Natural toxins最新文献

Monk seals in Cape Blanc (Western Sahara coast) suffered a mass mortality during May-July 1997 which was attributed to a morbillivirus. High performance liquid chromatography (HPLC) analysis on tissues of seals killed during the outbreak and on related fauna showed peaks with retention times coincident with those of some saxitoxin derivatives but their identity was not proved. Here we present results of further HPLC analyses that unambiguously prove the identity of these toxins by mass spectrometry (MS), supporting the hypothesis that this mortality of monk seals was caused by biotoxins rather than by a morbillivirus.

HPLC and Inhibitory Indirect ELISA (I.I. ELISA) methods for quantitation of aflatoxins (AF) in human urine were compared in terms of specificity, sensitivity, easiness and cost. I.I. ELISA was optimized in kind of antibody in use, type of plastic plate, adduct synthesis technique, peroxidase and antibody dilutions, etc. Both polyclonal (Cuban) and monoclonal (British) anti-AF antibodies were statistically studied and the process was standardized. HPLC and electrophoresis were performed while synthetizing AFB(1)-DNA and AFB(1)-Cl-Ovalbumin (AFB(1)-Cl-Ov) adducts. Costar polystyrene plate had the best adherence. Optimum coating dilution was 10 ng of AFB(1)-Cl-Ov per well. Dilutions of 1:1000 of monoclonal antibody from purified culture or 1:300 from monoclonal antibody from tissue culture and 1:1000 of peroxidase anti-mouse conjugate were the best. Optimum separation with HPLC was obtained isocratically with 60% MeOH and 40% distilled water mobile phase. ELISA had a sensitivity of 1 pg mL(-1) AFB(1) and HPLC sensitivity was 0.1 ng mL(-1) AFB(1) with fluorescence detector and 4.5 ng mL(-1) with UV detector. Monoclonal antibody gave more accurate results for determination of free and adducted AFB(1) in urine analysis.

Ochratoxin A is a carcinogen and nephrotoxin which can enter the food chain resulting in human exposure. As pig herds are exposed to ochratoxin A through their feed, their kidneys, livers and pork meat are considered as a possible route of exposure for humans. France, an important producer of pork and pork products, set up a national monitoring programme which included the training of six routine public laboratories in the analysis of ochratoxin A using an immunoaffinity step followed by a HPLC-fluorimetric detection. The programme randomly sampled 300 healthy and 100 nephropathic pig kidneys in 1997 and 710 healthy pig kidneys in 1998. Less than 10% of samples were significantly contaminated by ochratoxin A : in the 1997 survey, 1% of samples contained 0.40-1.40 microg kg(-1) of ochratoxin A and in the 1998 survey 7.6 % exhibited ochratoxin A levels in the range 0.5-5 microg kg(-1). In the case of nephropathic kidneys, only traces of ochratoxin A (0.16 to 0.48 microg kg(-1)) were detected in six samples out of 100. Even if not a major route of exposure for humans, pigs are clearly exposed to this mycotoxin and monitoring of pork products and of feed for swine is necessary.

A number of fungal cultures were screened to select an organism suitable to be used in the detoxification of aflatoxin B1. They were co-cultured in Czapek-Dox-Casamino acid medium with aflatoxin B1 producing Aspergillus flavus. Several fungal cultures were found to prevent synthesis of aflatoxin B1 in liquid culture medium. Among these Phoma sp., Mucor sp., Trichoderma harzianum, Trichoderma sp. 639, Rhizopus sp. 663, Rhizopus sp. 710, Rhizopus sp. 668, Alternaria sp. and some strains belonging to the Sporotrichum group (ADA IV B14(a), ADA SF VI BF (9), strain 720) could inhibit aflatoxin synthesis by > or =90%. A few fungi, namely ADA IV B1, ADA F1, ADA F8, also belonging to the Sporotrichum group, were less efficient than the Phoma sp. The Cladosporium sp. and A. terreus sp. were by far the least efficient, registering <10% inhibition. The cultures which prevent aflatoxin biosynthesis are also capable of degrading the preformed toxin. Among these, Phoma sp. was the most efficient destroying about 99% of aflatoxin B1. The cell free extract of Phoma sp. destroyed nearly 50 microg aflatoxin B1 100 ml(-1) culture medium (90% of the added toxin), and this was more effective than its own culture filtrate over 5 days incubation at 28+/-2 degrees C. The degradation was gradual: 35% at 24 h, 58% at 48 h, 65% at 72 h, 85% at 96 h and 90% at 120 h. The possibility of a heat stable enzymatic activity in the cell free extract of Phoma is proposed.

Eighteen maize samples were assayed for fumonisin B1 (FB1) and B2 content by immunoaffinity column coupled with high performance liquid chromatography (HPLC). The FumoniTest columns were used once for the isolation of fumonisins (single-use column method). In the second part of the assay the columns were regenerated. After elution with methanol, PBS solution was left on the column for one day at room temperature to regenerate the columns (regenerated column method). The efficiency of columns regenerated twice was tested by determining FB, recovery and the reproducibility of the determinations. The recovery rate of FB1 proved to be 82% by the single-use column method (RSD: 5.7%) and 82.6% (RSD: 5.6 %) by the regenerated column method; 500-8,000 ng FB1 loaded onto the columns did not affect column performances. Nearly identical values were obtained when the FB1 content of fumonisin-containing maize samples was determined by both methods. The results indicate that the FumoniTest columns can be regenerated by the method applied at least twice without decrease in column performance. The fumonisin affinity, capacity and specificity of the regenerated columns were not changed. Thus, columns regenerated in this way can be used for determining the fumonisin content of maize samples at least three times.

Using monoclonal anti-fumonisin B1 antibody (anti-FB1) and avidin-biotin-peroxidase system, liver and kidneys of broiler chicks were evaluated for the detection and distribution of fumonisins (FBs). One hundred and fifty micrograms of FB1 or culture extract of Fusarium moniliforme str. 113F containing 150 microg of FB1 and 4 microg of FB2 were administered into the vitelline sac of 1-day old, specific pathogen-free chicks. The animals were killed 24 h after injection, and renal and hepatic tissues submitted for immunohistochemical analysis. FBs were detected in the epithelial cells of convoluted distal and proximal tubules of the kidneys, as well as in the cytoplasm of hepatocytes. This novel immunohistochemical method developed is expected to be an efficient way for monitoring the target of the FB toxins in tissues.

Production of aflatoxin B1 and fumonisin B1 in pure and mixed cultures of Aspergillus flavus and Fusarium proliferatum were determined on irradiated maize seeds inoculated with different spore concentrations at 0.97 water activity (a(w)) and a temperature of 25 degrees C. The highest levels of aflatoxin B1 were produced by A. flavus at the lowest levels of inoculum (10(3) spore ml(-1)). There was no spore concentration influence on fumonisin B1 production after 10, 20 and 35 days of incubation. When A. flavus was co-inoculated with F. proliferatum, aflatoxin B1 production was inhibited. The higher the inocula levels of Fusarium produced, the higher the inhibition and this inhibition increased during the incubation period. Total inhibition was reached at 35 days of incubation. There was no interaction influence on fumonisin B1 production at all inoculum levels assayed. These results suggest that under optimal environmental conditions of substrate, water activity and temperature, the interaction between A. flavus and F proliferatum could produce inhibition of aflatoxin B1 and stimulation of fumonisin B1.

This article reviews current requirements for the analysis for mycotoxins in foods and identifies legislative as well as other factors that are driving development and validation of new methods. New regulatory limits for mycotoxins and analytical quality assurance requirements for laboratories to only use validated methods are seen as major factors driving developments. Three major classes of methods are identified which serve different purposes and can be categorized as screening, official and research. In each case the present status and future needs are assessed. In addition to an overview of trends in analytical methods, some other areas of analytical quality assurance such as participation in proficiency testing and reference materials are identified.

To develop an ultrasensitive immunoassay for microcystins (MCs), a group of heptapeptide hepatotoxins produced by cyanobacteria, we produced monoclonal antibodies (MAbs) which specifically recognize the immune complex (IC) formed by an anti-MC MAb (MC MAb) and MCs. The use of the anti-IC MAb (IC MAb) as the secondary antibody made it possible to develop a sandwich type immunoassay, which is theoretically superior to the widely used competitive immunoassay in sensitivity as well as accuracy. A MC MAb mixed with microcystin-LR (MCLR) to form the IC was immunized to mice. Three IC MAbs were obtained, all of which specifically reacted with the IC, but almost never reacted to MC MAb or MCLR in enzyme-linked immunosorbent assays (ELISAs). Binding kinetics study of one of the IC MAbs, 3F7, by a BIAcore biosensor technique revealed that 3F7 IC MAb could associate with free MC MAb as well as the IC, but the binding to free MC MAb was much more easily dissociated than that to the IC, thus resulting in about 300-fold higher affinity of 3F7 for the IC than for MC MAb alone (1.8 x 10(9) M(-1) and 4.6 x 10(6) M(-1) for the IC and MC MAb, respectively). Finally, 3F7 IC MAb was shown to react with the IC formed by the addition of MCLR to MC MAb-coated plates in a dose-dependent manner. Therefore, a new type sandwich immunoassay, anti-immune complex ELISA (IC ELISA) for MCs, was indeed established. The detection limit of the IC ELISA was 2 pg of MCLR ml(-1) (50 fg per assay), making it the most sensitive of all the methods for detecting MCs reported to date.

Two corn powder samples implicated in the human food poisoning that occurred in Guangxi province in 1989, and eight wheat and two barley samples linked to an episode that involved about 130,000 people in gastrointestinal disorders in Anhui province in 1991 were analyzed for trichothecenes including deoxynivalenol (DON), nivalenol (NIV) and their esters, zearalenone (ZEA) and fumonisins (FMs) by gas chromatography/mass spectroscopy and high performance liquid chromatography, and T-2 toxin by enzyme-linked immunosorbent assays. DON was detected in all samples as a major trichothecene (16-51,450 microg kg(-1)), and NIV was in one corn, one barley and all wheat at relatively low levels (10-6935 microg kg(-1)). ZEA was found in all corn and barley, and six wheat samples (46-3079 microg kg(-1)). In addition, 3-acetyl-DON (2544 microg kg(-1)) and 15-acetyl-DON (2537 microg kg(-1)) were detected separately in one corn and one wheat sample. The highest levels of these mycotoxins were found in one wheat sample associated with the human intoxication in Anhui province. FMs in corn were below 1000 microg kg(-1). Risks of DON and ZEA on the people who consumed the causative cereals were assessed.