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%.

Hormonal activity, cytotoxicity and developmental toxicity of UV filters.

Ultraviolet (UV) filters are commonly used compounds in personal care products and polymer based materials, as they can absorb solar energy in the UVA and UVB spectrum. However, they are able to bind to hormone receptors and have several and different types of hormonal activities determined by in vitro assays. One of the aims of this work was to measure the hormonal and cytotoxic activities of four frequently used UV filters using bioluminescence based yeast test organisms. Using Saccharomyces cerevisiae BLYES and BLYAS strains allowed the rapid and reliable detection of agonist and antagonist hormonal activities, whereas BLYR strain served to measure cytotoxicity. Results confirmed that all tested UV filters show multiple hormonal activities. Cytotoxicity is detected only in the case of benzophenone-3. Research data on the toxic effects of benzophenone-3, especially on aquatic organisms are scarce, so further investigations were carried out regarding its cytotoxic and teratogenic effects on bacteria and zebrafish (Danio rerio) embryos, respectively. Results revealed the cytotoxicity of benzophenone-3 not only to yeasts but to bacteria, as well as its ability to influence zebrafish embryo hatching and development.

Analysis of biofilm bacterial communities responsible for carbon removal through a reactor cascade treating wastewater.

In this study molecular microbiological and multivariate statistical analyses were carried out to determine the structure and dynamics of bacterial communities through a biofilm based, pilot-scale wastewater treatment cascade system comprised of eight reactors. Results indicated a vertical as well as horizontal differentiation of biofilm bacterial communities within individual reactors and through the reactor series, respectively. The richness of biofilm samples taken from dissolved oxygen rich sections of reactors were relatively lower than of samples taken from less oxygenized sections (one-way ANOVA P = 0.07). The Euclidean distance based one-way ANOSIM pointed out that in bacteriological point of view: (1) no statistically significant difference could be observed among the first five reactors (P ≥ 0.1); (2) the first seven reactors differed significantly from the last reactor, (P ≤ 0.03); (3) reactors 1 and 2 differed significantly from reactors 6 and 7 (P ≈ 0.02) and (4) reactor 3 from reactor 7 (P ≈ 0.03). 16S rRNA gene cloning revealed that through the cascade system the initially dominant heterotrophic bacteria (Acinetobacter, Acidovorax, Parabacteroides, Thauera, Desulfobacterium and Desulfomicrobium) were gradually replaced or supplemented by autotrophic nitrifying bacteria (Nitrosomonas, 'Candidatus Nitrotoga' and Nitrospira). Our results indicate that the vertical alteration of bacterial community structure within a particular reactor was driven by the alteration of dissolved oxygen concentration, while the horizontal alteration of bacterial community structure through the cascade system was driven mainly by the gradually decreasing dissolved organic matter content and increasing dissolved oxygen concentration.

Analysis of aflatoxin-B1-degrading microbes by use of a combined toxicity-profiling method.

To monitor cytotoxic and genotoxic effects of aflatoxin, a luminescent assay employing Aliivibrio fischeri as a test organism and a colorimetric assay based on the SOS-Chromotest were adapted to our needs. The aim of this method-developing work was to be able to select - from a collection of environmental isolates - microbes that degrade aflatoxin without production of harmful intermediates and by-products, in a fast and cost-effective way. By the combination of the two modified assays, microbes that met these criteria have been successfully selected. Among thirty-three isolates, the strain Rhodococcus rhodochrous NI2 proved to be the best aflatoxin-B1-degrading microbe, with the weakest harmful biological effects throughout aflatoxin-B1-degradation. Our findings underline the necessity to employ bio-tests in biodegradation assays, as cytotoxicity and/or genotoxicity may occur even after substantial degradation of the toxins.

Effects of Single and Repeated Oral Doses of Ochratoxin A on the Lipid Peroxidation and Antioxidant Defense Systems in Mouse Kidneys.

Ochratoxin-A (OTA) is a carcinogenic and nephrotoxic mycotoxin, which may cause health problems in humans and animals, and it is a contaminant in foods and feeds. The purpose of the present study is to evaluate the effect of oral OTA exposure on the antioxidant defense and lipid peroxidation in the kidney. In vivo administration of OTA in CD1, male mice (1 or 10 mg/kg body weight in a single oral dose for 24 h and repeated daily oral dose for 72 h or repeated daily oral dose of 0.5 mg/kg bodyweight for 21 days) resulted in a significant elevation of OTA levels in blood plasma. Some histopathological alterations, transcriptional changes in the glutathione system, and oxidative stress response-related genes were also found. In the renal cortex, the activity of the glutathione-system-related enzymes and certain metabolites of the lipid peroxidation (conjugated dienes, trienes, and thiobarbituric reactive substances) also changed.

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.

Development of a combined method to assess the complex effect of atrazine on sex steroid synthesis in H295R cells.

The aim of the study was to develop a rapid, cost-effective combined testing method to assess the indirect effect of compounds interfering with sex steroid synthesis and to determine complex effects of atrazine on estrogen and androgen synthesis in vitro on H295R human cell line. Steroidogenic assay was performed on H295R human adrenocortical carcinoma cell line. Instead of standard analytical methods, bioluminescence bioreporter assays (Saccharomyces cerevisiae BLYES and BLYAS) were used to measure estrogenic and androgenic effects of sex steroid hormones released by human cells in response to atrazine. Atrazine resulted in elevated estrogen production presumably due to its well documented inductive effect on aromatase on H295R cell line, detected by BLYES. Interestingly, results of BLYAS test showed concentration-dependent increase of androgen production in H295R cells. That indicates that atrazine can not only increase estrogen level via aromatase induction, but may interfere in androgen synthesis as well. The combined method allows us to assess the androgenic and estrogenic effect of sex steroids produced by human cells in increased or decreased quantity as a result of the different chemicals, without determining specific analytical measurement endpoints, by using the yeast based bioluminescent bioreporter test.

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.

A new ochratoxin A biodegradation strategy using Cupriavidus basilensis Or16 strain.

Ochratoxin-A (OTA) is a mycotoxin with possibly carcinogenic and nephrotoxic effects in humans and animals. OTA is often found as a contaminant in agricultural commodities. The aim of the present work was to evaluate OTA-degrading and detoxifying potential of Cupriavidus basilensis OR16 strain. In vivo administration of OTA in CD1 male mice (1 or 10 mg/kg body weight for 72 hours or 0.5 mg/kg body weight for 21 days) resulted in significant elevation of OTA levels in the blood, histopathological alterations- and transcriptional changes in OTA-dependent genes (annexinA2, clusterin, sulphotransferase and gadd45 and gadd153) in the renal cortex. These OTA-induced changes were not seen in animals that have been treated with culture supernatants in which OTA was incubated with Cupriavidus basilensis OR16 strain for 5 days. HPLC and ELISA methods identified ochratoxin α as the major metabolite of OTA in Cupriavidus basilensis OR16 cultures, which is not toxic in vivo. This study has demonstrated that Cupriavidus basilensis OR16 efficiently degrade OTA without producing toxic adventitious metabolites.

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.

A new zearalenone biodegradation strategy using non-pathogenic Rhodococcus pyridinivorans K408 strain.

Zearalenone (hereafter referred to as ZEA) is a nonsteroidal estrogenic mycotoxin produced by several Fusarium spp. on cereal grains. ZEA is one of the most hazardous natural endocrine disrupting chemicals (EDC) which induces hyper estrogenic responses in mammals. This can result in reproductive disorders in farm animals as well as in humans. Consequently, detoxification strategies for contaminated crops are crucial for food safety. In this study we have developed a bacterial based detoxification system using a non-pathogen Rhodococcus pyridinivorans K408 strain. Following 5 days treatment of ZEA with R. pyridinivorans K408 strain HPLC analyses showed an 87.21% ZEA-degradation efficiency of the bacterial enzyme systems. In another approach, the strain biotransformation ability has also been confirmed by a bioluminescent version of the yeast estrogen screening system (BLYES), which detected an 81.75% of biodegradability of ZEA, in a good agreement with the chemical analyses. Furthermore, the capacity of R. pyridinivorans to eliminate the estrogenic effects of ZEA was tested by using an immature uterotrophic assay. Prepubertal female rats were treated with vehicle (olive oil), 17ß-estradiol, ZEA (0.1-1-5-10 mg/kg body weight) and LB broth containing 500 mg/l ZEA that has already been incubated with or without Rhodococcus pyridinivorans K408 strain. Uterine weights were measured and the mRNA level changes relating to apelin, aquaporin 5, complement component 2, and calbindin-3 genes were measured by qRT-PCR. These genes represent the major pathways that are affected by estromimetic compounds. Zearalenone feeding significantly increased the uterus weight in a dose dependent manner and at the same time upregulated complement component 2 and calbindin-3 expression as well as decreased apelin and aquaporin 5 mRNA levels comparable to that seen in 17ß-estradiol exposed rats. In contrast, LB broth in which ZEA was incubated with Rhodococcus pyridinivorans K408 prior to the feeding did not display any estrogenic effect neither on uterine weight nor on the expression of estrogen-regulated genes. Consequently, the identification of Rhodococcus pyridinivorans K408 strain in ZEA biodegradation proved to be a very efficient biological tool that is able to eliminate the complete estrogenic effects of ZEA. It is also remarkable that this biotransformation pathway of ZEA did not result in any residual estrogenic effects.