M.J. Ashwood-Smith

Permeability of intact and dechorionated zebra fish embryos to glycerol and dimethyl sulfoxide

Intact developing embryos of the zebra fish Brachydanio rerio were exposed to [14C]DMSO and [3H]glycerol (1 M in Fish Ringer) to assess the degree of permeation of these cryoprotectants. Glycerol enters the embryo more easily, although reaching only about 8% of the expected equilibrium level after 2 hr at room temperature; DMSO reaches only about 2.5% of this level. In an attempt to identify the barrier to permeation, dechorionated embryos were similarly exposed to isotopic DMSO. Permeation increased severalfold, indicating that the chorion retards the free exchange of solute. Embryos are unaffected by exposure to 1 M DMSO in Fish Ringer at 23 degrees C for up to 1 hr. The number of embryos hatching after 1-hr exposure to DMSO at varying concentrations was significantly reduced at 1.5 and 2 M. Embryos exposed to 1 M glycerol for 1 hr at 23 degrees C showed disruption of periblast cells and separation of the blastoderm; it was impossible to remove glycerol either by abrupt or very slow dilution.

Cryoprotectant penetration and supercooling in the eggs of salmonid fishes

Abstract 1. 1. The freezing point of isolated, unfertilized eggs of rainbow trout ( Salmo gairdneri ) is −1.7 °C. 2. 2. Experiments using radiolabeled cryoprotectants show that the degree of permeation of methanol, dimethyl sulfoxide, and glycerol is inversely proportional to the molecular weights of the compounds; glycerol does not penetrate the egg, while methanol, which penetrates with the greatest rapidity, achieves no more than 23% of the expected equilibrium concentration after 2 hr exposure at 0 °C. 3. 3. Eggs treated with 10% DMSO and 10% sucrose in Fish Ringer resist intracellular freezing longer when cooled at 0.01 °C/min than do those in Fish Ringer alone; intracellular freezing is, however, inevitable with both cryoprotectants, although eggs that have remained unfrozen for several hours appear viable upon slow rewarming. These results make it likely that earlier studies reporting survival of “cryopreserved eggs” have in fact been dealing with supercooled eggs. 4. 4. Scanning electron micrographs of cooled and frozen DMSO-treated eggs show a progressive deterioration of the outermost layer of the zona radiata following cooling and freezing, and suggest that intracellular freezing is occurring following nucleation by way of pore canals in the zona that are exposed as cooling progresses.

Photoactive furocoumarins in fruits of some umbellifers

Abstract Fruits of Anethum graveolens, Angelica archangelica, Anthriscus cerefolium, Apium graveolens, Carum carvi, Coriandrum sativum, Cuminum cyminum, Daucus carota, Foeniculum vulgare, Heracleum sphondylium, Levisticum officinale, Pastinaca sativa, Petroselinum crispum and Pimpinella anisum were analysed for photoactive furocoumarins using HPLC analysis and photobiological bioassay. These sensitive methods revealed furocoumarins not previously detected.

Conversion of psoralen DNA monoadducts in E. coli to interstrand DNA cross links by near UV light (320-360 nm): inability of angelicin to form cross links, in vivo.

Both angelicin and psoralen monoadducts formed in vivo in E. coli by near UV light produce lethal and mutagenic effects. However psoralen monoadducts are converted to cross links by higher doses of UV; angelicin monoadducts are not. The relevance of these results to psoralen photosensitization is discussed.

Mutation induction in bacteria by freeze-drying.

The stability of the deoxyribonucleic acid (DNA) double helix to freezing and thawing both in vitro and in vivo has been well documented. Shikama (13), using thermal denaturation techniques, showed that purified DNA solutions were not affected by freeze-thaw procedures. Ashwood-Smith ( 1) presented evidence of the genetic stability of bacteria frozen and thawed under several different conditions. No mutation induction in Escherichiu coli was evident and no change in a wide spectrum of antibiotic resistance could be demonstrated after a long series of freeze-thaw selection procedures. Swartz (14), however, has published evidence that freezing and thawing ‘of E. coli produced single-strand breaks in DNA in vivo, but this has not been confirmed by other workers ( 1). The best evidence at this time, therefore, is that freezethaw procedures, per se, do not produce genetic damage, and this statement is substantiated by evidence presented in this paper. Genetic changes in bacteria induced by freeze-drying have not been reported, although Webb (16) has published data on the genetic effects of exposing E. co& in an aerosol to different degrees of humidity. The highest number of mutants was observed when the relative humidity of the aerosol was approximately 40%. Bridges (personal communication) has indicated that stocks of freeze-dried bacterial preparations have higher background reversion

Studies on the molecular weight and cryoprotective properties of polyvinylpyrrolidone and dextran with bacteria and erythrocytes

The distribution of molecules in various commercial samples of polyvinylpyrrolidone was studied by gel-exclusion chromatography on Sephadex columns. High-speed centrifugation on sucrose gradients was also used in this study. Unequivocal evidence is presented that the molecular weight spread in Plasdone C (PVP, K30) is much wider than the manufacturers published data. A continuous distribution of molecules associated with Plasdone C from about 1,000 to 200,000 daltons was demonstrated. This material is claimed to have an average molecular weight of 40,000 daltons with a range of 20,000–80,000 daltons. Radioactive 14C PVP gave essentially similar results. Estimates based on the elution pattern of bovine serum albumin (67,000 daltons) suggest that about 50% of the molecules in Plasdone C are larger than 67,000 daltons, and dialysis experiments indicate that about 12% of the molecules are less than about 12,000 daltons. Undialyzed PVP, not toxic per se, was toxic to Pseudomonas when this bacterium was frozen and thawed in its presence. Dialyzed PVP, however, gave good protection against freeze-thaw damage. Ultraviolet absorption spectra of PVP gave maxima at approximately 203 nm unaffected by dialysis or changes in pH from 4.0 to 7.0. Standard curves are published for the estimation of pure PVP samples by either uv spectroscopy or the iodine color reaction. Dextran samples of characterized molecular weight from 10,000 to 500,000 daltons were investigated for freeze-thaw protection with rabbit erythrocytes and Pseudomonas. Dialysis had no effect on cryoactivity. Higher concentrations of dextran were better than lower concentrations in both cell systems. With erythrocytes the influence of molecular weight on cryoprotection was negligible. With bacteria, however, there was a trend toward greater cryoprotective activity with higher molecular weight.

Low-temperature preservation of mammalian cells in tissue culture with polyvinylpyrrolidone (PVP), dextrans, and hydroxyethyl starch (HES)☆

Abstract The effects of freezing and thawing on Chinese hamster cells in tissue culture in the presence of PVP, HES, and various dextrans have been investigated. Cooling and thawing rates within a limited range (20–260 °C per min for cooling and 6–115 °C per min for thawing) were studied and best results were achieved with a cooling rate of 20 °C per min and a thawing rate of 115 °C per min in both 10% PVP, and 10% HES. Experiments demonstrated HES to be as good as, and possibly better than PVP. A number of dextrans of average molecular weight (10,000–500,000 daltons) were shown to be poor as cryoprotective agents in contrast to results obtained with this polymer with red cells and bacteria. The presence of 10% serum during all freezing and thawing procedures decreased day-to-day variability and with dextrans increased their limited effectiveness. Pinocytosis, as a mechanism by which polymers act to prevent freeze-thaw damage, is unlikely.

Cryoprotection of mammalian cells in tissue culture with polymers; possible mechanisms.

Summary Studies were undertaken to more clearly define the mechanism of cryoprotection by polymers. Significant cryoprotection of Chinese hamster cells in tissue culture was found in the presence of hydroxyethyl starch (HES), polyvinylpyrrolidone (PVP), and dextran. The addition of PVP to the medium after thawing did not increase the survival of these cells. The presence of PVP in the medium was shown to have no effect on the transport mechanism for alanine in unfrozen cells. The source of freeze-thaw injury did not appear to be due to a direct effect on this transport mechanism. Several physical parameters of polymeric solutions were monitored at subzero temperatures. The freezing point depression was found to increase dramatically at higher polymer concentrations. Tests on the NaCl concentration in the liquid fraction of partially frozen solutions showed that the increase in salt concentration with decreasing temperature was similar in the presence of 10% PVP or 2.5% DMSO, two agents which gave similar cryoprotection at these concentrations. NMR studies showed that polymers could retain water in the liquid state at temperatures as low as −35° C, and that the remaining water was highly structured. The cryoprotective properties of polymers appear to reside in their ability to alter the physical properties of solutions during the freezing process rather than in direct effects on cell membranes.

Furocoumarins in the cultivated carrot, Daucus carota

Abstract A combination of ultrasensitive bioassay and HPLC analysis was employed to analyse the garden carrot for furocoumarins. The earlier reported negative findings were due to the relatively insensitive methods of detection. Low levels of two furocoumarins, 8-MOP and 5-MOP were detected in all parts of fresh carrot plants for the first time. Occasionally one or two other unknown photoactive compounds were also present. Diseased carrot displayed increased levels of 8-MOP and 5-MOP; psoralen was also detected. In this respect carrot is capable of a phytoalexin response similar to celery.

Lethal and chromosomal effects of freezing, thawing, storage time, and x-irradiation on mammalian cells preserved at −196 ° in dimethyl sulfoxide

Abstract 1. 1. Tissue culture cells preserved at −196 °C in 10% glycerol or 10% PVP for periods of up to 6 months showed no gross chromosomal aberrations as disclosed by the micronucleus test. In one experiment cells preserved in 10% DMSO, which offered the best protection (91% survival after freezing and thawing), showed a statistically ( P = 0.05) greater number of micronuclei 20 hr after thawing. This was a transient effect and three more separate experiments failed to demonstrate any chromosomal damage associated with freezing, thawing and storage at −196 °C for periods of up to 2 years in 10% DMSO. 2. 2. Both the lethal and chromosomal effects of x-irradiating cells in 10% DMSO at −196 °C were reduced by a factor of approximately 3.5 compared to irradiation at +22 °C. 3. 3. It is estimated that a period of approximately 30,000 years of storage of cells in 10% DMSO at −196 °C would pass before an accumulated x-ray dose from background radiation would reach a level, which upon cellular resurrection, would result in the equivalent lethal and chromosomal damage of an accute D 10 dose. 4. 4. Cells cannot be kept at −196 °C ad infinitum as there is no escape from background radiation damage. Shielding would reduce the level but internal radiation from isotopic disintegration is inescapable.