A plant metabolic activation system from Persea americana with cytochrome P450-dependent and peroxidase activities.

Microsomal fractions from different tissues of various plants (potato, cauliflower, aubergine, avocado pear, courgette, cucumber, banana, kiwi and strawberry) were prepared and their content of cytochrome P-450 (cyt-P450) determined. A S117 fraction from Persea americana (avocado pear) presented the highest content of cyt-P450. As a consequence of these data, we have developed and characterized this fraction as a plant metabolic activation system. The P. americana S117, used in this work, contains 0.75 +/- 0.04 mg of protein per ml, 0.788 +/- 0.078 nmol of cyt-P450 per mg of protein and has a peroxidase activity of 0.036 +/- 0.005 (nmol tetraguaiacol/micrograms protein/min). The P. americana cyt-P450 remained stable during at least 60 days, stored at -80 degrees C. This fraction activated 2-aminofluorene to a mutagenic product in S. typhimurium TA98, while it had no effect on the benzo[a]pyrene activation. The treatment of the P. americana S117 with CO, the addition of diethyldithiocarbamate (DETC) or the absence of a NADPH-generating system in the activation mix, produced a partial inhibition of the 2-aminofluorene activation. Both peroxidase activity and a cyt-P450-dependent activity are assumed to be involved in the activation of this chemical mediated by P. americana S117.

Activation of 4-nitro-o-phenylenediamine by the S2 fraction of Zea mays to mutagenic product(s).

Studies on plant metabolic activation with the S2 fraction from Zea mays have been developed. The 4-nitro-o-phenylenediamine (NOP) activation by S2 has been analyzed with the Ames test as a short-term assay. The NOP mutagenic potency was increased two-fold by S2, while rat liver S9 produced the contrary effect. The presence of a NADPH-generating system and the treatment of S2 with CO do not modify S2 activation of NOP. In this fraction, neither cytochrome P450 nor some enzymatic activities depending on cyt-P450 (aniline hydroxylase and aminopyrine demethylase) were detected. Therefore, the enhancement of NOP mutagenic potency by S2 is independent of the mixed-function oxidase system. On the other hand, inhibitors of the peroxidase activity such as N-acetyl-p-aminophenol caused a partial inhibition of S2 activation of NOP. Likewise, diethyldithiocarbamate produced both a reduction of the S2 peroxidase activity in biochemical assays and a partial inhibition of S2 activation of NOP. Moreover, it was possible to find a direct correlation between the activity of peroxidase per plate of both the S2 fraction and horseradish peroxidase and the number of revertants induced by NOP in the TA98 strain. On the basis of these results, we report that a HRP-like peroxidase activity must be the main pathway of NOP activation by the plant metabolic activation system studied in this work.

Induction of ribonucleoside diphosphate reductase gene transcription by chemicals in Escherichia coli.

A fusion between the promoter of the nrdA gene of Escherichia coli and the lacZ gene has been constructed, and the induction of nrdA gene expression by 20 organic and 20 inorganic chemicals has been studied. The inducing compounds of the SOS genes, such as bleomycin, captan, ciprofloxacin, enoxacin, hydroxyurea, N-methyl-N'-nitro-N-nitrosoguanidine, mitomycin C, nalidixic acid, ofloxacin and hexavalent chromium compounds also trigger the expression of the nrdA gene. Other chemicals such as aluminium, manganese and zinc salts, reported as negative in the SOS Chromotest, are also inducers of the nrdA gene. These results suggest that ribonucleoside diphosphate reductase transcription is increased by chemicals able to either block DNA synthesis or to alter the enzymes participating in the DNA replication. Induction of nrdA gene is an effect to be further considered in the study of alterations produced by physical or chemical treatments which act upon DNA metabolism.

Induction of SOS genes in Escherichia coli and mutagenesis in Salmonella typhimurium by fluoroquinolones.

The induction of several SOS genes of Escherichia coli by fluoroquinolones has been studied. Three different SOS gene fusions (recA::lacZ, umuC::lacZ and sulA::lacZ) have been introduced into the E.coli MC1061 strain to study the induction of these SOS genes in the same genetic background. Data on the basal level of expression of these fusions, as well as their induction by mitomycin C and N-methyl-N'-nitro-N-nitrosoguanidine are presented. Using these strains, we have found that, like nalidixic acid, ofloxacin, enoxacin and ciprofloxacin are strong inducers of the SOS genes tested, umuC gene expression being the highest. Furthermore, fluoroquinolones produced a significant increase in the reversion of the base substitution hisG428 mutation in the TA102 Salmonella tester strain, while no effect was found in strains TA98, TA100, TA1537 and TA1535. These data indicate that the error-prone repair pathway can participate in mutagenesis induced by fluoroquinolones and also that the damage produced by these chemicals may be similar to that produced by nalidixic acid.