Neuroprotection against CCl4 induced brain damage with crocin in Wistar rats

Owing to its lipophilic property, carbon tetrachloride (CCl₄) is rapidly absorbed by both the liver and brain. We investigated the protective effects of crocin against brain damage caused by CCl₄. Fifty rats were divided into five groups of ten: control, corn oil, crocin, CCl₄ and CCl₄ + crocin. CCl₄ administration decreased glutathione (GSH) and total antioxidant status (TAS) levels, and catalase (CAT) activity, while significant increases were observed in malondialdehyde (MDA) and total oxidant status (TOS) levels and superoxide dismutase (SOD) activity. The cerebral cortex nuclear lamina developed a spongy appearance, neuronal degeneration was observed in the hippocampus, and heterochromatic and pyknotic neurons with increased cytoplasmic eosinophilia were observed in the hippocampus after CCl₄ treatment. Because crocin exhibits strong antioxidant properties, crocin treatment increased GSH and TAS levels and CAT activities, and decreased MDA and TOS levels and SOD activity; significant improvements also were observed in histologic architecture. We found that crocin administration nearly eliminated CCl₄ induced brain damage by preventing oxidative stress.     

Dengue virus detection by using reverse transcription-polymerase chain reaction in saliva and progeny of experimentally infected Aedes albopictus from Brazil

Oral susceptibility and vertical transmission of dengue virus type 2 (DENV-2) in an Aedes albopictus sample from Rio de Janeiro was estimated. The infection (36.7%) and transmission (83.3%) rates for Ae. albopictus were higher than those of an Ae. aegypti colony used as control, 32.8 and 60%, respectively. Fourth instar larvae and females descendants of 48.5 and 39.1% of experimentally infected Ae. albopictus showed to harbor the virus. The oral susceptibility and the high capacity to assure vertical transmission exhibited by Ae. albopictus from Brazil reinforce that this species may play a role in the maintenance of the virus in nature and be a threat for dengue control in the country.     

Cleavage of Zearalenone by Trichosporon mycotoxinivorans to a Novel Nonestrogenic Metabolite

Zearalenone (ZON) is a potent estrogenic mycotoxin produced by several Fusarium species most frequently on maize and therefore can be found in food and animal feed. Since animal production performance is negatively affected by the presence of ZON, its detoxification in contaminated plant material or by-products of bioethanol production would be advantageous. Microbial biotransformation into nontoxic metabolites is one promising approach. In this study the main transformation product of ZON formed by the yeast Trichosporon mycotoxinivorans was identified and characterized by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and LC-diode array detector (DAD) analysis. The metabolite, named ZOM-1, was purified, and its molecular formula, C₁₈H₂₄O₇, was established by time of flight MS (TOF MS) from the ions observed at m/z 351.1445 [M-H]⁻ and at m/z 375.1416 [M+Na]⁺. Employing nuclear magnetic resonance (NMR) spectroscopy, the novel ZON metabolite was finally identified as (5S)-5-({2,4-dihydroxy-6-[(1E)-5-hydroxypent-1-en-1-yl]benzoyl}oxy)hexanoic acid. The structure of ZOM-1 is characterized by an opening of the macrocyclic ring of ZON at the ketone group at C6′. ZOM-1 did not show estrogenic activity in a sensitive yeast bioassay, even at a concentration 1,000-fold higher than that of ZON and did not interact with the human estrogen receptor in an in vitro competitive binding assay.Zearalenone (ZON) is the main member of a growing family of biologically important “resorcylic acid lactones” (RALs), which have been found in nature. ZON is produced by several Fusarium species, which colonize maize, barley, oat, wheat, and sorghum and tend to develop ZON during prolonged cool, wet growing and harvest seasons (38). Maize is the most frequently contaminated crop plant, and therefore, ZON can be found frequently in animal feeding stuff. Occurrence, toxicity, and metabolism data of ZON were summarized by the European Food Safety Authority (EFSA) (5) and in recent reviews (12, 38).The potent xenohormone ZON leads to hyperestrogenism symptoms and in extreme cases to infertility problems, especially in pigs (15). Ovarian changes in pigs have been noted with toxin levels as low as of 50 μg/kg in the diet (1). Ruminants are more tolerant to ZON ingestion; however, hyperestrogenic syndrome, including restlessness, diarrhea, infertility, decreased milk yields, and abortion, have been well documented with cattle and sheep (4, 29).Because widespread ZON contamination in feed can occur in problematic years, efficient ways to detoxify are desirable. The transformation of mycotoxins to nontoxic metabolites by pure cultures of microorganisms or by cell-free enzyme preparations (3) is an attractive possibility. Microbial metabolization of ZON to alpha-ZOL and beta-ZOL cannot be regarded as detoxification, because both ZOL products are still estrogenic (14). Also, formation of ZON-glucosides and -diglucosides (8, 17) and ZON-sulfate (7) cannot be considered true detoxification but rather formation of masked mycotoxins, because the conjugates may be hydrolyzed during digestion (11, 23), releasing ZON again (2).As the estrogenic activity of ZON and its derivates can be explained by its chemical structure, which resembles natural estrogens (20), it can be expected that cleavage of the lactone undecyl ring system of ZON results in permanent detoxification.El-Sharkawy and Abul-Hajj (9) were the first to report inactivation of ZON after opening of the lactone ring by Gliocladium roseum. This filamentous fungus was capable of metabolizing ZON in yields of 80 to 90%. Also Takahashi-Ando et al. (31) described the degradation reaction of ZON with Clonostachys rosea (synonym of G. roseum). A hydrolase (encoded by a gene designated ZHD101) cleaves the lactone ring, and as recently proved (37; unpublished data) by subsequent decarboxylation of the intermediate acid, the compound 1-(3,5-dihydroxyphenyl)-10′-hydroxy-1′E-undecene-6′-one is formed. In contrast to ZON and 17β-estradiol, which showed potent estrogenic activity, this cleavage product did not show any estrogenic activity in the human breast cancer MCF-7 cell proliferation assay (16). Further details, e.g., on the conditions of the maximum activity of ZHD101 and its exploitation in genetically modified grains, can be found in later published work of this research group (32, 33).Only a few authors reported the loss of estrogenicity in microbial metabolites of ZON, which are based on reactions other than cleavage of the lactone undecyl ring system. El-Sharkawy and Abul-Hajj demonstrated (10) that binding to rat uterine estrogen receptors requires a free 4-OH phenolic group (devoid of methylation or glycosylation). Loss of estrogenicity was, for instance, observed with 2,4-dimethoxy-ZON, one of the metabolites produced by Cunninghamella bainieri ATCC 9244B. Nevertheless, this rule cannot be generalized, as 8′-hydroxyzearalenone formed by Streptomyces rimosus NRRL 2234, despite having a free 4-phenolic hydroxyl group, did not bind to the estrogen receptor. Also, other authors reported that 8′-hydroxyzearalenone and 8′-epi-hydroxyzearalenone are nonestrogenic (13). However, so far, no practical application in feed or food detoxification has been found for the microorganisms producing these compounds.It has been shown previously that the yeast Trichosporon mycotoxinivorans has a very high capability to degrade both ochratoxin A (OTA) and ZON (22, 26, 27). When T. mycotoxinivorans is used as a feed additive preparation, microbial degradation of the mycotoxins is assumed to take place in the gastrointestinal tract of the animal after consumption of contaminated feed. The protective effect of T. mycotoxinivorans against OTA toxicity has already been shown with broiler chicken (24).In the present study we report the isolation, analytical characterization, and structure elucidation, as well as the evaluation, of the estrogenic activity of the main degradation product of ZON produced by T. mycotoxinivorans.     

Investigation of various adsorbents for their ability to bind aflatoxin B1

The contamination of animal feed with mycotoxins represents a worldwide problem for the animal industry. The most applied method for protecting animals against aflatoxicosis is the utilization of clay minerals. In the course of a research project adsorption experiments were performed in buffer solutions in order to evaluate the ability to bind Aflatoxin B₁ (AfB₁) at various pH-values. In order to investigate the strength of binding, the chemisorption index was calculated. Isothermal analysis was used to determine the values for the maximum adsorption capacity. Adsorption experiments in simulated gastrointestinal fluid and real gastric juice were carried out. Furthermore binding capability of the materials regarding selected vitamins was examined. Special attention was paid to the formation of AfB_2a during experimental conditions. Based on the obtainedin vitro results, highly promising sorbent materials were ranked for furtherin vivo studies. Some adsorbing bentonites were also analysed mineralogically, but the results did not indicate which smectite property influences the adsorption process for AfB₁ Presented at the 28^th Mykotoxin-Workshop, Bydgoszcz, Poland, May 29–31, 2006 Financial support: Christian Doppler Society     

A new approach to dengue fatal cases diagnosis: NS1 antigen capture in tissues

Abstract/Background

Dengue is the most important arthropod borne viral disease worldwide in terms of morbidity and mortality and is caused by any of the four serotypes of dengue virus (DENV-1 to 4). Brazil is responsible for approximately 80% of dengue cases in the Americas, and since the introduction of dengue in 1986, a total of 5,944,270 cases have been reported including 21,596 dengue hemorrhagic fever and 874 fatal cases. DENV can infect many cell types and cause diverse clinical and pathological effects. The goal of the study was to investigate the usefulness of NS1 capture tests as an alternative tool to detect DENV in tissue specimens from previously confirmed dengue fatal cases (n = 23) that occurred in 2002 in Brazil.

Methodology/Principal Findings

A total of 74 tissue specimens were available: liver (n = 23), lung (n = 14), kidney (n = 04), brain (n = 10), heart (n = 02), skin (n = 01), spleen (n = 15), thymus (n = 03) and lymph nodes (n = 02). We evaluated three tests for NS1 antigen capture: first generation Dengue Early ELISA (PanBio Diagnostics), Platelia NS1 (BioRad Laboratories) and the rapid test NS1 Ag Strip (BioRad Laboratories). The overall dengue fatal case diagnosis based on the tissues analyzed by Dengue Early ELISA, Platelia NS1 and the NS1 Ag Strip was 34.7% (08/23), 60.8% (14/23) and 91.3% (21/23), respectively. The Dengue Early ELISA detected NS1 in 22.9% (17/74) of the specimens analyzed and the Platelia NS1 in 45.9% (34/74). The highest sensitivity (78.3%; 58/74) was achieved by the NS1 Ag Strip, and the differences in the sensitivities were statistically significant (p<0.05). The NS1 Ag Strip was the most sensitive in liver (91.3%; 21/23), lung (71.4%; 10/14), kidney (100%; 4/4), brain (80%; 8/10), spleen (66.6%, 10/15) and thymus (100%, 3/3) when compared to the other two ELISA assays.

Conclusions/Significance

This study shows the DENV NS1 capture assay as a rapid and valuable approach to postmortem dengue confirmation. With an increasing number of DHF and fatal cases, the availability of new approaches useful for cases confirmation plays an important tool for the disease surveillance.     

Capability of yeast derivatives to adhere enteropathogenic bacteria and to modulate cells of the innate immune system

Yeast derivatives including yeast cell wall components are promising alternatives to antibiotics with respect to the promotion of health and performance in livestock, based on their capacity to bind enteropathogenic bacteria and to beneficially modulate the immune system. However, these mode(s) of action both in vitro and in vivo are still not well understood. Furthermore, standardization and reproducibility of in vitro techniques (microbiology, cell culture assays) are critical features for the application of yeast derivatives as well as for the proof of effectiveness. Yeast cell wall products are suggested as anti-adhesive agents and are thus proposed to prevent attachment of certain intestinal bacteria by providing alternative adhesion sites to enterobacteria, which contain mannose-specific type I fimbriae such as Escherichia coli or Salmonella spp. and which is well documented. Various in vitro assay techniques have become of paramount importance for biotechnological research since they allow for determination and quantification of potential mode(s) of action. However, in vitro assays may be criticized by product end users as not accurately reflecting in vivo responses. Pro and cons of different assays and their bias will be discussed specifically regarding yeast cell wall components and adhesion of enteropathogenic bacteria. Immunomodulation is a therapeutic approach intervening in auto-regulating processes of the defense system. Yeast derivatives such as beta-glucans are proposed to interact with cells of the innate immune system by receptor recognition. Controversial data in literature and mode(s) of action are reviewed and discussed here.     

An outbreak of dengue virus at Rio de Janeiro--1986

Dengue virus type 1 has been isolated in Aedes albopictus cell strain, from sera of patients living in the Nova Igua?u county, by Rio de Janeiro. The clinical picture was characterized by fever, headache, retrobulbar pain, backache, pains in the muscles and the joints and prostration. Studies in paired sera confirmed the presence of recent infection by dengue virus type 1. The outbreak reached adjacent areas, including Rio de Janeiro city (May, 1986).