Long term preliminary studies on toxic and carcinogenic effect of individual or simultaneous exposure to ochratoxin A and penicillic acid in mice.

Carcinogenic effects of ochratoxin A (OTA) on liver, kidneys, intestine, muscles and subcutaneous tissue of BALB/c albino mice divided in three experimental and one control groups (30 mice in each group - 15 males and 15 females) and exposed to 10 ppm OTA and/or 50-60 ppm penicillic acid (PA) in the diet were seen. A total 22 neoplasias were found to be induced in the mice during 20 months' experimental period. Among them 14 were malignant and 8 benign. The number of neoplasias was significantly higher in the mice treated simultaneously with OTA and PA (14) compared to those in mice treated with OTA only (8). The number of malignant neoplasias was also higher (14) compared to benign neoplasias (8). Nine of a total fourteen malignant neoplasis and five of a total eight benign neoplasias were seen in the male mice. Pathological changes in mice after two weeks' experimental period were characterized by degenerative changes in kidneys, liver and gastrointestinal tract, which were better expressed in the mice exposed simultaneously to OTA and PA. A strong synergistic effect was found between OTA and PA towards tumorogenesis. It seems that mice are not a good experimental model for humans with regard to OTA-induced tumourigenesis, because the target organ of OTA-toxicity in humans or pigs is mainly the kidney, but not the liver, intestines, subcutaneous tissue or muscles as seen in this study.

Cytotoxic activity of fungal strains isolated from the ascidian Eudistoma vannamei.

The cytotoxic activity at 50 µg/ml of extracts obtained from eleven fungal strains associated to Eudistoma vannamei, an endemic ascidian from Northeast Brazil, against two cell lines, i.e., the HCT-8 (colon cancer) and the MDA-MB-435 (melanoma) cell lines, was investigated. The most promising extract (EV10) was obtained from a fungus identified as Aspergillus sp. by molecular analysis and was selected for bioassay-guided isolation of its active principals. Large-scale fermentation of EV10 in potato-dextrose broth followed by chromatographic purification of the active extract from the liquid medium allowed the isolation of the isocoumarins mellein, cis-4-hydroxymellein, and trans-4-hydroxymellein, besides penicillic acid. All isolated compounds were tested for their cytotoxicity against the tumor cell lines MDA-MB-435 and HCT-8 and revealed penicillic acid as the only cytotoxic compound (cell growth inhibitions >95%).

Mycotoxic nephropathy in Bulgarian pigs and chickens: complex aetiology and similarity to Balkan endemic nephropathy.

Spontaneous nephropathy in Bulgaria, which is observed frequently during meat inspection and which differs morphologically from the classical description of mycotoxic porcine/chicken nephropathy as made in Denmark, was found to have a multi-mycotoxic aetiology being mainly provoked by a combined effect of ochratoxin A, penicillic acid and fumonisin B1 in addition to a not-yet-known metabolite. Mean contamination levels of ochratoxin A were consecutively low (188.8 and 376.4 microg kg(-1)) in contrast to high contamination levels of fumonisin B1 (5564.1 and 3254.5 microg kg(-1)) and penicillic acid (838.6 and 904.9 microg kg(-1)) for 2006 and 2007, respectively. Some other mycotoxins with lower importance such as citrinin, penitrem A, etc., may also influence clinicopathological picture of this nephropathy. A heavy contamination with Gibberella fujikuroi var. moniliformis (Fusarium verticillioides) and Penicillium aurantiogriseum complex (mainly Penicillium polonicum) was observed in almost all examined feed samples coming from pig and chick farms with nephropathy problems from Bulgaria. In contrast, low contamination with Aspergillus ochraceus, Penicillium verrucosum and Penicillium citrinum was observed in the same feed samples and these species were isolated as very rare components of the mycobiota.

Cytotoxicity assessment of gliotoxin and penicillic acid in Tetrahymena pyriformis.

Various studies have documented the associations between mold exposure and effects on health. Mycotoxins, which occur in spores and mold fragments, can be involved in processes that have pathological effects, such as adynamia of the immune system, recurrent infections of the respiratory tract, or asthma. Using Tetrahymena pyriformis, a single-cell organism well established as a suitable model for human respiratory epithelium-cell functionalities, we investigated dose-response relationships of the mycotoxins gliotoxin and penicillic acid. Our study focused on the viability (cell count, MTT assay), energy levels (adenosine-5'-triphosphate content), energy-providing processes (MTT reduction per cell), and cell respiration (oxygen consumption). Both mycotoxins acted as cytotoxins in a dose-dependent manner. Gliotoxin had a stronger inhibitory effect (EC50 0.38 microM) than did penicillic acid (EC50 343.19 microM). The energy-providing processes were not inhibited or were only weakly inhibited under the influence of gliotoxin, whereas penicillic acid caused stimulation of the physiological parameters. Summarizing the results, it is clear that the two investigated mycotoxins must have different modes of action. They are not only different in the strength of their toxic effects but also in a variety of physiological aspects. In addition, T. pyriformis showed differences in its ability to overcome the negative effects of particular mycotoxin exposures.

Experimental mycotoxicosis in chickens induced by ochratoxin A and penicillic acid and intervention with natural plant extracts.

The combined toxic effect of ochratoxin A (OTA) and penicillic acid (PA) on the body mass, the weight and pathomorphology of some internal organs was studied in 85 broiler chickens fed a mouldy diet containing 130, 300 or 800 ppb OTA and 1000-2000 ppb PA. The main pathomorphological changes were cloudy swelling and granular degeneration in the epithelium and mononuclear cell proliferation and activation of capillary endothelium in the kidney and liver; degenerative changes and depletion of lymphoid cells in lymphoid organs (bursa of Fabricius, thymus and spleen) were also seen. Protective effects of 5% total water extract of artichoke and a new natural phytosubstance Rosallsat against these pathomorphological changes were observed. A significant decrease in body mass and relative weight of lymphoid organs was found after 6 weeks of exposure and a greater decrease after 10 weeks of exposure to OTA and PA, and a protective effect of artichoke extract and a slight effect of Rosallsat against that decrease was observed. A significant increase in relative weight of liver and kidneys was also observed as well as a protective effect of artichoke extract against that increase. The quantity of OTA and the percentage of positive samples were significantly lower in tissues of chickens treated with artichoke extract or Rosallsat in addition to OTA than in those treated with only OTA.

The mycotoxin penicillic acid inhibits Fas ligand-induced apoptosis by blocking self-processing of caspase-8 in death-inducing signaling complex.

Upon engagement with Fas ligand (FasL), Fas rapidly induces recruitment and self-processing of caspase-8 via the adaptor protein Fas-associated death domain (FADD), and activated caspase-8 cleaves downstream substrates such as caspase-3. We have found that penicillic acid (PCA) inhibits FasL-induced apoptosis and concomitant loss of cell viability in Burkitt's lymphoma Raji cells. PCA prevented activation of caspase-8 and caspase-3 upon treatment with FasL. However, PCA did not affect active caspase-3 in FasL-treated cells, suggesting that PCA primarily blocks early signaling events upstream of caspase-8 activation. FasL-induced processing of caspase-8 was severely impaired in the death-inducing signaling complex, although FasL-induced recruitment of FADD and caspase-8 occurred normally in PCA-treated cells. Although PCA inhibited the enzymatic activities of active recombinant caspase-3, caspase-8, and caspase-9 at similar concentrations, PCA exerted weak inhibitory effects on activation of caspase-9 and caspase-3 in staurosporine-treated cells but strongly inhibited caspase-8 activation in FasL-treated cells. Glutathione and cysteine neutralized an inhibitory effect of PCA on caspase-8, and PCA bound directly to the active center cysteine in the large subunit of caspase-8. Thus, our present results demonstrate that PCA inhibits FasL-induced apoptosis by targeting self-processing of caspase-8.

Experimental mycotoxic nephropathy in pigs provoked by a diet containing ochratoxin A and penicillic acid.

Mycotoxic nephropathy was induced in 18 young pigs by diets contaminated with strains of Aspergillus ochraceus containing ochratoxin A (OTA) and penicillic acid (PA) at levels corresponding to those naturally encountered in animal feeds in Bulgaria. Haematological and biochemical parameters, as well as the morphological and ultrastructural changes in various internal organs, and especially in the kidneys, were examined at different stages of development of the disease. A mottled surface of the kidneys was only seen in pigs exposed to a mouldy diet containing 180 ppb OTA for 3 months, but microscopic lesions, as well as changes in various haematological and biochemical parameters, were observed in all groups exposed to the same mouldy diet containing only 90 or 180 ppb OTA. Histological examination showed two types of change: degenerative changes affecting the epithelial cells of the proximal tubules, which predominated at the initial stage, and proliferative changes in the interstitium, which predominated at the later stage of the disease. Telangiectasis and lymph stasis were also seen, as well as degenerative changes in the capillary endothelium. The characteristic renal lesions were similar to those observed in spontaneous cases of mycotoxic porcine nephropathy in Bulgaria, but they were a little different from the classic Danish porcine nephropathy. The enhanced toxicity of OTA in our study may be due to a synergistic effect between OTA and PA or to some other unknown metabolites produced by the same ochratoxinogenic strains of A. ochraceus.

Influence of ochratoxin A and an extract of artichoke on the vaccinal immunity and health in broiler chicks.

The combined effect of ochratoxin A (at diet levels of 130, 305 and 790 ppb) and penicillic acid was studied in 100 broiler chicks. Serological investigations revealed significantly lower haemagglutination inhibiting antibody titers in the experimental chicks immunized with vaccine against Newcastle disease. A statistically significant decrease of the body weight and the relative weight of lymphoid organs as well as a significant increase of the relative weight of kidneys and liver were seen. The main degenerative changes were observed in the proximal convoluted tubules in kidneys and slight degenerative changes were found in the hepatocytes. Degenerative changes and depletion of lymphoid cells were observed in the bursa Fabricii, thymus, spleen and Peyer's patches of intestinal mucosa. Serum analyses revealed significant decreases of the total protein and cholesterol, and significant increases of the uric acid and glucose. Haematological analyses showed a slight anaemia, leucocytosis and slightly decompensated metabolic acidosis. A statistically significant protective effect of 5% total water extract of artichoke on humoral immune response (increase of haemaglutination inhibiting antibody titer), relative organ weight as well as on pathomorphological, haematological and biochemical changes induced by ochratoxin A, was established.

Penicillic acid as Na+,K+ and Ca2+ channel blocker in isolated frog's heart at toxic levels.

The effect of penicillic acid on isolated frog's heart has been studied along with ions of Na+,K+ and Ca2+. Penicillic acid has been found to inhibit the entry of these ions into cardiac tissue thereby arresting the action of the heart. The blockage can be washed away by perfusion with Ringer's solution.

Bacterial tests as indicators for the detoxification of the mycotoxin penicillic acid by ammonia treatment.

The detoxification of penicillic acid by reaction with ammonia was examined by means of a polymerase assay using two strains of Escherichia coli (pol A+ and pol A-1) and a recombination assay using two strains of Bacillus subtilis (rec+ and rec-). A 100-fold surplus of ammonia added to penicillic acid abolished the cytotoxic and genotoxic effects of penicillic acid towards the bacteria under the test conditions. The study presents the possibility of detoxifying mycotoxins in feeds by ammonia treatment and demonstrates the suitability of bacterial assays as indicators for mycotoxins.

Toxicity of penicillic acid for rat alveolar macrophages in vitro.

Penicillic acid (PA) is a polyketide mycotoxin produced by several species of Aspergillus and Penicillium. This mycotoxin is toxic in experimental animals and has also been reported to be carcinogenic. The cytotoxicity of penicillic acid was studied in rat alveolar macrophages (AM) in vitro. The effects of penicillic acid on membrane integrity were studied by measuring cell volume changes and 51Cr release. There was significant 51Cr release after 2 hr exposure to 1.0 mM penicillic acid, but not after 1 hr exposure. There was a significant decrease in adenosine triphosphate (ATP) in cell cultures exposed to 1.0 mM penicillic acid for 4 hr. Inhibition of the incorporation of [3H]leucine into protein was both dose- and time-dependent and protein synthesis was inhibited significantly after 2 hr exposure to greater than or equal to 0.1 mM penicillic acid. RNA synthesis was inhibited to a lesser extent than protein synthesis. Although there was a significant inhibition of RNA synthesis at 1.0 mM PA after 4 hr, there was no inhibition of RNA synthesis even after 4 hr at any concentration less than 1.0 mM. The ED50 dose after 2 hr exposure was 0.18 and 0.60 mM for protein and RNA synthesis, respectively. There was significant inhibition of phagocytosis after 2 hr exposure at greater than or equal to 0.3 mM penicillic acid and the ED50 for phagocytosis was 0.09 mM. Thus phagocytosis was more sensitive to the toxic effects of penicillic acid than any other cellular process studied. The results reported in this study are similar to those observed for patulin in an earlier study from our laboratory except that patulin was generally more toxic to alveolar macrophages than penicillic acid. The data demonstrate that penicillic acid is toxic to rat alveolar macrophages in vitro and suggest the possibility of a respiratory hazard to agricultural workers exposed to contaminated grain.

Induction of DNA single-strand breaks and DNA synthesis inhibition in CHO and AWRF cells after exposure to sterigmatocystin and penicillic acid.

Sterigmatocystin is 12 times more toxic to transformed rat fibroblasts (AWRF) than to Chinese hamster ovary cells (CHO). In contrast, penicillic acid is twice more toxic to CHO cells than to AWRF cells. The ability of sterigmatocystin and penicillic acid to inhibit DNA synthesis correlated well with the differences in cytotoxicity of these mycotoxins in the cell lines used. Sterigmatocystin at a concentration of 10 micrograms/ml inhibited DNA synthesis in AWRF cells during a 3-h exposure to 60% of that found in controls, but did not inhibit DNA synthesis in CHO cells. Within the same time interval penicillic acid inhibited DNA synthesis in AWRF cells at concentrations higher than 5 micrograms/ml and in CHO cells at concentrations over 0.5 microgram/ml. Induction of DNA single-strand breaks (SSB) during a 3-h exposure to sterigmatocystin and penicillic acid was comparable in both cell types. The results suggest that sterigmatocystin is metabolized to reactive metabolites that are responsible for the toxicity and DNA synthesis inhibition at a more rapid rate in AWRF cells than in CHO cells. The observed ability to induce SSB indicates that penicillic acid is potentially carcinogenic.

Acute toxicity of penicillic acid and rubratoxin B in dogs.

The effect of intraperitoneally administered penicillic acid, a mycotoxin produced by several species of Penicillium and Aspergillus, on female dogs of mixed breeding was determined by serum tests, by observation of clinical signs and survival times, and by evaluation of gross and microscopic lesions. Combination studies employing penicillic acid and a second mycotoxin, rubratoxin B, also were undertaken. Post mortem examination disclosed hemorrhaging of the serosal surfaces of the abdomen of dogs receiving penicillic acid. The most significant histologic change observed in penicillic-acid-treated dogs was congestion and dilatation of hepatic sinusoids. Extensive hepatic changes of the liver were noted only in the dog receiving 20 mg/kg penicillic acid. There was no evidence of parenchymal necrosis in any of the liver samples examined from animals given penicillic acid. A predominently peripheral lobular depletion of glycogen in parenchymal cytoplasm also was seen in liver sections from animals exposed to penicillic acid. Although slight decreases in lactic dehydrogenase were observed, no trends were detected in the several blood enzymes and serum constituents examined that could be specifically related to penicillic acid intoxification. Glutamic-oxaloacetic transaminase, lactic dehydrogenase and alkaline phosphatase activities and survival time varied in relation to duration of exposure and total dose of rubratoxin B administered. The lesions in animals injected with 1.0 mg/kg rubratoxin B consisted of mild hepatic necrosis and degenerative changes in renal tubular epithelium. An additive effect due to the combined administration of penicillic acid and rubratoxin B was observed only by an elevation in serum sodium and chlorine levels.