EPG RECORDINGS AS A NEW TOOL FOR
TOXICOLOGY IN C. elegans
USING THE SCREENSHIP SYSTEM - BY DR. JANIS C. WEEKS, PH.D
PURPOSE
The NemaMetrix ScreenChip System is a microfluidics platform for recording electropharyngeograms
(EPGs) from the nematode worm Caenorhabditis
elegans and other species. C. elegans is a well-validated experimental subject for toxicological research1
with which to investigate 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. 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
NemaMetrix, Inc.
44 W 7th Ave
Eugene, OR 97401
1-844-663-8749
nemametrix.com
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 DISCUSSIONS
Experiment 1 was performed using 8-channel
microfluidic EPG chips with perfusion capability5,6. Fig.
1A shows pharyngeal pump 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).
CONCLUSION
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/copp
er.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/strainm
aintain.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-pumpin
g-c-elegans-using-screenchip/
10. http://nemametrix.com/downloads/
11.http://nemametrix.com/wp-content/uploads/2016/08/Screen
Chip_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.
NemaMetrix, Inc.
44 W 7th Ave
Eugene, OR 97401
1-844-663-8749
nemametrix.com