Comparison Evaluation of the Biological Effects of Sterigmatocystin and Aflatoxin B1 Utilizing SOS-Chromotest and a Novel Zebrafish (Danio rerio) Embryo Microinjection Method.

Aflatoxin B1 (AFB1) is a potent mycotoxin and natural carcinogen. The primary producers of AFB1 are Aspergillus flavus and A. parasiticus. Sterigmatocystin (STC), another mycotoxin, shares its biosynthetic pathway with aflatoxins. While there are abundant data on the biological effects of AFB1, STC is not well characterised. According to published data, AFB1 is more harmful to biological systems than STC. It has been suggested that STC is about one-tenth as potent a mutagen as AFB1 as measured by the Ames test. In this research, the biological effects of S9 rat liver homogenate-activated and non-activated STC and AFB1 were compared using two different biomonitoring systems, SOS-Chromotest and a recently developed microinjection zebrafish embryo method. When comparing the treatments, activated STC caused the highest mortality and number of DNA strand breaks across all injected volumes. Based on the E. coli SOS-Chromotest, the two toxins exerted the same genotoxicities. Moreover, according to the newly developed zebrafish microinjection method, STC appeared more toxic than AFB1. The scarce information correlating AFB1 and STC toxicity suggests that AFB1 is a more potent genotoxin than STC. Our findings contradict this assumption and illustrate the need for more complex biomonitoring systems for mycotoxin risk assessment.

Evaluation of the Multimycotoxin-Degrading Efficiency of Rhodococcus erythropolis NI1 Strain with the Three-Step Zebrafish Microinjection Method.

The multimycotoxin-degrading efficiency of the Rhodococcus erythropolis NI1 strain was investigated with a previously developed three-step method. NI1 bacterial metabolites, single and combined mycotoxins and their NI1 degradation products, were injected into one cell stage zebrafish embryos in the same doses. Toxic and interaction effects were supplemented with UHPLC-MS/MS measurement of toxin concentrations. Results showed that the NI1 strain was able to degrade mycotoxins and their mixtures in different proportions, where a higher ratio of mycotoxins were reduced in combination than single ones. The NI1 strain reduced the toxic effects of mycotoxins and mixtures, except for the AFB1+T-2 mixture. Degradation products of the AFB1+T-2 mixture by the NI1 strain were more toxic than the initial AFB1+T-2 mixture, while the analytical results showed very high degradation, which means that the NI1 strain degraded this mixture to toxic degradation products. The NI1 strain was able to detoxify the AFB1, ZEN, T-2 toxins and mixtures (except for AFB1+T-2 mixture) during the degradation experiments, which means that the NI1 strain degraded these to non-toxic degradation products. The results demonstrate that single exposures of mycotoxins were very toxic. The combined exposure of mycotoxins had synergistic effects, except for ZEN+T-2 and AFB1+ZEN +T-2, whose mixtures had very strong antagonistic effects.

Qualifying the T-2 Toxin-Degrading Properties of Seven Microbes with Zebrafish Embryo Microinjection Method.

T-2 mycotoxin degradation and detoxification efficiency of seven bacterial strains were investigated with zebrafish microinjection method in three steps ((1) determination of mycotoxin toxicity baseline, (2) examination of bacterial metabolites toxicity, (3) identification of degradation products toxicity). Toxicity of T-2 was used as a baseline of toxic effects, bacterial metabolites of strains as control of bacterial toxicity and degradation products of toxin as control of biodegradation were injected into one-cell stage embryos in the same experiment. The results of in vivo tests were checked and supplemented with UHPLC-MS/MS measurement of T-2 concentration of samples. Results showed that the Rhodococcus erythropolis NI1 strain was the only one of the seven tested (R. gordoniae AK38, R. ruber N361, R. coprophilus N774, R. rhodochrous NI2, R. globerulus N58, Gordonia paraffinivorans NZS14), which was appropriated to criteria all aspects (bacterial and degradation metabolites of strains caused lower toxicity effects than T-2, and strains were able to degrade T-2 mycotoxin). Bacterial and degradation metabolites of the NI1 strain caused slight lethal and sublethal effects on zebrafish embryos at 72- and 120-h postinjection. Results demonstrated that the three-step zebrafish microinjection method is well-suited to the determination and classification of different bacterial strains by their mycotoxin degradation and detoxification efficiency.

Mycotoxin Biodegradation Ability of the Cupriavidus Genus.

The biodegradation and biodetoxification ability of five prominent mycotoxins, namely aflatoxin B1 (AFB1), ochratoxin-A (OTA), zearalenone (ZON), T-2 toxin (T-2) and deoxynivalenol (DON) of Cupriavidus genus were investigated. Biological methods are the most appropriate approach to detoxify mycotoxins. The Cupriavidus genus has resistance to heavy metals and can be found in several niches such as root nodules and aquatic environments. The genus has 17 type strains, 16 of which have been investigated in the present study. According to the results, seven type strains can degrade OTA, four strains can degrade AFB1, four strains can degrade ZON and three strains can degrade T-2. None of the strains can degrade DON. The biodetoxification was measured using different biotests. SOS-chromotest was used for detecting the genotoxicity of AFB1, the BLYES test was used to evaluate the oestrogenicity of ZON, and the zebrafish embryo microinjection test was conducted to observe the teratogenicity of OTA, T-2 and their by-products. Two type strains, namely C. laharis CCUG 53908T and C. oxalaticus JCM 11285T reduced the genotoxicity of AFB1, whilst C. basilensis DSM 11853T decreased the oestrogenic of ZON. There were strains which were able to biodegrade more than two mycotoxins. Two strains degraded two mycotoxins, namely C. metalliduriens CCUG 13724T (AFB1, T-2) and C. oxalaticus (AFB1, ZON) whilst two strains C. pinatubonensis DSM 19553T and C. basilensis degraded three toxins (ZON, OTA, T-2) and C. numazuensis DSM 15562T degraded four mycotoxins (AFB1, ZON, OTA, T-2), which is unique a phenomenon amongst bacteria.

Biological evaluation of microbial toxin degradation by microinjected zebrafish (Danio rerio) embryos.

The use of microinjection of newly fertilized zebrafish eggs as an appropriate tool for qualifying the biodetoxification properties of toxin-degrading microbes was investigated. Ochratoxin A (OTA), bacterial degradation products of OTA and bacterial metabolites of the Cupriavidus basilensis OR16 strain were microinjected. Results showed that variations in the injected droplet size, and thus treatment concentrations, stayed within ±20%, moreover embryo mortality did not exceed 10% in controls, that is in accordance with the recommendations of the OECD 236 guideline. The highest lethality was caused by OTA with a significantly higher toxicity than that of bacterial metabolites or OTA degradation products. However, toxicity of the latter two did not differ statistically from each other showing that the observed mortality was due to the intrinsic toxicity of bacterial metabolites (and not OTA degradation products), thus, the strain effectively degrades OTA to nontoxic products. Sublethal symptoms also confirmed this finding. RESULTS: confirmed that microinjection of zebrafish embryos could be a reliable tool for testing the toxin-degrading properties of microbes. The method also allows comparisons among microbial strains able to degrade the same toxin, helping the selection of effective and environmentally safe microbial strains for the biodetoxification of mycotoxins in large scale.

Aflatoxin B1 and Zearalenone-Detoxifying Profile of Rhodococcus Type Strains.

Aflatoxin B1 (AFB1) and zearalenone (ZON) are dangerous mycotoxins due to their carcinogenicity or oestrogenicity. To alleviate negative effects on humans and animals, successful detoxification tools are needed. The application of microorganisms to biodegrade mycotoxins can be an effective way in food and feed industry enhancing food safety. Several Rhodococcus strains are effective in the degradation of aromatic mycotoxins and their application in mycotoxin biodetoxification processes is a promising field of biotechnology. In this study, we investigated the AFB1 and ZON detoxification ability of 42 type strains of Rhodococcus species. Samples were analysed by high-performance liquid chromatograph equipped with fluorescence detector for mycotoxin concentration and SOS-chromotest was used for monitoring remaining genotoxicity. Out of the 42 Rhodococcus strains, 18 could eliminate more than 90% of the applied AFB1 and the genotoxicity was ceased by 15 strains in 72 h (R. imtechensis JCM 13270T, R. erythropolis JCM 3201T, R. tukisamuensis JCM 11308T, R. rhodnii JCM 3203T, R. aerolatus JCM 19485T, R. enclensis DSM 45688T, R. lactis DSM 45625T, R. trifolii DSM 45580T, R. qingshengii DSM 45222T, R. artemisiae DSM 45380T, R. baikonurensis DSM 44587T, R. globerulus JCM 7472T, R. kroppenstedtii JCM 13011T, R. pyridinivorans JCM 10940T, R. corynebacterioides JCM 3376T). In case of ZON, only R. percolatus JCM 10087T was able to degrade more than 90% of the compound and to reduce the oestrogenicity with 70%.

Aflatoxin B1 detoxification by cell-free extracts of Rhodococcus strains.

Aflatoxin B1 (AFB1) produced by Aspergillus molds is a genotoxic and carcinogenic mycotoxin. For the elimination of mycotoxins from food and feed, biodetoxification can be a successful tool. The aim of this study was to reveal biodetoxification with the cell-free extracts of Rhodococcus erythropolis NI1 and Rhodococcus rhodochrous NI2, which have been already proved to detoxify AFB1. Extracellular matrices of cultures and also intracellular extracts were applied for detoxification. In both cases, media containing constitutively produced and AFB1-induced enzymes were tested, respectively. The pH tolerance of enzymes in the detoxification was examined at pH 7, 7.5, and 8. The remained genotoxicity was detected by SOS-Chromotest and the AFB1 concentration was measured by high performance liquid chromatography with fluorescence detection. In the extracellular matrix, no reduction of genotoxicity was observed. However, detoxification was completed by intracellular enzymes. In intracellular extracts of both strains, genotoxicity was ceased by the constitutive enzymes within 6 h but induced and constitutive enzymes collectively achieved this result within minutes. Moreover, total biodetoxification was observed at every pH adjustment. Analytical results confirmed >84% degradation potential in each sample. Our results indicate a uniquely fast way for the detoxification of AFB1 with intracellular enzymes of R. erythropolis NI1 and R. rhodochrous NI2.

Aflatoxigenic Aspergillus flavus and Aspergillus parasiticus strains in Hungarian maize fields.

Due to the climate change, aflatoxigenic Aspergillus species and strains have appeared in several European countries, contaminating different agricultural commodities with aflatoxin. Our aim was to screen the presence of aflatoxigenic fungi in maize fields throughout the seven geographic regions of Hungary. Fungi belonging to Aspergillus section Flavi were isolated in the ratio of 26.9% and 42.3% from soil and maize samples in 2013, and these ratios decreased to 16.1% and 34.7% in 2014. Based on morphological characteristics and the sequence analysis of the partial calmodulin gene, all isolates proved to be Aspergillus flavus, except four strains, which were identified as Aspergillus parasiticus. About half of the A. flavus strains and all the A. parasiticus strains were able to synthesize aflatoxins. Aflatoxigenic Aspergillus strains were isolated from all the seven regions of Hungary. A. parasiticus strains were found in the soil of the regions Southern Great Plain and Southern Transdanubia and in a maize sample of the region Western Transdanubia. In spite of the fact that aflatoxins have rarely been detected in feeds and foods in Hungary, aflatoxigenic A. flavus and A. parasiticus strains are present in the maize culture throughout Hungary posing a potential threat to food safety.

Application of a yeast estrogen reporter system for screening zearalenone degrading microbes.

The aim of this study was to screen microbes for their zearalenone degrading potential and to select microbes whose activities do not create toxic or endocrine disrupting metabolites. Bioluminescent bioreporters (Saccharomyces cerevisiae BLYES and BLYR) were successfully used to monitor toxin degradation; the results of zearalenone biodegradation experiments were confirmed by parallel chemical analysis (HPLC-FLD) and immunoanalytical (ELISA) tests. Using the BLYES/BLYR bioreporters, the most appropriate microbes (ones that produced minimal toxic products and products with lower estrogenic potential) could be selected. The most promising strains belong to Streptomyces and Rhodococcus genera. Our findings demonstrate the benefit of using biological tests beside the analytical method, since bioreporters were able to monitor the samples for toxicity and estrogenic potential even after substantial degradation. We conclude that the BLYES/BLYR bioreporter system is a cost effective, fast and reliable tool for screening zearalenone-degrading microbes.