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This study identified secondary metabolites produced by Alternaria alternata, Colletotrichum gloeosporioides, and Penicillium digitatum in fruits of two blood orange cultivars before harvest. Analysis was performed by UHPLC–Q-TOF-MS. Three types of fruits were selected, asymptomatic, symptomatic showing necrotic lesions caused by hail, and mummified. Extracts from peel and juice were analyzed separately. Penicillium digitatum was the prevalent species recovered from mummified and hail-injured fruits. Among 47 secondary metabolites identified, 16, 18, and 13 were of A. alternata, C. gloeosporioides, and P. digitatum, respectively. Consistently with isolations, indicating the presence of these fungi also in asymptomatic fruits, the metabolic profiles of the peel of hail-injured and asymptomatic fruits did not differ substantially. Major differences were found in the profiles of juice from hail-injured and mummified fruits, such as a significant higher presence of 5,4-dihydroxy-3,7,8-trimethoxy-6C-methylflavone and Atrovenetin, particularly in the juice of mummified fruits of the Tarocco Lempso cultivar. Moreover, the mycotoxins patulin and Rubratoxin B were detected exclusively in mummified fruits. Patulin was detected in both the juice and peel, with a higher relative abundance in the juice, while Rubratoxin B was detected only in the juice. These findings provide basic information for evaluating and preventing the risk of contamination by mycotoxins in the citrus fresh fruit supply chain and juice industry.

To elucidate the mechanism underlying rubratoxin B toxicity, the effects of rubratoxin B on the secretion of CCR5 chemokines, CCL3, CCL4, and CCL5, in a human promyelocytic leukemia cell line, HL60, were investigated. In addition, to examine whether the molecular chaperone 90-kDa heat shock protein (Hsp90) contributes to rubratoxin B toxicity, the effects of Hsp90-specific inhibitors, radicicol and geldanamycin, were investigated. Exposure to rubratoxin B for 24h induced secretion of each CCR5 chemokine, although the effect on CCL5 secretion was modest, and it enhanced secretion of proinflammatory cytokines tumor necrosis factor-α, CXCL8, and CCL2. Concomitant treatment with radicicol abolished the rubratoxin-induced secretion of all cytokines investigated. Geldanamycin antagonized the rubratoxin B-induced effects on CCL3 and CCL5, but not CCL4; the effects of geldanamycin were less than that of radicicol. Taken together, the results suggest that rubratoxin B, with the contribution of Hsp90, induces secretion of CCR5 chemokines.

Rubratoxin B is a mycotoxin that causes hypoglycemia and fatty liver. We investigated the effect of rubratoxin B on hepatic glycogen content and regulation, because blood glucose levels are associated with hepatic glycogen storage. Mice were treated with 1.5 mg/kg rubratoxin B for 24 h. Stomachs of treated mice became extremely swollen, and the contents were significantly heavier than those of controls. Hypoglycemia stimulates appetite; therefore, rubratoxin B may perturb satiation. Rubratoxin B evidently depleted hepatic glycogen stores. Phosphoenolpyruvate carboxykinase (PEPCK) activity and mRNA levels in treated mice were reduced, indicating that rubratoxin B caused hepatic glycogen depletion by inhibiting PEPCK. PEPCK activity and mRNA levels were reduced to similar degrees; it appears that PEPCK activity is regulated transcriptionally. Levels of the PEPCK gene trans-activators phospho-CREB (active form) and C/EBPα were significantly reduced in the livers of treated mice, suggesting that these factors are important for PEPCK gene transcription. Rubratoxicosis and fatty acid oxidation disorders (FAODs) share characteristic signs, such as robust appetite, hypoglycemia, hepatic glycogen depletion, and fatty liver. Although FAODs are generally considered genetic deficiencies, our results indicate that a chemical can also cause FAOD-like signs in mice.

One new nonadride derivative rubratoxin C ( 1 ) together with a known compound rubratoxin B ( 2 ) were isolated from an endophytic fungus Penicillium sp. F-14. Their structures were elucidated by various spectroscopic methods. Bioassays showed that 2 had moderate cytotoxic effect against HCT-8, BEL-7402, Ketr3, A2780, MCF-7 and BGC-823 human cancer cell lines, and 1 exhibited weak activity.

Rubratoxin B is a mycotoxin that causes hepatic fatty changes. We examined whether white adipose tissue (WAT) contributes to rubratoxin B toxicity through effects on interleukin (IL)-6. Rubratoxin B was intraperitoneally injected into mice at 1.5 mg/kg. Urinary albumin and macrophage inflammatory protein (MIP)-2 secretion were increased 24 h after treatment with rubratoxin B. Rubratoxin B was previously reported to induce IL-6 secretion, although the secreting tissue was unknown. Here, rubratoxin B prominently augmented IL-6 transcription in epididymal WAT and to a lesser extent in perirenal WAT and liver. Rubratoxin B may thus exert its toxicity partly through IL-6 secretion from WATs. In contrast, MIP-2 gene expression increased only in liver. To examine the specific involvement of adipocytes, we used mouse 3T3-L1 cells, an in vitro differentiation model of adipocytes. Expression of IL-6 and MIP-2 mRNA in 3T3-L1 adipocytes after 24 h of rubratoxin B treatment increased dose-dependently. Rubratoxin B also increased IL-6 and MIP-2 secretion from 3T3-L1 adipocytes. The increase in IL-6 secretion was markedly higher than the increase in IL-6 gene transcription, indicating that rubratoxin B-induced secretion of IL-6 from 3T3-L1 adipocytes is chiefly controlled post-transcriptionally. Rubratoxin B is thus the first mycotoxin known to exert its toxicity through effects on WATs.

肝毒性を示すマイコトキシンのルブラトキシンB は脂肪肝を引き起こす．そこで脂肪蓄積メカニズムを解明するために，ルブラトキシンB で24 時間処理したマウス肝臓において蓄積した脂肪滴のタイプや，脂質合成系酵素であるグルコース－ 6 －リン酸脱水素酵素（G6PD）と脂肪酸合成酵素の活性を調べた．オイルレッドO 染色では，ルブラトキシンB 処理したマウスから多数の小滴性の脂肪滴が観察された．脂肪酸合成のためのNADPH を供給するペントースリン酸回路において極めて重要な働きをするG6PD の活性は，ルブラトキシンB 処理によって顕著に上昇した．予想に反して，ルブラトキシンB は脂肪鎖を伸長させる脂肪酸合成酵素の活性を下げた．脂肪酸合成酵素は脂肪酸合成における律速酵素ではないので，ルブラトキシンB で処理されたマウスの活性でも脂肪蓄積には十分なのかもしれない．

Terrestrial fungi are a prolific source of metabolites with significant biological activities. Many important anticancer, antifungal and antibacterial chemotherapeutics are either microbial metabolites or semisynthetic derivatives. Investigating the metabolites of fungi may increase the chance of finding novel compounds. A bioassay-guided fractionation of soil fungus Penicillium purpurogenum fermentation, yielded the metabolite Rubratoxin B using morphological deformation of Pyricularia oryzae mycelia. Analysis by flow cytometry showed that Rubratoxin B inhibited the cell cycle progression of tsFT210 cells in the G2/M phase at the concentration of 24 microM. Cytotoxic activity analysis showed that Rubratoxin B has a cytotoxic activity and IC50 was 67.3 +/- 1.4 microM on human fibrosarcoma cell (HT1080), and Rubratoxin B exhibited inhibitory activities against matrix metalloproteinase (MMP)-2 and 9 on HT 1080 cells with an inhibitory rate of 61.5% and 74.7% at 30 microM, respectively.

The induction of insulin-like growth factor binding protein-1 (IGFBP-1) secretion by rubratoxin B was investigated using human hepatoma cell line HepG2; we also documented the involvement of stress-activated MAP kinases [c-Jun-N-terminal kinases (JNKs) and p38s] in this process. Rubratoxin B dramatically enhanced IGFBP-1 secretion, which peaked at a concentration of 40 microg/ml. The amount of IGFBP-1 mRNA increased with time and plateaued at 6 h. Compared with the amounts of IGFBP-1 secreted, the induction ratios of transcription were much smaller, indicating that IGFBP-1 secretion is regulated chiefly post-transcriptionally. The result of concomitant treatment with rubratoxin B and JNK inhibitor indicated that JNKs do not affect rubratoxin B-induced IGFBP-1 secretion. Alternatively, rubratoxin B-associated induction of IGFBP-1 secretion was marked in the absence of p38 inhibitor but attenuated in its presence. Therefore, p38s appear to stimulate rubratoxin B-induced IGFBP-1 secretion. Treatment with p38 inhibitor slightly increased the amount of rubratoxin B-induced IGFBP-1 mRNA. However this induction ratio was smaller than that of rubratoxin B-induced secretion, suggesting that p38s regulate IGFBP-1 secretion both transcriptionally and post-transcriptionally. In this study, we showed that rubratoxin B induces IGFBP-1 levels in HepG2 cells and p38s contribute to this process.

Activities of mycotoxins--citrinin, ochratoxin B, rubratoxin B, and zearalenol beta--that affected superoxide anion (O2-) production were studied on human neutrophils with regard to hypochlorous acid (HOCl) generation, nitric oxide (NO) formation, and chemotaxis of isolated cells. At the doses of 10(-8), 10(-6), 10(-4), or 10(-2) mg/mL, in stimulated cells the mycotoxins inhibited HOCl production proportionally to that of O2- because HOCl production is directly dependent on O2- generation. But unlike the others, zearalenol beta decreased HOCl production much more than one of O2-, indicating an effect also on myeloperoxidase secretion. All mycotoxins inhibited NO generation but only at the dose of 10(-2) mg/mL. Formyl-met-leu-phe-induced chemotaxis of neutrophils was deeply affected as well, as all mycotoxins at all doses used decreased it. All considered effects were not dose-dependent, likely connected to high variability of individual sensitivity to these compounds and because they already are present in blood introduced with foods. These activities of neutrophils confirm and extend the danger for immune systems by mycotoxins also in very small quantities, since the smaller concentrations we used are easily reachable in blood.

The interaction of rubratoxin B (RB) with bovine serum albumin (BSA) was investigated. Circular dichroism spectroscopy shows the interaction of RB with BSA as conformational change of BSA. Fluorescence quenching study suggests that RB binds to BSA at a few sites. At low concentration of RB, RB firstly binds to BSA at any binding sites far from Trp residues. By increasing RB concentration, RB binds to BSA at any binding sites near Trp residues.

Epigallocatechin-3-gallate (EGCG), exhibited protective effect against rubratoxin B (RB)-induced apoptosis in cultured rat hepatocytes. In this study, the mechanism of hepatoprotection by EGCG was investigated. Hepatocytes were concomitantly treated with RB and EGCG and then the expression levels of mitogen-activated protein kinase (MAPK) p38, caspase-3 and caspase-8 genes were measured by reverse transcriptase-polymerase chain reaction (RT-PCR). p38 was induced by RB, indicating that RB-induced apoptosis was mediated by p38 pathway. On the contrary, the elevation of p38 by RB was diminished by EGCG co-treatment. The analysis of caspase-3 mRNA expression showed the same tendency as observed for the p38. To further elucidate, the expression level of mitogen-activated kinase kinase 6 (MKK6) gene, upstream signaling factor of p38, was analyzed by RT-PCR. EGCG also suppressed the expression of MKK6 mRNA. These results suggest that EGCG protects hepatocytes from RB toxicity by the suppression of the gene expression in p38 and caspase pathway at any points examined.

To improve an animal model used in toxicological studies, we investigated the effects of genetics, sex, and age on rubratoxin B toxicity in mice. Treatment with rubratoxin B for 24 h increased serum alanine aminotransferase (ALT) activity, a common indicator of hepatic injury, in C3H/He, BALB/c, DBA/2, and C57BL/6 mice. The increase in serum ALT levels was most pronounced in rubratoxin B-treated C3H/He mice, as was the decrease in serum glucose concentration. In addition, these effects were greater in C3H/He male mice than female. Serum ALT activities were more elevated in rubratoxin B-exposed 9- and 12-week-old animals than that of 6-week-olds. The effect of rubratoxin B on the serum glucose concentration was greatest in 9-week-old male C3H/He mice. Therefore, our findings show that the genetics, sex, and age of the mice treated all contributed to the toxicity of rubratoxin B and that 9-week-old male C3H/He mice were the most susceptible of the animals we tested. Moreover, rubratoxin B-exposed mice had fatty livers and increased levels of triglycerides (the typical lipid involved in the development of fatty liver).

Despite the numerous known beneficial effects of flavonoids, knowledge regarding their actions on hepatotoxicity is scanty. We therefore investigated whether the flavonoids quercetin and apigenin could protect human hepatoma cell line HuH-7 against rubratoxin B-induced toxicity. These flavonoids alone markedly retarded cell proliferation in a dose-dependent manner. Quercetin significantly lessened the inhibitory effects of rubratoxin B on cell proliferation at all concentrations tested (30-100 μM), however apigenin was protective only at low concentration (9 μM). Rubratoxin B stimulated the secretion of tumor necrosis factor- α and macrophage colony stimulating factor, and quercetin and apigenin dose-dependently inhibited the rubratoxin B-induced secretion of these cytokines. Apigenin appears to be a more potent inhibitor of cytokine secretion than is quercetin. Together our results suggest that quercetin and apigenin are protective against rubratoxin B-induced cytotoxicity.

The effects of anthocyanins on hepatotoxin rubratoxin B-treated HL60 cells were investigated. Anthocyanins are known to have an anti-oxidant activity and corresponding protective effects on hepatic injury. While cyanidin 3-O-β-D-glucoside (Cy 3-glc) and peonidin 3-O-β-D-glucoside (Pn 3-glc) slightly impaired the inhibitory effect of rubratoxin B on cell proliferation, their effects were not significant. In contrast, malvidin 3-O-β-D-glucoside (Mv 3-glc) drastically strengthened the effect of rubratoxin B. Structural difference between Mv 3- glc and the others is the presence of an R group in the 5' position at the B ring, suggesting that the group in this position affected the proliferation of rubratoxin B-treated cells. The results for cytokine secretion were apparently different from those for cell proliferation. The effects of the three anthocyanins on rubratoxin B-induced secretion of interleukin-8 and tumor necrosis factor-α were not substantial. At 100 μM, all the anthocyanins markedly boosted the secretion of monocyte chemotactic protein (MCP)-1, indicating that the common structure shared by the three anthocyanins affected rubratoxin B-induced MCP-1 secretion. In this study, we thus found that anthocyanins exert their activities in ways that either do or do not depend on the identity of the R group in the 5' position at their B ring.

Activity of mycotoxins citrinin, ochratoxin B, rubratoxin B, and zearalenol beta was studied on human neutrophils with regard to O2- generation. At the doses of 10(-8), 10(-6), 10(-4), or 10(-2) mg/mL the mycotoxins stimulated the superoxide anion production in resting neutrophils in whole blood but inhibited it in isolated cells. In neutrophils stimulated with zymosan or PMA, mycotoxins showed inibiting effect both on isolated and in whole blood cells. The behavior of resting isolated neutrophils, identical to that of stimulated cells, was caused by manipulations to isolate cells that activated O2- production. Activity of mycotoxins was no dose-dependent permitting to consider their effect immunotoxic rather than immunomodulator; the absence of dose-dependence was connected to high variability of individual sensitivity to these compounds. Since the smaller concentrations by us used (10(-8) and 10(-6) mg/mL) are easily reachable in blood, it is important to point out the danger represented for immune system by mycotoxins also in very small quantities.

To elucidate the hepatoprotective effects of green tea catechins, the following experiments were conducted utilizing (−)-epigallocatechin-3-gallate (EGCG), the major component of green tea catechin, together with other catechins. The protective effects of catechins against hepatotoxins, bromobenzene or rubratoxin B, were examined in primary cultures of rat hepatocytes. Bromobenzene and rubratoxin B are known to induce necrosis and apoptosis of cells, respectively. After 24-h treatment with toxin, EGCG and (−)-epigallocatechin-3-(3″- O -methyl)gallate (EGCg-3″-OMe) suppressed the bromobenzene-induced morphological change and dose-dependently prevented bromobenzene-induced cell death. Both catechins also prevented apoptotic cell death caused by rubratoxin B. In rubratoxin B-treated cells, both catechins were found to suppress the activation of caspase-3 by rubratoxin B. The results in the present study suggest that EGCG and EGCg-3″-OMe are potent hepatoprotective agents. This report is the first to show that catechins suppress cytotoxin-induced cell death.

Induction of cytokine secretion by rubratoxin B was investigated using HL60 cells. Tumor necrosis factor (TNF)-alpha and interleukin (IL)-8 were secreted from 40 and 80 microg/ml rubratoxin B-treated cells. In 20 and 40 microg/ml rubratoxin B-treated samples, monocyte chemotactic protein (MCP)-1 was released. These rubratoxin B-induced cytokines are known to promote liver myelocytic cell infiltration, and activate cytokine-recruited cells. As a result, recruited myelocytic cells are considered to contribute to hepatic injury. We investigated the effects of tyrosine kinase inhibitors genistein and emodin. Genistein reduced the release of all three cytokines from rubratoxin B-treated cells. Likewise, emodin diminished the secretion of MCP-1. Alternatively, emodin reversed on the secretion of TNF-alpha, and the release of IL-8 was not affected. Since emodin did not impede rubratoxin B-caused TNF-alpha and IL-8 secretion, they appeared to be regulated differently from MCP-1 secretion, suggesting that rubratoxin B exerts its toxicity using two or more signal transduction pathways.

The effects of prenatal rubratoxin-B (RB) exposure on 8 behavioral parameters in JCL:ICR mice were assessed. Pregnant mice were injected intraperitoneally with 0.1 or 0.2 mg/kg/day of RB dissolved in propylene glycol water solution on days 7-9 (Group A) or 10-12 (Group B) of gestation. Controls received the vehicle similarly on days 7-12 of gestation. Before weaning, the offspring of both sexes were examined to test their the surface righting reflex (5 days of age), cliff avoidance response (6 days), negative geotaxis response (7 days), and swimming development (8, 10, and 12 days). After weaning, male animals were examined using the rotarod test (6 weeks of age), the open-field test (7 weeks), the shuttle-box-avoidance-learning test (9 weeks), and the water E-maze test (10 weeks). The preweanling offspring in the 0.2 mg/kg-B group showed significantly lower success rates and longer response times than controls in the cliff-avoidance response. In swimming development, the offspring in the 0.2 mg/kg B group had significantly lower scores than controls for swimming angle at 10 and 12 days of age. The avoidance learning of the mice in all RB-exposed A and B groups was significantly poorer than that of controls. These results indicate that prenatal exposure to RB produced a delay of early response development and impaired learning ability in the offspring of mice exposed to RB during middle pregnancy.

The effects of calcium channel blockers (CCBs) verapamil and diltiazem on the toxicity of rubratoxin B in HL60 cells were investigated. Treatment of rubratoxin B caused a considerable rate of blebbing, fragmentation and condensation of cells. The rate of this morphological change was much lower in the concomitant rubratoxin B and CCBs-treated cells than in the cells treated only with rubratoxin B. Cell viability was determined by measuring mitochondrial succinic dehydrogenase activity and the rate of cell proliferation. The results of these assays were of the same tendency as that of the morphological study. CCBs attenuated rubratoxin B's toxicity on cell viability because of their protective action. Rubratoxin B induced apoptosis in the presence of internucleosomal fragmentation. In contrast, concomitant rubratoxin B and CCBs treatment did not. Taken together, the above results indicated that CCBs impaired the toxicity including morphological change, cell viability and apoptosis caused by rubratoxin B.

The influence of inoculum size on the production of rubratoxin-B by P. purpurogenum was estimated on unirradiated and irradiated corn stored for prolonged periods at 28 °C and at a moisture content of 25%. Irradiation at doses of 1.0–3.0 kGy reduced the level of mould growth greatly relative to unirradiated controls. Rubratoxin-B production in corn grains at all doses after 40 days was less than the unirradiated control level and was below the detection limit above 4.0 kGy. Non-sterile corn that was inoculated with conidia of the fungus and then irradiated with 2.0 kGy gamma rays, produced lower rubratoxin-B levels compared with unirradiated controls. When corn was inoculated with 105 spores per gram after irradiation, the spore count and rubratoxin-B level were higher than the unirradiated control after 60 days. The total viable counts of endogenous fungal flora in the corn were reduced by the 2-kGy radiation dose, but after 20 days of incubation the numbers of P. purpurogenum increased with greater enhancement of rubratoxin-B production.

Based on the DNA fragmentation profile in gel electrophoresis and the morphological changes in electron microscopy, the induction of apoptotic nuclear changes by mycotoxins and other microbial products, in total 31 chemicals, was investigated in HL-60 human promyelotic leukemia cells, along with the cytotoxicity tests with 3-[4,5-dimethylthiazol-zyl]-2,5-diphenyltetrazolium bromide (MTT) and trypan blue exclusion. Among the chemicals tested, trichothecenes (T-2 toxin, roridin A, nivalenol, deoxynivalenol), certain anthraquinones (luteoskyrin, skyrin, 2-hydroxyemodin), diketopiperazines (emethallicin A, emestrin), isocoumarins (ochratoxin A, citrinin), lactone (penicillic acid), dihydrobisfuran (aflatoxin B1), potassium ionophore (valinomycin), and an inhibitor of interleukin-2 synthesis (cyclosporin A) were positive for the induction of DNA fragmentation. No DNA fragmentation was observed under the present conditions with fumonisin B1, cyclic peptides (cyclochlorotine, phalloidin, microcystin-LR), certain anthraquinones (emodin, chrysophanol, rugulosin), and others (sterigmatocystin, cytochalasin A, griseofulvin, fusaric acid, kojic acid, rubratoxin B, butenolide, wortmannin, FK506, and sphingosine). The apoptotic changes in the cells exposed to T-2 toxin and luteoskyrin were confirmed by electron microscopic observation. Detailed experiments on dose and time dependencies revealed that T-2 toxin induced the apoptosis at 10 ng/ml (= 4 x 10(-8) M) levels within 2-6 hr without significant cytotoxicity evaluated by the dye exclusion and MTT.

The immune system is primarily responsible for defense against invading organisms. The effects of several mycotoxins on the immune responses have been investigated; however, most data concern laboratory animals. In some instances, farm animals and cells derived from livestock species have been employed to evaluate the immunotoxicity of mycotoxins. Immune responses are highly variable, and cost considerations usually preclude the use of dairy cattle as experimental models. Immunosuppression caused by aflatoxin B1 has been demonstrated in various livestock species (e.g., turkeys, chickens, and pigs) and also in laboratory animals (mice, guinea pigs, and rabbits). The response of bovine lymphocytes to aflatoxin in vitro is similar to that of other laboratory animals. Trichothecenes are potent immunosuppressive agents that directly affect immune cells and also modify immune responses as a consequence of tissue damage elsewhere. Sheep and calves treated with fusarium T-2 toxin develop leukopenia and decreased functioning of peripheral lymphocytes. Immunosuppressive effects of ochratoxin A, rubratoxin B, and patulin have been reported. Citrinin produced lymphopenia but stimulated responses against antigens. Antibodies against mycotoxins conjugated to proteins have been produced and are useful for analytical purposes.

Although cytostatin analog protein phosphatase 2A (PP2A)-specific inhibitors are promising candidates of a new type of anticancer drug, their development has been hindered because of their liability. To find new classes of PP2A-specific inhibitors, we conducted a screening with microbial metabolites and found that rubratoxin A, a classical mycotoxin, is a highly specific and potent inhibitor of the enzyme. While rubratoxin A inhibits PP2A at Ki = 28.7 nm, it hardly inhibited any other phosphatases examined. Rubratoxin B, a close analog, also specifically but weakly inhibits PP2A at Ki = 3.1 microM. The inhibition of intracellular PP2A in cultured cells is obviously observed with 20 microM rubratoxin A treatment for 3 h, inducing the overphosphorylation in PP2A substrate proteins. Although rubratoxins and cytostatin differ in the apparent structures, these compounds share similarities in the structures in detail and PP2A-binding manners. Rubratoxin A showed higher suppression of tumor metastasis and reduction of the primary tumor volume than cytostatin in mouse experiments. As a successor of cytostatin analogs, rubratoxin A should be a good compound leading to the development of antitumor drugs targeting PP2A.

Mycotoxins are secondary metabolites of moulds that which exert adverse effects in humans and animals. It is known that direct cellular toxicity is often associated with increased cellular accumulation of toxic compounds, and membrane transport may be the first fundamental stage in the development of the cytotoxicity. To elucidate the entry pathway for mycotoxins into cells, we have investigated the interactions of human and rat organic anion transporters (hOATs/rOats) and human organic cation transporters (hOCTs) with mycotoxins using cells stably expressing hOATs/rOats/hOCTs. The mycotoxins tested were aflatoxin B1, α-zearalenol, citrinin, citrioveridine, cyclopiazonic acid, fumonisin B1, gliotoxin, patulin, penicillic acid, rubratoxin B, and zearalenone. These mycotoxins inhibited organic anion uptake mediated by hOAT1-4, and organic cation uptake mediated by hOCT1-2. By comparing the IC50values of mycotoxins for hOATs, it was found that hOAT1 and hOAT3 exhibited higher affinity interactions with mycotoxins than hOAT2 and hOAT4. There was no interspecies difference between humans and rats for the interactions of OATs with mycotoxins except that of OAT3 with rubratoxin B. Finally, we observed that hOAT1-4 and hOCT1-2 mediated the uptake of aflatoxin B1. In conclusion, hOATs and hOCTs interacted with various mycotoxins. Considering the localization of hOATs/rOats and hOCTs, it was suggested that these transporters were the possible entrance pathway for mycotoxins in kidney and liver, leading to the induction of adverse effects in humans and rats. Keywords:: organic anion transporter, organic cation transporter, SLC22, mycotoxin, aflatoxin

One hundred and fifty random poultry and animal feed samples were collected and analyzed for Penicillium toxins. The effect of gamma irradiation on the production of mycotoxins in feeds was studied. The results revealed the isolation of 12 Penicillium species. Out of 179 pure cultures of Penicillium species only 104 isolates (58%) were mycotoxin-producers. The isolates have the ability to produce, citrinin, patulin, ochratoxin A, rubratoxin B and cyclopiazonic acid with variable levels. Of the 150 samples examined, 70 were positive for Penicillium toxins. Analysis of feed samples revealed the occurrence of citrinin, ochratoxin A, cyclopiazonic acid and rubratoxin B. Irradiation at a dose 4.0 kGy reduced the Penicillium growth greatly relative to unirradiated controls and there was no growth and mycotoxin production at dose 5.0 kGy. Treatment of feed samples with 10 kGy detoxified citrinin by 92.5 to 97.5% and ochratoxin A by 68.8 to 78.5%. A dose of 15 kGy was sufficient for complete destruction of citrinin and by 90 to 95% for ochratoxin A. Patulin, cyclopiazonic acid and rubratoxin B were not detect at 5.0 kGy. Egyptian Journal of Biotechnology Vol. 25 (1) 2007: pp. 130-144

The effects of stress-activated MAP kinases (SAPKs) on biological phenomena in HepG2 cells caused by the hepatotoxin rubratoxin B were investigated. The amounts of phosphorylated (active) SAPKs (c-Jun N-terminal kinases (JNKs) and p38s) were significantly increased after treating cells with rubratoxin B, suggesting that rubratoxin B exerts its toxicity through SAPK signal transduction pathways. Compared with rubratoxin B-treatment alone, treatment with both rubratoxin B and the JNK inhibitor SP600125 decreased cell morphology changes and the activity of the apoptosis-related enzymes caspase-3 and caspase-7, indicating that JNKs are involved in rubratoxin B-induced apoptosis. The p38 inhibitor SB203580 had the same general effects as SP600125; however, its effects were rather weak. The percent inhibition of cell proliferation by SAPKs were nearly the same with or without rubratoxin B, suggesting that the regulation of SAPKs is independent of rubratoxin B effects. SAPK inhibitors decreased rubratoxin B-induced secretion of interleukin-8 and macrophage colony stimulating factor; SP600125 impaired rubratoxin B-induced granulocyte-macrophage colony stimulating factor secretion, but SB203580 enhanced this secretion. The effects of SAPK inhibitors on the levels of cytokine mRNAs showed basically the same pattern as their effects on cytokine secretion, except that their relative effects on mRNA levels was smaller. Thus, SAPKs play important roles in rubratoxin B-induced cytokine secretion, mainly post-transcriptionally.

The induction of cytokine secretion by rubratoxin B was investigated using human hepatoma cell lines HepG2 and HuH-7. Interleukin (IL)-8, macrophage colony stimulating factor (M-CSF) and granulocyte-macrophage (GM)-CSF were detected in the media of rubratoxin B-treated both cell lines, and their levels peaked at about 40 microg/ml. Rubratoxin B-induced cytokine secretion was enhanced by tumor necrosis factor (TNF)-alpha in HepG2 cells. While emodin increased GM-CSF secretion, the secretion of the others was decreased, indicating that they are regulated differently in rubratoxin B-treated HepG2 cells. To our knowledge, this is the first report that an exogenous stimulus induced the secretion of M-CSF and GM-CSF in hepatocyte-derived hepatoma cells, suggesting that rubratoxin B is an excellent model compound to study the mechanisms of M-CSF and GM-CSF secretion. Our results showed that hepatotoxin rubratoxin B has a potential to induce the secretion of these two CSFs, implying that these factors play roles in rubratoxin B-caused hepatic injury.

Disease-preventing activities of plant products were investigated. Augmentation of vascular smooth muscle cell (SMC) proliferation is known to be a typical phenomenon of atherosclerosis. Genistein and (-)-epigallocatechin gallate impaired proliferation of cultured aortic SMC A-10 cells concentration-dependently. Also, high concentrations of emodin apparently inhibited proliferation. Conversely, quercetin impaired proliferation moderately. The inhibitory effects of the chemicals on mitochondrial enzyme activity were temperate in A-10 cells. Cell death rates increased according to the concentrations of the chemicals. However, their rates were much smaller than the inhibition rates of cell proliferation, indicating that the chemicals retarded aortic SMC proliferation. Rubratoxin B, a potent hepatotoxic mycotoxin, is considered to retard cell proliferation and induce apoptosis and corresponding internucleosomal fragmentation (DNA ladder). Genistein and emodin significantly impaired rubratoxin B-caused retardation of proliferation and DNA ladder in HL60 cells. Therefore, genistein and emodin may prevent rubratoxin B-caused hepatic injury. In conclusion, plant products used in this study could effectively prevent atherosclerosis and chemical-caused hepatic injury.

The antitumor effect of rubratoxin B, a mycotoxin, has been studied. Yoshida ascites sarcoma cells, in vitro in contact for 30 min with the drug and injected immediately afterwards into young rats by the intraperitoneal route, produced the survival of 80% of the animals and the doubling of the median survival time of the rats developing neoplasia. In vivo administration of the drug by the intraperitoneal route 24 h after implantation of tumor cells determined the prolongation of median survival time; supplied by other routes both against T8 sarcoma of Guerin and Yoshida ascites sarcoma it was ineffective. The differences between in vitro and in vivo results can be explained by a rapid neutralization of the drug on behalf of the organism’s enzymes or by being bound to proteins and peritoneal cells unlike tumor cells.