Antifungal and antimycotoxigenic effect of Lactobacillus plantarum CRL 778 at different water activity values.

Ochratoxin A (OTA) is a mycotoxin produced by filamentous fungi with high impact in food safety due to its toxicity. In the last decade, the presence of OTA was widely reported in different foods. In this study, the ability of Lactobacillus (L.) plantarum CRL 778 to control growth and OTA production by Aspergillus (A.) niger 13D strain, at different water activity (aw) values (0.955, 0.964, 0.971, 0.982, and 0.995) was determined in vitro. Both parameters were significantly (p<0.05) reduced by the lactobacilli and the effect depended on aw. Greatest growth rate inhibition (46.9%) was obtained at aw=0.995, which is the most suitable value for growth and production of antifungal metabolites (lactic acid, acetic acid, phenyllactic and hydroxyl-phenyllactic acids) by L. plantarum CRL 778. Besides, morphological changes and inhibition of melanin synthesis were observed in colonies of A. niger 13D in presence of L. plantarum CRL 778 at aw ranged between 0.971 and 0.995. In addition, maximum reduction (90%) of OTA production took place at aw=0.971, while inhibition of fungi growth was more evident at aw=0.995. These findings suggest that L. plantarum CRL 778 could be used for control of ochratoxigenic fungal growth and OTA contamination in different fermented foods with aw values between 0.971 and 0.995.

Quercetin protects human peripheral blood mononuclear cells from OTA-induced oxidative stress, genotoxicity, and inflammation.

Ochratoxin A (OTA) is one of the most abundant food-contaminating mycotoxins world wide, and is detrimental to human and animal health. This study evaluated the protective effect of quercetin against OTA-induced cytotoxicity, genotoxicity, and inflammatory response in lymphocytes. Cytotoxicity determined by MTT assay revealed IC20 value of OTA to be 20 µM, which was restored to near control values by pretreatment with quercetin. Oxidative stress parameters such as antioxidant enzymes, LPO and PCC levels indicated that quercetin exerted a protective effect on OTA-induced oxidative stress. Quercetin exerted an antigenotoxic effect on OTA-induced genotoxicity, by significantly reducing the number of structural aberrations in chromosomes and comet parameters like, % olive tail moment from 2.76 ± 0.02 to 0.56 ± 0.02 and % tail DNA from 56.23 ± 2.56 to 12.36 ± 0.56 as determined by comet assay. OTA-induced NO, TNF-α, IL-6, and IL-8 were significantly reduced in the quercetin pretreated samples indicating its anti-inflammatory role. Our results demonstrate for the first time that quercetin exerts a cytoprotective effect against OTA-induced oxidative stress, genotoxicity, and inflammation in lymphocytes. © 2014 Wiley Periodicals, Inc. Environ Toxicol 31: 855-865, 2016.

Efficiency of Artemisia nilagirica (Clarke) Pamp. essential oil as a mycotoxicant against postharvest mycobiota of table grapes.

BACKGROUND: In order to get a potent botanical fungicide for the management of fungal decay of table grapes, an experiment was conducted in which 20 essential oils of higher plants were screened at 0.33 µL mL(-1) against dominant fungi causing decay of table grapes, including Aspergillus flavus, A. niger and A. ochraceus. Furthermore, the minimum inhibitory/fungicidal concentration, fungitoxic spectrum and mycotoxin inhibition activity of the most potent oil were determined. The efficacy of the most potent oil in preservation of table grapes, along with organoleptic evaluation, was also carried out by storing 1 kg of grapes in the oil vapour. RESULTS: Artemisia nilagirica oil was found to be most toxic, exhibiting 100% mycelia inhibition of all test fungi. Moreover, 0.29 µL mL(-1) A. nilagirica oil was fungistatic and 0.58 µL mL(-1) was fungicidal for all tested species of Aspergillus. The oil exhibited a broad range of fungitoxicity against other grape berry-rotting fungi. Artemisia nilagirica oil completely suppressed the growth and mycotoxin (AFB1 and OTA) secretion of aflatoxigenic and ochratoxigenic strains of Aspergillus at 1.6 µL mL(-1) . During the in vivo experiment, fumigation of 1 kg of table grapes with 200 and 300 µL dosage of A. nilagirica oil enhanced the shelf life for up to 9 days. The oil did not show any phytotoxic effect. Besides, oil application did not substantively change the sensory properties of the fruits. CONCLUSION: Artemisia nilagirica oil can be used as an alternative botanical fungicide for the control of fruit-rotting fungi of stored grapes.

Evaluation of berberine efficacy in reducing the effects of aflatoxin B1 and ochratoxin A added to male broiler rations.

Many types of mycotoxins are found in food sources contaminated with fungi, and if these are ingested in large quantities or over a long period, they can affect the health of humans and domestic animals. Berberine (BBR) is a plant alkaloid with multiple pharmacological functions. This study aimed to investigate the effect of different levels of the plant alkaloid BBR on reducing toxic effects of aflatoxin B1 (AFB) and ochratoxin A (OTA) in broilers by examining performance characteristics, blood biochemistry, antioxidant systems, ileum morphology, and histopathology of the liver. The experiment was performed with 288 Ross 308 broilers reared in floor pens for 42 d in a randomized design with 9 treatments. Each treatment was replicated 4 times, and each replicate contained 8 chicks. Experimental treatments included (1) negative control diet with no additives (NC); (2) NC + 2 ppm AFB (positive control AFB; PCAFB); (3) NC + 2 ppm OTA (positive control OTA; PCOTA); (4) PCAFB + 200 mg/kg BBR; (5) PCAFB + 400 mg/kg BBR; (6) PCAFB + 600 mg/kg BBR; (7) PCOTA + 200 mg/kg BBR; (8) PCOTA + 400 mg/kg BBR; and (9) PCOTA + 600 mg/kg BBR. Compared with NC, feeding PCAFB and PCOTA diets reduced average daily feed intake, weight gain, serum concentrations of superoxide dismutase, glutathione peroxidase, and the length and width of ileum villi (P < 0.05). At the same time, these parameters increased in birds fed PCAFB or PCOTA diets supplemented with 600 mg/kg of BBR (P < 0.05). Feeding PCAFB and PCOTA diets increased feed conversion ratio (FCR), serum aspartate aminotransferase (AST), lactate dehydrogenase (LDH), alanine aminotransferase (ALT), and gamma-glutamyl transferase (GGT) activities, serum urea, and liver lesions compared with NC. By contrast, compared with PCAFB and PCOTA, adding 600 mg/kg BBR decreased FCR, AST, LDH, ALT, and GGT activities, urea, and liver lesions (P < 0.05). Overall, supplementation with 600 mg/kg BBR may improve growth performance, liver function, and antioxidant status of broilers fed diets contaminated with AFB and OTA.

Enterococcus faecium: a promising protective culture to control growth of ochratoxigenic moulds and mycotoxin production in dry-fermented sausages.

Moulds positively contribute to the development of typical characteristic flavour and aroma of dry-fermented sausages. However, some mould species, such as Penicillium nordicum and Penicillium verrucosum, may contaminate this product with ochratoxin A (OTA). For this reason, the control of toxigenic moulds is needed. Strategies based on the use of antifungal microorganisms present in the native microbial population in the dry-fermented sausage processing could be a promising strategy. The aim of this work was to study the effect of Enterococcus faecium strains on P. nordicum and P. verrucosum growth and OTA production in a dry-fermented sausage-based medium at conditions of temperature and water activity similar to those occurring during the ripening of these meat products. Six strains were screened to evaluate their growth capacity and antifungal activity against P. nordicum and P. verrucosum at three fixed temperatures related to the sausage ripening. The two E. faecium strains that decreased growth of both species were chosen to further evaluate their effect on growth of P. verrucosum and P. nordicum and their mycotoxin production under conditions simulating the dry-fermented sausage ripening. The presence of E. faecium SE920 significantly reduced OTA production of P. nordicum although it did not affect P. verrucosum. E. faecium SE920, isolated from dry-fermented sausages, could be a good candidate to reduce OTA production by P. nordicum in dry-fermented sausages.

Nanobody affinity improvement: Directed evolution of the anti-ochratoxin A single domain antibody.

The characteristics of single domain and ease of gene manipulation of the single domain antibody (sdAb) make it suitable for affinity maturation in vitro. Since the affinity of antibodies can influence the immunoassays' sensitivity, a nanobody (Nb), the anti-ochratoxin A sdAb (AOA-sdAb), was herein selected as the model antibody to explore feasible approach for improving its affinity. Homology modeling and molecular docking were used to analyze the interaction between OTA and the AOA-sdAb. After alanine scanning verification, Gly53, Met79, Ser102, and Leu149 were determined as the key amino acids of the AOA-sdAb. Two site-directed saturated mutation libraries were constructed by two-site mutation against those four key amino acids. After biopanning and identification, a mutant Nb-G53Q&S102D was obtained with a half maximal inhibition concentration (IC50) of 0.29 ng/mL and a KD value of 52 nM, which is 1.4-fold and 1.36-fold lower than that of the original sdAb, respectively. The computer simulation analysis indicated that the hydrogen bond, hydrophobic interaction, and side chain steric hindrance of amino acid residues are critical for the binding affinity of the AOA-sdAb. Overall, the techniques shown in this study are effective ways at 'identifying residues involved in antigen binding' that can be altered by site-directed mutation.

Capsaicin, an inhibitor of Ochratoxin A production by Aspergillus section Nigri strains in grapes (Vitis vinifera L.).

Food decay by spoilage fungi leads to significant economic losses and hazards to consumers' health due to the potential of mycotoxin occurrence. Ochratoxin A (OTA) is a mycotoxin known as nephrotoxic and carcinogenic to humans. Natural capsaicin was evaluated for its effectiveness against the growth of five Aspergillus section Nigri strains and accumulation of OTA in inoculated black grapes. Results showed that capsaicin was effective in inhibiting fungal growth and OTA production by new four Aspergillus section Nigri strains (ATHUM 6997, 6998, 6999, 7000) and by Aspergillus carbonarius as well. Moreover, capsaicin addition exhibited maximum inhibition of OTA produced by ATHUM 6997, 6998, 6999, and 7000 in black grapes at 28.9%, 8.6%, 68.4%, and 78.1%, respectively. Inhibition percentage of OTA production by A. carbonarius in grapes treated with capsaicin was estimated at 61.5%. These results suggest that capsaicin influences the OTA biosynthesis pathway of all Aspergillus section Nigri strains and therefore could be used as an effective natural preservative against OTA contamination of vineyards. Risk assessment revealed that when grapes are treated with capsaicin, consumers are less exposed to OTA.

Inula crithmoides extract protects against ochratoxin A-induced oxidative stress, clastogenic and mutagenic alterations in male rats.

Ochratoxin A (OTA) is a mycotoxin often found in cereals and agricultural products. There is unequivocal evidence of renal carcinogenicity of OTA in male rats, although the mechanism of action is unknown. Several reports suggest that exposure to OTA resulted in oxidative stress, genotoxicity and DNA damage. Therefore, the aim of the current study was to evaluate the protective effects of aqueous extract of Inula crithmoides growing in Egypt against OTA-induced mutagenicity and oxidative stress. Forty male Sprague-Dawley rats were divided into four groups and treated for 15 days as follows: control group and the groups treated with OTA (3 mg/kg b.w), I. crithmoides extract alone (370 mg/kg b.w) and OTA+I. crithmoides extract. Blood and tissue samples were collected for different biochemical analyses. Bone marrow micronucleus test and blood for random amplified polymorphism DNA-PCR (RAPD-PCR) method were performed to assess the antigenotoxic effect of the extract. The results indicated that OTA induced toxicological effects typical to those reported in the literature and increased the frequencies of MnPCEs in bone marrow. The RAPD-PCR analysis revealed the appearance of new bands in DNA resulting from genetic alteration. The extract alone was safe and succeeded in counteracting the oxidative stress and protect against the cytotoxicity resulting from OTA.

Efficacy of a new ochratoxin-binding agent (OcraTox) to counteract the deleterious effects of ochratoxin A in laying hens.

An experiment was conducted to evaluate the efficacy of a new ochratoxin-binding agent (Ocra-Tox, 5 g/kg of feed) in offsetting the toxic effects of ochratoxin A (OTA, 2 mg/kg of feed) in laying hen diets. Performance, serum biochemistry, OTA residue in the liver and eggs, and egg quality parameters were evaluated. Twenty-eight Hisex Brown laying hens, 47 wk of age, were allocated to 1 of 4 experimental treatments for 3 wk: control, OTA (containing 2 mg of OTA/kg of feed), OcraTox (containing 5 g of OcraTox/kg of feed), and OTA + OcraTox (containing 2 mg of OTA and 5 g of OcraTox/kg of feed). Laying hens fed OcraTox showed results similar to the control hens (P > 0.05). The OTA diet significantly (P < 0.05) reduced daily feed consumption, egg mass production, and serum triglyceride concentrations, and increased the relative liver weight, the serum activity of alkaline phosphatase, and the serum concentration of uric acid as compared with the control diet. Addition of OcraTox to the contaminated diet alleviated (P < 0.05) the negative effects resulting from OTA, reaching values not significantly different from the control diet for most of the parameters except the relative weight of the liver. Birds fed the OTA treatment showed a greater content of OTA in the liver (15.1 microg/kg) than those fed the control diet (<0.05 microg/kg). Supplementing the contaminated diet with OcraTox (OTA + OcraTox) reduced the values to 12.0 microg/kg. Residues of OTA were not detected above our detection limit (0.05 microg/kg) in any of the analyzed eggs. In conclusion, our results indicated that addition of OcraTox can counteract the deleterious effects caused by OTA in laying hens.

Dietary L-carnitine and vitamin-E; a strategy to combat ochratoxin-A induced immunosuppression.

This study aimed to evaluate the effect of dietary ochratoxin A (OA), in the presence and absence of L-carnitine (LC) and vitamin E (VE), on the humoral immune responses of White Leghorn cockerels (WLC). One-day old white male Leghorn chicks were divided into 12 groups, having 20 birds each and were offered ration contaminated with OA (1.0 or 2.0 mg/kg feed) alone and concurrently with LC (1.0 g/kg) and/or VE (0.2 g/kg), for 42 days. The humoral immune responses were accessed by lymphoproliferative response to avian tuberculin, in-vivo phagosomes activity to carbon particles and antibody response to the sheep red blood cells (SRBCs). The dietary addition of OA alone suppressed the humoral immune responses, however, the exposure of birds to 1.0 mg/kg OA in the presence of LC and/or VE showed a significant reduction in OA induced immunotoxicity. This protective response was absent in the birds fed 2.0 mg/kg OA in the presence and absence of LC and/or VE. Histopathological and morphometric examination of the bursa of Fabricius exhibited a decrease in the severity and frequency of OA induced lesions in the presence of dietary LC and/or VE. The use of LC and VE as dietary supplement, can effectively overcome OA (≤1.0 mg/kg) induced immunosuppression.

Taurine attenuates OTA-promoted PCV2 replication through blocking ROS-dependent autophagy via inhibiting AMPK/mTOR signaling pathway.

Previous research found that ochratoxin A (OTA) could promote PCV2 replication by inducing autophagy. The aim of this study is to evaluate the effect of dietary amino acid derivative taurine on OTA-promoted PCV2 replication and explore the underlying mechanism. The results showed that taurine could inhibit OTA-promoted PCV2 replication in PK-15 cells. The effect of taurine could be mediated by its ability to attenuate ROS level and block OTA-promoted autophagy. Indeed, induction of autophagy by rapamycin could suppress the inhibitory effect of taurine on OTA-promoted PCV2 replication. Furthermore, taurine supplementation inhibited 5'AMP-activated protein kinase (AMPK) and activated mammalian target of rapamycin (mTOR). Activation of AMPK by acadesine (AICAR) could suppress the effect of taurine. In conclusion, taurine treatment suppresses autophagy by regulating the ROS/AMPK/mTOR signaling axis, thereby inhibiting OTA-promoted PCV2 replication. These findings provide the rationale for the use of taurine as an intervention against PCV2 infection.

Activation of AMPK-dependent SIRT-1 by astragalus polysaccharide protects against ochratoxin A-induced immune stress in vitro and in vivo.

Recent studies have highlighted the immune stress caused by ochratoxin A (OTA), but little attention was paid to its alleviation. In the present study, the protective effects of astragalus polysaccharide (APS) against OTA-induced immune stress in vitro and in vivo and its mechanism/(s) involved were investigated. The in vitro results showed that APS (20 µg/ml) induced a significant decrease in cytotoxicity, apoptosis and pro-inflammatory cytokine expressions elevated by OTA (1.5 µg/ml) in porcine alveolar macrophages (PAMs). In vivo, APS (200 mg/kg b.w.) significantly alleviated OTA-induced (75 µg/kg b.w.) spleen damages and decreased the expressions of OTA-promoted apoptosis-related protein and pro-inflammatory cytokine. Further study indicated that APS caused significant enhancement of AMPK/SIRT-1 and inhibition of NFκB in PAMs and mice. The down-regulation of SIRT-1 by EX527 in vivo or EX527 and SIRT-1 knockdown in vitro abolished the inhibitory effects of APS on OTA-induced cytotoxicity, apoptosis, spleen damages and pro-inflammatory cytokine expressions. Taken together, these findings indicate that APS could attenuate the immune stress induced by OTA in vitro and in vivo via activation of the AMPK/SIRT-1 signaling pathway.

Catechins: natural free-radical scavengers against ochratoxin A-induced cell damage in a pig kidney cell line (LLC-PK1).

Besides aflatoxin B1, recent findings suggested that oxidative stress plays an important role in the toxicity of an other mycotoxin: ochratoxin A (OTA). The protective effect of two catechins (epigallocatechin gallate, EGCG, and epicatechin gallate, ECG) against OTA-induced cytotoxicity was investigated in a pig kidney cell line (LLC-PK1). The ability of the catechins to reduce ROS production and DNA fragmentation induced by OTA was also investigated. Our experiments proved the significant cytoprotective effects of the molecules in vitro from OTA-induced cell damage. In particular a 24h pre-treatment with EGCG or ECG restored cell viability with respect to OTA alone. Pre-treatment with EGCG at low concentration for 8 days protected cells from OTA-induced cell death. Moreover both catechins reduced OTA-induced ROS production. A reduction of OTA-induced DNA fragmentation was found for LLC-PK1 cells pre-treated with EGCG and ECG. The free-radical scavenging capacity of both catechins was tested with the Briggs-Rauscher oscillating method (pH approximately 2) and the TEAC assay (pH 7.4). The results show a good scavenging power according with inhibition of ROS production. Catechins could be useful to develop alimentary strategies for both humans and animals to prevent OTA-induced cytotoxicity.

Ochratoxin A-induced DNA damage in human fibroblast: protective effect of cyanidin 3-O-beta-d-glucoside.

Ochratoxin A (OTA), a mycotoxin produced by Aspergillus ochraceus and other moulds, has recently received growing attention because of its carcinogenic, teratogenic and nephrotoxic properties in both humans and farm animals. Nevertheless, with regard to the mechanism of toxicity, the data in the literature are inconclusive. The aim of our work was to verify in human fibroblasts treated with different OTA dosages the involvement of oxidative pathway in the damage mechanism of this mycotoxin and the possible protective effect exerted by cyanidin 3-O-beta-D-glucoside (C3G), an anthocyanin present in pigmented oranges, red wines, fruits and vegetables. The addition of OTA at 25 and 50 microM concentrations for 48 h determined only a slight but significant (P<.05) increase in radical oxygen species, whereas a substantial increase in their production was observed at longer exposure, in particular, when the fibroblasts were treated with 50 microM OTA for 72 h. Under the same experimental conditions, our data showed a significant (P<.05) increase in the rupture of cellular membrane and high damage to genomic DNA, evaluated by single-cell gel electrophoresis (comet assay), thus confirming the involvement of oxidative stress in the OTA genotoxicity in agreement with other studies. Diversely, mitochondrial functionality does not appear influenced by OTA treatment. C3G (0.125, 0.250 mM) added to the cells treated with 50 microM OTA significantly reduced free radical species production and prevented genomic DNA damage.

Effect of vitamin E and polyphenols on ochratoxin A-induced cytotoxicity in liver (HepG2) cells.

It has been shown that oxidative damage contributes to the wide range of toxic effects of the mycotoxin ochratoxin A (OTA). Therefore, we examined the effects of alpha-tocopherol (alpha-TOC) and different polyphenols--catechin (CAT), daidzein (DAI), epicatechin (EC), epigallocatechin gallate (EGCG), genistein (GEN), and quercetin (QUE)--on OTA-induced cytotoxicity in HepG2 liver cells. Incubation of HepG2 cells with increasing concentrations of OTA resulted in a dose- and time-dependent cytotoxicity as measured by the neutral red assay. Half lethal concentrations (LC50) of OTA were 35 and 10 microM after 48 and 72 h incubation, respectively. Incubation of HepG2 cells with alpha-TOC as well as with different polyphenols (exhibiting different antioxidant potency as determined by the FRAP, TEAC and DPPH assays) did not counteract OTA-induced cytotoxicity. These findings indicate that OTA may exert its toxic effects by affecting other hepatic mechanisms than those directly modulated by alpha-TOC and polyphenols.

Hepatotoxic effect of ochratoxin A and citrinin, alone and in combination, and protective effect of vitamin E: In vitro study in HepG2 cell.

Ochratoxin A (OTA) and citrinin (CTN) are the most commonly co-occurring mycotoxins in a wide variety of food and feed commodities. The major target organ of these toxins is kidney but liver could also be a target organ. The combined toxicity of these two toxins in kidney cells has been studied but not in liver cell. In this study HepG2 cells were exposed to OTA and CTN, alone and in combination, with a view to compare the molecular and cellular mechanisms underlying OTA, CTN and OTA + CTN hepatotoxicity. OTA and CTN alone as well as in combination affected the viability of HepG2 cells in a dose-dependent manner. OTA + CTN, at a dose of 20% of IC50 of each, produced effect almost similar to that produced by either of the toxins at its IC50 concentration, indicating that the two toxins in combination act synergistically. The cytotoxicity of OTA + CTN on hepatocytes is mediated by increased level of intracellular ROS followed/accompanied by DNA strand breaks and mitochondria-mediated intrinsic apoptosis. Co-treatment of vitamin E (Vit E) with OTA, CTN and OTA + CTN reduced the levels of ROS and the cytotoxicity. But the genotoxic effect of OTA and OTA + CTN was not completely alleviated by Vit E treatment whereas the DNA damage as caused by CTN when treated alone was obviated, indicating that OTA induces DNA damage directly whereas CTN induces ROS-mediated DNA damage and OTA + CTN combination induces DNA damage not exclusively relying on but influenced by ROS generation. Taken together, these findings indicate that OTA and CTN in combination affect hepatocytes at very low concentrations and, thereby, pose a potential threat to public and animal health.

Ochratoxin A promotes porcine circovirus type 2 replication in vitro and in vivo.

Ochratoxin A (OTA), a worldwide mycotoxin found in food and feeds, is a potent nephrotoxin in animals and humans. Porcine circovirus-associated disease (PCVAD), including porcine dermatitis and nephropathy syndrome, is a worldwide swine disease. To date, little is known concerning the relationship between OTA and porcine circovirus type 2 (PCV2), the primary causative agent of PCVAD. The effects of OTA on PCV2 replication and their mechanisms were investigated in vitro and in vivo. The results in vitro showed that low doses of OTA significantly increased PCV2 DNA copies and the number of infected cells. Maximum effects were observed at 0.05 µg/ml OTA. The results in vivo showed that PCV2 replication was significantly increased in serum and tissues of pigs fed 75 µg/kg OTA compared with the control group and pigs fed 150 µg/kg OTA. In addition, low doses of OTA significantly depleted reduced glutathione and mRNA expression of NF-E2-related factor 2 and γ-glutamylcysteine synthetase; increased reactive oxygen species, oxidants, and malondialdehyde; and induced p38 and ERK1/2 phosphorylation in PK15 cells. Adding N-acetyl-L-cysteine reversed the changes induced by OTA. Knockdown of p38 and ERK1/2 by their respective specific siRNAs or inhibition of p38 and ERK1/2 phosphorylation by their respective inhibitors (SB203580 and U0126) eliminated the increase in PCV2 replication induced by OTA. These data indicate that low doses of OTA promoted PCV2 replication in vitro and in vivo via the oxidative stress-mediated p38/ERK1/2 MAPK signaling pathway. This suggests that low doses of OTA are potentially harmful to animals, as they enhance virus replication, and partly explains why the morbidity and severity of PCVAD vary significantly in different pig farms.

Ochratoxin binding to phenylalanyl-tRNA synthetase: computational approach to the mechanism of ochratoxicosis and its antagonism.

Ochratoxin A (OA) is a toxic isocoumarin derivative released by various species of mold which grow on grain, coffee, and nuts, representing a serious worldwide health problem. Among other mechanisms of toxicity, it has been suggested that OA inhibits phenylalanyl-tRNA synthetase (PheRS), thereby reducing protein synthesis. Using the crystal structure of PheRS from Thermusthermophilus, we have modeled its interactions with OA as well as with phenylalanyl adenylate (FAMP), the high-affinity intermediate substrate of PheRS. Our results indicate that while OA may be capable of weakly inhibiting PheRS, the OA-PheRS complex cannot adopt the same conformation as does FAMP-PheRS, contrary to previous assumptions. Relative to FAMP, the phenylalanyl moiety is found to bind more shallowly and in a different overall conformation. Free-energy perturbation calculations of the relative free energies of binding of OA with the phenolic moiety protonated versus deprotonated suggest that the protonated form binds significantly more strongly. Two alternative binding modes were also identified which cannot be discounted on the basis of these calculations. Our results, however, do not suggest binding stronger than millimolar for any of the binding modes, a conclusion which is in agreement with more recent experimental findings. This, in turn, suggests that the previously observed antagonistic effects of aspartame and piroxicam are more likely due to their prevention of OA binding to human serum albumin than to PheRS, which is in agreement with binding studies as well as with preliminary simulations performed in our laboratory.

Effect of superoxide dismutase and catalase on the nephrotoxicity induced by subchronical administration of ochratoxin A in rats.

Ochratoxin A (OTA) is a mycotoxin produced by Aspergillus ochraceus as well as other molds. It is a natural contaminant of mouldy food and feed. OTA has a number of toxic effects, the most prominent being nephrotoxicity. Furthermore, OTA is immunosuppressive, genotoxic, teratogenic and carcinogenic. OTA inhibits protein synthesis by competition with phenylalanine in the phenylalanine-tRNA aminoacylation reaction. Recently, lipid peroxidation induced by OTA has been reported, indicating that the lesions induced by this mycotoxin could be also related to oxidative pathways. It was then interesting to study effects of the superoxide dismutase (SOD) and catalase on the nephrotoxicity induced by OTA in rats. The two enzymes (20 mg/kg body weight each) were given to rats by subcutaneous injection, every 48 h, 1 h before gavage by OTA (289 micrograms/kg b.w. every 48 h), for 3 weeks. SOD and catalase prevented most of the nephrotoxic effects induced by ochratoxin A, observed as enzymuria, proteinuria, creatinemia and increased urinary excretion of OTA. Altogether these results indicate (i) that superoxide radicals and hydrogen peroxide are likely to be involved in the damaging processes of OTA in vivo, (ii) that SOD and catalase might be used for prevention of renal lesions in cases of ochratoxicosis.

Phenylalanine prevents acute poisoning by ochratoxina in mice.

Ochratoxin-A (OT-A) a mycotoxin isolated from Aspergillus ochraceus is toxic for animals and man causing a fatal chronic kidney disease and liver damages. OT-A is a competitive inhibitor of phenylalanine in the phenylalanyl-tRNA synthetase-catalyzed reaction thus inhibiting protein synthesis. This inhibition can be reversed by phenylalanine. When injected intraperitoneally (i.p.) to mice a dose of 0.8 mg of OT-A is lethal within 24h. However, when this lethal dose is injected together with 1 mg of phenylalanine 100% of the animals survive. When phenylalanine was injected 30 min after OT-A the doses required for the survival of 92% of the animals had to be about 25 times higher.