Abstract # 2356: American Pediatric Society & Society for Pediatric Research - April 28 - May 1, 2001, Baltimore
Genetic Assay for Oxidant Stress in C. elegans
Farhang Payvar, Charles A. DeMatteo, Thomas A. Hazinski Department of Pediatrics, Vanderbilt University Medical School, Nashville, TN, 37232
Objective: To identify C. elegans mutants with
altered phenotypic responses to two forms of oxidant stress.
Background: Genetic events triggered by inducers of oxidative
stress (OS), such as nitroprusside (NP) and hyperoxia, are complex. Thus, we are employing the nematode Caenorhabditis
elegans, an organism amenable to forward genetic analysis. Previously, we found that 3.5 days of NP
treatment reduces survival rates in wild-type C. elegans (LD50
~ 6.0 mM at 20o C)
but hyperoxia does not. Other
investigators have reported on discrete signaling pathways regulating both
lifespan/aging and sensitivity to some forms of OS. We reasoned that if mutagenized C. elegans are screened
with high doses of NP - depending on whether NP-, hyperoxia-, and
lifespan-signaling pathways are independent, dependent, or mixed - mutants with
altered sensitivity to NP and/or hyperoxia and aging could be identified.
Design/Methods: To produce mutants with altered OS sensitivity, wild type C. elegans (N2) were mutagenized with 47 mM ethylmethanesulfonate by conventional methods. The F2 animals arising from 4800 F1 individuals were screened on plates containing 20 mM NP at 20o C. Synchronized clones of young adult C. elegans mutants surviving multiple rounds of NP-selection were characterized and compared to wild type animals: (i) Animals were exposed to increasing doses of NP for 3.5 days at 20o C in air. Survival rates were measured, and the NP LD50 were calculated; and (ii) Animals were cultured on standard plates at 20o C in air or under hyperoxia (95 % oxygen), and the number of live animals was determined daily over the course of 30 days. Median lifespan was calculated.
Results: Thus far, our independent clonal
isolates fall into three phenotypic
classes: Mutant 4.12b, termed OS-resistant, exhibits marked
resistance to NP (75% increase) and is increased in its lifespan in both air
and hyperoxia (13%). Mutant 4.12c,
termed OS-sensitive (25-% decreased sensitivity to NP), has markedly
shortened median lifespan in air, and under hyperoxic condition (25% & 45%
reductions, respectively). In contrast
to these mutants, mutant 4.10b is mixed with respect to OS responses: It
is more resistant to NP (210%), and has an increased lifespan in air (20 %
increase), whereas its lifespan is unaffected by hyperoxia.
Conclusions: These data indicate that the genetic events underlying OS sensitivity are likely to involve both dependent (e.g., mutants 4.12b and 4.12c) and mixed (mutant 4.10b) pathways. Subsequent experiments will identify the genes associated with these different phenotypic responses (Supported by HL56636). 12/12/00.