A two-step reversible-irreversible model can account for a negative activation energy in an Arrhenius plot.

Arrhenius plots of the rate of inactivation (killing) of mammalian cells and Drosophila embryos have "apparent negative" activation energies at low temperatures. This can be explained by assuming that the rate-limiting event resulting in killing is a two-step process or mechanism, where the first step is reversible and the second irreversible. Two examples, consistent with this model, are suggested as possible mechanisms of hypothermic killing: (i) a membrane lipid liquid crystalline-to-gel transition followed by a metabolic block or event which kills the cell and (ii) cold denaturation of a protein followed by protein aggregation.

Evidence for two modes of hypothermia damage in five cell lines.

The Arrhenius plots of inactivation (killing) rates of five mammalian cell lines, V-79 Chinese hamster lung, mouse L-929, mouse neuroblastoma, human 18LU, and human erythrocytes, exposed to hypothermia contain a break somewhere between 5 and 10 degrees C caused by a change from positive to negative slope, which corresponds to the minimum inactivation rate. This implies that there are two distinct mechanisms of hypothermic damage above and below the minimum inactivation rate temperature in a system that is uncomplicated by previous or simultaneous hypoxia. Hence, having two distinct mechanisms for hypothermia damage is not unique to the Chinese hamster cell line. This suggests that the optimum aerobic hypothermic storage temperature for human cells and tissues is not 0 degree C, but somewhere in the range of 5 to 10 degrees C.

Factors influencing survival of mammalian cells exposed to hypothermia. VI. Effects of prehypothermic hypoxia followed by aerobic or hypoxic storage at various hypothermic temperatures.

The Arrhenius plot of inactivation (killing) rates of V-79 Chinese hamster cells exposed to hypothermia in air-equilibrated (aerobic) medium contains a break at about 8 degrees C, which corresponds to the minimum inactivation rate, implying that there are distinct hypothermic damage mechanisms above (range I, 8 to 25 degrees C) and below (range II, 0 to 8 degrees C) 8 degrees C. Prehypothermic hypoxia (PHH) for 75 min at room temperature sensitizes cells to subsequent aerobic hypothermia at both 5 and 10 degrees C (range II and I). However, PHH followed by severe hypoxia (0.03 microM oxygen in the medium) protected cells during 10 degrees C (range I) storage by increasing the shoulder, but not the slope, of the cell survival curve compared to the PHH plus 10 degrees C aerobic hypothermia case. On the other hand, PHH plus severe hypoxia during 5 degrees C storage (range II) protected cells by decreasing the slope, but not the shoulder, of the cell survival curve compared to the PHH plus 5 degrees C aerobic hypothermia case. Furthermore, PHH plus severe hypoxia during 5 degrees C storage was not significantly worse than aerobic storage without PHH at 5 degrees C. With or without severe hypoxia, 10 degrees C storage is preferable to 5 degrees C storage in this cell line. Extrapolated to organ storage, the results may imply that if warm ischemia (PHH) has occurred, subsequent hypoxic hypothermic perfusion storage may be preferable to aerobic hypothermic perfusion storage.

Induction of tolerance to freeze-thaw (FT) damage in mammalian cells by pre-FT hypothermia treatment.

Attached asynchronous exponential phase V79 Chinese hamster cells were pretreated by hypothermic cycling in Hepes growth medium by Method 3 (48 h at 25 degrees C + trypsin) or Method 4 (48 h at 25 degrees C + 3 h at 37 degrees C + trypsin) prior to a freeze-thaw (FT) cycle in Hepes growth medium. Pretreatment by Method 3 or 4 increased the FT survival by a factor of 3.4. This implies that the 3 h at 37 degrees C, after the 25 degrees C exposure, is not necessary to confer resistance to the subsequent FT cycle in the case of V79 cells. However, with RIF-1 mouse cells, the 3 h at 37 degrees C confers increased resistance. The increase in FT survival of V79 cells after the above pretreatments cannot be accounted for by changes in cell cycle age distribution. No heat shock proteins are produced by this pretreatment. Since pretreatment by Method 3 or 4 also makes the cells resistant to hyperthermia, three other pretreatments, making the cells thermotolerant, were tried. None of these pretreatments resulted in a change in FT survival of the cells. Interaction analysis of FT data, when pretreatment by Method 4 is combined with the presence of DMSO during the FT cycle, indicates that the pretreatment and DMSO act synergistically whether exponential or stationary phase cells are used. Furthermore, the pretreatments and L-glutamine also act synergistically. These pretreatments also increase the FT survival of the RIF-1 mouse cell line; again, pretreatment and DMSO act synergistically. Hence, the method is not limited to cells of hibernating mammals.

Induction of tolerance to hypothermia and hyperthermia by a common mechanism in mammalian cells.

Pretreatment by hypothermic (25 degrees C) cycling (PHC) of attached exponential-phase V79 Chinese hamster cells by Method 4 (24 hr at 25 degrees C + 1.5 hr at 37 degrees C + 24 hr at 25 degrees C + trypsin + 3 hr at 37 degrees C) or by Method 3 (48 hr at 25 degrees C + trypsin + 3 hr at 37 degrees C) make mammalian V79 cells significantly more resistant to 43 degrees C hyperthermia. There is no significant difference in the 43 degrees C curves whether Method 3 or 4 is used for pre-exposure. If pre-exposure at 15 or 10 degrees C, the resistance to hyperthermia is significantly reduced. PHC by Method 4 significantly increases survival of cells exposed to 5 degrees C and, to a lesser extent, to 10 degrees C. The increase in hyper- and hypothermic survival after PHC cannot be accounted for by changes in cell cycle distribution. Heat-shock protein synthesis is not induced by PHC; hence, protection does not result from newly synthesized proteins. When cells are made tolerant to hyperthermia by a pretreatment in 2% DMSO for 24 hr at 37 degrees C (Method 8), the cells are not more resistant to subsequent exposures to hypothermia, either at 5 or 10 degrees C. The results imply that there may be two mechanisms of inducing resistance to hyperthermia, only one of which also confers resistance to hypothermia.

Further evidence for two modes of hypothermia damage.

The Arrhenius plot of inactivation (killing) rates of V-79 Chinese hamster cells exposed to hypothermia contains a break at about 8 degrees C, which corresponds to the minimum inactivation rate, implying that there are distinct hypothermic damage mechanisms above (Range I = 8 to +25 degrees C) and below (Range II = 0 to +8 degrees C) 8 degrees C. Several membrane-permeable hydroxyl free radical scavengers, N-acetylhomocysteinethiolactone (citiolone), dimethylthiourea (DMTU), and dimethyl sulfoxide (DMSO), were tested for their ability to protect cells exposed to hypothermic temperatures of 10 degrees C (Range I) or 5 degrees C (Range II) as a function of time in a system that is uncomplicated by previous hypoxia. Citiolone (3 mM) protected cells in Range I, but not in Range II. To date, citiolone is the only agent that protects in Range I. Adenosine was of no benefit in Range I. Glycine (5 mM) protected cells in Range II, but not in Range I. DMSO (10 mM) was ineffective in Range II, while DMTU (10 mM) protected cells in Range II, but not in Range I. The combination of DMTU and citiolone had a synergistic protective effect on the cells during 10 degrees C exposure (Range I). However, the combination of DMTU and citiolone is neither synergistic nor additive at 5 degrees C (Range II).

Influence of transition rates and scan rate on kinetic simulations of differential scanning calorimetry profiles of reversible and irreversible protein denaturation.

The thermodynamic parameters characterizing protein folding can be obtained directly using differential scanning calorimetry (DSC). They are meaningful only for reversible unfolding at equilibrium, which holds for small globular proteins; however, the unfolding or denaturation of most large, multidomain or multisubunit proteins is either partially or totally irreversible. The simplest kinetic model describing partially irreversible denaturation requires three states: Formula [see text] We obtain numerical solutions for N, U, and D as a function of temperature for this model and derive profiles of excess specific heat (Cp) in terms of the reduced variables v/ki and k1/k3, where v is the scan rate. The three-state model reduces to the two-state reversible or irreversible models for very large or very small values of k1/k3, respectively. The apparent transition temperature (Tapp) is always reduced by the irreversible step (U-->D). For all values of k3, Tapp is independent of v/k1 at sufficiently slow scan rates, even when denaturation is highly irreversible, but increases identically for all models at fast scan rates in which case the excess specific heat profile is determined by the rate of unfolding. Accurate values of delta H and delta S can be obtained for the reversible step only when k1 is more than 2000-50,000 times greater than k3. In principle, approximate values for the ratio k1/k3 can be obtained from plots of fraction unfolded vs fraction irreversibly denatured as a function of temperature; however, the fraction irreversibly denatured is difficult to measure accurately by DSC alone.(ABSTRACT TRUNCATED AT 250 WORDS)

Protective effects of amino acids against freeze-thaw damage in mammalian cells.

Amino acids were tested for their effectiveness as cryoprotectants. From the results of this study, the mean fractional area loss of amino acid residues upon incorporation in globular proteins, a measure of hydrophobicity, is generally inversely proportional to the freeze-thaw protection by these free amino acids. However, the pattern of protection ("fingerprint") of cells by various amino acids is different from that of the enzymes liver alcohol dehydrogenase and calcium ATPase of the sarcoplasmic reticulum. Furthermore, unlike the case with these enzymes, for cells glutamine is the best cryoprotective agent of the amino acids tested.

Site of freeze-thaw damage and cryoprotection by amino acids of the calcium ATPase of sarcoplasmic reticulum.

The Ca2+,Mg(2+)-ATPase of skeletal muscle sarcoplasmic reticulum (SR) is irreversibly inactivated by a freeze-thaw (FT) cycle. The membrane does not become more permeable to calcium after a FT cycle, suggesting that the reduced uptake is due to damage to the Ca2+,Mg(2+)-ATPase. Several amino acids, in addition to standard cryoprotectants provide good protection of calcium uptake against FT damage. The amount of protection given by the amino acids is generally inversely proportional to a measure of hydrophobicity, the mean fractional area loss upon incorporation in globular proteins of the amino acid side chain. Unlike the case for cells, glutamine and dimethyl sulfoxide do not act independently as cryoprotectants for SR calcium ATPase. When the protein is exposed to multiple FT cycles, the amount of inactivation is exponentially proportional to the number of FT cycles. This is true for both protected and unprotected samples. Some SR vesicles fuse during FT. Fusion of vesicles cannot account for the observed inactivation of the enzyme. Fluorescence studies, using intrinsic tryptophan and extrinsic FITC and NCD-4, suggest that FT does not damage the transmembrane region of the Ca2+,Mg(2+)-ATPase or the calcium binding sites, but only the mechanism coupling ATPase activity to calcium translocation. Differential scanning calorimetry (DSC) studies suggest that this region comprises less than 15% of the whole enzyme.

Factors influencing survival of mammalian cells exposed to hypothermia. V. Effects of hepes, free radicals, and H2O2 under light and dark conditions.

Cytotoxicity resulting from the interaction of fluorescent light from a flow hood with Hepes-buffered cell culture medium at room temperature was demonstrated. Toxicity was prevented by keeping both cells (V79 Chinese hamster) and medium shielded from direct fluorescent light ("dark conditions") or by supplementing the medium with 10 micrograms/ml catalase; this suggests that extracellular hydrogen peroxide is a major cause of the lethal effect under "lighted conditions." No sensitization resulted from the exposure of cells in a sodium bicarbonate (SBC)-buffered medium to fluorescent light, nor in a catalase supplemented SBC-buffered medium. The Hepes/light reaction during routine cell manipulations presensitized cells to hypothermia damage in the dark with the presensitization being more severe for 5 than for 10 degrees C hypothermic exposure. Presensitization was prevented by performing the complete experiment under dark conditions or by supplementing the medium with 10 micrograms/ml catalase. However, catalase did not improve the hypothermic survival when experiments were performed under dark conditions. Hence, 10 micrograms/ml catalase does not protect cells from hypothermic (5 and 10 degrees C) damage per se, but rather from Hepes/light sublethal damage which interacts with hypothermic sublethal damage to result in lethal lesions. Additionally, under dark conditions, superoxide dismutase (SOD), allopurinol, catalase plus SOD, DMSO, or mannitol did not improve survival when present during hypothermic storage, suggesting that extracellular superoxide anion, hydrogen peroxide, or hydroxyl radicals are not the cause of cell killing under conditions of pure hypothermia uncomplicated by prehypothermic ischemia or hypoxia.

Mechanism of freeze-thaw damage to liver alcohol dehydrogenase and protection by cryoprotectants and amino acids.

Multiple freeze-thaw (FT) cycles, with complete melting between cycles, resulted in an exponential decline in liver alcohol dehydrogenase (LADH) enzyme activity. The reduction in activity of LADH as a result of FT damage was proportional to the decrease in the intensity of the tryptophan fluorescence of the enzyme. Treatment with urea resulted in a similar relationship between tryptophan fluorescence intensity and inactivation. Evidence from fluorescence and activity studies from the same sample, as well as gel electrophoresis, indicates that damage to LADH from a FT cycle, resulting in inactivation, is likely an unfolding of the enzyme rather than separation of subunits or aggregation of enzymes at the enzyme concentrations and cooling rates used. A nonexponential decline in enzyme activity, as a function of the number of FT cycles, can be achieved if complete melting between cycles is not allowed or if the samples are stored at +4 degrees C for 24 hr following the last FT cycle, prior to assay. In the latter case, a partial recovery in enzyme activity is seen. "Seeding," while lowering the enzyme activity, is desirable to achieve consistent results without the artifacts that are introduced if not used. Amino acids were tested for their effectiveness as cryoprotectants. From the results of this study, the mean fractional area loss of amino acid residues upon incorporation in globular proteins (f) is inversely proportional to the FT protection by these free amino acids. Thus, amino acid residues which tend to be found at the surface of proteins (e.g., glutamate) improve the FT survival of LADH, when added as the free amino acid, while those amino acids which are found in the interior of proteins (e.g., valine, leucine) sensitize LADH to FT damage. The pattern of protection ("fingerprint") of LADH by various amino acids is different from that of living cells. Furthermore, unlike the case with cells, glutamine and DMSO do not act independently when protecting LADH.

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.

Increased thermostability of thermotolerant CHL V79 cells as determined by differential scanning calorimetry.

Heat shock denatures cellular protein and induces both a state of acquired thermotolerance, defined as resistance to a subsequent heat shock, and the synthesis of a category of proteins referred to as heat-shock proteins (HSPs). Thermotolerance may be due to the stabilization of thermolabile proteins that would ordinarily denature during heat shock, either by HSPs or some other factors. We show by differential scanning calorimetry (DSC) that mild heat shock irreversibly denatures a small fraction of Chinese hamster lung V79-WNRE cell protein (i.e., the enthalpy change, which is proportional to denaturation, on scanning to 45 degrees C at 1 degree C/min is approximately 2.3% of the total calorimetric enthalpy). Thermostability, defined by the extent of denaturation during heat shock and determined from DSC scans of whole cells, increases as the V79 cells become thermotolerant. Cellular stabilization appears to be due to an increase in the denaturation temperature of the most thermolabile proteins; there is no increase in the denaturation temperatures of the most thermally resistant proteins, i.e., those denaturing above 65 degrees C. Cellular stabilization is also observed in the presence of glycerol, which is known to increase resistance to heat shock and to stabilize proteins in vitro. A model is presented, based on a direct relationship between the extent of hyperthermic killing and the denaturation or inactivation of a critical target that defines the rate-limiting step in killing, which predicts a transition temperature (Tm) of the critical target for control V79-WNRE cells of 46.0 degrees C and a Tm of 47.3 degrees C for thermotolerant cells. This shift of 1.3 degrees C is consistent with the degree of stabilization detected by DSC.

Statistical methods for measuring interactions between protectors and sensitizers for freeze-thaw survival data.

A statistical model is developed to generate the survival probabilities of cells subjected to freeze-thaw treatments using various sensitizing and/or protective agents. The purpose is to determine whether different freeze-thaw protective agents act independently or whether there is an interaction between the agents. The model permits a statistical analysis of the data to yield an objective quantitative assessment of the size and nature of the interaction.

Protein degradation in CHL V79 cells during and after exposure to 43 degrees C.

Cellular protein degradation during and following hyperthermia should be altered due to increased enzymatic activity at elevated temperatures, inhibition of protein synthesis, and denaturation of proteins. We have previously demonstrated by differential scanning calorimetry that approximately 1-2% of total CHL V79 cellular protein denatures during a 10- to 15-min exposure to 43 degrees C (J. E. Lepock et al., J. Cell. Physiol. 137, 14-24 (1988)). Proteolysis was measured during and after exposure to 43 degrees C. The decay curves of the degradation of [3H]Leu-labeled proteins are fit well by a double exponential; however, each component is the sum of the decay curves of a large number of proteins, probably with a distribution of rates of degradation. At 37 degrees C a fast-decaying component (T1/2 congruent to 1.3 h), representing short-term proteins, and a slow-decaying component (T1/2 congruent to 50 h), representing long-term proteins, are observed. At 43 degrees C the rate of degradation of the fast-decaying component is stimulated three- to fivefold (to T1/2 = 0.27-0.45 h). After return to 37 degrees C, the rate of degradation of the slow-decaying component is depressed twofold (to T1/2 = 109-141 h). The period of depression is dose dependent (i.e., time at 43 degrees C) and recovers at approximately the same time as resumption of protein synthesis and growth. Overall stimulation of degradation lasts for approximately 15 min at 43 degrees C and, coupled with an inhibition of synthesis, leads to the loss of at least a small percentage of total cellular protein. It is likely that the initial stimulated degradation is in part due to increased substrate in the form of denatured protein, further supporting the denaturation of proteins during hyperthermia.

Thermal analysis of CHL V79 cells using differential scanning calorimetry: implications for hyperthermic cell killing and the heat shock response.

Differential scanning calorimetry (DSC) was used to assay thermal transitions that might be responsible for cell death and other responses to hyperthermia or heat shock, such as induction of heat shock proteins (HSP), in whole Chinese hamster lung V79 cells. Seven distinct peaks, six of which are irreversible, with transition temperatures from 49.5 degrees C to 98.9 degrees C are detectable. These primarily represent protein denaturation with minor contributions from DNA and RNA melting. The onset temperature of denaturation, 38.7 degrees C, is shifted to higher temperatures by prior heat shock at 43 degrees and 45 degrees C, indicative of irreversible denaturation occurring at these temperatures. Thus, using DSC it is possible to demonstrate significant denaturation in a mammalian cell line at temperatures and times of exposure sufficient to induce hyperthermic damage and HSP synthesis. A model was developed based on the assumption that the rate limiting step of hyperthermic cell killing is the denaturation of a critical target. A transition temperature of 46.3 degrees C is predicted for the critical target in V79 cells. No distinct transition is detectable by DSC at this temperature, implying that the critical target comprises a small fraction of total denaturable material. The short chain alcohols methanol, ethanol, isopropanol, and t-butanol are known hyperthermic sensitizers and ethanol is an inducer of HSP synthesis. These compounds non-specifically lower the denaturation temperature of cellular protein. Glycerol, a hyperthermic protector, non-specifically raises the denaturation temperature for proteins denaturing below 60 degrees C. Thus, there is a correlation between the effect of these compounds on protein denaturation in vivo and their effect on cellular sensitivity to hyperthermia.

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 Eagle's basal medium is sufficient to account for cryoprotection by this medium.

Hyperthermia-induced inhibition of respiration and mitochondrial protein denaturation in CHL cells.

Respiration of Chinese hamster lung V79 cells, as assayed by O2 consumption, increases linearly from 8 to 40 degrees C when plotted in the Arrhenius fashion but is strongly inhibited above 40 degrees C. The protein of mitochondria isolated from V79 cells undergoes structural transitions at 28 and 40 degrees C. This is supported by changes in the fluorescence excitation spectrum of conjugated pyrene maleimide and, to a lesser extent, intrinsic protein fluorophores. Electron spin resonance labelling studies with a derivative of tempo maleimide imply that extensive protein unfolding coincides with the 40 degrees C transition. The structural transition at 40 degrees C correlates well with inhibition of O2 consumption, is irreversible and is probably due to protein denaturation, while the change at 28 degrees C is reversible and has no effect on O2 consumption. Previous studies indicate the presence of a broad lipid transition extending from approximately 8 to 30 degrees C in mitochondrial membranes with all lipids being in the liquid-crystalline state above 30 degrees C. Thus, the onset of the lipid transition may induce the observed protein conformational change at 28 degrees C, but inhibition of respiration above 40 degrees C can be explained by protein denaturation alone. The region from 28 to 40 degrees C of stable protein conformation corresponds to the temperature range of V79 cell growth.

Sensitization of rat hepatocytes to hyperthermia by calcium.

The viability of isolated rat hepatocytes, as assayed by trypan blue exclusion, decreases in a dose-dependent fashion during exposure to hyperthermia (D0 [43 degrees C] = 105 +/- 10 min, D0 [45 degrees C] = 24 +/- 4 min). Hyperthermic sensitivity varies as a function of extracellular Ca2+ concentration in a biphasic manner; optimum survival occurs at 1-5 mM Ca2+, with sensitization in the absence of Ca+ and increasing sensitization at Ca2+ concentrations greater than 10 mM. Ca influx does not correlate well with loss of viability for hepatocytes in 4 mM extracellular Ca2+; influx does not occur until viability decreases to less than 1%. Under sensitizing conditions, Ca2+ influx proceeds loss of viability. Influx begins within 15 min at 45 degrees C in 15 mM Ca2+, and the ionophore A23187 is a potent hyperthermic sensitizer in the presence of extracellular Ca2+. Thus, Ca2+ influx, whether caused by high extracellular Ca2+ or A23187, increases cellular damage caused by supraoptimal temperatures, although some Ca2+ is necessary for maximum resistance, probably because of stabilization of Ca2+ binding proteins against thermal denaturation or possibly to Ca2+-induced decrease in lipid fluidity.

Effects of butylated hydroxytoluene on membrane lipid fluidity and freeze-thaw survival in mammalian cells.

Butylated hydroxytoluene (BHT) increases the fluidity of membrane lipids in the hydrocarbon but not the polar regions, as measured by electron spin resonance spin label probes. BHT also sensitizes nucleated mammalian cells to freeze-thaw damage as measured by colony formation survival assays. Furthermore, the membranes of BHT-exposed cells are more susceptible to physical stress, as reflected by the BHT-induced sensitization to hypotonic stress. Since others have shown that BHT induces hexagonal phase lipids in lipid bilayers, this phenomenon may also influence the above survival results.