Worm Breeder's Gazette 15(3): 22 (June 1, 1998)
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.
Department of Biology/Toxicology, Ashland University, Ashland, Ohio 44805
It has long been known that exposure to certain stresses (e.g.,
hyperthermia or hypoxia) can induce the expression of a set of genes
known as the heat shock proteins. Sodium azide, which inhibits
cytochrome oxidase c and ATP synthase, creates a chemical hypoxia in
cells and can induce certain heat shock proteins. It has been used as a
worm anesthetic but the mechanism by which C. elegans can survive
prolonged exposure to this chemical is not known. We hypothesized that
chemicals affecting energy metabolism will also induce the heat shock
response in C. elegans. We have found that exposure to low levels of
sodium azide confers thermotolerance in the nematode (see WBG 15(1): 38
for our first report on this project).
Our studies have shown that worms exposed to 10 mM sodium azide
for 60 minutes will show a significantly elevated survival probability
after exposure to 37 oC. The survival probability for control (no azide
or heat exposure) worms taken directly to 37 oC is 0.07, while worms
exposed to 10 mM sodium azide have a survival probability of 0.68
(p<0.05). This compares well with worms exposed to 33 oC and then taken
to 37 oC. Their survival probability is 0.92 (p<0.05).
We then hypothesized that the molecular mechanism of the
response to azide is the same as the response to elevated temperatures,
specifically regarding induction of the heat shock proteins. Snutch and
Baillie (Can. J. Biochem. Cell Biol 61: 480-487, 1983) demonstrated
that exposing C. elegans to 33 oC resulted in the induction of hsp70 and
hsp 16. Using SDS-PAGE and Coomassie Brilliant Blue staining, we
observed a 250% induction of hsp70 after exposure to 33 oC, but only a
25% induction after exposure to sodium azide. Using a more sensitive
silver staining technique, this pattern was still observed. Neither
staining technique was able to resolve induction of a low molecular
weight hsp. Using an anti-hsp16 antibody kindly provided by Peter
Candido, we determined via Western Blot analysis that hsp16 was induced
after exposure to sodium azide. The level of induction, when compared to
the 33 oC pre-treated worms was significantly lower, and it took 4 hours
after exposure to azide before hsp16 could be detected on a Western
Blot. In conjunction with the SDS-PAGE results, Western blots did not
demonstrate an induction of any other hsp examined (hsp90 and hsp60).
To further elucidate the mechanism, we also tested the only
known hsp mutant in C. elegans, daf-21 (JT6130). Elizabeth Malone and
Jim Thomas reported at the 1997 Worm Meeting that this constitutive
dauer forming mutant strain carries a conserved glu -> lys change in
hsp90. We tested this temperature sensitive mutant and found when grown
at the permissive temperature, it survived heat shock after receiving
pre-treatment with either azide or 33 oC. However, these worms showed a
reduction in survival probability when compared to N2. At the
restricted temperature, the worms pre-treated with either azide or 33oC
did not survive the heat shock. These results strongly suggest that
while hsp90 is not induced by hyperthermia or sodium azide, it is an
essential part of the worm's response to stress.
Our data indicate that while the mechanism of the worm!s
response to sodium azide is similar to its response to elevated
temperatures, there are significant differences in the degree of the
response. We will be reporting these results at the Midwest Worm
meeting.