Guy Dirheimer

Factors determining the specificity of the tRNA aminoacylation reaction: Non-absolute specificity of tRNA-aminoacyl-tRNA synthetase recognition and particular importance of the maximal velocity

Summary It is generally believed that the specificity of tRNA aminoacylation results solely from a specific recognition between the aminoacyl-tRNA synthetase and the cognate tRNA. In fact, this specificity is not absolute: this is supported by the following observations (1) the existence of tRNA mischarging in homologous systems under usual aminoacylation conditions, (2) the existence of inhibitions produced by « non-cognatetRNA species in correct aminoacylation reactions, (3) the lack of specificity of AMP- and PPi- independent aminoacyl-tRNA synthetase catalysed deacylation of aminoacyl-tRNA species, (4) the isolation of complexes between aminoacyl-tRNA synthetases and non-cognate tRNA species. The affinities between aminoacyl-tRNA synthetases and non-cognate tRNA species, estimated by the Km measurements in mischarging reactions, have been found only diminished by 1 or 2 orders of magnitude as compared to the values found in specific systems, whereas the Vmax values for mischarging have been found diminished by 3 or 4 orders of magnitude. This suggests that tRNA aminoacylation depends more upon the maximal velocity of the reaction than upon the recognition between aminoacyl-tRNA synthetase and tRNA. Furthermore, we found that the recognition of a tRNA by an aminoacyl-tRNA synthetase does not seem to require the 3′ terminal part of the amino acid acceptor stem. As the importance of this part of the tRNA molecule during the aminoacylation process has been well established, it is possible that it is involved in determining the Vmax of the aminoacylation reaction, probably by positioning the 3′ terminal adenosine in the catalytic site of the enzyme. In conclusion, it appears that the specificity of the tRNA aminoacylation reaction proceeds through two discrimination mechanisms: the first one, measured by the Km, acts at the recognition level; the second one, which is more effective, is measured by the Vmax values. Competition phenomena have been observed between cognate and non-cognate tRNA species. They enhance the specificity of the tRNA aminoacylation, but their contribution to the specificity is low compared to that brought by Km and Vmax. Finally we found that a more rapid enzymatic deacylation of mischarged tRNA species (as compared to correctly charged ones) cannot be considered as a general mechanism for correction of misaminoacylation.

Genotoxicity of ochratoxin A in mice: DNA single-strand break evaluation in spleen, liver and kidney.

Ochratoxin A, a natural contaminant of feed and food, has been shown to induce experimental liver and kidney tumours. In vitro experiments on mice spleen cells showed evidence of DNA single-strand breaks induced by ochratoxin A. We measured single-strand breaks in the DNA of spleen, liver and kidney of ochratoxin A-treated animals. Ochratoxin A induced DNA damage in vivo. This damage reversed with time. The appearance and extent of the damage varied in different tissues. Except for spleen our data correlate with the tumours induced by ochratoxin in mouse liver and kidney. With regard to the spleen, there has been no report to date of experimental leukemia induced by ochratoxin A. Thus our results indicate that this possibility has to be considered.

Differential DNA adduct formation and disappearance in three mouse tissues after treatment with the mycotoxin ochratoxin A

Ochratoxin A (OTA) is a mycotoxin which has been implicated in Balkan endemic nephropathy, a disease characterized by a high incidence of urinary tract tumors. It induces DNA single-strand breaks and has been shown to be carcinogenic in two rodent species. For a better understanding of the OTA genotoxic effect, OTA-DNA adduct formation and disappearance has been measured using the 32P-post-labelling method after oral administration of 2.5 mg/kg of OTA to mice. In kidney, liver and spleen, several modified nucleotides were clearly detected in DNA, 24 h after administration of OTA, but their level varied significantly in a tissue and time dependent manner over a 16-day period. Total DNA adducts reached a maximum at 48 h when 103, 42 and 2.2 adducts per 10(9) nucleotides were found respectively in kidney, liver and spleen, indicating that kidney is the main target of the genotoxicity and likely carcinogenicity of OTA. The major adduct differed between kidney and liver. All adducts disappeared in liver and spleen 5 days after compound administration, whereas some adducts persisted for at least 16 days in the kidney. Some adducts were organ specific. The finding that the adducts are not quantitatively and qualitatively the same in the three organs examined is likely due to differences of metabolism in these organs, leading to different ultimate carcinogens and may also result from differences in the efficiency of repair processes.

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.

Distribution of the [3H]-label from low doses of radioactive ochratoxin A ingested by rats, and evidence for DNA single-strand breaks caused in liver and kidneys.

The distribution of a single low dose of [3H]ochratoxin A (OTA) in different tissues of male Wistar rats, after administration by intubation, was investigated after 5 h, 24 h and 48 h. This dose corresponds to concentrations encountered in naturally contaminated feed (4 ppm). The distribution of [3H]-label varied with the time elapsed after administration; at 5 h the highest specific label was found in the stomach contents and in decreasing order in: intestinal contents, lung, liver, kidney, heart, fat, intestine, testes, and the lowest in muscles, spleen and brain. With exception of brain, fat, stomach and lung, all tissues showed maximum levels at 24 h, after which time the label decreased steadily, whereas in fat it increased.After a 12-week feeding experiment, with doses of 288.8 μg/kg corresponding to an intake of 4 ppm in feed each 48 h, the DNA in liver and kidneys was investigated for damage. By the alkaline elution method combined with micro-spectrofluorimetric determinations of DNA, evidence for DNA single-strand breaks was obtained. These findings support reports on the carcinogenic action of OTA.

Effect of superoxide dismutase and catalase on the nephrotoxicity induced by subchronical administration of ochratoxin A in rats

Ochratoxin A (OTA) is a mycotoxin produced by Aspergillus ochraceus as well as other molds. It is a natural contaminant of mouldy food and feed. OTA has a number of toxic effects, the most prominent being nephrotoxicity. Furthermore, OTA is immunosuppressive, genotoxic, teratogenic and carcinogenic. OTA inhibits protein synthesis by competition with phenylalanine in the phenylalanine-tRNA aminoacylation reaction. Recently, lipid peroxidation induced by OTA has been reported, indicating that the lesions induced by this mycotoxin could be also related to oxidative pathways. It was then interesting to study effects of the superoxide dismutase (SOD) and catalase on the nephrotoxicity induced by OTA in rats. The two enzymes (20 mg/kg body weight each) were given to rats by subcutaneous injection, every 48 h, 1 h before gavage by OTA (289 micrograms/kg b.w. every 48 h), for 3 weeks. SOD and catalase prevented most of the nephrotoxic effects induced by ochratoxin A, observed as enzymuria, proteinuria, creatinemia and increased urinary excretion of OTA. Altogether these results indicate (i) that superoxide radicals and hydrogen peroxide are likely to be involved in the damaging processes of OTA in vivo, (ii) that SOD and catalase might be used for prevention of renal lesions in cases of ochratoxicosis.

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.

Formation of ochratoxin a metabolites and DNA-adducts in monkey kidney cells

Monkey kidney cells (named Vero cells) were incubated with increasing doses of ochratoxin A (10-100 microM). The inhibiting concentration 50% (IC50) on protein synthesis was about 14 microM in the presence of 5% fetal calf serum and 37 microM in the presence of 10% fetal calf serum. Some metabolites of ochratoxin A, including the chlorinated dihydroisocoumarin moiety of OTA (OT alpha), 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 microM 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.

Codon reading patterns in Saccharomyces cerevisiae mitochondria based on sequences of mitochondrial tRNAs

The sequences of Saccharomyces cerevisiae mitochondrial tRNA1 Arg, tRNA2 Arg, tRNAGly, tRNA2 Lys tRNALeu and tRNAPro are reported. Special structural features were found in tRNAPro, which has A8, C21, A48 instead of the constant residues U8, A21 and pyrimidine 48, and in tRNA2 Lys, which has a U excluded from basepairing and bulging out from the TΨC stem. The tRNA1 Arg, tRNA2 Lys and tRNALeu, which belong to two‐codon families ending in a purine, have a modified uridine in the wobble position, which prevents misreading of C and U. It is likely to be 5‐carboxymethylaminomethyluridine. tRNAGly and tRNAPro have an unmodified uridine in the wobble position allowing the reading of all four codons of a four‐codon family. However, tRNA2 Arg, which is a minor species and belongs to the CGN four‐codon family, has an unmodified A in the wobble position. This very unusual feature raises the problem of the mechanism by which the codons CGA, CGG and CGC are recognized.

Induction of micronuclei with ochratoxin A in ovine seminal vesicle cell cultures

Abstract 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.