Effects of four cryoprotectants in combination with two vehicle solutions on cultured vascular endothelial cells.

The necessary first step in successful organ cryopreservation will be the maintenance of endothelial cell integrity during perfusion of high concentrations of cryoprotective agents (CPAs). In this report we compare the effects of incubation on cultured porcine endothelial cells at 10 degrees C for 1 h with the CPAs glycerol, dimethyl sulfoxide (Me2SO), ethanediol (EG), and propane-1,2-diol (PG) in the vehicle solutions RPS-2 (high potassium, high glucose) and HP-5NP (low potassium, high sodium), both with and without added colloids. Tritiated adenine uptake and acid phosphatase estimation of cell number were used as indicators of cell viability. HP-5NP was superior to RPS-2 except with Me2SO when the differences in viability were not significant. Adding Haemaccel to HP-5NP improved the results, but adding albumin to RPS-2 was of no significant benefit. Osmotic stress appeared to be the major problem with glycerols use. Beyond 3.0 M the toxicity of Me2SO increased dramatically but it could not be determined if this was osmotic or chemical toxicity. PG was remarkably well tolerated to 3.0 M but a sharp decrease in cell viability beyond this concentration suggests that PG may be most useful with mixtures of other CPAs. Overall, EG appeared to be the least toxic CPA and in the context of vascular preservation warrants further investigation.

Vascular changes following hypothermic preservation.

This study identifies ultrastructural changes in the endothelium due to hypothermic HP-5 perfusion. These changes appear to be reversible and a manifestation of hypothermic hypoxia. This data provides a basis for improving perfusion techniques in both hypothermic preservation and cryoprotectant introduction and removal for cryopreservation.

An isolated perfused rat mesentery model for direct observation of the vasculature during cryopreservation.

An intact vasculature is essential for successful hypothermic perfusion and cryopreservation of solid organs, but few studies have specifically assessed the vascular effects of these procedures. A technique was therefore developed for continuous, direct observation of an isolated vascular bed during hypothermic perfusion with cryoprotectants, and during freezing and thawing. The isolated rat mesentery was spread across a controlled low temperature microscope stage and perfused with solutions containing fluorescein isothiocyanate (FITC)-Dextran 70 as an indicator of macromolecular permeability of the vessels. Hypertonic citrate washout, HP-5 perfusion (23), rapid and slow addition and removal of glycerol, and freezing/thawing were studied. Control perfusion with HP-5 produced slow FITC-Dextran leakage, reflecting normal physiological macromolecular permeability of vessels. Rapid addition of glycerol dramatically increased vascular permeability, consistent with osmotic damage to vessels. Rapid removal stopped flow through capillaries and decreased vascular dimensions, suggesting overhydration of endothelial cells and extravascular tissue. Venules and capillaries were the most susceptible vessels to osmotic stress. Slow addition and removal of glycerol (80 mmol/liter/min) produced results similar to control perfusions. During slow freezing (0.5 degree C/min to -5 degrees C) extravascular ice compressing vessels was more obvious than intravascular ice. Glycerol afforded some protection to the microvasculature during freeze/thaw cycles since flow was reestablished in venules and arterioles after thawing, although FITC-Dextran leakage indicated that damage had occurred.

Rat hepatic microsomal enzyme induction by pretreatment with toxaphene and toxaphene fractions.

The levels of hepatic microsomal induction caused by toxaphene were determined. Young Sprague-Dawley rats (70 g) were administered toxaphene (ip injection, daily for 5 d) at 0, 5, 25, and 100 mg/kg. All doses caused increases in liver/body weight ratio, cytochrome P-450 level, aminopyrine demethylation, and aldrin epoxidation. The aldrin epoxidase activity increased almost 700% at the 100-mg/kg dose. Toxaphene was separated into nonpolar (S-A) and polar (S-B) fractions and administered as before at 25 mg/kg. All treatments caused significant increases in cytochrome P-450, aminopyrine demethylation, and aldrin epoxidation. A comparison of the treatments, however, did not reveal any significant differences between the treatments.

Effect of toxaphene exposure on immune responses in mice.

Toxaphene was fed to female weanling Swiss-Webster mice at dosages of 10, 100, and 200 ppm for 8 wk. Immunologic assays revealed depressed IgG antibody formation in those animals receiving 100 and 200 ppm toxaphene, as compared to controls. Cell-mediated immune responses were not affected in the toxaphene-exposed mice. In another experiment, mature female mice fed the same amounts of toxaphene were mated 3 wk after feeding began and were maintained on the diets until 3 wk after parturition, at which time the pups were weaned onto the control ration. Assays performed on the offspring 8 wk after their birth revealed suppressed antibody formation in the 100-ppm-toxaphene group and enhanced antibody formation in the 200-ppm group. The cell-mediated immune response was suppressed in the offspring from the 100-ppm group, while no change from the controls occurred in the other groups. Phagocytic ability of macrophages was significantly reduced in all toxaphene-treated groups, but to a greater extent in the offspring of the mice that consumed 100 ppm toxaphene.

The subchronic toxicity and teratogenicity of alternariol monomethyl ether produced by Alternaria solani.

Alternariol monomethyl ether (AME), a metabolite of species of Alternaria, was studied for subchronic toxicity to rats and teratogenic effects in golden hamsters. In rats no toxic effects were observed at the highest dosage tested (3.75 mg given by oral gavage daily for 30 days). However, AME was maternally toxic and foetotoxic to Syrian golden hamsters when given ip at 200 mg/kg body weight on day 8 of gestation. No such effects were observed at dose levels of 50 or 100 mg/kg.

The elimination, distribution, and metabolism of 14C-toxaphene in the pregnant rat.

Pregnant Sprague-Dawley rats were orally administered 14C-toxaphene in olive oil on day 15 of pregnancy and housed in glass metabolism cages. Urine, feces, and tissues were collected and assayed for radioactivity. The elimination was similar to that in virgin females with the majority of activity excreted in the feces (38.4%; five days) and less in the urine (23.7%; five days). The fetuses contained the lowest levels of radioactivity of all tissues tested (28 ppb; five days) and fat contained the highest levels (7476 ppb; five days). A comparison of the activity in the fetuses with that in the dam's fat showed slight differences, indicating the presence of more polar compounds (perhaps metabolites).

The distribution, elimination, and metabolism of 14C-alternariol monomethyl ether.

Adult male Sprague-Dawley rats were orally administered 14C-alternariol monomethyl ether (AME) in olive oil and housed in glass metabolism cages. Urine, feces, and expired CO2 were monitored for the presence of radioactivity. The majority of activity was excreted in the feces apparently unabsorbed. Urine excretion accounted for less than 10% of the administered dose. A substantial amount of "absorbed" activity was excreted in expired CO2. TLC autoradiography of urine extracts revealed extensive metabolism, but little metabolism was evident in feces extracts. Incubation of AME with rat liver post-mitochondrial supernatant also resulted in substantial metabolism.

Toxicities and description of some toxaphene fractions: isolation and identification of a highly toxic component.

Toxaphene was separated into 13 fractions and the toxicity of each fraction was determined. The acute toxicities (LD50) to houseflies (topical in acetone) ranged from 21 to greater than 246 mg/kg (toxaphene LD50 = 33 mg/kg) and the relative toxicities ranged from 0.6 to greater than 7.5. A similar pattern was found in mice when the toxicities of several fractions were determined. The acute toxicities (LD50, ip injection in dimethyl sulfoxide) in mice ranged from 20 to 67 mg/kg (toxaphene LD50 = 33 mg/kg) for the fractions tested. The most toxic fraction was further separated into six subfractions and their toxicities (housefly LD50) were found to range from 10 to 74 mg/kg. The most toxic subfraction appeared to be an almost pure compound and was purified for further identification. It was found to be identical to a previously reported highly toxic C10H10Cl8 mixture of predominantly two components. The components were reported to be 2,2,5-endo,6-exo, 8,8,9,10-octachlorobornane and 2,2,5-endo,6-exo,8,9,9,10-octachlorobornane. This highly toxic mixture has now been isolated independently by three research teams using different separation schemes.

Excretion and storage of [14C]toxaphene and two isolated [14C]toxaphene fractions.

The 7-d urinary and fecal excretions of [14C]toxaphene and two isolated [14C]toxaphene fractions (polar fraction 7 and nonpolar fraction 2) were determined in orally dosed rats. The urinary, fecal, and total excretions of toxaphene were, respectively, 22.5, 35.7, and 58.2% of the administered dose. The total excretions of fractions 2 and 7 were, respectively, 69.4 and 65.0%, and the overall order of excretion was fraction 2 greater than toxaphene greater than fraction 7. All three groups had low toxaphene levels (below 0.2 ppm) in all tissues analyzed except for fat, where significant levels were detected. Hexane and chloroform extracts of the urine revealed that the activity was more polar than the parent material for all three groups. Apparently, toxaphene must be metabolized before it can be excreted in the urine. When fat extracts were analyzed by thin-layer chromatography and autoradiography, differences were found between the parent material and the extracted activity. There was an increase in polar activity in the residue obtained from toxaphene-treated rats. The fat from the fraction 2 group contained fraction 2 and two additional more polar spots, which represented about 11% of the total activity. The fat from the fraction 7 group also contained two additional spots, but they were less polar than fraction 7. Apparently, the metabolism of fraction 7 results in some products that are less polar and, perhaps, more persistent.