Metabolism of aflatoxin, ochratoxin, zearalenone, and three trichothecenes by intact rumen fluid, rumen protozoa, and rumen bacteria

The effect of rumen microbes on six mycotoxins (aflatoxin B1, ochratoxin A, zearalenone, T-2 toxin, diacetoxyscirpenol, and deoxynivalenol ) considered to be health risks for domestic animals was investigated. The mycotoxins were incubated with intact rumen fluid or fractions of rumen protozoa and bacteria from sheep and cattle in the presence or absence of milled feed. Rumen fluid had no effect on aflatoxin B1 and deoxynivalenol . The remaining four mycotoxins were all metabolized, and protozoa were more active than bacteria. Metabolism of ochratoxin A, zearalenone, and diacetoxyscirpenol was moderately or slightly inhibited by addition of milled feed in vitro. The capacity of rumen fluid to degrade ochratoxin A decreased after feeding, but this activity was gradually restored by the next feeding time. Ochratoxin A was cleaved to ochratoxin alpha and phenylalanine; zearalenone was reduced to alpha-zearalenol and to a lesser degree to beta-zearalenol; diacetoxyscirpenol and T-2 toxin were deacetylated to monoacetoxyscirpenol and HT-2 toxin, respectively. Feeding of 5 ppm (5 mg/kg) of ochratoxin A to sheep revealed 14 ppb (14 ng/ml) of ochratoxin A and ochratoxin alpha in rumen fluid after 1 h, but neither was detected in the blood. Whether such conversions in the rumen fluid may be considered as a first line of defense against toxic compounds present in the diet is briefly discussed.     

Metabolism of aflatoxin, ochratoxin, zearalenone, and three trichothecenes by intact rumen fluid, rumen protozoa, and rumen bacteria.

The effect of rumen microbes on six mycotoxins (aflatoxin B1, ochratoxin A, zearalenone, T-2 toxin, diacetoxyscirpenol, and deoxynivalenol ) considered to be health risks for domestic animals was investigated. The mycotoxins were incubated with intact rumen fluid or fractions of rumen protozoa and bacteria from sheep and cattle in the presence or absence of milled feed. Rumen fluid had no effect on aflatoxin B1 and deoxynivalenol . The remaining four mycotoxins were all metabolized, and protozoa were more active than bacteria. Metabolism of ochratoxin A, zearalenone, and diacetoxyscirpenol was moderately or slightly inhibited by addition of milled feed in vitro. The capacity of rumen fluid to degrade ochratoxin A decreased after feeding, but this activity was gradually restored by the next feeding time. Ochratoxin A was cleaved to ochratoxin alpha and phenylalanine; zearalenone was reduced to alpha-zearalenol and to a lesser degree to beta-zearalenol; diacetoxyscirpenol and T-2 toxin were deacetylated to monoacetoxyscirpenol and HT-2 toxin, respectively. Feeding of 5 ppm (5 mg/kg) of ochratoxin A to sheep revealed 14 ppb (14 ng/ml) of ochratoxin A and ochratoxin alpha in rumen fluid after 1 h, but neither was detected in the blood. Whether such conversions in the rumen fluid may be considered as a first line of defense against toxic compounds present in the diet is briefly discussed.     

Metabolism of ochratoxin B and its possible effects upon the metabolism and toxicity of ochratoxin A in rats

A metabolic product was formed from ochratoxin B by rat liver microsomal fractions in the presence of NADPH. It was isolated from the incubation mixture by extraction, thin-layer chromatography, high-pressure liquid chromatography, and crystallization. On the basis of mass and nuclear magnetic resonance spectroscopy, the structure is suggested to be 4-hydroxyochratoxin B. The Km for the formation of 4-hydroxyochratoxin B was determined, and the hydroxylation of ochratoxin A was not altered by the presence of ochratoxin B. Rats were given ochratoxin A or B, or a mixture of both intraperitoneally. The ratios of the three metabolites, ochratoxin A, (4R)-4-hydroxyochratoxin A, and ochratoxin alpha, excreted in the urine did not change in the presence of ochratoxin B. Ochratoxin B was metabolized to 4-hydroxyochratoxin B and ochratoxin beta, but in a different ratio than for the ochratoxin A metabolites. When given intraperitoneally, ochratoxin beta was excreted within 24 h. In rats treated with ochratoxin A alone, the food intake was reduced by 50%, and histologically severe lesions, degeneration, and necrosis were observed in the proximal tubules. When ochratoxin A and B given in combination, the animals were clinically unaffected and histologically there was only slight damage of proximal tubules. These observations indicate that ochratoxin B considerably reduces the toxic effects of ochratoxin A.     

Metabolism of ochratoxin B and its possible effects upon the metabolism and toxicity of ochratoxin A in rats.

A metabolic product was formed from ochratoxin B by rat liver microsomal fractions in the presence of NADPH. It was isolated from the incubation mixture by extraction, thin-layer chromatography, high-pressure liquid chromatography, and crystallization. On the basis of mass and nuclear magnetic resonance spectroscopy, the structure is suggested to be 4-hydroxyochratoxin B. The Km for the formation of 4-hydroxyochratoxin B was determined, and the hydroxylation of ochratoxin A was not altered by the presence of ochratoxin B. Rats were given ochratoxin A or B, or a mixture of both intraperitoneally. The ratios of the three metabolites, ochratoxin A, (4R)-4-hydroxyochratoxin A, and ochratoxin alpha, excreted in the urine did not change in the presence of ochratoxin B. Ochratoxin B was metabolized to 4-hydroxyochratoxin B and ochratoxin beta, but in a different ratio than for the ochratoxin A metabolites. When given intraperitoneally, ochratoxin beta was excreted within 24 h. In rats treated with ochratoxin A alone, the food intake was reduced by 50%, and histologically severe lesions, degeneration, and necrosis were observed in the proximal tubules. When ochratoxin A and B given in combination, the animals were clinically unaffected and histologically there was only slight damage of proximal tubules. These observations indicate that ochratoxin B considerably reduces the toxic effects of ochratoxin A.     

Mechanism of ochratoxin A-induced immunosuppression

Ochratoxin A (OA) has been reported to affect immune function both at the level of antibody synthesis and natural killer (NK) cell activity. In the present study we demonstrate that exposure of purified human lymphocyte populations and subpopulations to the toxin will abrogate the cells' ability to respond to activating stimuliin vitro. Thus, both IL-2 production and IL-2 receptor expression of activated T lymphocytes are severely impaired. When the cells are preincubated with the analogue ochratoxin B (OB) prior to OA exposure, the inhibitory effect of OA is reversed. Furthermore, the inhibitory effect of OA on antibody production is not only due to blocking of T helper cell function. Highly purified B lymphocytes will not respond to polyclonal activatorsin vitro after a brief pulse with OA. The results strongly suggest that the toxin causes its immunosuppression through interference with essential processes in cell metabolism irrespective of lymphocyte population or subpopulation.     

The digestion in the small intestine of young bulls of the protein of rapeseed meal treated or untreated with formaldehyde

Twelve bulls of approximately 140 kg bodyweight were each fitted with simple cannulae in the rumen and abomasum and a re-entrant cannula in the terminal ileum. They were kept in individual pens and given daily 1.8 kg of a basal diet containing dried maize meal (whole plant), dried sugarbeet pulp and a urea/mineral preparation in the proportions 0.80, 0.18 and 0.02. Four animals received this diet alone, four received in addition 198 g daily (11 g N) of rapeseed meal by infusion into the abomasum and the remaining four the basal diet plus the same amount of formaldehyde-treated rapeseed also by infusion.The concentration of ammonia-N in the rumen fluid of animals given the untreated rapeseed meal was higher (P < 0.05) than for those on the other two treatments. The pH of the rumen and abomasal fluid was similar on all treatments. Coefficients for the apparent disappearance in the small intestine of the dry matter and nitrogen of untreated rapeseed meal infused into the abomasum were 0.76 and 0.75, respectively. In contrast formaldehyde-treated rapeseed meal was poorly digested. It is suggested that procedures for the treatment of rapeseed meal that will minimise the degradability of its protein in the rumen, yet prevent depression of its digestion and absorption in the abomasum and small intestine have still to be defined.     

Metabolism of ochratoxin A by primary cultures of rat hepatocytes.

Association of ochratoxin A with cultured rat hepatocytes occurs at 4 degrees C, and the saturation level in the medium is 0.3 mM ochratoxin A, with maximal binding after 60 min. At 37 degrees C the level of cell-associated ochratoxin A increased up to 6 h and remained at 2 nmol of toxin per mg of cell protein for 30 h. With increasing concentrations of ochratoxin A, increasing amounts of the toxin accumulated in the cells; saturation occurred at a concentration of 0.3 mM. Ochratoxin A was metabolized by hepatocytes at 37 degrees. (4R)-4-Hydroxyochratoxin A appeared in the medium at a maximal level (about 30 nmol/mg of cell protein) at an ochratoxin A concentration of 0.25 mM after 48 h of incubation. Small amounts of (4S)-4-hydroxyochratoxin A were detected only after incubation for 22 h or longer.     

The occurrence of ochratoxin A in dust collected from a problem household

Accumulated dust samples were collected from the heating ducts in a household where signs resembling ochratoxin poisoning in animals occurred. Several Penicillium spp. and Aspergillus ochraceous had been identified previously from air samples taken from this house. A composite sample from six collected samples was examined by HPLC, and it was determined that 58 ppb of ochratoxin A was present in this sample. A second set of six samples was collected and determinations were made by HPLC of the ochratoxin content in each sample. All samples, including one sample of dirt from a crawl space, yielded at least a trace of ochratoxin A; however, one sample of dust collected from the heating ducts yielded over 1500 ppb of ochratoxin A, and another sample of dust from a different heating duct yielded 306 ppb of ochratoxin A. Ochratoxin A was confirmed in all samples by LC-MS, and ochratoxin was evident in the samples by TLC analysis. This is believed to be the first report of finding ochratoxin inhouse dust. This revised version was published online in June 2006 with corrections to the Cover Date.     

Penicillium verrucosum in feed of ochratoxin A positive swine herds

Ochratoxin A contamination of cereal feed grain was monitored during October 1989–September 1990 by analysis of blood samples from slaughter swine in Sweden. The detection of ochratoxin A in swine blood was used as a method to identify swine herds fed ochratoxin A contaminated feed. The contamination level of ochratoxin A in the blood of the positive herds was in the range 2–45 ng/ml with the mean concentration 5.2 ng/ml. Feed samples for mycological analysis were collected from both ochratoxin A positive herds (2 ng/ml blood) and ochratoxin A negative herds (<2 ng/ml blood). From the ochratoxin A positive herds and the ochratoxin A negative herds 22 and 21 feed samples were collected, respectively. No quantitative differences in mould content, as determined by colony forming units, were observed between the two groups. However, there were differences in the mycoflora. The incidence of storage fungi (Penicillium and Aspergillus spp.) was significantly higher (p < 0.05) in feed from ochratoxin A positive herds. Particularly, Penicillium verrucosum was found to be significantly more common (p < 0.001). Altogether 274 isolates were screened for their ability to produce ochratoxin A. Ochratoxin A producers were found only within P. verrucosum; 38% of the 63 isolates produced detectable amounts of ochratoxin A. Ochratoxin A producing isolates of P. verrucosum were found in 60% of the feed samples collected from ochratoxin A positive swine herds and in one sample (5% ) of the feed samples collected from the ochratoxin A negative herds.     

Immunochemical detection of ochratoxin A in black Aspergillus strains

One hundred and fifty-seven strains belonging to Aspergillus section Nigri were tested for ochratoxin A production using three different methods: a relatively new immunochemical method based on an enzyme-linked immunosorbent assay (ELISA), thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC). The monoclonal antibody-based ELISA technique was successfully used to screen for low levels of ochratoxin A in the black Aspergilli without concentrating the culture filtrates. The results were confirmed by TLC and HPLC analysis and chemical derivatization. These latter methods required concentrated filtrates. Ochratoxin A was detected in the culture filtrates of five of the 12 A. carbonarius strains, none of the 45 A.japonicus strains and three of the 100 isolates in the A. niger aggregate (A. foetidus, A. awamori and A. niger).Abbreviations ELISA enzyme-linked immunosorbent assay - HPLC high-performance liquid chromatography - OA ochratoxin A - TLC thin-layer chromatography     

Effect of dietary sodium bicarbonate supplementation on the toxicokinetics of ochratoxin A in pigs

The mycotoxin ochratoxin A (OA) is regarded as a causative agent for endemic nephropathy in farm animals and humans. Reabsorption of OA along the nephron results from nonionic diffusion and by carrier-mediated mechanisms indicating that urine alkalinization may help to accelerate OA excretion and thus reduce its toxicity. The aim of the present study was to investigate the effect of a dietary sodium bicarbonate (NaHCO₃) supplementation as a means to increase urinary pH on the systemic availability and excretion of OA in pigs. Dietary supplementation of 2% NaHCO₃ increased urinary pH (5.7±0.2 to 8.3±0.1) and daily urine volume (1108±276 to 2479±912ml) significantly. The systemic availability of OA and its dechloro-analog Ochratoxin B (OB) in the NaHCO₃ group calculated as the area under the serum concentration-time curve (AUC) was reduced to 75 and 68%, respectively, of the control (P<0.05). This effect was mainly due to an accelerated elimination of OA and OB in the urine. The faster renal elimination might be explained by a reduced reabsorption of the ochratoxins by nonionic diffusion, and other H⁺-dependent mechanisms. Thus, urinary alkalinization might be an efficient means to partially reduce the toxic effects and carry-over of OA in pigs. Presented at the 26^th Mykotoxin Workshop in Herrsching, Germany, May 17–19, 2004     

Transformation of the mycotoxin ochratoxin A in plants. 2. Time course and rates of degradation and metabolite production in cell-suspension cultures of different crop plants

Ochratoxin A, one of the most toxic mycotoxins, can be metabolized nearly completely by suspension cultures of various plant cells. The transformation products identified in this study were almost the same in the cell-suspension cultures of maize, carrot, tomato, potato, soybean, wheat and barley, but the quantitative distribution differed strongly depending on incubation time and species of plant-cell culture. The compounds were extracted with ethyl acetate and detected by reversed-phase HPLC with gradient elution. From the result it is supposed that besides ochratoxin A also ochratoxin derivatives may occur in food and feedstuff of plant origin.     

In vitro study of dietary factors affecting the biohydrogenation shift from trans-11 to trans-10 fatty acids in the rumen of dairy cows

On the basis of the isomer-specific effects of trans fatty acids (FA) on human health, and the detrimental effect of t10,c12-conjugated linoleic acid (CLA) on cows' milk fat production, there is a need to identify factors that affect the shift from trans-11 to trans-10 pathway during ruminal biohydrogenation of FA. This experiment was conducted in vitro and aimed at separating the effects of the diet of the donor cows from those of the fermentative substrate, which is necessary to prevent this shift. A total of four dry Holstein dairy cows were used in a 4 × 4 Latin square design. They received 12 kg of dry matter per day of four diets based on maize silage during four successive periods: the control diet (22% starch, <3% fat); the high-starch diet, supplemented with wheat plus barley (35% starch, <3% crude fat); the sunflower oil diet, supplemented with 5% of sunflower oil (20% starch, 7.6% crude fat); and the high-starch plus oil diet (33% starch, 7.3% crude fat). Ruminal fluid of each donor cow was incubated for 5 h with four substrates having similar chemical composition to the diets, replacing sunflower oil by pure linoleic acid (LA). The efficiency of isomerisation of LA to CLA was the highest when rumen fluids from cows receiving dietary oil were incubated with added LA. The shift from trans-11 to trans-10 isomers was induced in vitro by high-starch diets and the addition of LA. Oil supplementation to the diet of the donor cows increased this shift. Conversely, the trans-10 isomer balance was always low when no LA was added to incubation cultures. These results showed that a large accumulation of trans-10 FA was only observed with an adapted microflora, as well as an addition of non-esterified LA to the incubation substrate.     

In vitro inhibition of rat platelet aggregation by ochratoxin A

The mycotoxin ochratoxin A (OA) consists of 5-chloro-3-methyl-3,4-dihydro-8-hydroxyisocoumarin moiety linked by an amide bond to β-L-phenylalanine. When added to washed rat platelets in vitro, OA caused a dose-dependent inhibition of aggregation induced by agonists such as adenosine diphosphate (ADP) or thrombin. The aggregatory response induced by prior addition of an agonist was also reversed in a dose-dependent manner by OA. Inhibition of aggregation appeared to be irreversible since exposure of platelets to OA followed by several washings removed most of the mycotoxin associated with the platelets but did not diminish the inhibitory response. Serotonin secretion from dense granules and arachidonic acid release from membrane phospholipid (especially phosphatidylcholine) as well as its further metabolism were also inhibited by OA. These results suggest that a disruption of the platelet plasma membrane structure by OA is probably responsible for inhibition of the primary and secondary phases of aggregation.     

Spontaneous occurrence of ochratoxin A residues in porcine kidney and serum samples in Poland

During the period 1 April 1983 to 31 July 1984, 214,700 swine were processed in a slaughterhouse in Poznań, Poland. Of these pigs, 122 (0.057%) exhibited macroscopical kidney changes typical for mycotoxic porcine nephropathy. Ochratoxin A was found in kidneys from 52 of these pigs. Porcine serum samples not biased for nephropathy were collected at random in the same slaughterhouse. Of 388 samples, 148 exhibited ochratoxin A residues from 1 to 520 ng/ml. Significant increases in nephropathy and ochratoxin A frequencies were observed during the spring of 1984.     

Spontaneous occurrence of ochratoxin A residues in porcine kidney and serum samples in Poland.

During the period 1 April 1983 to 31 July 1984, 214,700 swine were processed in a slaughterhouse in Poznań, Poland. Of these pigs, 122 (0.057%) exhibited macroscopical kidney changes typical for mycotoxic porcine nephropathy. Ochratoxin A was found in kidneys from 52 of these pigs. Porcine serum samples not biased for nephropathy were collected at random in the same slaughterhouse. Of 388 samples, 148 exhibited ochratoxin A residues from 1 to 520 ng/ml. Significant increases in nephropathy and ochratoxin A frequencies were observed during the spring of 1984.     

In vitro inhibition of rat platelet aggregation by ochratoxin A.

The mycotoxin ochratoxin A (OA) consists of 5-chloro-3-methyl-3,4-dihydro-8-hydroxyisocoumarin moiety linked by an amide bond to beta-L-phenylalanine. When added to washed rat platelets in vitro, OA caused a dose-dependent inhibition of aggregation induced by agonists such as adenosine diphosphate (ADP) or thrombin. The aggregatory response induced by prior addition of an agonist was also reversed in a dose-dependent manner by OA. Inhibition of aggregation appeared to be irreversible since exposure of platelets to OA followed by several washings removed most of the mycotoxin associated with the platelets but did not diminish the inhibitory response. Serotonin secretion from dense granules and arachidonic acid release from membrane phospholipid (especially phosphatidylcholine) as well as its further metabolism were also inhibited by OA. These results suggest that a disruption of the platelet plasma membrane structure by OA is probably responsible for inhibition of the primary and secondary phases of aggregation.     

Kinetics of ochratoxin A production in different Aspergillus species

Kinetics of ochratoxin A production was examined in a number of ochratoxin producing isolates representing different sections of the Aspergillus genus. Both weak and high ochratoxin producers were tested using immunochemical or high-performance liquid chromatograhic methods. All isolates were found to produce the highest amounts of ochratoxin A after 7-10 days of incubation. Ochratoxin production varied between 30 - 5 x l0(5) ng ml(-1) among the Aspergillus isolates tested. The A. albertensis and A. melleus isolates examined were found to produce ochratoxin A constitutively. A. albertensis produced the highest amounts of ochratoxin A at 30 degrees C after 7 days' incubation in YES liquid medium. Ergosterol content and ochratoxin production of A. albertensis cultures were in good correlation.     

Digestion of epithelial tissue of the rumen wall by adherent bacteria in infused and conventionally fed sheep.

Comparisons were made, by light and electron microscopy, of the rumen epithelium of sheep fed conventionally and fed by infusion of volatile fatty acids and buffer into the rumen and casein into the abomasum. Similar bacterial colonization of the epithelium was observed in each case. The mitotic index of epithelial cells in infused sheep was high, as it was in barley-fed animals, while the mitotic index of cells from animals receiving roughage was low. The bacterial flora appeared to be actively digesting the epithelial cells. The fate of sloughed epithelial cells in the rumen fluid of sheep fed by infusion was also studied. The sloughed cells were rapidly digested, probably by their attached flora of facultatively anaerobic, highly proteolytic bacteria, leaving abundant highly keratinized remnants in rumen fluid. The importance of epithelial cell turnover and of proteolysis by partially facultative bacteria in the rumen is discussed.     

Metabolites of ochratoxins in rat urine and in a culture of Aspergillus ochraceus.

We studied the metabolic profile of ochratoxin A (OA) in rats and in a culture of OA-producing Aspergillus ochraceus. Ochratoxin alpha (O alpha), ochratoxin beta (O beta), 4-R-hydroxyochratoxin A (4-R-OH OA), 4-R-hydroxyochratoxin B (4-R-OH OB), and 10-hydroxyochratoxin A (10-OH OA) were isolated from a culture of A. ochraceus and structurally characterized by 1H nuclear magnetic resonance spectroscopy, mass spectrometry and high-pressure liquid chromatography. 4-R-OH OA and O alpha were consistently produced and were the dominant biotransformed metabolites in the fungal culture and in rats treated with OA and ochratoxin C (OC), while the formation of 10-OH OA was conditional in the fungal system. Green fluorescent biomacromolecules were isolated by detergent extraction of the fungal culture followed by cold-acetone precipitation and gel filtration. Acid hydrolysis of the fluorescent macromolecules resulted in the release of several ochratoxins, including O alpha (80%), OA (2%), and OC (5%), and other unidentified fluorescent compounds but not OB and O beta. Cross-reactivity studies of the natural macromolecule conjugates of OA with anti-OA polyclonal antibodies indicated that they were covalently linked to the macromolecules via a group other than the carboxyl group. These studies demonstrated that a fungus can produce some of the same metabolites of OA as the rat and that O alpha, OA, and OC may be covalently linked to fungal macromolecules.