| The use of C. elegans for determining toxicity of environmental contaminants has been reported in earlier studies (Candido and Jones, 1996; Cioci et al. 2000; Custodia et al, 2001; Donkin and Dusenbery, 1994; Powers et al, 1998). The reasons for the use of the nematode are many and focus on the simplicity of the nematodes biological structure, the sequencing of its genome, ease of culturing and commonality of toxic findings with higher organisms (WormBase web site, http://www.wormbase.org, release WS159, date June 25, 2006, and Caenorhabditis elegans Genetics and Genomics, http://elegans.swmed.edu/genome) and, WormBook, ed. Eisenmann, D.M., Wnt signaling (June 25, 2005) The C. elegans Research Community, WormBook, ed. doi/10.1895/wormbook.1.7.1, http://www.wormbook.org.; The purpose of this research was to determine whether C. elegans could be used as a biomarker for environmental lead based upon changes in the metabolic patterns found when the nematode is exposed to lead and other metals. The research protocol required use of mixed population cultures (multi-generational), and defined age-fractionated cultures in order to determine effects of soluble lead in both a long-term and short-term exposure scenario and to determine whether the younger populations of the nematode were more sensitive to lead's toxicity than the older age fractions of post-reproductive nematodes. The results of the experiment showed that there were several biochemical changes occurring within the nematode that could be used as a signal or marker reflecting the effects of the lead. First, the analysis of the chromatogram peaks obtained from the CoulArray showed the ratio of the key metabolite tryptophan to its counterpart tyrosine as measured by the peak area under the curve (AUC) was reduced as a result of the addition of increasing concentrations of bioavailable lead. This finding suggested that the metabolite tryptophan, whose peak areas were reduced by 10x with doses of lead acetate, was more sensitive to the effects of lead than tyrosine. Second, the data from the mixed populations after treatment with several forms of soluble and insoluble lead showed dose dependent and bioavailability dependent reductions in the concentrations of several key tryptophan metabolites, 5-hydroxytryptophan (5-OHT), 3-hydroxyanthranilate (3-OHA), and serotonin (SER). There was a third significant change in the metabolic profiles analyzed through the CoulArray, and this was the finding of anthranilic acid which occurred only after treatments with soluble lead. The concentrations of the anthranilic acid were dependent on the concentration and bioavailability of the lead compound used. Anthranilic acid was not found in negative control experiments. Minimal amounts of anthranilic acid were noted with insoluble lead compounds at the limits of detection only when the dosage used was twice the Lc50 for the nematode. Anthranilic acid was not found after treatments with 4 other soluble metals (Cd, Sn, Zn, As) common in the environmental waste up to the limits of detection (1x10-4 ug). Therefore, anthranilic acid in addition to the changes in the other tryptophan metabolites seems to provide a basis for identifying soluble lead compounds in environmental settings.; The changes noted in the mixed populations also were seen in the nematode cultures that were age fractionated. The age fractionated also demonstrated that the effects of lead were more pronounced in the juvenile (L2) and pre-reproductive (L3) nematodes. In case of the other metals tested, the metabolic profiles and the TRP:TYR ratios were much different than the metabolic profiles and TRP:TYR ratios of the lead dosed nematode. The major markers of lead toxicity, anthranilic acid, and the changes in the specific tryptophan pathway metabolites seemed to be dependent on unique characteristics of lead's toxicity and therefore should allow for identification of lead and a determination of the availability of the lead in th... |
