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 A by rats.

Albino rats were given ochratoxin A (6.6 mg/kg body weight) intraperitoneally or per os. Independent of route administration, 6% of a given dose was excreted as the toxin, 1 to 1.5% as (4R)-4-hydroxyochratoxin A, and 25 to 27% as ochratoxin alpha in the urine. The metabolite (4S)-4-hydroxyochratoxin A, which is formed by rat liver microsomes in the presence of NADPH, was not detected. Only traces of ochratoxins A and alpha were found in feces. Identical experiments were carried out with brown rats, since the Km value for the formation of the 4S epimer was considerably lower when brown rat microsomes were used. About the same ratios of metabolites and metabolite recoveries as those found for albino rats were found for brown rats. Brown rats were also given the two hydroxylated metabolites and ochratoxin alpha (0.66 mg/kg body weight) intraperitoneally. The three compounds were excreted in the urine; within 48 h, 90% recovery of ochratoxin alpha and 54 and 35%, respectively, of the 4R and 4S isomers were observed.     

Formation of (4R)- and (4S)-4-hydroxyochratoxin A from ochratoxin A by liver microsomes from various species.

Two metabolic products were formed from ochratoxin A by human, pig, and rat liver microsomal fractions in the presence of reduced nicotinamide adenine dinucleotide phosphate. They were isolated from the incubation mixture in the presence of pig liver microsomes by extraction, thin-layer chromatography, and high-pressure liquid chromatography Their structures are suggested to be (4R)- and (4S)-4-hydroxyochratoxin A on the basis of mass and nuclear magnetic resonance spectroscopy. Km and the maximum velocity for the formation of the two metabolites by human, pig, and rat microsomes were determined. Their formation was inhibited by carbon monoxide and metyrapone. The results indicate that the microsomal hydroxylation system is a cytochrome P-450 and that different species are involved in the formation of the two epimeric forms of 4-hydroxyochratoxin A.     

Formation of (4R)- and (4S)-4-hydroxyochratoxin A and 10-hydroxyochratoxin A from Ochratoxin A by rabbit liver microsomes.

Three metabolites were formed from ochratoxin A in the presence of rabbit liver microsomal fractions and NADPH. They were isolated by extraction, thin-layer chromatography, and high-pressure liquid chromatography. Two of them were identified as (4R)- and (4S)-4-hydroxyochratoxin A. It is suggested on the basis of mass and nuclear magnetic resonance spectroscopy that the third metabolite is 10-hydroxyochratoxin A. The formation of the metabolites was inhibited by carbon monoxide and metyrapone and was stimulated when microsomes from phenobarbital-treated animals were used. The results suggest that cytochrome P-450 catalyzes the formation of these metabolites.     

Oxidation of two hydroxylated ochratoxin A metabolites by alcohol dehydrogenase

(4R)-4-hydroxyochratoxin A, (4S)-4-hydroxyochratoxin A, and 10-hydroxyochratoxin A, all formed from ochratoxin A, were incubated with alcohol dehydrogenase in the presence of NAD. Only (4R)-4-hydroxyochratoxin A and 10-hydroxyochratoxin A acted as substrates for the enzyme. K(m) and turnover number for 10-hydroxyochratoxin A were 110 muM and 0.1 s(-1), respectively.     

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.     

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.     

Oxidation of Two Hydroxylated Ochratoxin A Metabolites by Alcohol Dehydrogenase

(4R)-4-hydroxyochratoxin A, (4S)-4-hydroxyochratoxin A, and 10-hydroxyochratoxin A, all formed from ochratoxin A, were incubated with alcohol dehydrogenase in the presence of NAD. Only (4R)-4-hydroxyochratoxin A and 10-hydroxyochratoxin A acted as substrates for the enzyme. K_m and turnover number for 10-hydroxyochratoxin A were 110 μM and 0.1 s⁻¹, respectively.     

Effects of ochratoxin A metabolites on yeast phenylalanyl-tRNA synthetase and on the growth and in vivo protein synthesis of hepatoma cells

The ochratoxin A (OTA) metabolite (4R)-4-hydroxyochratoxin A [4R)-OTA) inhibits the aminoacylation of phenylalanine tRNA catalyzed by phenylalanyl-tRNA synthetase (PheRS) with a Ki-value of 0.9 mM as compared to 1.3 mM for OTA. It also inhibits protein synthesis and cell growth in the same manner as OTA. Ochratoxin alpha (OT alpha) does not affect either protein synthesis or cell growth.     

Formation of 4-hydroxyochratoxin A from ochratoxin A by rat liver microsomes.

Hydroxyochratoxin A was isolated and identified from the urine of rats after injection with ochratoxin A. By incubating ochratoxin A with rat liver microsomes and reduced nicotinamide adenine dinucleotide phosphate, one major (90%) and two minor metabolites, more polar than ochratoxin A, were formed. Thin-layer chromatography revealed that the major metabolite had Rf values identical to those of hydroxyochratoxin A in six different solvent systems. Formation of the metabolites in vitro was inhibited by carbon monoxide and by metyrapone, and the rate of formation increased after pretreatment of the rats with phenobarbital. A type I spectrum appeared upon binding of ochratoxin A to microsomes with a spectral dissociation constant (Ks) of 37.6 microM. These findings strongly suggest the involvement of a cytochrome P-450 in the hydroxylation of ochratoxin A by rat liver microsomes. Apparent Km and Vmax values for the formation of hydroxyochratoxin A were determined to 50 microM and 5.5 nmol/mg of protein per h, respectively.     

Biochemical characterization of ochratoxin A-producing strains of the genus Penicillium

In order to explore the biochemical scope of ochratoxin A-producing penicillia, we screened 48 Penicillium verrucosum isolates for the production of secondary metabolites. Fungal metabolites were analyzed by high-pressure liquid or gas chromatography coupled to diode array detection or mass spectrometry. The following metabolites were identified: ochratoxins A and B, citrinin, verrucolones, verrucines, anacines, sclerotigenin, lumpidin, fumiquinazolines, alantrypinones, daldinin D, dipodazine, penigequinolines A and B, 2-pentanone, and 2-methyl-isoborneol. By use of average linking clustering based on binary (nonvolatile) metabolite data, the 48 isolates could be grouped into two large and clearly separated groups and a small outlying group of four non-ochratoxin-producing isolates. The largest group, containing 24 isolates, mainly originating from plant sources, included the type culture of P. verrucosum. These isolates produced ochratoxin A, verrucolones, citrinin, and verrucines and had a characteristic dark brown reverse color on yeast extract-sucrose agar medium. Almost all of a group of 20 isolates mainly originating from cheese and meat products had a pale cream reverse color on yeast extract-sucrose agar medium and produced ochratoxin A, verrucolones, anacines, and sclerotigenin. This group included the former type culture of P. nordicum. We also found that P. verrucosum isolates and three P. nordicum isolates incorporated phenylalanine into verrucine and lumpidin metabolites, a finding which could explain why those isolates produced relatively lower levels of ochratoxins than did most isolates of P. nordicum.     

Metabolism of sodium oestrone [35S]sulphate in the guinea pig

Intraperitoneal administration of sodium oestrone [(35)S]sulphate to male and female free-ranging guinea pigs is followed by excretion of most of the radioactivity mainly as inorganic [(35)S]sulphate in the urine within 72h. The remainder of the radioactivity in the urine was found in oestrone [(35)S]sulphate, two unidentified metabolites (A and B) and traces of oestradiol-17beta 3-[(35)S]sulphate. When injected intraperitoneally into animals with bile-duct and bladder cannulae, most of the dose was excreted in the bile. Unchanged oestrone [(35)S]sulphate was the main biliary component excreted in males and females, but the latter also excreted appreciable amounts of oestradiol-17beta 3-[(35)S]sulphate and metabolites A and B. The urine from these animals also contained these metabolites, inorganic [(35)S]sulphate and also oestrone [(35)S]sulphate, but in small amounts. Metabolite A was present only in samples from males. Whole body radioautography pinpointed the liver and kidney as the possible sites of metabolism of the ester. The ester underwent little desulphation in the isolated perfused female guinea-pig liver and in animals in which kidney function had been eliminated, and was excreted unchanged in the bile. These results and the observed low oestrogen sulphatase and arylsulphatase C activities found in guinea-pig liver and kidney support the view that the two enzymes are identical.     

Simultaneous analysis of ochratoxin A and its major metabolite ochratoxin alpha in plasma and urine for an advanced biomonitoring of the mycotoxin

Ochratoxin A (OTA) is a frequent mycotoxin contaminant found worldwide in foods and feedstuffs. Biomonitoring has been used to assess internal OTA exposure resulting from dietary intake and from other sources. Mycotoxin levels in blood and/or urine provide good estimates of past and recent exposure since OTA binds to serum proteins and is also partly excreted via the kidney. But, measuring OTA alone does not reflect its biotransformation. In light of scarce data on its metabolites in humans, it was the aim of this study to develop a method that allows analysis of OTA and its detoxication product ochratoxin alpha (OTα) in urine and in blood plasma. The method involves enzymatic hydrolysis of conjugates, liquid–liquid extraction, and analysis of sample extracts by liquid chromatography with fluorescence detection. Application of the validated method in a pilot study with 13 volunteers revealed the presence of OTA and OTα in all samples (limit of quantification: 0.05 ng/mL in urine, and 0.1 ng/mL in plasma). In line with negative findings of others, an OTA glucuronide was not detected, neither in urine nor in plasma. By contrast, conjugates of OTα (glucuronide and/or sulfate) are major products in these samples. This was confirmed by mass spectrometry detection. As OTα represents a large fraction of ingested mycotoxin, we propose to include analyses of this metabolite in future biomonitoring studies, also in light of the observed variations for urine OTα-levels that suggest different interindividual abilities for OTA-detoxification in humans.     

Assessment of ochratoxin A and tenuazonic acid in Canadian ice-wines

Twenty-six samples of commercial ice-wine made from late-harvested grapes were assayed for the mycotoxins ochratoxin A and tenuazonic acid. Canadian wines originated in British Columbia (18) and Ontario (8). For comparison two German wines from Hesse were also studied. Four additional samples of research ice-wine originating in were also studied. In all wine samples, assays using immuno-affinity chromatography and fluorescence liquid chromatography indicated ochratoxin A below 0.15 μg/L, the limit of determination of the method. Tenuazonic acid was determined by solidphase micro-extraction and liquid chromatography and was below the limit of determination (70 μg/L) in all samples. The European Union food tolerance limit for ochratoxin A in wine is 2 μg/L. A tolerance for tenuazonic acid has not yet been established.     

Citrinin,ochratoxin A and iron. Possible implications for their biological function and induction of nephropathy

Experiments with Neisseria meningitidis have shown that Fe³⁺ to some extent can reverse the toxicity of ochratoxin A and citrinin, as measured by inhibition zones around impregnated paper discs. Similar phenomena were observed with the less toxic ochratoxin B. Zearalenone also inhibited growth, but its effect was not counteracted by iron. The mycotoxins aflatoxin B₁ and deoxynivalenol did not inhibit bacterial growth at all. Desferal (deferoxamine) also inhibited growth of meningococci, but iron totally abolished this inhibition. The results indicate that ochratoxin A and citrinin interfere with iron metabolism in this organism but that other additional toxic mechanisms are involved as well since a marked growth inhibition by both toxins was also observed in the presence of iron. One function of ochratoxin A and citrinin in nature could consequently be to affect the iron uptake of other competing microorgansms.Since both toxins interfere with iron and both cause nephropathy, a possible connection between these properties and lipid peroxidation is also briefly discussed.Abbreviations DON deoxynivalenol - OA ochratoxin A - OB ochratoxin B     

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     

Sterol and ochratoxin biosynthesis and miconazole action onAspergillus ochraceus activity

Miconazole (MC), an effective drug used in medicine as a topical broad-spectrum antifungal agents for treating tinea, aspergilloses and candidoses, was used for studying its effect on sterols, ochratoxin A and B biosynthesis byAspergillus ochraceus in vitro. Sterol biosynthesis and production of ochratoxin A and B were inhibited by MC when added into the culture medium together with the inoculum. MC, addition 2 d after culture inoculation, inhibited sterol biosynthesis after 5, 9 and 14 d, ochratoxin A production after 5 and 9 d and ochratoxin B production after 9 d of incubation. However, at 10, 50 and 100 ppm concentrations it induced a significant accumulation of both toxins after 14 d of incubation.     

Detection of ochratoxin A in porcine kidneys by a monoclonal antibody-based radioimmunoassay

By using an indirect enzyme-linked immunosorbent assay, eight monoclonal antibodies (MAbs) were selected. Mice were immunized with ochratoxin A that was conjugated to bovine serum albumin. The hybridoma cell line designated 10G2 was grown in tissue culture and as an ascites tumor. The MAb was characterized to be specific to ochratoxin A and of the immunoglobulin G (IgG) class. Subsequently, the ascites fluid of this hybridoma was used in a competitive solid-phase IgG radioimmunoassay on protein A-Sepharose CL-4B, with [14C]ochratoxin A as tracer. Porcine kidneys were extracted with 0.5% phosphoric acid in chloroform. A two-step cleanup was achieved on a Sep-Pak C18 cartridge and a Sep-Pak silica cartridge. Radioimmunoassay with MAbs coupled to protein A-Sepharose CL-4B allowed the detection of ochratoxin A in porcine kidneys at a concentration as low as 0.2 ng/g.     

Conversion of ochratoxin C into ochratoxin A in vivo.

The conversion of ochratoxin C to ochratoxin A was studied in rats after oral and intravenous administration. The concentration of ochratoxin A in the blood as a function of time was the same after oral administration of equivalent amounts of either ochratoxin C or ochratoxin A. The maximum ochratoxin A concentrations were measured 60 min after administration. Given intravenously, ochratoxin C was also converted to ochratoxin A. Maximum concentrations were reached after 90 min. It is concluded that ochratoxin C is readily converted to ochratoxin A after both oral and intravenous administration. There is reason to believe that a comparable toxicity of the two toxins is based upon this conversion and that only interference with the biotransformation mechanisms may cause a difference in their toxicity.     

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.