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Aflatoxin contamination in the field is known to be influenced by numerous factors. Drought and high temperatures are conducive to Aspergillus flavusinfection and aflatoxin contamination. This article will review the application of new molecular tools to study host resistance to biotic and abiotic factors affecting preharvest aflatoxin contamination in corn and peanut. We will also summarize recent studies conducted in our laboratories to evaluate the relationship of drought tolerance and aflatoxin contamination, and discuss the progress in using genetic engineering approaches to control preharvest aflatoxin contamination. The application of molecular tools, such as proteomics, DDRTPCR differential display reverse transcriptionpolymerase chain reaction, expressed sequence tag EST and gene chip technology macromicroarray to study gene expression in response to drought stress, and genetic transformation, will be reviewed. We have used DDRTPCR to display genes expressed in peanut and corn grown under drought stress vsirrigation condition. A new program has been initiated to use ESTmicroarray technology to study the whole genome as influenced by drought stress in corn and peanut. We are also studying A. flavusESTs to better understand the genetic control and regulation of toxin biosynthesis. Because of the complexity of the Aspergillusplant corn and peanut interactions, better understanding of the genetic mechanisms of resistance will be needed using both conventional and molecular breeding for crop improvement and control of preharvest aflatoxin contamination. Genetic improvement of crop resistance to drought stress is one component and will provide a good perspective on the efficacy of control strategy through genetic improvement.

The roles of superoxide and H2O2 in the cytotoxicity of paraquat were assessed in Chinese hamster ovary cells. Neither catalase nor superoxide dismutase inhibited the loss of ability to form colonies when added to the medium. When introduced into the cells, superoxide dismutase but not catalase inhibited the toxicity of paraquat. That superoxide dismutase acted by its known catalytic action is shown by the loss of inhibition when the enzyme was inactivated by H2O2 before being introduced into the cells. The lack of inhibition by catalase, by dimethyl sulfoxide, and by desferoxamine suggests that the toxicity is not mediated by a reaction between H2O2 and superoxide to engender the hydroxyl radical. Exposure of Chinese hamster ovary cells to paraquat may be a suitable means to determine the effects of superoxide anion in cultured cells and the ways in which cells can resist this toxic action.

To establish some of the necessary steps in the toxic action of paraquat for cultured mammalian cells, we isolated paraquat-resistant HeLa cells after lethal increments in concentration of paraquat in the medium. The paraquat-resistant cells had increased the cellular content of both the Mn-containing and the CuZn-containing superoxide dismutases. The effect of paraquat on the consumption of oxygen by the wild-type and the resistance cells was similar; in both cases exposure to paraquat for 24 or more hours produced similar proportions of cyanide-resistant consumption of oxygen, suggesting that paraquat entered both cells, underwent reduction, and donated electrons to molecular oxygen. When cultivated for 5 months in the absence of paraquat the paraquat-resistant cells maintained the increased cellular content of superoxide dismutases and remained resistant to paraquat. This observation suggested that resistance to paraquat might be caused by enrichment for the two superoxide dismutases and, further, that the increased cellular content of the two enzymes was not a response to growth under stressful conditions. NIH/3T3 cells whose content of superoxide dismutase was increased by transcription of the transfected cDNA for the human CuZn superoxide dismutase were also resistant to paraquat, suggesting strongly that paraquat promotes the formation of O2- as a necessary part of its cytotoxic effects in two types of cultured mammalian cells.

Evidence has been presented both that metallothionein does and does not produce resistance to cisplatin. The metallothionein-enriched cells described in most previous studies have been selected for resistance to heavy metals, such as cadmium, or have been maintained in a medium enriched for the metals. Exposure to toxic metals could alter the cells in many ways. This report addresses the effect of metallothionein content alone, independent of exposure to metals, on cellular resistance to cisplatin. The toxicity of cisplatin was compared in NIH/3T3 cells that vary in their content of metallothionein as a consequence of transfection with a plasmid that results in the constitutive expression of metallothionein. The plasmid contains the bovine papillomavirus genome and the mouse metallothionein-I gene; it is driven by a glucose-regulated protein of 78 kD. Control cells were transfected with a similar plasmid in which the coding sequences for metallothionein were inverted and separated from the promoter, thereby abolishing expression. Expression of metallothionein required neither selection nor maintenance of cells in the presence of heavy metals. Despite large differences between the two types of cells in their cellular content of metallothionein and in their resistance to the toxicity of cadmium, no differences in resistance to cisplatin were observed.

We assessed the roles of intrabacterial catalase and superoxide dismutase in the resistance of Escherichia coli to killing by neutrophils. E. coli in which the synthesis of superoxide dismutase and catalase were induced by paraquat 10-fold and 5-fold, respectively, did not resist killing by neutrophils. When bacteria were allowed to recover from the toxicity of paraquat for 1 h on ice and for 30 min at 37 degrees C, they still failed to resist killing by neutrophils. Induction of the synthesis of catalase 9-fold by growth in the presence of phenazine methosulfate did not render E. coli resistant to killing by either neutrophils or by H2O2 itself. The lack of protection by intrabacterial catalase from killing by neutrophils could not be attributed to an impermeable bacterial membrane; the evolution of O2 from H2O2 was no less rapid in suspensions of E. coli than in lysates. The failure of intrabacterial catalase or superoxide dismutase to protect bacteria from killing by neutrophils might indicate either that the flux of O-2 and H2O2 in the phagosome is too great for the intrabacterial enzymes to alter or that the site of injury is at the bacterial surface.