Janis Weeks, Ph.D.

NemaMetrix logo - C. elegans research and phenomics


Purpose

C. elegans is a well-validated experimental subject for toxicological research1 including the physiological effects of specific toxic substances and genes that influence sensitivity or resistance to these substances. C. elegans also provides a whole-animal system for assaying the toxicity of environmental soil or water samples. The U.S. National Institute of Environmental Health Sciences has highlighted C. elegans as a valuable system for toxicological research2.

The NemaMetrix ScreenChip System is a microfluidics platform for recording electropharyngeograms (EPGs) from the nematode worm Caenorhabditis elegans and other species. Various endpoints have been used to detect toxic effects, including development, reproduction, induction of stress-related genes, lethality and—relevant to the ScreenChip system—pharyngeal pumping. Toxicants that inhibit pumping in C. elegans include heavy metals, insecticides, organophosphate pesticides and cyanobacterial toxins. Here we used EPG recordings to investigate toxic effects of the heavy metal, copper (Cu2+), which inhibits pharyngeal pumping3. Exposure to high levels of Cu2+, e.g., from corrosion of copper pipes by acidic water, is likewise toxic to humans and can damage the liver and kidneys4.

We found that microfluidic EPG recordings detected quantitatively the presence of Cu2+ in aqueous samples at concentrations within the range found in contaminated home water supplies.

Results and Discussion

Pump frequency over time of C. elegans after exposure to 50 mg/L Cu2+. Pump frequency is shown for different Cu2+ dosages as well.

Experiment 1 was performed using 8-channel microfluidic EPG chips with perfusion capability5,6. Fig. 1A shows pharyngeal pumpPump frequency of C. elegans after exposure to 50 mg/L Cu2+ frequency in control
solution (K medium + 10 mM 5HT), followed at
t = 0 min by switching the perfusate to the same solution containing a range of Cu2+ concentrations. In control worms (black line), pump frequency remained steady over time, at ~ 4 Hz (pumps/s). In response to Cu2+ exposure, pump frequency decreased in a concentration–dependent manner, with inhibition apparent within 5 min at the higher concentrations. Steady-state pump frequency, defined as the mean frequency between t = 30 and 60 min, was plotted in Fig. 1B to derive an IC50 value (the concentration that caused a 50% reduction in pump frequency) of 49 mg/L Cu2+. For comparison, a prior study reported an IC50 of 3.32 mg/L Cu2+ when pumping was counted visually after 24 h exposure on Cu2+-containing agar plates3. Thus—not unexpectedly—Cu2+-induced inhibition of pumping depends on both the concentration and duration of exposure.

Experiment 2. In these experiments, the ScreenChip system was used to compare pump frequency in worms exposed to control or 50 mg/L Cu2+ solutions (Fig. 2; n = 26-28 worms/group; mean ± S.E.M.). EPG recordings (2 min per worm) were started 30 min after the onset of Cu2+ exposure, during the steady-state inhibition of pumping (see Fig. 1A). The Cu2+-exposed group showed a significant reduction in pump frequency of ~68% compared to controls (P < 0.00001; 2-tailed Mann-Whitney Wilcoxon Test).

Conclusions

These data demonstrate the use of microfluidic EPG recordings to quantify concentration- and time-dependent effects of Cu2+ on pharyngeal pumping. To our knowledge, this is the first demonstration of a rapid, electrophysiological effect of Cu2+ on C. elegans. EPG recordings detected [Cu2+] as low as 10 mg/L (Fig. 1), well within the range of copper-contaminated water in the United States, which can exceed 30 mg/L4; for comparison, the U.S. Environmental Protection Agency’s upper limit for safe drinking water is 1.3 mg/L Cu2+. We conclude that the ScreenChip system provides rapid and sensitive detection of the toxic heavy metal, Cu2+, in a concentration range suitable for testing environmental samples.

Methods

Synchronized N2 (wild-type) worms were cultivated at 20 oC to the first day of adulthood on plates containing nematode growth medium (NGM) seeded with E. coli OP50, using standard methods7,8. For microfluidic EPG recordings, reagent-grade Cu2+ solutions were prepared in K medium (32 mM KCl, 51 mM NaCl in dH2O) containing 10 mM 5HT to stimulate pumping5,6,9. EPG recordings were acquired using Spike2 software (Cambridge Electronic Design Ltd.) for 8-channel chips or NemAcquire software10 for the ScreenChip system. Detailed ScreenChip methods are available elsewhere11. Recordings were analyzed using custom software in IGOR Pro6, soon to be superseded by the release of NemaMetrix’s NemAnalysis software10.

 

References

  1. Leung MC et al. (2008) Caenorhabditis elegans: an emerging model in biomedical and environmental toxicology. Toxicol Sci. 106(1):5-28
  2. C. elegans: a medium-throughput screening tool for toxicology (2006). http://ntp.niehs.nih.gov/ntp/factsheets/wormtoxfs06.pdf
  3. Jiang Y et al. (2016) Sublethal toxicity endpoints of heavy metals to the nematode Caenorhabditis elegans. PLoS One, Jan 29;11(1):e0148014
  4. Donohue J (2004) Copper in drinking-water: background document for development of WHO guidelines for drinking-water quality. http://www.who.int/water_sanitation_health/dwq/chemicals/copper.pdf
  5. Lockery SR et al. 2012. A microfluidic device for whole-animal drug screening using electrophysiological measures in the nematode C. elegans. Lab Chip 12:2211-2220
  6. Weeks et al. Microfluidic platform for electrophysiological recordings from host-stage hookworm and Ascaris suum larvae: a new tool for anthelmintic research.  Int J Parasitol: Drugs Drug Res, in press
  7. Stiernagle T. 2006. Maintenance of C. elegans. WormBook, http://www.wormbook.org/chapters/www_strainmaintain/strainmaintain.html
  8. Porta-de-la-Riva M et al. 2012. Basic Caenorhabditis elegans methods: synchronization and observation J Vis Exp 64:4019
  9. http://nemametrix.com/serotonin-induced-pharyngeal-pumping-c-elegans-using-screenchip/
  10. http://nemametrix.com/downloads/
  11. http://nemametrix.com/wp-content/uploads/2016/08/ScreenChip_System_QuickStart_Guide.pdf; http://nemametrix.com/tech-notes

Acknowledgements

Unpublished data were provided by JC Weeks, KJ Robinson and WM Roberts. Funding from Oregon BEST.