Ochratoxin A (OTA), a mycotoxin that induces nephrotoxicity and urinary tract tumors, is genotoxic and can be metabolized not only by different cytochromes P450 (CYP) but also by peroxidases involved in the arachidonic cascade, although the exact nature of the metabolites involved in the genotoxic process is still unknown. In order to establish the relation between OTA genotoxicity and the formation of metabolites, we chose three experimental models: kidney microsomes from rabbit, human bronchial epithelial cells, and microsomes from yeast that specifically express the human cytochrome P450 2C9 or 2B6 genes. OTA-DNA adducts were analyzed by (32)P postlabeling and the OTA derivatives formed were isolated by HPLC after incubation of OTA in the presence of: (1) kidney microsomes from rabbit pretreated or not with phenobarbital (PB); (2) human pulmonary epithelial cells simultaneously pretreated (or not) with PB alone or in the presence of ethacrynic acid (EA); (3) microsomes expressing CYP 2B6 and 2C9. PB pretreatment significantly increased DNA adducts formed after OTA treatment, both in the presence of kidney microsomes and bronchial epithelial cells, and induced the formation of new adducts. Ethacrynic acid, which inhibits microsomal glutathione-S-transferase, reduced DNA adduct level. DNA adducts were detected when OTA were incubated with microsomes expressing human CYP 2C9 but not with those expressing CYP 2B6. Several metabolites detected by HPLC were increased after PB treatment. Some of them could be related to DNA-adduct formation. In conclusion, OTA biotransformation, enhanced by PB pretreatment, increased DNA-adduct formation through pathways involving microsomal glutathion-S-transferase and CYP 2C9.
SummaryBiological properties and deduced amino acid sequences of the overlapping coat and 17kDa proteins were compared for 12 barley yellow dwarf virus PAV‐type isolates collected at different locations in Morocco. The biological variability of the 12 isolates was investigated by inoculation to a set of as many as eight barley cultivars and an oat cultivar. Significant biological variability was observed among the Moroccan PAV–type isolates, with the isolates falling into two clusters. Cluster 1 contained isolates MA9501, MA9502, MA9504, MA9512 and MA9513, and cluster 2 the MA9415, MA9505, MA9508, MA9511, MA9514, MA9516 and MA9517. Isolates of cluster 2 produced more severe symptoms and stunting than did those of cluster 1 when inoculated to appropriate index plants. In parallel, the nucleic acid sequences were obtained for the region containing the cistron coding for the coat protein (CP) and 17 kDa protein for all 12 isolates by reverse transcription followed by the polymerase chain reaction. Sequence homology grouping yielded the same two clusters of the isolates as found by measuring biological variability. Comparisons of the CP sequences between the two clusters revealed 87.4% to 90.5% nucleotide identity and 82.6% to 87.6% amino acid identity. Comparison with other sequenced isolates demonstrated that the CP's of cluster 1 were closely related to the Australian (Vic‐PAV), the North American (NY–PAV, P–PAV), and Japanese (JPN–PAV) PAV–type isolates. The CP's of cluster 2, on the other hand, were closely related to the North American PAV–129 isolate. Finally, an RFLP test on CP gene–derived PCR products has been developed which distinguishes between the two clusters of the PAV–type isolates.
<p style="text-align: justify;">The levels of free and bound ABA (<em>cis</em>-ABA and <em>cis</em>-ABA-GE) were quantified weekly during the dormancy phase of three years in leaves, in latent buds and in internodes grapevine (<em>Vitis vinifera</em> L. cv. Merlot) by high pressure liquid chromatography (HPLC). An inverse variation between <em>cis</em>-ABA and <em>cis</em>-ABA-GE levels was observed in buds and in internodes during some phases of their development. This result seems suggest a possible interconversion phenomenon between these two ABA forms as described in others organs by some authors. The development of ABA in leaves, in buds and in internodes, showed three successive maximums of <em>cis</em>-ABA. The first maximum was observed in leaves during onset of dormancy, the second maximum in buds during dormancy and the last in internodes during the leaf fall. Maximums of <em>cis</em>-ABA in leaves and in buds are approximatively the some values (around of 150 µg/100 g Dry Weight) whereas in internodes is lower (60 µg/100 g Dry Weight). An inverse variation between <em>cis</em>-ABA of leaves and <em>cis</em>-ABA of buds was observed suggesting that the increase of <em>cis</em>-ABA in buds was caused by the <em>cis</em>-ABA translocation from leaves but also from roots. The removal of leaves on september 1997 during the dormancy phase, before leaf fall period, induced an increase of <em>cis</em>-ABA content in buds and internodes. These results seem suggest that before leaf fall period, ABA of buds and internodes were translocated to leaves. During this same period, the removal of leaves and buds induced a slightly soften of ABA content increase in internodes, that seems indicate that ABA was greatly translocated from roots. During the period of leaf fall, the leaf removal traitment induced a decrease of <em>cis</em>-ABA levels in buds and in internodes while the same traitment associated to buds removal stabilized the ABA levels in internodes. During this period, ABA seems exported from leaves to buds and internodes and the roots don't appear to operate significantly in the development of ABA in these two organs.</p>
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