We describe a method for cryopreservation of milt from individual Brachydanio rerio using methanol and powdered milk as cryoprotectants. Motility was positively correlated with hatching, which averaged 51 ± 35.6% in a typical experiment. Variablity in motility and hatching was not correlated with sperm volume or age of the fish, and is believed to be due to differences in sperm quality between individuals, as well as technical constraints imposed by the short duration of motility in thawed spermatozoa.

The present study was designed to evaluate the effects of a series of natural coumarins on ethoxyresorufin O-dealkylase (EROD) and pentoxyresorufin O-dealkylase (PROD) activities in vitro using hepatic tissues from SENCAR mice. Fifteen different coumarins were examined for potential modulating activities. Several naturally occurring coumarins, found in the human diet, were effective inhibitors of hepatic EROD activity in vitro, including coriandrin, bergamottin, isoimperatorin, and ostruthin. Notably, coriandrin and bergamottin were approximately as potent as 7,8-benzoflavone, a relatively selective inhibitor of cytochrome P450 1A1. Several naturally occurring coumarins were also potent inhibitors of hepatic PROD activity, including imperatorin, bergamottin, isopimpinellin, and angelicin. Kinetic studies of the type of inhibition revealed that these compounds inhibited hepatic EROD and PROD activity by a variety of modes rather than by a uniform one. Furthermore, experiments using a two-stage incubation assay revealed that coriandrin, imperatorin, ostruthin, and several other natural coumarins inactivated hepatic EROD activity (i.e., predominantly cytochrome P450 1A1-mediated) and that isopimpinellin inactivated hepatic PROD activity (i.e., predominantly cytochrome P450 2B1-mediated). Finally, the results indicate that some coumarins had selective inhibitory effects for EROD vs PROD and preliminary analyses suggested a possible structural basis for the observed differences. The current data suggest that certain naturally occurring coumarins, to which humans are exposed in the diet, are potent modulators of cytochrome P450. Furthermore, these compounds may be capable of influencing the metabolic activation of other xenobiotics, including chemical carcinogens.

Diseased celery infected with the fungus Sclerotinia sclerotiorum had greatly increased levels of three phytoalexin furocoumarins, namely psoralen, 5-MOP, and 8-MOP, which are responsible for skin photosensitivity. Storage of freshly harvested celery at 4 °C resulted in clear signs of fungal infection, from latent fungus, appearing within 23-29 days, with concomitant increases in total furocoumarin levels from 1.84 ppm (wet weight) to 43.82 ppm and with occasional samples as high as 95.52 ppm. Psoralen, the most active of the DNA photoalkylating furocoumarins, increased during storage from <0.06 to 14.14 ppm and on occasions 24.24 ppm. A combination of HPLC, TLC, and an extremely sensitive photobiological assay was used to obtain these results, which are discussed in relation to possible health consequences.

Many furocoumarins found in several species of plant are potent photosensitizing agents known to cause lethal and mutagenic effects in a wide range of organisms, from viruses to man. Their role in the aetiology of cancer is debatable, but work has focused on the PUVA (psoralen-UVA) treatment of psoriasis with 8-methoxypsoralen (8-MOP) and near UV radiation. Bergaptene (5-methoxypsoralen, 5-MOP) is a major constiutent of oil of bergamot, and might be expected to have qualitatively similar photosensitizing properties to 8-MOP. Although 5-MOP is widely used as a stimulus to melanin deposition in several suntan preparations surprisingly little is known about its basic photobiology. We report here that 5-MOP has the expected properties of other biologically active furocoumarins. These properties include lethal and mutagenic photosensitization of bacteria, 'dark' induced frameshift mutagenesis in bacteria, and lethal and clastogenic effects on mammalian cells in tissue culture.

Observations made during the freezing and thawing of mouse and human oocytes and mouse embryos with the cryomicroscope suggest that physical factors as well as physicochemical factors may play a role in the development of lethal damage upon thawing. The point of contact with the approaching ice front may predispose that area to the appearance of future cytoplasmic blebbing. The ice front distorts the oocyte and this distortion remains during its subsequent thermal history and is unrelated to desiccation distortion. Ice initiates the formation of both intra- and extracellular gas bubbles which are apparent upon thawing; with the progression of the thawing process they can be seen to grow in volume. Growth of these bubbles can give rise to expanding vesicles which can totally destroy an embryo. The consequences of these physical factors for the successful cryopreservation of oocytes, embryos, tissues and organs are discussed.