Suspended animation of C. Elegans embryos using carbon monoxide

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					  Suspended animation of C.
Elegans embryos using carbon

       By Cyndi Edwards
•   Normal air: ~21 kPa O2 (101 kPa = 1 atm)
•   Mild hypoxia: 0.25-1.0 kPa O2
•   Intermediate hypoxia: 0.001-0.25 kPa O2
•   During mild hypoxia:
    – HIF-1 (Hypoxia Inducible Factor-1)
    – Anaerobic energy production genes up-
       • glycolytic enzymes, glucose transporters
    – Antioxidant genes upregulated
       • catalase, superoxide dismutase
• <0.001 kPa O2
• HIF-1 response alone is not sufficient to
  protect the cells from free radicals
• Oxidative phosphorylation ceases
• ATP demand must also decrease
• Cells of some organisms enter into a state
  called “suspended animation”
       Suspended Animation
• No observable movement is visible
• Developmental progression ceases
• Turtle hepatocytes in this state are
  capable of reducing their ATP demand by
  – limit protein synthesis, ion channel activity,
    and anabolic pathways
• Zebrafish embryos and C. elegans
  embryos stop moving and developing
C. Elegans Embryo Viability
  Carbon Monoxide: A Competitive
    Inhibitor of Oxygen Binding

• CO binds and inhibits cytochrome c oxidase
  (complex IV), causing free radical production
• Thus CO effectively lowers the effective amount
  of oxygen electron carriers
Can carbon monoxide induce
  suspended animation in
   damaging low oxygen
               Experimental Setup

Two-cell embryos     Embryos put in     Embryos exposed
were harvested       30 mL leak-proof   to various levels of
from adults of the   glass syringes     hypoxia or to
Bristol N2 strain    with gas inlets    anoxia by changing
                                        gas inflow

Embryos were         Embryos were       Embryos were
exposed to the       then removed and   moved to culture
same oxygen          immediately        plates to assay
level for 24 hours   photographed       their viability
 Intermediate Hypoxic Exposure
Resulted in Abnormal Development
Carbon monoxide was effective in
 inducing suspended animation
        Take Home Message
• C. elegans embryos are capable of surviving in
  anoxia and mild hypoxia, but intermediate
  hypoxic conditions are lethal

• CO induced suspended animation in embryos
  experiencing intermediate hypoxia

• Embryos that had entered CO-induced
  suspended animation were highly viable
• Nystul TG, Roth MB. Proc Natl Acad Sci USA. 2004 Jun
  15;101(24):9133-6. Epub 2004 Jun 7. Carbon
  monoxide-induced suspended animation protects
  against hypoxic damage in Caenorhabditis elegans.