Effect of rHu-EPO on cold-induced apoptosis of cultured rat hepatocytes. (A) rat hepatocytes were pre-incubated in the presence or absence of rHu-EPO (10 or 100 U/ml) in L-15 medium for 18 hours at 37°C (74% N2/21% O2/5% CO2). Cells were then exposed to hypothermia (4°C) in University of Wisconsin solution with or without rHu-EPO. To prevent iron-dependent cold-induced apoptosis, the iron chelator 2,2'-dipyridyl (2,2'-DPD, 100 μM) was added prior to cold incubation. In (B), the effect of rHu-EPO on the iron-independent component of cold-induced apoptosis is shown. Cells were pre-incubated with rHu-EPO for 18 hours and then exposed to hypothermia in L-15 medium with or without rHu-EPO and/or 2,2'-DPD (100 μM). The occurrence of cell injury (including late apoptosis) was assessed by the release of lactate dehydrogenase (LDH). Values represent means ± S.D. of 3 independent experiments. (A) Open squares: hypothermia/rewarming, no addition; closed circles: hypothermia/rewarming, 100 U rHu-EPO/ml; closed squares: hypothermia/rewarming, 10 U rHu-EPO/ml; open circles: hypothermia/rewarming, 100 μM 2,2'-DPD. (B) Open squares: hypothermia/rewarming, no addition; open circles: hypothermia/rewarming, 100 μM 2,2'-DPD; closed circles: hypothermia/rewarming, 100 μM 2,2'-DPD and 10 U rHu-EPO/ml; closed triangles: hypothermia/rewarming, 100 μM 2,2'-DPD and 100 U rHu-EPO/ml.