Influence of the preimplantation embryo development (Ped) gene on embryonic platelet-activating factor (PAF) levels.

PURPOSE: A major gene responsible for the control of preimplantation cleavage rate is the Ped gene, the product of which is the Qa-2 protein. Fast, but not slow developing mouse embryos express the Qa-2 protein. Platelet-activating factor (PAF) is a novel and potent signaling phospholipid that has unique pleiotropic properties in addition to platelet activation. PAF plays a significant role in virtually every reproductive event, including ovulation, fertilization, implantation, and parturition. The role of the Ped gene in PAF production by preimplantation embryos is yet to be established. The presence of this gene provides embryos with a reproductive advantage over those that are Ped negative, and may also serve as a regulator of PAF synthesis. The study hypothesis is that the amount of PAF produced is dependent upon the presence or absence of the Ped gene. METHODS: B6.K1 (Ped negative) and B6.K2 (Ped positive) mouse embryo-conditioned culture media were assayed for PAF content by a PAF-specific radioimmunoassay. RESULTS: There was a significant (p < 0.001) difference in blastocyst development rates between the Ped+ B6.K2 (61.0%) and the Ped- B6.K1 (25.3%) embryo culture groups. There was a significant difference (p < 0.05) in PAF production between the Ped+ B6.K2 (4.70+/-0.46 pmol per embryo) embryo culture group and the Ped- B6.K1 (10.02+/-3.49 pmol per embryo) embryo group. The B6.K1 (Ped-) embryo group produced >2x more PAF than did the B6.K2 (Ped+) group. CONCLUSIONS: The Ped gene plays a role in PAF production and release in preimplantation stage embryos. The use of two mouse identical strains, except for the Ped gene, show that its presence is associated with an increase in developmental potential. Embryos where the Ped gene was absent produced significantly higher levels of PAF, which may aid in their survival.

The hemotoxicity of para-substituted aniline analogs in dog and rat erythrocytes: a species comparison.

INTRODUCTION: Hemolytic anemia, the early removal of erythrocytes from the circulation, has been recognized as a side effect of drugs and other environmental chemicals. Formation of methemoglobin (MetHb) following chemical exposure is the first hemotoxic response in the induction of hemolytic anemia. The purpose of this study was to compare the species differences in the chemical induction of MetHb in dog and rat erythrocytes exposed to para-substituted halogenated aniline analogs (phenylhydroxylamine, p-bromo-, p-fluoro-, and p-iodo-phenyhydroxylamine). METHODS: Whole blood was collected from a healthy, male Dalmatian dog that weighed 51 lbs and male Sprague-Dawley rats that weighed 100-125 g. Cells were washed (x3) with phosphate-buffered saline supplemented with glucose (pH 7.4). Methemoglobin (MetHb) induction was determined by treating aliquots with pre-specified micromoles of the test agents. Aliquots (75 microL) were removed from each treatment group at specific time points and mixed with cold hemolysis buffer for MetHb determination. Methemoglobin (MetHb) was determined spectrophotometrically at 635 nm. RESULTS: Methemoglobin (MetHb) levels in dog erythrocytes treated with the four analogs increased continuously over 180 minutes and showed no signs of declining. Methemoglobin (MetHb) levels in rat erythrocytes, however, immediately increased and continued to rise and fall before gradually approaching control levels. CONCLUSIONS: Our data demonstrated the species differences in the MetHb-inducing ability of the analogs tested in both dog and rat erythrocytes. The differences in the patterns associated with MetHb induction in the animal models used may be attributed to variations in the MetHb reductase enzyme in both species.

Aniline derivative-induced methemoglobin in rats.

Methemoglobinemia and hemolysis are the most prominent side-effects of exposure to a wide variety of arylamine drugs, including agricultural and industrial chemicals. Recent studies with aniline and dapsone have identified N-hydroxyl metabolites as the red blood cell (RBC) mediators. This study examines the time-course methemoglobinemic potential of several halogenated aniline phenylhydroxylamines. Symptoms of aniline poisoning include headache, fatigue, dizziness, respiratory and cardiac arrest, and possibly death. Initial studies indicated that the parent compounds are converted to their toxic metabolites (N-hydroxylamine), which enter the RBC and react with oxyhemoglobin. Consequent reduction of molecular oxygen to active oxygen species occurs, leading to RBC damage. Our laboratory is investigating the role of redox cycling and an alternative hypothesis--that a "hydroxylamine-centered" radical formed during arylhydroxylamine-oxyhemoglobin reaction results in RBC injury. The methemoglobinemic capacities of several structurally related N-hydroxy derivatives of aniline--phenylhydroxylamine (PHA), p-fluoro-, p-chloro-, p-bromo-, and p-iodo-PHA--were studied spectrophotometri-cally by treating washed rat RBC at concentrations ranging from 30 to 300 microM of the test compounds for up to 240 minutes. The results showed dose- and time-dependent changes in the induction of methemoglobin (MetHb) by aniline derivatives. The MetHb levels peaked to as high as 75% and remained elevated up to 240 minutes, depending on the electronegativity of halogenated phenylhydroxylamine aniline. This study supports the previous findings that there may be several aniline-derived metabolites other than PHA that are capable of inducing MetHb. The minimum dose required to induce this effect and duration of the MetHb may vary with the test agent.

Hemolytic potential of structurally related aniline halogenated hydroxylamines.

This study was undertaken to investigate the hemolytic potential of several structurally related aniline halogenated phenylhydroxylamines based on their decreasing electro negativity. The compounds compared are phenylhydroxylamine (PHA) and para-fluoro-, para-bromo-, and para-iodo-phenylhydroxylamines. Red blood cells of male Sprague-Dawley rats were labeled with radioactive chromium-51 and exposed to the test agent before being infused into the tail vein of isologous rats. The time course of blood radioactivity was monitored. The stability of some selected halogenated aniline analogs was also determined in blood. All four tested hydroxylamines produced dose-dependent reduction in the circulating labeled red blood cells indicating their destruction and loss. The most pronounced reduction was observed at doses from 175 to 250 microM. The dose of 100 microM appeared to be the threshold limit. The para-iodo-PHA was two times more toxic than para-fluoro-PHA in the destruction of red blood cells in rats.

Embryonic platelet-activating factor: temporal expression of the ligand and receptor.

PURPOSE: Preimplantation embryos synthesize platelet-activating factor (PAF) and this embryo-derived PAF is required for development. PAF's signal transduction is receptor-mediated and PAF-receptor mRNA is present in early embryos. The study objective was to determine the relationship between PAF production and PAF-receptor mRNA expression levels in mouse preimplantation embryos. METHODS: Embryo-derived PAF levels were determined by radioimmunoassay. Embryonic PAF-receptor mRNA levels were determined by semiquantitated reverse transcription-polymerase chain reaction. RESULTS: Embryonic-PAF increased as time progressed at a relatively constant rate (1.4-1.9 x) except between the eight- and morula-cell stages where levels increased sevenfold. Embryonic PAF-receptor expression was highest at the two-cell stage and decreased steadily until the morula stage before increasing again. Regression analysis of embryo-derived PAF on PAF-receptor expression does not demonstrate a significant relationship. CONCLUSIONS: PAF-receptor expression (mRNA) levels decrease, while embryo-derived PAF levels decrease, as the preimplantation embryo develops. Embryonic-PAF and PAF-receptor mRNA expression do not appear related. Therefore, embryonic-PAF does not appear to regulate expression of its own receptor in vitro. The data provide a clue to the complicated cell signaling system involving PAF production and receptor expression that may help our understanding of developmental events.