Ochratoxin A (OTA), is a mycotoxin contaminating food and feed stuffs, consisting of a chlorinated dihydroisocoumarin linked through a 7-carboxyl group tol-phenylalanine by an amide bond. When OTA (0.12–1.4 mM) is incubated with freshly isolated rat hepatocytes, it inhibits both the hydroxylation of phenylalanine (0.05 mM) to tyrosine, catalyzed by phenylalanine hydroxylase and the subsequent metabolism of tyrosine as measured by homogentisate oxidation. The IC50 of OTA for phenylalanine hydroxylation is 0.43 mM. OTα, (0.5–1.0 mM), the dihydroisocoumarin moiety of OTA, does not inhibit phenylalanine hydroxylase activity under these conditions. During incubations of hepatocytes with uniformly labelled [3H]-OTA and unlabelled phenylalanine, tyrosine-ochratoxin A is formed (up to 6% of the total mycotoxin added), indicating that ochratoxin can act as a substrate for phenylalanine hydroxylase. In vivo tyrosine-OTA is also found in liver of poisoned animals.

Ochratoxin A (OTA) was, up to now, believed to be non-mutagenic in the classical Salmonella typhimurium reverse mutation test. This was confirmed using rat liver microsomal fractions with the strains, TA1535, TA1538 and TA98, and up to 1210 micrograms/plate, utilizing an Ames microtest. However, using mice kidney microsomal fractions as metabolic activators, reverse mutations were obtained with the three strains used, in the presence of either NADP or arachidonic acid as cofactors. The mutagenicity was higher with arachidonic acid than with NADP using the TA1535 strain. This lends support to the results concerning the DNA or dGMP adducts obtained in vitro which were also higher in the presence of arachidonic acid, and indicate that several metabolic pathways of OTA can lead to genotoxic compounds. In addition, both base pair substitutions and frameshift mutations can be caused by OTA after metabolic activation.

Ribosome-inactivating proteins (RIP) are RNA-N-glycosidases widely diffused in plants which depurinate ribosomal RNA at a specific universally conserved position, A4324 in rat ribosomes. A small group of RIPs (cofactor-dependent RIPs) require ATP and tRNA to reach maximal activity on isolated ribosomes. The tRNA which stimulates gelonin was identified as tRNATrp. The present paper reports the identification of three other tRNAs which stimulate agrostin (tRNAAla), barley RIP (tRNAAla, tRNAVal) and PAP-S (tRNAGly), while for tritin-S no particular stimulating tRNA emerged. The sequences of tRNAVal and tRNAGly correspond to the already known ones (rabbit and man, respectively). The tRNAAla (anticodon IGC) identifies a new isoacceptor. Only the stimulating activity of the tRNAAla for agrostin approaches the specificity previously observed for the couple gelonin-tRNATrp.

The genotoxic potential of the carcinogenic mycotoxin ochratoxin A (OTA) has been investigated by means of an in vitro micronucleus assay, an endpoint for genotoxicity which has not been studied previously for OTA. OTA was found to induce dose-dependently micronuclei (MN) in cytokinesis-blocked binucleated ovine seminal vesicle (OSV) cell cultures, which had been treated with the mycotoxin (12–30 μM) for 6 h in medium containing 10% fetal calf serum. For comparison, OSV cells were treated with colcemid (0.02–0.06 μg/ml), or 4-nitroquinoline N-oxide (NQO; 0.5 μM), a typical aneugen and clastogen, respectively. All test compounds increased the frequency of MN in OSV cells, the highest level being induced by 30 μM OTA. When MN were characterized by indirect immunofluorescence microscopy using anti-kinetochore (CREST) antibodies, the majority of MN in colcemid-treated cells was CREST-reactive (>70% kinetochore positive); as expected, this fraction was <10% for the NQO-treatment group. In cells treated with OTA the fraction of kinetochore positive MN was similar (33–40%) to that observed in solvent controls (38%). These data indicate that OTA induces MN apparently by a mixed, although predominantly clastogenic mode of action. OSV cells lack monooxygenase activity but express high prostaglandin H synthase (PGHS) activity. When cells were treated with OTA in the presence of indomethacin (10 and 50 μM), a well known inhibitor of PGHS, the frequency of MN induced by OTA was not decreased, but rather increased. This indicates that metabolic activation of OTA by PGHS seems not to be required for genotoxicity. The increased MN induction in OSV cell cultures is most likely due to competition of indomethacin with OTA for binding to serum proteins thus raising the fraction of free mycotoxin.

Ochratoxin A (OTA), and zearalenone (ZEN), two mycotoxins, have been implicated in numerous mycotoxicoses in farm animals and are genotoxic. Several adducts were detected in mouse and rat kidney after a single administration of OTA and in mice organs after zearalenone treatment which induces hepatocellular adenomas. The effects of some vitamins such as retinol (A), ascorbic acid (C) and alpha-tocopherol (E), which are known to act as superoxide anion scavengers, were tested on OTA genotoxicity. Pretreatment of mice by vitamin E decreased DNA adducts by 80% in kidney. Vitamin A decreased DNA adduct levels by 70% and Vitamin C by 90% in kidney. In the same way, pretreatment of female mice with alpha-tocopherol before administration of zearalenone inhibited significantly DNA adduct formation in liver and in kidney. The total DNA adduct level after E treatment was decreased by 45% and 58% in liver and kidney respectively.

Elements that confer identity to a tRNA in the cellular environment, where all aminoacyl-tRNA synthetases are competing for substrates, may be delineated by in vivo experiments using suppressor tRNAs. Here we describe the selection of active Escherichia coli tRNAAsp amber mutants and analyze their identity. Starting from a library containing randomly mutated tRNA(CUA)Asp genes, we isolated four amber suppressors presenting either lysine, alanine, or glutamine activity. Two of them, presenting mainly alanine or lysine activity, were further submitted to a second round of mutagenesis selection in order to improve their efficiency of suppression. Eleven suppressors were isolated, each containing two or three mutations. Ten presented identities of the two parental mutants, whereas one had switched from lysine to arginine identity. Analysis of the different mutants revealed (or confirmed for some nucleotides) their role as positive and/or negative determinants in AlaRS, LysRS, and ArgRS recognition. More generally, it appears that tRNAAsp presents identity characteristics closely related to those of tRNALys, as well as a structural basis for acquiring alanine or arginine identity upon moderate mutational changes; these consist of addition or suppression of the corresponding positive or negative determinants, as well as tertiary interactions. Failure to isolate aspartic acid-inserting suppressors is probably due to elimination of the important G34 identity element and its replacement by an antideterminant when changing the anticodon of the tRNAAsp to the CUA triplet.

Ochratoxin A (OTA) is a ubiquitous nephrotoxic mycotoxin which was shown to be carcinogenic to laboratory animals and may be responsible for kidney pelvis, ureter and urinary bladder tumors associated with Balkan endemic nephropathy in man. Previous evidence from this laboratory demonstrated that OTA exposure results in adduct formation on kidney, testicles, liver and spleen DNA. We show in this study that after a single oral administration of OTA to mice (2 mg/kg body weight) a high level of DNA adducts (150 per 10(9) nucleotides) is also detected in the urinary bladder. The metabolic pathway of OTA leading to genotoxic compounds is not yet known. We demonstrate here that two inhibitors of the prostaglandin H synthase, indomethacin and aspirin, administered to mice before OTA treatment, dramatically reduce the amounts of DNA adducts, particularly in the urinary bladder and kidney. This suggests a role of protaglandin H synthase in the metabolism of OTA leading to active metabolites which react with DNA.

The nucleotide analysis of a cytoplasmic tRNA(Leu) isolated from bovine liver revealed the presence of an unknown modified nucleotide N. The corresponding N nucleoside was isolated by different enzymatic and chromatographic protocols from a partially purified preparation of this tRNA(Leu). Its chemical characterization was determined from its chromatographic properties, UV-absorption spectroscopy and mass spectrometric measurements, as well as from those of the borohydride reduced N nucleoside and its etheno-trimethylsilyl derivative. The structure of N was established as 2'-O-methyl-5-formylcytidine (f5CM), and its reduced derivative as 2'-O-methyl-5-hydroxy-methylcytidine (om5Cm). By sequencing the bovine liver tRNA(Leu), the structure of the anticodon was determined as f5CmAA. In addition, the nucleotide sequence showed two primary structures differing only by the nucleotide 47c which is either uridine or adenosine. The two slightly differing bovine liver tRNAs-Leu(f5CmAA) are the only tRNAs so far sequenced which contain f5Cm. The role of such a modified cytidine at the first position of the anticodon is discussed in terms of decoding properties for the UUG and UUA leucine codons. Recently, precise evidence was obtained for the presence of f5Cm at the same position in tRNAs(Leu)(NAA) isolated from rabbit and lamb liver. Therefore, the 2'-O-methyl-5-formyl modification of cytidine at position 34 could be a general feature of cytoplasmic tRNAs(Leu)(NAA) in mammals.

Bolesatine is a toxic glycoprotein isolated from the mushroom Boletus satanas Lenz, which has been shown to inhibit protein synthesis in cell-free systems and cell culture. It is toxic to rodents, the LD50% 24 h being 1 mg kg-1 (i.p.) and 0.15 mg kg-1 (i.v) in the rat in which it induces hepatic blood stasis. Bolesatine possesses lectinic properties with in parti cular a sugar binding site for D-galactose and mitogenic activity toward lymphocytes. Tested for cell agglutination on red blood cells and platelets, bolesatine agglutinates both human and rat platelets from threshold concentrations of 30 and 300 nM respectively. EDTA and PGI2 (aggregation inhibitors) do not decrease the agglutination induced by bolesatine, indicating that the process does not involve platelet activation. In contrast, fibrinogen decreases platelet agglutination in duced by bolesatine, most likely by masking the binding sites on platelets or by interacting with the toxin. Bolesatine agglutinates all red blood cells without any blood group specificity in the concentration range of 20 to 40 nM. This haemagglutination cannot be prevented by sugars, including D-galactose at a concentration of 0.5 M.

Bolesatine is a potent cytotoxic glycoprotein purified from Boletus satanas Lenz, which has previously been shown to be an inhibitor of protein synthesis in several in vitro systems and in vivo. For a better understanding of its mechanism of action on protein synthesis at the ribosomal level, rat liver ribosomes were pretreated with bolesatine ( 1 to 10 μg) added to in vitro polyuridylic acid (poly(U)) translation systems before and after washing. The fact that ribosomes were still active confirmed that bolesatine cannot be included in the group of protein synthesis inhibitors of plant origin, known as ribosome-inactivating proteins (RIPs). The effect of bolesatine on the EF-2 elongation factor and post-ribosomal fraction was then studied in vitro. The results indicated that bolesatine does not have a direct effect on elongation factors, but hydrolyses the nucleoside triphosphates, GTP (80% to 90%, respectively for 1 to 10 μg) and ATP (10% to 40%, respectively for 1 to 10 μg), with consequent inhibition of protein synthesis. Thus, bolesatine should be classified as a nucleoside triphosphate phosphatase, rather than as a direct inhibitor of protein synthesis. The study of the effect of bolesatine on the EF-2 factor revealed that the mechanism whereby bolesatine affects protein synthesis probably involves GTP hydrolysis rather than EF-2 inhibition.

Journal Article Cytochrome P-450 Isoforms Implicated in Ochratoxin A Genotoxicity Determined by DNA Adduct Formation Get access Clinical Chemistry, Volume 41, Issue 12, 1 December 1995, Pages 1927–1929, https://doi.org/10.1093/clinchem/41.12.1927 Published: 01 December 1995

Two single-strand-specific nucleases, discovered in plants, have been used to investigate the secondary and tertiary structures of the native bovine liver selenocysteine tRNA(Sec). To check the possible influence of nucleotide modifications on these structures, we compared the results obtained with the fully modified tRNA to the unmodified transcript prepared by in vitro T7 transcription of the Xenopus laevis tRNA(Sec) gene. We found that the structures in solution of the native tRNA(Sec) and the transcript are very similar despite some differences in accessibility to the enzymatic probes. Indeed, the modified anticodon-loop of native bovine tRNA(Sec), containing 5-methylcarboxymethyluridine (mcm5U34) and N6-isopentenyladenosine (i6A37), is less accessible to Rn nuclease than that of the transcript: the intensity of bands representing cuts at A36 and A38 is much lower as compared to those of the transcript, whereas no cuts were found at the level of i6A37 in the anticodon loop of the native molecule. Surprisingly, the variable arm of the native molecule has been found to be more susceptible to single-strand-specific nuclease action, suggesting a looser structure of the variable arm in native bovine tRNA(Sec) than in the transcript.

A single-strand-specific nuclease from wheat chloroplasts (ChS nuclease) was tested as a tool for RNA secondary and tertiary structure investigations, using yeast tRNA(Phe) and yeast tRNA(Asp) as models. In tRNA(Phe) the nuclease introduced main primary cleavages at positions U33, A35 and A36 in the anticodon-loop and G18 and G19 in the D-loop. In tRNA(Asp) the main primary cleavages occurred at positions U33, G34 and U35 in the anticodon-loop and the lower one at position C20:1 in the D-loop. No primary cleavages were observed within the double-stranded stems. Because ChS nuclease has (i) a low molecular weight, (ii) a wide pH range of action (5.0 to 7.5) (iii) no divalent cation requirement in the reaction mixture and (iv) can be obtained as a pure protein in rather large quantities it appeared to be a very good tool for secondary and tertiary structural studies of RNAs.

Monkey kidney cells (named Vero cells) were incubated with increasing doses of ochratoxin A (10–100 μM). The inhibiting concentration 50% (IC50) on protein synthesis was about 14 μM in the presence of 5% fetal calf serum and 37 μM in the presence of 10% fetal calf serum. Some metabolites of ochratoxin A, including the chlorinated dihydroisocoumarin moiety of OTA (OTα), 4-[S]-hydroxy-OTA and 4-[R]-hydroxy-OTA were detected by HPLC in the mixture of cell homogenate after a 24 h incubation with 10 and 25 μM of OTA. Using the 32P-postlabelling method, several DNA-adducts, similar to those formed in mouse kidney after OTA treatment, were detected in monkey kidney cells. Thus, Vero cells are suitable for genotoxic and cytotoxic studies in relation to the metabolism of nephrotoxic xenobiotics such as OTA.