The severity of the toxic side effects of chemotherapy varies among patients, and much of this variation is likely genetically based. Here, we use the model system Drosophila melanogaster to genetically dissect the toxicity of methotrexate (MTX), a drug used primarily to treat childhood acute lymphoblastic leukemia and rheumatoid arthritis. We use the Drosophila Synthetic Population Resource, a panel of recombinant inbred lines derived from a multiparent advanced intercross, and quantify MTX toxicity as a reduction in female fecundity. We identify three quantitative trait loci (QTL) affecting MTX toxicity; two colocalize with the fly orthologs of human genes believed to mediate MTX toxicity and one is a novel MTX toxicity gene with a human ortholog. A fourth suggestive QTL spans a centromere. Local single-marker association scans of candidate gene exons fail to implicate amino acid variants as the causative single-nucleotide polymorphisms, and we therefore hypothesize the causative variation is regulatory. In addition, the effects at our mapped QTL do not conform to a simple biallelic pattern, suggesting multiple causative factors underlie the QTL mapping results. Consistent with this observation, no single single-nucleotide polymorphism located in or near a candidate gene can explain the QTL mapping signal. Overall, our results validate D. melanogaster as a model for uncovering the genetic basis of chemotoxicity and suggest the genetic basis of MTX toxicity is due to a handful of genes each harboring multiple segregating regulatory factors.
While dihydrofolate reductase (DHFR) is an important drug target in mammals, bacteria and protozoa, no inhibitors of this enzyme have been developed as commercial insecticides. We therefore examined the potential of this enzyme as a drug target in an important ectoparasite of livestock, the Australian sheep blow fly, Lucilia cuprina (Diptera: Calliphoridae) (Wiedemann). The non-specific DHFR inhibitors aminopterin and methotrexate significantly inhibited the growth of L. cuprina larvae, with IC50 values at µg levels. Trimethoprim and pyrimethamine were 5–30-fold less active. Relative IC50 values for the inhibition of recombinant L. cuprina DHFR by various inhibitors were in accordance with their relative effects on larval growth. The active-site amino acid residues of L. cuprina DHFR differed by between 34% and 50% when compared with two mammalian species, as well as two bacteria and two protozoa. There were significant charge and size differences in specific residues between the blow fly and human DHFR enzymes, notably the L. cuprina Asn21, Lys31 and Lys63 residues. This study provides bioassay evidence to highlight the potential of blow fly DHFR as an insecticide target, and describes differences in active site residues between blow flies and other organisms which could be exploited in the design of blow fly control chemicals.
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