Changes in urinary and renal tubular enzymes caused by subchronic administration of ochratoxin A in rats.

The activities of 5 enzymes in urine and renal tubules were measured after administration to male Wistar rats of small doses of ochratoxin A (145 micrograms/kg per day for 8-12 weeks, corresponding to 2 ppm in the feed) by intubation. These doses are in the range of natural contaminations found in food and feed. The enzymes examined were gamma-glutamyl transferase (gamma-GT), alkaline phosphatase (ALP), leucine aminopeptidase (LAP), lactate dehydrogenase (LDH), and N-acetyl-beta-D-glucosaminidase (NAG). The doses employed caused increased enzymuria and lower activities of tubular enzymes after 1 week of feeding. This suggests tubular injury. The change of the enzyme activities in the urine and in the tubules appeared in a cyclic way (degeneration and regeneration). Phenylalanine (20 ppm) partially prevented this action of ochratoxin A. The p-[14C]aminohippurate accumulation was inhibited by 60% in the second week but returned to almost normal level 6 weeks after the beginning of the treatment, suggesting an adaptation of the organism or a substitution of damaged cells.

Evidence for an enterohepatic circulation of ochratoxin A in mice.

The distribution and elimination of [3H]ochratoxin A (OTA) from stomach content and tissue, intestine content and tissue, liver, bile, serum and urine of Swiss male mice which had received a single low dose of OTA by intubation was followed as a function of time. The profiles of radioactivity do not show a smooth decline after the absorption period, but an oscillating pattern with rapid declines followed by increases which favour the assumption of an enterohepatic circulation. Between 28% and 68% of conjugated OTA together with OTA cleavage products were found in bile giving evidence for biliary excretion of OTA and its metabolites in mice. When given i.m. to mice [3H]OTA is already found after 30 min in bile and intestine contents and its elimination patterns show several peaks confirming the biliary excretion and the enterohepatic circulation. Cholestyramine, which is known to prevent the enterohepatic circulation of drugs and toxins, changes the profile of elimination of OTA which no longer presents the cyclic pattern. This result is also in favour of an enterohepatic circulation of OTA. When phenylalanine is given together with OTA by oral gavage the toxicokinetics of the mycotoxin change completely in the different body fluids, in stomach and intestine content and tissues. Phenylalanine seems to facilitate the gastric absorption of OTA and the gastro-intestinal transit. It increases also its early excretion into urine and bile. However, its elimination pattern no longer shows the oscillating pattern. Thus phenylalanine seems to inhibit the intestinal reabsorption of OTA conjugates.

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.

Phenylalanine prevents acute poisoning by ochratoxina in mice.

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

Combined action of citrinin and ochratoxin A on hepatoma tissue culture cells.

Citrinin (CTN) and ochratoxin A (OT-A) may occur simultaneously in mould-contaminated commodities. Both are cytotoxic to hepatoma tissue culture (HTC) cells. The effect of both mycotoxins, eiter alone or in association on cellular protein, RNA and DNA synthesis was tested. In the presence of ochratoxin A protein synthesis is first inhibited 30 min after the addition of the toxin and RNA synthesis after 150 min. No inhibition of DNA synthesis occurs for at least 5 h, whereas citrinin inhibits first the RNA synthesis after 10 min, second protein synthesis after 20 min and third DNA synthesis after 120 min. When both mycotoxins are added simultaneously to HTC cells the inhibition of RNA and protein synthesis occurs immediately, that of DNA synthesis after a short lag time. This suggests a cooperative effect of both mycotoxins.

Comparative study of the effect of ochratoxin A analogues on yeast aminoacyl-tRNA synthetases and on the growth and protein synthesis of hepatoma cells.

Ochratoxin A (OTA), a naturally occurring mycotoxin of Aspergillus and Penicillium species, consists of a 5' chlorinated dihydromethyl isocoumarin linked to L,beta-phenylalanine by an alpha-amide bond. 8 analogues of OTA were prepared in which the phenylalanine was always substituted by another amino acid. The effects of these analogues on yeast tRNA amino acylation reaction and on growth and protein synthesis of hepatoma culture cells were compared with those of OTA. In addition, Ochratoxin B (OTB) and ochratoxin alpha (OT alpha) were examined. All the analogues of OTA had inhibitory effects in the 3 test systems, although to a lesser degree than OTA. The degree of inhibition depended on the kind of substituted amino acid, the tyrosine, valine, serine and alanine analogues being most effective, in contrast to the proline analogue. OTB and OT alpha were ineffective.

Influence of ochratoxin B on the ochratoxin A inhibition of phenylalanyl-tRNA formation in vitro and protein synthesis in hepatoma tissue culture cells.

Ochratoxin B (OTB), the dechloro-analogue of ochratoxin A (OTA), was studied separately and in combination with OTA on the aminoacylation of phenylalanine tRNA (tRNAPhe) catalysed by mice liver phenylalanyl-tRNA synthetase. OTB was neither a significant inhibitor of the reaction nor an antagonist of OTA. OTB was also assayed for its possible antagonistic effect on the in vivo protein synthesis inhibition caused by OTA in hepatoma tissue culture cells. No prevention of OTA inhibition could be found for OTB. It rather showed a slight additional inhibitory activity when mixed (100-180 microM) with low concentrations of OTA (40-60 microM). In conclusion, these results are not in favor of an antagonistic effect of OTB with respect to OTA action, at least on the level of cellular protein synthesis.

Inhibition of protein synthesis in mice by ochratoxin A and its prevention by phenylalanine.

The sensitivity of protein synthesis to ip doses of ochratoxin A ranging from 1 to 15 mg/kg body weight has been determined in the livers, kidneys and spleens of mice. The incorporation of 14C-labelled amino acids into protein was measured in the total tissue homogenate, in the 105,000-g supernatant fraction and in the fraction of thermostable soluble proteins. The inhibition of protein synthesis was greatest in spleen and kidney and least in liver. The percentage inhibition of protein synthesis by any one dose of ochratoxin A was similar in all of the protein fractions from any one organ. The degree of inhibition of protein synthesis 5 hr after administration of 1 mg ochratoxin A/kg was 26% in liver, 68% in kidney and 75% in spleen. Phenylalanine (100 mg/kg) injected together with ochratoxin A prevented the inhibition of protein synthesis by a dose of 10 mg OTA/kg in all of these organs.

Strand scissions of DNA by patulin in the presence of reducing agents and cupric ions.

In the presence of NADPH and CuCl2, patulin induced the cleavage of ColE1 DNA and lambda phage DNA in vitro. The DNA-cleaving activity of patulin was concentration dependent. At the lowest concentration of patulin, ColE1 supercoiled DNA was relaxed and the highest concentration induced linearization of the DNA. This activity was inhibited by superoxide dismutase, catalase and radical scavengers, showing involvement of free radicals in the DNA-cleavage. lambda Phage DNA was also degraded by patulin under the same conditions.

beta-D-phosphogalactoside galactohydrolase of Streptococcus faecalis and the inhibition of its synthesis by glucose.

The lactose hydrolysing system of Streptococcus faecalis is described. It is closely related to that one of the group N streptocci as it consists of a beta-D-phosphogalactoside galactohydrolase (beta-Pgal). The uptake of methyl-beta-D-thiogalactoside (TMG), lactose, and glucose is maintained by the phosphoenolpyruvate-dependent phosphotransferase system (PTS) but the uptake of galactose is not. The induction time is 6--7 min. Inducers are lactose and galactose but not isopropyl-beta-D-galactoside (IPTG) and TMG. In the presence of glucose, mannose, and maltose no induction of beta-Pgal occurs but pyruvate and glycerol allow induction. The competitive inhibition of uptake of TMG by glucose suggests inducer exclusion by this sugar. TMG accumulates in the cells exclusively as a derivative.

Induction of autolysis in Bacillus subtilis by ochratoxin A.

Ochratoxin A (OTA) added during the exponential growth phase at a concentration higher than 12 microgram/ml caused autolysis of Bacillus subtilis. Optical density of cultures decreased, and at higher concentrations the cultures became sterile. Optimum OTA-induced lysis was about pH 5. At concentrations below 10 microgram/ml, protein synthesis was inhibited more strongly than RNA synthesis. Cell wall synthesis was also strongly inhibited. A fraction extracted from the lysates had the property of a lysis inhibitor. The relevance of this fraction in respect to autolysis is discussed.

DNA-damaging activity of patulin in Escherichia coli.

At a concentration of 10 micrograms/ml, patulin caused single-strand DNA breaks in living cells of Escherichia coli. At 50 micrograms/ml, double-strand breaks were observed also. Single-strand breaks were repaired in the presence of 10 micrograms of patulin per ml within 90 min when the cells were incubated at 37 degrees C in M9-salts solution without a carbon source. The same concentration also induced temperature-sensitive lambda prophage and a prophage of Bacillus megaterium. When an in vitro system with permeabilized Escherichia coli cells was used, patulin at 10 micrograms/ml induced DNA repair synthesis and inhibited DNA replication. The in vivo occurrence of DNA strand breaks and DNA repair correlated with the in vitro induction of repair synthesis. In vitro the RNA synthesis was less affected, and overall protein synthesis was not inhibited at 10 micrograms/ml. Only at higher concentrations (250 to 500 micrograms/ml) was inhibition of in vitro protein synthesis observed. Thus, patulin must be regarded as a mycotoxin with selective DNA-damaging activity.

Immunosuppression by ochratoxin A and its prevention by phenylalanine.

Ochratoxin A, a toxin of Aspergillus ochraceus, suppresses the immune response to sheep erythrocytes in BALB/c mice at doses as low as 0.005 microgram/kg of body weight. This effect is prevented by phenylalanine.

The inhibition of bacterial growth by ochratoxin A.

A series of bacterial species was examined for their sensitivity to ochratoxin A. Only grampositive bacteria could be inhibited, generally at a pH lower than 7.0. Bacillus subtilis did not show any reduction of growth rates in presence of ochratoxin A, but had a prolonged lag phase. With Staphylococcus pyogenes var. aureus and Streptococcus faecalis, a prolonged lag phase and a reduction of the growth rate was observed. Most sensitive was Streptococcus faecalis in the exponential-growth phase. The inhibition could be diminished by changing the pH to neutral, or by addition of yeast extract, tetrahydrofolate, or MgSO4. With MgSO4 a complete abolition of the inhibitory effect was achieved, but not with CaCl2. During growth inhibition, protein and RNA synthesis were reduced simultaneously, but not DNA synthesis. Even with the very high concentration of 1 mg/ml, no lethal effect was observed.

[Preventive action of phenylalanine on acute intoxication of ochratoxin-A]. / Action préventive de la phenylalanine sur l'intoxication aiguë par l'ochratoxine-A.

Ochratoxin-A, a mycotoxin causing kidney and liver damage, inhibits protein synthesis by competition with phenylalanine in the phenylalanyl-tRNA synthetase catalysed reaction. A dose of 0,8 mg ochratoxine per Mouse is 100% lethal within 24 hrs, when injected intraperitoneally. When phenylalanine is injected simultaneously with ochratoxine-A, the survival of the animals was 100% for 1 mg of phenylalanine per Mouse. Ten times higher doses of phenylalanine are necessary to obtain a 85% survival if the amino acid is injected 30 min. after the mycotoxin. Phenylalanine gives a very low protection if injected 1 hr or more ochratoxine-A.