Long-term Behavioral and Reproductive Consequences of Embryonic Exposure to Low-dose Toxicants

Exposure to environmental toxicants can acutely impact development with long-term effects. Detailed protocols are provided to illustrate a strategy using an effective lab model to study the effect of early embryonic exposure to bisphenol A. We provide fecundity and behavioral assays to monitor the effectiveness of our toxicant exposure bio-assays.

Transcript

The overall goal of these procedures is to investigate the long-term effects of low-dose BPA exposure in early embryogenesis. C.elegans embryos are exposed to BPA for a limited period and tested upon reaching adulthood for fecundity and learning behavior. This method can help answer key questions regarding the longterm developmental effects of exposure to toxicants such as bisphenols and other endocrine disrupting compounds.

The main advantage of this technique is that it allows testing of long term and subtle effects of toxicants on development through quantitative behavioral assays on an easy to use invertebrate lab model. The implications of this technique extends towards understanding the developmental and neural basis of deleterious health effects of exposure to toxicants even at low doses. We first had the idea for these experiments when we realized that a number of complex or multifactorial disorders seemed to have some or the other developmental component.

Which may exert it's influence in part through anomalous expression of critical genes. Visual demonstration of this method is critical as the interior touch habituation steps can be difficult to learn solely through text based instructions. The day before synchronization, transfer N2 worms onto 100 millimeter plates containing NGM media, spread with E.coli OP50.

By chunking slab of approximately one centimeter square. Allow the worms to grow for three days at 20 degrees Celsius. After three days, when the plates are populated with well-fed, mature adults containing visible embryos, begin synchronization by first washing each plate with five millimeters of M9 buffer, and transferring the liquid to a sterile 15 milliliter conical centrifuge tube.

Centrifuge at 3, 000 times gravity for four minutes at room temperature. Following the certrifugation, use a ten milliliter pipette to carefully remove the supernatant from each loose pellet of adult worms. Then add 5 milliliters of freshly prepared hypochlorite solution to each pellet of worms, and gently mix.

Centrifuge at 3, 000 times gravity at room temperature for four minutes. It is critical to obtain synchronized populations. Preparing a fresh hypochlorite solution helps in avoiding contamination, and unsynchronized animals.

Next, use a ten milliliter pipette to remove the supernatant, and wash the seven milliliters of M9 buffer. Repeat this step twice. After the last wash, add approximately 100 microliters of M9 buffer to the eggs.

Then transfer about 50 microliters of eggs to two milliliter microfuge tubes containing BPA diluted in S buffer. Adjust the amount of S buffer to ensure the correct final concentration of BPA per tube, as shown in the table. Place the tubes on a rocking, or rotary shaker, moving at 25 tilts per minute, or 25 RPM for four hours at 20 degrees Celsius.

After four hours, place the tubes into tube holders and allow the worms to settle. Discard the supernatant and transfer the worms to NGM plates seeded with OP50 E.coli. Allow the worms to grow for 60 hours at 20 degrees Celsius.

Examine the plates every day for contamination. Contaminated NGM plates are usually discolored and accompanied by individual colonies. Check worms under the microscope for overcrowding.

First, use a fire-steriled 30 gauge platinum wire pick to transfer approximately ten synchronized young adult worms to new unseeded NGM plates. Leave the worms undisturbed for five minutes to allow them time to acclimatize to the new plate. Then sterilize an eyebrow hair attached to the end of a wooden skewer or toothpick by dipping in 70%ethanol.

Wipe with a clean, lint-free tissue and wait for one minute for the ethanol to evaporate. Gently touch the worm on the head using the eyebrow hair. Repeat the touches, allowing ten seconds in between touches to allow the worm to recover.

Continue to touch, allow ten second interstimulus intervals until the worm no longer moves backwards. Record the number of touches required for this habituation to occur. Allowing a ten second interval between touches is important as this allows the animal recovery time and helps with obtaining consistent habituation behavior assays.

Adult animals that were embryonically exposed to one micromolar or higher concentration of BPAs laid significantly less eggs compared to the control represented by the horizontal line. Adult animals derived from embryos that were exposed to BPA concentrations as low as 0.1 micromolar display non-associative deficits based on the increased number of anterior touches required for them to habituate. While preparing embryos for BPA exposure, it is important to choose a plate with well-fed C.elegans adults with mature eggs visible under the dissection microscope in order to have a sufficient sample size.

This technique paves the way for researchers in the field of environmental toxicology to evaluate the effects of toxins and other endocrine disrupting compounds on behavior and design mechanistic experiments. After watching this video, you should have a good understanding of how to expose early stage C.elegans embryos to BPA and how to test habituation behavior in the resulting adults derived from the BPA exposed embryos.

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Journal (Developmental Biology)

Journal (Developmental Biology)

Long-term Behavioral and Reproductive Consequences of Embryonic Exposure to Low-dose Toxicants

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