D.J. Glofcheski

Relationship between hyperthermic cell killing and protein denaturation by alcohols

The monohydric alcohols methanol, ethanol, 2-propanol, and tert.-butanol dramatically sensitize V79 Chinese hamster lung cells to hyperthermia. for concentrations less than 3% alcohol by weignt, the rate of cell inactivation (k) appears to vary exponentially as a function of alcohol concentration. The degree of sensitization increases with increasing chain length, and for a constant concentration the ratio of the ks is 1:1.3:1.8:3.1 for methanol, ethanol, 2-propanol, and t-butanol, respectively. Mono-, di-, and trihydric alcohols have similar effects upon the rate of protein denaturation. Membrane lipid fluidity of V79 cells was determined from 23 to 63/sup 0/C by measuring the rotational correlation time of the spin label 2,2-dimethyl-5-dodecyl-5-methyloxazolidine-N-oxide (2N14). A straight line was found for the Arrhenius plot of tau/sub c/, with an activation energy of 4.82 +- 0.07 kcal/mole. From the Arrhenius plot of the rate of cell killing from 42.0 to 46.8/sup 0/C, the enthalpy (..delta..H) and entropy (..delta..S) of activation were found to be 146 +- 9 kcal/mole and 388 cal/mole K, respectively. These values are consistent with protein denaturation (or possibly the denaturation of some other macromolecule) being the rate-limiting step in hyperthermic cell killing.

Protective effect of l-glutamine against freeze-thaw damage in mammalian cells

L-Glutamine at 18 mM protects mammalian cells against freeze-thaw (FT) damage by a factor of about 6, depending on FT conditions, in balanced salt solutions. While not nearly as effective a cryoprotectant as dimethyl sulfoxide (DMSO) or propylene glycol (PG), the mechanism of protection by glutamine appears to be independent from that of DMSO or PG; thus, 18 mM glutamine is effective at reducing FT damage in combination with these agents. These combinations allow lower concentrations of the more toxic agents DMSO and PG to be used in FT medium. There is no pre-FT or post-FT effect of glutamine when cells are exposed to a FT cycle in balanced salt solutions. Hence, protection is due to its presence during the FT-cycle. The presence of 2 mM L-glutamine in Eagles basal medium is sufficient to account for cryoprotection by this medium.

Factors influencing survival of mammalian cells exposed to hypothermia: IV. Effects of iron chelation

Survival of V-79 Chinese hamster cells was assessed by colony growth assay after hypothermic exposure in the presence of iron chelators. At 5 degrees C, maximum protection from hypothermic damage was achieved with a 50 microM concentration of the intracellular ferric iron chelator Desferal. A 3-hr prehypothermic incubation with 50 microM Desferal followed by replacement with chelator-free medium at 5 degrees C also provided some protection. This was not observed when the extracellular chelator DETA-PAC (50 microM) was used prior to cold storage. Treating 5 degrees C-stored cells with Desferal just prior to rewarming was ineffective, but treating cells with Desferal during hypothermia exposure after a significant period of unprotected cold exposure ultimately increased the surviving fraction. Submaximal protection during hypothermia was achieved to various degrees with extracellular chelators at 5 degrees C, including 50 microM DETAPAC and 110 microM EDTA. EGTA (110 microM) had little effect. The sensitization of cells at 5 degrees C with 200 microM FeCl3 could be reduced or eliminated with Desferal in accordance with a 1:1 binding ratio. At 10 degrees C, 50 microM Desferal, 50 microM DETAPAC, and 110 microM EDTA were as or less effective in protecting cells than at 5 degrees C. An Arrhenius plot of cell inactivation rates shows a break at 7-8 degrees C, corresponding to maximum survival for control cells and cells in 50 microM Desferal; however, the amount of protection offered by the chelator increases with decreasing temperature below about 19 degrees C, and sensitization increases above that point. It has not previously been shown that iron chelators protect against cellular hypothermia damage which is uncomplicated by previous or simultaneous ischemia. This may be relevant to the low-temperature storage of transplant organs, in which iron of intracellular origin and in the perfusate may be active and damaging.

Interactions between cryoprotectors and cryosensitizers

Abstract When cryoprotected or unprotected mammalian cells are exposed to multiple freeze-thaw (FT) cycles, survival decreases exponentially, even when combinations of cryoprotector (CP) agents are used. This enables one to calculate independence or synergistic interactions between different CP (and/or cryosensitizers) agents. However, theoretical and practical considerations restrict the accuracy and interpretation of these experiments to a limited range of survivals. For example, low concentrations of CPs act independently until the concentration is increased so that competition for the “FT targets” occurs. While the standard CP agents, DMSO, propylene glycol (PG), HES, and glycerol interact with each other as cryoprotectants, glutamine acts independently when used in combination with any of the above CP agents. If cryosensitizers (CS) are used, some differences between standard CPs are observed. For example, DMSO and PG are better protectants than HES against FT sensitization by protein denaturants like ethanol or guanidine HCl. Some of the CSs (ethanol, A23187, BHT, zinc sulfate) interact with CPs, while others (guanidine HCl, DTNB, europium) act independently. These interactions, or lack of interaction, may help us determine how various CPs protect the many different targets of FT damage.