Abstract
Organisms have developed many protective systems to reduce the toxicity from heavy metals. The nematode Caenorhabditis elegans has been widely used to determine the protective mechanisms against heavy metals. Responses against heavy metals can be monitored by expression of reporter genes, while sensitivity can be determined by quantifying growth or survival rate following exposure to heavy metals.
Keywords: Caenorhabditis elegans, Arsenic, Cadmium, Copper
Background
Some heavy metals, such as arsenic, cadmium and mercury, are known to be harmful to the majority of organisms including humans (Valko et al., 2005). To reduce the toxicity by these metals, the organisms have developed various protective systems. The nematode Caenorhabditis elegans has been used to understand the mechanisms of protection against heavy metals. Previous studies have revealed that many genes, such as detoxification enzymes, transcription factors and signaling factors, are involved in the protection from heavy metals in this organism (Broeks et al., 1996; Mizuno et al., 2004; Inoue et al., 2005; Schwartz et al., 2010). Determinations of viability and growth, in addition to measurements of reporter gene expression, are usually used to monitor the effects of heavy metals in C. elegans. In this protocol, we describe the methods for assays for arsenic, copper and cadmium using C. elegans.
Materials and Reagents
Equipment
Software
Procedure
Data analysis
In Acute assays, take pictures by appropriate camera attached to the fluorescent microscope. Then the pictures can be used to quantify the intensity of GFP fluorescence with the ImageJ program. The frequency of growth into adulthood can be calculated by dividing the number of adult animals 4 days after egg laying by the number of eggs hatched on the day after egg laying. The difference between the heavy metal treatment and control data can be tested for statistical significance by Fisher’s or chi-square test using for example GraphPad QuickCalcs.
Notes
Recipes
Acknowledgments
This work was supported by grants from the Ministry of Education, Culture and Science of Japan. Many other researchers have utilized similar protocols to test sensitivity to heavy metals. The protocols are based on the way that we measure heavy metal sensitivity in our lab. This protocol is essentially adopted previous works (Mizuno et al., 2004; Inoue et al., 2005).
References
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