

食品中桔霉素控制方法的研究进展

桔霉素(Citrinin,CIT)是由青霉、曲霉和红曲霉属产生的一种具有肾毒性的真菌毒素。许多食品和饲料中均含有桔霉素,污染范围十分庞大。桔霉素可与其他真菌毒素发生协同作用,如展青霉素(Patulin,PAT)、赭曲霉毒素(Ochratoxin,OTA)等,从而增强其毒性作用,对人及动物健康造成更大的危害。现阶段常用的控制手段主要有物理、化学和生物方法,均取得了一定的成就。本文简单介绍了桔霉素的毒性及污染状况,对桔霉素控制方法的研究进展进行了综述。     

Detection of citrinin in ochratoxin A-containing products by a new HPLC method

A new method for citrinin was developed and validated, which is based on solid phase extraction with polyamide columns and HPLC with fluorescence detection. Sufficient skill with the method given, precise results, i.e. variation coefficients <10%, will be achieved. The mean recovery rates were in the range 74 – 90%. The detection limits of the method determined according to DIN 32645, at good precision, were 1 μg/kg for wheat, rye, barley, maize, and oats. The analysis of several samples containing ochratoxin A (OTA) showed that citrinin is present in brans, wheatings and shorts containing a higher ratio of the outer layers of the grain kernel; both OTA and citrinin were found in in cocoa shells and raisins. Citrinin was detected in 14 OTA-containing samples (1–8 μg/kg). Furthermore, it was demonstrated that citrinin also can be determined in red mold rice according to the new method. Presented at the 25^th Mykotoxin Workshop in Giessen, Germany, May 19–21, 2003     

Effects of citrinin on iron-redox cycle

The ability of the mycotoxin citrinin to act as an inhibitor of iron-induced lipoperoxidation of biological membranes prompted us to determine whether it could act as an iron chelating agent, interfering with iron redox reactions or acting as a free radical scavenger. The addition of Fe3+ to citrinin rapidly produced a chromogen, indicating the formation of citrinin-Fe3+ complexes. An EPR study confirms that citrinin acts as a ligand of Fe3+, the complexation depending on the [Fe3+]:[citrinin] ratios. Effects of citrinin on the iron redox cycle were evaluated by oxygen consumption or the o-phenanthroline test. No effect on EDTA-Fe2+-->EDTA-Fe3+ oxidation was observed in the presence of citrinin, but the mycotoxin inhibited, in a dose-dependent manner, the oxidation of Fe2+ to Fe3+ by hydrogen peroxide. Reducing agents such as ascorbic acid and DTT reduced the Fe3+-citrinin complex, but DTT did not cause reduction of Fe3+-EDTA, indicating that the redox potentials of Fe3+-citrinin and Fe3+-EDTA are not the same. The Fe2+ formed from the reduction of Fe3+-citrinin by reducing agents was not rapidly reoxidized to Fe3+ by atmospheric oxygen. Citrinin has no radical scavenger ability as demonstrated by the absence of DPPH reduction. However, a reaction between citrinin and hydrogen peroxide was observed by UV spectrum changes of citrinin after incubation with hydrogen peroxide. It was also observed that citrinin did not induce direct or reductive mobilization of iron from ferritin. These results indicate that the protective effect on iron-induced lipid peroxidation by citrinin occurs due to the formation of a redox inactive Fe3+-citrinin complex, as well as from the reaction of citrinin and hydrogen peroxide.     

Pigments and citrinin biosynthesis by fungi belonging to genus Monascus

Citrinin is a mycotoxin, which is produced by fungi belonging to the genus Monascus, known in biotechnology as producers of azaphilone pigments. The relation between biosynthesis of these secondary metabolites was investigated in different species of the genus Monascus in batch-culture at the following cultivation conditions: T = 28 degrees C, agitation 220 rpm, and a medium, which induce citrinin production, containing ethanol as a carbon source. The screening was carried out with 16 fungal strains and the biosynthesis of citrinin and pigments was monitored quantitatively at the standard conditions mentioned above. Some kinetic parameters of the process have been determined. The values of the growth yield coefficient Y(X/C) were between 0.32 and 0.57. The amount of the extracellular red and orange pigments at the end of cultivation varied for the different strains between 0.09 and 1.33 OU/ mg dry weight, and 0.15 and 0.96 OU/mg dry weight, respectively. The amount of the total pigments measured was between 0.16 and 3.6 OU/mg dry weight, and between 0.21 and 3.39 OU/mg dry weight. The determined ratio 500 nm/400 nm, characterizing the pigment production, ranged between 0.60 and 1.06. Twelve of the investigated strains produced citrinin and pigments, two of them produced only pigments. Two strains were not able to produce neither pigments nor citrinin. Thus, the biosynthesis of citrinin appeared to be strain-specific and does not correlate with the pigments' biosynthesis by the fungal strains belonging to the genus Monascus.     

An indirect enzyme immunoassay for the mycotoxin citrinin.

An indirect competitive enzyme immunoassay using rabbit antisera could detect citrinin in buffer solutions at 1 to 13 ng/ml (0.05 to 0.65 ng per assay). Cross-reactivity with austdiol, alternariol, ochratoxin A, and deoxynivalenol was < 0.1% relative to citrinin. Recovery of citrinin added to wheat flour at 200 to 2,000 ng/g was 89 to 104%, with a coefficient of variation of 6.9 to 13%.     

Redoxcitrinin, a biogenetic precursor of citrinin from marine isolate of fungus Penicillium sp

A chemical analysis of the fermentation of the marine-derived fungus Penicillium sp. led to the isolation of a biogenetic precursor of citrinin, redoxcitrinin (1), together with polyketide mycotoxins, phenol A (2), citrinin H2 (3), 4-hydroxymellein (4), citrinin (5), and phenol A acid (6). The structures of compounds 1-6 were determined on the basis of physicochemical data analyses. Among them, compounds 1-3 exhibited a potent radical scavenging activity against 1,1-diphenyl-2-picrylhydrazyl (DPPH) with IC50 values of 27.7, 23.4, and 27.2 microM, respectively.     

Angelmarin, a novel anti-cancer agent able to eliminate the tolerance of cancer cells to nutrient starvation

The CH(2)Cl(2)-soluble extract of Angelica pubescens was found to kill PANC-1 cancer cells preferentially under nutrition starvation at a concentration of 50 microg/ml, with virtually no cytotoxicity under nutrient-rich conditions. Further bioassay-guided fractionation and isolation led to the isolation of a novel compound named angelmarin as the primary compound responsible for the preferential cytotoxicity; the compound exhibited 100% preferential cytotoxicity against PANC-1 cells at a concentration of 0.01 microg/ml.     

Stability of citrinin and deoxynivalenol during germination process of barley

The stability of citrinin and deoxynivalenol during germination process of barley spiked with these mycotoxins at a level of 2/ig/g was investigated. Germinated barley was analyzed after 1, 3, and 5 days to follow the stability of citrinin and deoxynivalenol during the germination process. Two thin layer chromatographic methods were used for determinations of citrinin and deoxynivalenol. An average of 93.6% of citrinin and 77.1% of deoxynivalenol were destroyed within 5 days during the germination process of barley.     

Anti-enterovirus activity and structure-activity relationship of a series of 2,6-dihalophenyl-substituted 1H,3H-thiazolo[3,4-a]benzimidazoles

Despite the fact that enteroviruses are implicated in a variety of human diseases, there is no approved therapy for the treatment of enteroviral infections. Here, a series of 2,6-dihalophenyl-substituted 1H,3H-thiazolo[3,4-a]benzimidazoles with anti-enterovirus activity is reported. The compounds elicit potent activity against coxsackievirus A9, echovirus 9 and 11 and all six strains of coxsackievirus B. A structure-activity relationship analysis revealed that the presence of substituents at position 6 of the tricyclic system positively influences the antiviral activity, whereas substitutions at position 7 are less favorable. In particular a 6-trifluoromethyl substitution leads to a substantial improvement of the antiviral activity as compared to the unsubstituted structure. Furthermore, an additional introduction of a 2-Cl, 6-F substitution on the phenyl at C-1 results in a further increase of the antiviral activity. Hence, 1-(2-chloro-6-fluorophenyl)-6-trifluoromethyl-1H,3H-thiazolo[3,4-a]benzimidazole results in a dose-dependent inhibition of viral replication with a 50% effective concentration (EC50) of 0.41 microg/ml without any detectable cytotoxicity at the highest concentration (100 microg/ml) tested.     

Antiproliferative effects of two amides, piperine and piplartine, from Piper species

The present work evaluated the cytotoxicity of piplartine {5,6-dihydro-1-[1-oxo-3-(3,4,5-trimethoxyphenyl)-trans-2-propenyl]-2(1H)pyridinone} and piperine {1-[5-(1,3)-benzodioxol-5-yl)-1-oxo-2,4-pentadienyl]piperidine}, components obtained from Piper species. The substances were tested for their cytotoxicity on the brine shrimp lethality assay, sea urchin eggs development, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay using tumor cell lines and lytic activity on mouse erythrocytes. Piperine showed higher toxicity in brine shrimp (DL50 = 2.8 +/- 0.3 microg/ml) than piplartine (DL50 = 32.3 +/- 3.4 microg/ml). Both piplartine and piperine inhibited the sea urchin eggs development during all phases examined, first and third cleavage and blastulae, but in this assay piplartine was more potent than piperine. In the MTT assay, piplartine was the most active with IC50 values in the range of 0.7 to 1.7 microg/ml. None of the tested substances induced hemolysis of mouse erythrocytes, suggesting that the cytotoxicity of piplartine and piperine was not related to membrane damage.     

Does the mycotoxin citrinin function as a sun protectant in conidia from Penicillium verrucosum

Our results demonstrate high concentrations of the UV absorbing mycotoxin citrinin in the outer layer of spores from three citrinin-producing strains of Penicillium verrucosum, which is released in an aqueous environment. An important function of the toxin could be to act as a sun protectant in order to create favorable conditions during the initial germination process. When spores from these strains of P. verrucosum were examined by confocal microscopy, a clearly visible fluorescent layer associated with the cell wall was observed. The strains were grown on agar plates, and the mycelial mat was washed with saline. This suspension contained at least 95% of the spores and particulate material, which was removed by filtration after counting the conidia. An aliquot of this filtrate was extracted and citrinin was purified by high pressure liquid chromatography. The absorbance at 319 nm was used to calculate the amount of UV absorbing material released from the spores. Based on the spore numbers in the suspension of the saline extract, we estimated that this material released was 1.4–4.1 pg per spore or 8–24% of the spore weight. Citrinin (and minor amounts of ochratoxin A and some other unidentificable fluorescent compounds) were observed in the filtrate when subjected to thin layer chromatography. This revised version was published online in June 2006 with corrections to the Cover Date.     

Natural occurrence of ochratoxin A and citrinin in food stuffs in Egypt

Natural occurrence of ochratoxin A (OA) and citrinin in cereals (274 samples) and animal tissues (250 samples) have been investigated during a period of more than 2 years. OA was found in cereals and animal tissues while citrinin was found in cereals only. The highest level of OA (up to 80.0 μg/kg) was found in yellow corn, 52.8% of contaminated samples while respectively 55.9% and 39.4% of barley and rice samples were contaminated with citrinin, with the highest level up to 100.0 and 27.92 μg/kg for barley and rice respectively. The frequent contamination of animal kidney with OA (28% positive out of 150 tested) average concentration 12.33 μg/kg. 2% of liver and 4% of muscles tissue were observed.