Radiation Research

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Radiation Research 168(5):552-559. 2007
doi: 10.1667/RR1009.1

Ethyl Pyruvate, a Potentially Effective Mitigator of Damage after Total-Body Irradiation

Michael Epperlya, ShunQian Jina, Suhua Niea, Shaonan Caoa, Xichen Zhanga, Darcy Franicolaa, Hong Wanga, Mitchell P. Finkbcd, and Joel S. Greenberger1a

aDepartments of aRadiation Oncology,

bCritical Care Medicine,

cSurgery

dPharmacology, University of Pittsburgh Cancer Institute, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania 15213

1Address for correspondence: Department of Radiation Oncology, University of Pittsburgh Medical Center, 200 Lothrop Street, Pittsburgh, PA 15213;

Abstract

Epperly, M., Jin, S., Nie, S., Cao, S., Zhang, X., Franicola, D., Wang, H., Fink, M. P. and Greenberger, J. S. Ethyl Pyruvate, a Potentially Effective Mitigator of Damage after Total-Body Irradiation. Radiat. Res. 168, 552–559 (2007).

Ethyl pyruvate (EP), a simple aliphatic ester of pyruvic acid, has been shown to improve survival and ameliorate organ damage in animal models of sepsis, ischemia/reperfusion injury and hemorrhagic shock. Incubating IL3-dependent mouse hematopoietic progenitor cell 32Dcl3 cells before or after irradiation with 10 mM EP increased resistance to radiation as assessed by clonogenic radiation survival curves, decreased release of mitochondrial cytochrome C into the cytoplasm, and decreased apoptosis. EP inhibited radiation-induced caspase 3 activation and poly(ADP-ribose) polymerase (PARP) cleavage in 32Dcl3 cells in a concentration-dependent fashion. EP was given i.p. to C57BL/6NHsd mice irradiated with 9.75 Gy total-body irradiation (TBI). This treatment significantly improved survival. The survival benefit was apparent irrespective of whether treatment with EP was started 1 h before TBI and continued for 5 consecutive days after TBI or the compound was injected only 1 h before or only for 5 days after TBI. In all of the in vitro and in vivo experiments, ethyl lactate, an inactive analogue of EP, had no detectable radioprotective or mitigating effects. EP may be an effective radioprotector and mitigator of the hematopoietic syndrome induced by TBI.

Received: March 1, 2007; Accepted: August 3, 2007



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FIG. 1. EP protects 32Dcl3 cells from radiation. EP at concentrations of 1 mM (panel A) or 10 mM (panel B) was added to cultures of 32Dcl3 cells before, after or both before and after irradiation. Survival curves were fitted with linear-quadratic and single-hit, multitarget models. Curves fitted with the linear-quadratic model are shown. Cells incubated in 1 mM EP prior to irradiation were more radiation resistant (panel A) (P = 0.0046). At 10 mM EP, the 32Dcl3 cells were more resistant to radiation when incubated in EP before (P = 0.0003), after (P = 0.0001), or before and after (P = 0.016) irradiation (panel B)

FIG. 2. Inhibition of radiation-induced mitochondrial cytochrome C release by ethyl pyruvate. 32Dcl3 cells were incubated in 10 mM EP or EL for 1 h before irradiation with 10 Gy (IR). Cytoplasm was isolated 0–24 h after irradiation and Western blot analyses were performed for cytochrome C. Densitometric analysis (panel B) revealed a significant increase in release of cytochrome C into the cytoplasm beginning 3 h after irradiation in control cells or cells preincubated in EL but not in cells preincubated in EP (*P < 0.05 compared to unirradiated cells). Expression is presented as the ratio of cytochrome C density to actin density

FIG. 3. Ethyl pyruvate inhibits radiation-induced caspase 3 activation and PARP cleavage. 32Dcl3 cells were incubated with EP and EL for 1 h and irradiated with 10 Gy (IR). The cells were collected 24 h later and Western blot analysis was performed with antibodies to cleaved caspase 3 and PARP (panel A). Densitometric analysis demonstrated a decrease in cleavage of PARP (panel B) and caspase 3 (panel C) for EP compared to the control irradiated cells or cells preincubated in EL (*P < 0.05 compared to unirradiated control cells; #P < 0.05 compared to EP-treated cells)

FIG. 4. Ethyl pyruvate inhibits cleavage of caspase 3 and PARP after 10 or 20 Gy irradiation. 32Dcl3 cells were incubated for 1 h with 10 mM EP or EL before irradiation (IR). Western blot analysis was performed using antibodies against the cleaved products of caspase 3 or PARP (panel A). Densitometric analysis showed that treatment with EP before irradiation significantly decreased PARP (panel B) and caspase 3 activation (panel C). (*P < 0.05 compared to unirradiated control cells; #P < 0.05 compared to EP-treated cells at same radiation dose)

FIG. 5. Ethyl pyruvate inhibits radiation-induced apoptosis. 32Dcl3 cells were incubated with EP (10 mM) for 1 h before irradiation with 10 Gy. Cells were collected 24 h later and fixed. 3′ DNA strand breaks were labeled using a TUNEL assay, and nuclei were stained with propidium iodide. Cells undergoing apoptosis fluoresced green while the nuclei fluoresced red. Cells incubated with EP had a significant decrease in apoptotic cells after irradiation compared to the control irradiated cells (P < 0.0001)

FIG. 6. Ethyl pyruvate both protects and mitigates against the lethal effect of TBI. C57BL/6NHsd mice were injected with EP (panel A) or EL (panel B) (70 mg/kg) before, after (daily for 5 days), or both before and after irradiation with 9.75 Gy. Mice injected with EP before (P = 0.017) irradiation and both before and after irradiation (P = 0.006) had significantly improved survival. Mice injected after irradiation had a nonsignificant increase in survival (P = 0.123). Mice injected with EL had no significant change in survival after irradiation (panel B)

Scheme 1

table

TABLE 1 Ethyl Pyruvate Increases Radiation Resistance of 32Dcl 3 Cells

table

TABLE 2 EP is Comparable to Overexpression of MnSOD Transgene Product in Increasing the Radioresistance of 32Dcl 3 Cells

Cited by

Xichen Zhang, Michael W. Epperly, Mark A. Kay, Zhi-Ying Chen, Tracy Dixon, Darcy Franicola, Benjamin A. Greenberger, Paavani Komanduri, Joel S. Greenberger. (2008) Radioprotection In Vitro and In Vivo by Minicircle Plasmid Carrying the Human Manganese Superoxide Dismutase Transgene. Human Gene Therapy 19:8, 820-826
Online publication date: 1-Sep-2008.
CrossRef
Mitchell P Fink. (2008) Ethyl pyruvate. Current Opinion in Anaesthesiology 21:2, 160-167
Online publication date: 1-May-2008.
CrossRef
 
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