Worm Breeder's Gazette 12(5): 90 (February 1, 1993)

These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.

Transgenic hsp16 -lacZStrains of C. Elegans as Biological Monitors of Environmental Stress

Eve G. Stringham, E. Peter M. Candido

Dept. of Biochemistry, University of British Columbia. Vancouver, Canada, V6 T

The heat shock or stress response is a universal phenomenon characterized by the rapid and coordinate induction of a set of proteins in response to elevated temperature or other types of environmental stress, such as exposure to certain harmful chemicals. Since stress proteins are highly conserved in all organisms, play a protective role in the cell and are induced by a wide variety of biologically harmful agents, the stress response has recently come under intense investigation as a basis for generating biomonitors to assess environmental contamination.

We have derived stable transgenic nematode lines which carry fusions of the hspl6 genes to the E. coli lacZ reporter gene. The availability of transgenic strains which produce ß-galactosidase as a stress protein has provided a unique opportunity to examine the effects of a wide variety of chemicals and environmental conditions on the stress response by simply assaying for enzymatic activity. ß-galactosidase activity is detected in situ by the commonly used histochemical stain (X-gal) which provides qualitative information as to which tissues in the animal have been stressed. Alternatively, enzymatic cleavage of the colourless substrate ONPG produces a soluble yellow product which can be quantified spectrophotometrically, hence dosage response curves can be determined (example below).

This system allows the rapid and convenient assay of large numbers of samples for agents which induce the stress response. Salt and fresh water, or soil samples can be assayed following short-term (1-4 hr.) or long-term (2-4 day) exposures. The responses of this system to a variety of compounds in pure form, as well as mixtures and contaminated water samples from the environment are currently being assessed.

Agents tested to date which induce the hsp 16-lacZ transgene in these animals include heavy metals such as arsenite, cadmium, copper, lead, mercury and zinc, and the herbicide, paraquat. These agents yield different tissue patterns of stress induction: mercury and paraquat induce only intestinal expression, lead affects pharyngeal muscle, especially at the base of the terminal bulb, copper induces in neurons and muscle at the anterior end of the pharynx, and cadmium throughout the pharynx or occasionally in the intestine. Arsenite exposure produces the most tissue general expression, in a manner reminiscent of classic heat shock induction. These results suggest that classification of stress agents in complex mixtures may be a useful feature of this biomonitoring system.

Somewhat surprisingly, for each of these inducers only a fraction of the tested animals (15- 50 %) expressed the transgene. In contrast, virtually 100 % of the animals stain after a two hour heat shock. This suggested that worms might avoid exposure to nasty chemicals by "clamming up". To determine whether the worms were feeding, we added some non-toxic red acrylic paint to the formula containing E. coli and mercuric chloride. After 24 hours, all of the animals contained particles of red paint in their pharynx, intestine or anus, but when stained with Xgal, only 30 % of the animals stained in the intestine. Control animals which were fed paint alone did not stain with Xgal.

Apparently the animals continued to pump in the chemicals, so why did only a fraction respond to the stress? One possibility is that some of the animals had not consumed a threshold dose of the chemical to induce the response, while others may have died before a stress response could be mounted.

The stress-reporter gene induction always occurred at metal concentrations below the LC50 (concentration at which 50 % of the animals die within a specified time period) suggesting that this assay is a more sensitive and rapid indicator of stress than current LC50 assays using C. elegans. Moreover, these experiments suggest that the stress response in general may be a valuable indicator of sub-lethal toxicity in environmental risk assessments.

Supported by StressGen Biotechnologies Corp., the B.C. Science Council and the Medical Research Council of Canada.

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