Organ preservation solutions attenuate accumulation and nuclear translocation of hypoxia-inducible factor-1alpha in the hepatoma cell line HepG2.

ABSTRACT

Hypoxia-inducible factor-1alpha (HIF-1alpha) is a key transcription factor orchestrating hypoxic and inflammatory reactions. Here, we determined the impact of organ preservation solutions (Celsior; histidine-tryptophan-ketoglutarate solution, HTK; University of Wisconsin solution; UW), oxygen supply, and temperature on HIF-1alpha accumulation, recorded by Western blotting and immunocytochemistry, in the human hepatoma cell line HepG2. Generation of reactive oxygen species (ROS), NO, and cell viability were concomitantly assessed. At 4 degrees C, HIF-1alpha accumulation was not detectable. In normothermic (37 degrees C) cell culture medium (Dulbecco's Modified Eagle's Medium, DMEM), HepG2 cells accumulated HIF-1alpha even in normoxia (21% O(2)) which was not observed in either of the preservation solutions. This correlated to high generation of NO, a normoxic stabilizer of HIF-1alpha, and L-arginine content (substrate for NO synthesis) in DMEM, and low NO production and absence of L-arginine in preservation solutions. In normothermic hypoxia up to 24 h, intracellular HIF-1alpha accumulated in all conditions, but less in preservation solutions compared to DMEM. The inhibitory effect on accumulation and nuclear translocation was most prominent for HTK, the only solution containing the activator of HIF-1alpha degradation, alpha-ketoglutarate. Addition of other intermediates of the tricarbon acid cycle-succinate, fumarate, malate-did not alter HIF-1alpha accumulation, although succinate exhibited a beneficial effect on cell viability in cold storage. In conclusion, preservation solutions attenuate accumulation and nuclear translocation of the transcription factor HIF-1alpha, and this property is seemingly related to their chemical composition (L-arginine, alpha-ketoglutarate). Thus, it appears feasible to design preservation solution specifically to modify HIF-1alpha accumulation and nuclear translocation.