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Survey of the mycoflora and mycotoxins of cotton seeds and cotton seed products in Egypt.

Thirty-nine species and 16 fungal genera were isolated from Egyptian cotton seeds, cotton seed meal and cotton seed cake on 1% glucose-Czapek's agar medium incubated at 28 degrees C. Aspergillus was the most frequent genus and it emerged in 87-100% of the samples contributing 70-98% of total fungi in the three substrates tested. The most common species were A. niger, A. flavus, A. fumigatus, A. terreus and Rhizopus stolonifer; A. niger, A. fumigatus and Penicillium corylophilum; and A. niger, A. flavus, A. terreus, A. nidulans and Rhizopus stolonifer, respectively. Cotton seeds and cotton seed products were naturally contaminated by aflatoxin B1 and B2. About 16% of the different substrates tested were positive for aflatoxin contamination. No citrinin, ochratoxin A, patulin, sterigmatocystin, diacetoxyscirpenol, T-2 toxin or zearalenone were detected in the samples assayed.

Aspergillus↗

Mycoflora and mycotoxins of peanut (Arachis hypogaea L.) seeds in Egypt. 1--Sugar fungi and natural occurrence of mycotoxins.

Sixty-four species and 2 varieties which belong to nineteen genera of fungi were identified from 40 peanut seed samples collected from different places in Egypt by using a dilution-plate method on glucose-Czapek's medium. The most frequent genera were Aspergillus (21 species & 2 varieties), Penicillium (16 species) and Fusarium (6 species). A. flavus, A. fumigatus, A. niger, P. chrysogenum and F. oxysporum were the most common fungal species. Forty seven percent of the samples proved to be toxic to brine shrimp (Artemia salina) larvae. Thin-layer chromatographic analysis revealed that peanuts were contaminated by aflatoxins (11 samples), trichothecene-toxins (10 samples), zearalenone (one sample) and citrinin (one sample). We believe that this is the first report of the natural occurrence of zearalenone and trichothecene-toxins in peanuts.

Animals↗

Mycoflora and mycotoxins of peanut (Arachis hypogaea L.) seeds in Egypt. III. Cellulose-decomposing and mycotoxin-producing fungi.

From 40 peanut seed samples collected in Egypt, forty-three species and one variety of fungi, belonging to 16 genera, were collected. The most dominant genera were Aspergillus (11 species + one variety), Penicillium (11 species) and Fusarium (4 species). From the preceding genera A. fumigatus, A. flavus, A. niger, P. chrysogenum and F. oxysporum were the most frequent species. Forty-nine isolates belonging to 12 species and one variety were tested for production of mycotoxins, after growth on liquid medium containing two carbon sources (sucrose or cellulose). Thin layer chromatographic analysis revealed that the quality and quantity of mycotoxins was higher on sucrose than cellulose. Mycotoxins identified were aflatoxins B1, B2, G1 & G2, citrinin; fumagillin; diacetoxyscirpenol T-2 toxin; satratoxin H; and zearalenone.

Arachis↗

Mycotoxin formation in moist wheat under controlled temperatures.

One-kilogram parcels of wheat with 20.5% moisture content were maintained at 15 degrees and 22 degrees C for 10 weeks to study quality changes. Temperature, moisture, oxygen and carbon dioxide levels, microflora incidence and abundance, seed germination, fat acidity values, aflatoxins, sterigmatocystin, ochratoxin A, penicillic acid, citrinin and zearalenone were monitored. By two weeks, trace levels of ochratoxin had formed at both temperatures. By 10 weeks, the wheat contained at least three times more ochratoxin A at 22 degrees C than at 15 degrees C. Strains of Penicillium verrucosum var. cyclopium were associated with ochratoxin A production. No other mycotoxins were detected. The effect of temperature was significant for all variables (greater effect at 22 degree C) except A. glaucus gr. and Penicillium (P less than .01). The effect of time was significant for all variables except bacteria (P less than .01). The shape of the response was fully characterized by the linear and quadratic terms, except for % moisture which was linear only, and for bacteria for which time was not significant. The interaction between time and temperature was significant (P less than .01) for total fungal propagule count, % moisture, and Aspergillus versicolor, indicative of the steeper rise in slope for 22 degrees C.

Food Contamination↗

Chromatography of mycotoxins on precoated reverse-phase thin-layer plates.

Reverse-phase thin-layer chromatography of 18 common mycotoxins was carried out on Merck C18 and Whatman C18 and diphenyl bonded phases. The mobile phases included neutral and acidified binary mixtures of water and methanol, acetonitrile or tetrahydrofuran. Mycotoxins studied included aflatoxins B1, B2, G1, G2, sterigmatocystin, ochratoxin A, citrinin, penicillic acid, patulin, zearalenone, and the trichothecenes diacetoxyscirpenol, HT-2 toxin, nivalenol, neosolaniol, fusarenone-X, T-2 toxin, deoxynivalenol and 3-acetyl-deoxynivalenol. Both C18-bonded phases performed well, and could serve as a convenient confirmation for mycotoxins appearing in normal-phase (silica) thin-layer chromatographic screening procedures.

Chromatography, Thin Layer↗

High-performance liquid chromatographic screening method for mycotoxins using new retention indexes and diode array detection.

A reversed-phase high-performance liquid chromatographic (RP-HPLC) screening method for the determination of mycotoxins has been developed. The toxins were characterized by retention indexes and on-line UV spectra produced with a diode array detector (DAD). Retention indexes of mycotoxins exhibiting a wide range of polarities and chemical structures were determined during linear gradient elution with an acetonitrile/water solvent system. The retention index scale was based on the use of a new homologous series of 1-[4-(2,3-dihydroxypropoxy)phenyl]-1-alkanones (D-compounds) as internal standards. The mycotoxins studied were: five trichothecenes of group A: T-2 toxin (T-2), HT-2 toxin (HT-2), diacetoxyscirpenol (DAS), neosolaniol (NEO), and an isomer of neosolaniol (NEO isomer); three trichothecenes of group B: nivalenol (NIV), deoxynivalenol (DON), and fusarenon-X (FUS-X); four aflatoxins: B1, B2, G1, and G2; and five other mycotoxins: sterigmatocystin (STE), zearalenone (ZEA), ochratoxin A (OCH A), citrinin (CIT), and patulin (PAT).

Chemical Phenomena↗

Fungal flora and mycotoxins of six kinds of nut seeds for human consumption in Saudi Arabia.

A wide range of moulds representing several genera and species, was recorded in this study from 5 seed samples of each almond, cashew nut, chestnut, hazelnut, pistachio nut and walnut collected from different markets in Ar' Ar, Saudi Arabia. The total counts of fungi were widely fluctuated between 1960-7704 and 1948-7434 colonies/g dry seeds on glucose-Czapek's and glycerol agar media at 28 degrees C, respectively, and represented twenty genera, 53 species and 2 varieties of fungi. The prevalent fungi on the 2 agar media were Aspergillus flavus, A. niger and Penicillium chrysogenum. On glucose-Czapek's agar, Rhizopus stolonifer and Aspergillus flavus var. columnaris were isolated from all 6 kinds of nut, A. parasiticus from 5 kinds and A. fumigatus from 4 kinds with high frequencies. Eurotium species were completely absent on glucose-Czapek's agar but they were isolated in high frequency from all kinds of nut on glycerol agar medium. The different nut samples were analyzed by thin layer chromatography for the presence of aflatoxins B1, B2, G1 & G2, citrinin, ochratoxins, patulin, sterigmatocystin, diacetoxyscirpenol, T-2 toxin and zearalenone. Aflatoxins B1 & G1 were detected in 3 out of the 5 samples tested of chestnut at concentrations ranging between 20 to 60 micrograms/kg. All other samples of almond, cashew nut, hazelnut, pistachio nut, and walnut that were analyzed were mycotoxin free.

Food Microbiology↗

Natural occurrence of mycotoxins in different spices in Egypt.

A total of 120 different samples belonging to 24 kinds of species collected from different places at Assiut Governorate (Egypt) were examined for the natural occurrence of mycotoxins. TLC analysis of spice extracts revealed the presence of aflatoxins (8-35 micrograms/kg) in 16 samples of anise, black pepper, caraway, black cumin, fennel, peppermint, coriander and marjoram, sterigmatocystin (10-23 micrograms/kg) in ten samples of red pepper, caraway, cumin and marjoram and citrinin (8-12 micrograms/kg) in two samples of black cumin, while ochratoxin A and zearalenone could not be detected.

Aflatoxins↗

Mycotoxin-producing potential of fungi associated with qat (Catha edulis) leaves in Yemen.

Forty-four fungal species belonging to 20 genera were isolated from 30 samples of qat leaves. The most frequent genera were Aspergillus, Alternaria, Penicillium, and Cladosporium followed by Fusarium, Drechslera, Chaetomium, and Mucor. The most prevalent species in above genera were Aspergillus niger, A. flavus, A fumigatus, Alternaria alternata, Penicillium chrysogenum, P. citrinum, Cladosporium cladosporioides, and Fusarium verticillioides. From these fungi, 17 species (39%) related to 7 genera (35%) proved to be true endophytes. Eleven out of 75 isolates were mycotoxigenic. A. alternata produced alternariol and alternariol monomethyl ether whereas A. flavus produced aflatoxins B1 and B2. Ochratoxin A, sterigmatocystin, citrinin and T-2 toxin were produced by A. ochraceus, A. versicolor, P. citrinum and F. oxysporum, respectively. The presence of such toxigenic fungi associated with qat leaves is considered to be a threat to public health.

Catha↗

Detection of ochratoxin A-induced DNA damage in MDCK cells by alkaline single cell gel electrophoresis (comet assay).

The mycotoxin ochratoxin A (OTA), a widespread contaminant of food and feedstuffs, is nephrotoxic, immunosuppressive and carcinogenic in domestic and laboratory animals. Additionally, it is suspected as being responsible for urinary tract tumours in patients suffering from Balkan endemic nephropathy. Moreover, evidence has accumulated that OTA is a genotoxic carcinogen, although the mechanism that results in DNA damage has not been fully resolved. In this study, the induction of DNA damage by OTA and the subsequent DNA repair was investigated by alkaline single cell gel electrophoresis (comet assay) in cells originally derived from the kidney, a target organ of OTA. With modifications of the method, the influence of OTA uptake into the cells and of DNA repair on the genotoxic effect of OTA should be investigated. In Madin-Darby canine kidney (MDCK) cells, OTA induced single-strand breaks in a concentration dependent manner. When an external metabolising enzyme system (S9-mix from rat liver) was added, this genotoxic effect was significantly stronger. By co-incubation with methotrexate or with the mycotoxin citrinin, a substrate of the organic anion transporter, the adverse effect of OTA was inhibited. When DNA repair was inhibited by addition of cytosine arabinoside and hydroxyurea, the tail length increased dramatically and all treated cells showed single-strand breaks. A further culture of the damaged cells in the absence of any supplement resulted in a complete repair of the DNA damage within 2 h. Adverse effects on the mechanisms of DNA repair, or exposure to OTA in periods of reduced DNA repair capacity may influence the genotoxic potency of OTA and have to be regarded as a further mechanism by which genotoxic effects of OTA can be performed.

Animals↗

In vivo hypolipidemic effects and safety of low dosage Monascus powder in a hamster model of hyperlipidemia.

Monascus or more commonly known as red mold rice is fermented rice on which Monascus purpureus has been grown. It has been a traditional Chinese food additive for thousands of years in China. Secondary metabolite product of Monascus, monacolin K, has been proven that it could be used as an antihypercholesterolemic agent. In this study, M. purpureus NTU568 mutated and selected from a monacolin K productivity strain-M. purpureus HM105 produced high quantities of monacolin K at a level of 9,500 mg kg(-1). This research focused on the effect of adding red mold rice powder of M. purpureus NTU568 to a hamster diet on total cholesterol (TC), triglyceride (TG), high-density lipoprotein cholesterol, and low-density lipoprotein cholesterol (LDL-C). In the results, the oral administration of Monascus powder in hyperlipidemia hamster was indeed proven to decrease TC, TG, and LDL-C levels. Plasma TC levels in hamster fed with Monascus powder at one-fold dosage [10.78 mg (day 100 g bw)(-1)] for 4 and 8 weeks were significantly lower (31.2 and 22.0%, respectively) than that in hyperlipidemia hamster. Plasma TG (30.1 and 17.9%) and LDL-C levels (36.0 and 20.7%) were also significantly lowered by feeding Monascus powder at one-fold dosage for 4 and 8 weeks compared to hyperlipidemia hamster. In addition, examinations of liver TC and TG levels of hyperlipidemia hamster were also performed and showed similar effects on lipid-lowering action by oral administration of Monascus powder. Since citrinin is a mycotoxin that possesses nephrotoxic and hepatoxic effects, it has a negative impact on the safety of red mold rice for people. This study examined the liver somatic index [plasma glutamyl oxaloacetic transaminase (GOT) and glutamyl pyruvic transaminase (GPT) levels] and liver biopsy to investigate whether Monascus powder induced damage in liver. It was found that the plasma GOT and GPT levels were not significantly increased by feeding Monascus powder. There was no difference in the results of the liver biopsy between the Monascus powder-treated groups and the control group.

Animals↗

Chemical response of Picea glehnii seed-epiphytic Penicillium species to Pythium vexans under in vitro competitive conditions for mycelial growth.

The potential protection of Picea glehnii seedlings from damping-off by seed-epiphytic Penicillium species was investigated. We studied the chemical response of seed-epiphytic Penicillium species (Pen. cyaneum, Pen. damascenum, and Pen. implicatum) to Pythium vexans, a damping-off fungus, in vitro. Penicillium species were cultured singly or cocultured with Pyt. vexans for 14 or 18 d, and mycelial growth, pH of culture filtrate, antifungal activity of the culture filtrate against Pyt. vexans, and the amount of antifungal compound produced by each Penicillium species, were examined. The filtrate of both the single culture of Penicillium and the coculture of Penicillium and Pyt. vexans showed antifungal activity against Pyt. vexans. In a coculture with Pyt. vexans, Pen. cyaneum produced an antifungal compound (patulin) as in the single culture. Pen. damascenum cocultured with Pyt. vexans produced an antifungal compound (citrinin), as it did in the single culture and in larger amounts on day 10. Pen. implicatum produced two antifungal compounds, frequentin and palitantin, and the ratio of frequentin (with higher antifungal activity than palitantin) to palitantin was higher in the coculture with Pyt. vexans than in the single culture. Our results indicate that these Penicillium species have the ability to produce antifungal compounds and to keep anti-fungal activity under competitive condition with Pyt. vexans. The chemical response of these Penicillium species to Pyt. vexans may contribute to protect P. glehnii seedlings from damage by Pyt. vexans.

Antifungal Agents↗

Fungal populations and mycotoxins in silage in Assiut and Sohag governorates in Egypt, with a special reference to characteristic Aspergilli toxins.

Forty silage samples were collected from Assiut and Sohag governorates in Egypt to measure the presence of fungal population in silage. Forty-three species and 2 species varieties belonging to 17 genera were isolated using glucose Czapek's and Sabouraud's dextrose agar media at 28 degrees C. The most prevalent genera were Aspergillus (57.5 and 100 of the samples), Penicillium (100 and 55%) on the two mentioned media, respectively. Also, Fusarium oxysporum and Gibberella fujikurori were recovered in moderate incidences. Mycotoxin profiles were also determined in these samples: Aflatoxins showed the highest incidence rates of occurrence, it occurred in 22.5% of all samples analyzed. Other mycotoxins were detected from all samples (T2 toxins and sterigmatocystin at incidence of 7.5 and 5%, respectively). The screening of the characteristics mycotoxins of different isolates of Aspergillus isolated from silage samples was tested. The results clarified that some mycotoxins (aflatoxins-aspergillic acid-beta nitro propionic acid-cyclopiazonic acid-kojic acid and sterigmatocystin) were produced by some isolates of A. flavus. Some isolates of A.fumigatus could produce gliotoxin and verrucologen. All of A. niger isolates tested were able to produce kojic acid. One isolate of A. ochraceous formed ochratoxin A and other isolate produced penicillic acid. Concerning A. terreus isolates, the results showed that 5 isolates were able to produce citrinin and 4 isolates had ability to produce patulin. A. versicolor isolates showed the ability to produce ochratoxin A.

Animals↗

Structure-activity studies in E. coli strains on ochratoxin A (OTA) and its analogues implicate a genotoxic free radical and a cytotoxic thiol derivative as reactive metabolites.

Ochratoxin A (OTA), its major metabolite in rodents, ochratoxin alpha, and seven structurally related substances were assayed for SOS DNA repair inducing activity in Escherichia coli strain PQ37. At concentrations of 0.1-4 mM, OTA, chloroxine, 5-chloro-8-quinolinol, 4-chloro-meta-cresol and chloroxylenol induced SOS DNA repair in the absence of an exogenous metabolic activation system. Ochratoxin B, ochratoxin alpha, 5-chlorosalicylic acid and citrinin were inactive, but all except ochratoxin alpha were cytotoxic. Thus, the presence of chlorine at C-5 appears to be one determinant of genotoxicity in these substances. Amino oxyacetic acid, an inhibitor of the cysteine conjugate beta-lyase, decreased the cytotoxicity of OTA but did not alter its genotoxic activity, suggesting the formation of a cytotoxic thiol-containing derivative. The mechanisms by which OTA and some of its active analogues induce SOS DNA repair activity was further investigated in E. coli PQ37 and in three derived strains (PQ300, OG100 and OG400), containing deletions within the oxy R regulon. The response in strain PQ37 was measured in the absence and presence of Trolox C, a water-soluble form of vitamin E. Trolox C completely quenched the genotoxicity of OTA, and the effect was similar in the mutant and wild-type strains. These results implicate an OTA-derived free radical rather than reduced oxygen species as genotoxic intermediate(s) in bacteria.

Escherichia coli↗

Mutagenicity to Salmonella typhimurium of some Aspergillus and Penicillium mycotoxins.

17 mycotoxins produced by various Aspergillus and Penicillium species were screened for their mutagenic activity to Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537, both with and without metabolic activation. Austdiol, austocystins A and D, kojic acid and viridicatumtoxin were found to be mutagenic after metabolic activation, while austdiol was also mutagenic per se. Aflatoxin B1, sterigmatocystin and versicolorin A, which were used as positive controls were also mutagenic. No mutagenic activity was evident in the case of citrinin, cyclopiazonic acid, fumitremorgen B, griseofulvin, luteoskyrin, O-methylsterigmatocystin, mycophenolic acid, ochratoxin A, patulin, penicillic acid, secalonic acid D and TR2-toxin. A good relationship was found between the mutagenic activity, or lack of it, of most of the mycotoxins with existing data on carcinogenicity. Inadequate information on the carcinogenicity of austdiol, austocystins A and D, kojic acid and viridicatumtoxin precluded correlations with mutagenicity to S. typhimurium. The relationship between chemical structure and mutagenicity of the mycotoxins is discussed.

Aspergillus↗

Mycotoxin-producing potential of fungi isolated from amaranth seeds in Argentina.

To evaluate the potential for mycotoxin production by molds in amaranth grains, the mycoflora was determined both before and after surface disinfection on dichloran-chloramphenicol-peptone agar (DCPA) and dichloran-18% glycerol agar (DG18). On both media Aspergillus, Penicillium, Fusarium and Alternaria were the predominant genera. A flaus and A. parasiticus were the Aspergillus species most frequently isolated. P. chrysogenum was the species most common among the penicillia. F. equiseti was the predominant Fusarium species. Isolates of Aspergillus, Penicillium and Fusarium were screened for mycotoxin production on sterile rice substrate and also using a simple screening method. Toxinogenic strains of A. flavus and A. parasiticus (aflatoxins), A. versicolor (sterigmatocystin), P. citrinum (citrinin), P. viridicatum (penicillic acid), F. moniliforme, F. equiseti and F. semitectum (zearalenone), were encountered. The simple screening method for toxinogenic molds showed good performance for the detection of molds producing aflatoxins and zearalenone compared with mycotoxins production on the natural substrate.

Aflatoxins↗

In vivo and in vitro changes in renal function caused by ochratoxin A in the rat.

Ochratoxin A has been suggested to be an important, environmentally prominent nephrotoxin. Possible involvement in both human and animal disease states has been indicated. This study was designed to examine under controlled laboratory conditions, the nephrotoxic potential of ochratoxin A in the rat. Daily administration of ochratoxin A in doses of 0.75 and 2.0 mg/kg caused persistent urinary hypoosmolality coupled with excessive glucose and protein excretion. In addition the renal slice transport of several organic compounds also was altered. Renal slice tissue electrolytes and water were unaffected by ochratoxin A pretreatment. Direct addition of ochratoxin A to fresh renal cortex slices also depressed transport of some organic compounds. Although rats pretreated with ochratoxin A lost large amounts of body weight, the weight loss alone did not account totally for the changes in renal function. These data indicate that ochratoxin A has the capability of producing alterations in renal function suggestive of nephrotoxicity. However, the pattern of response is different than that observed with citrinin.

Aminobutyrates↗

Potential involvement of renal transport mechanisms in nephrotoxicity.

Among the various physiological factors involved in the development of a nephrotoxic insult, certain renal transport systems may be important. The movement of exogenous organic anions and cations from the blood to the tubular fluid is well recognized. The anion transport system, which has been extensively characterized for the transport of p-aminohippuric acid, may have particular relevance. The nephrotoxic effect of citrinin, certain cephalosporin antibiotics and cysteine conjugates in rats and in isolated renal cells can be blocked by probenecid, a drug known to block the organic anion transporter competitively. The reduction in toxic response is correlated with the renal and cellular content of the nephrotoxic chemical.

Animals↗