The Effect of Trypan Blue on the Serum Proteins of the Fetal Rat.

Pregnant rats were injected intraperitoneally with a teratogenic dose of trypan blue (14 mg/100 g or 20 mg/100 g body weight) on the eighth day of gestation. Fetal blood was collected from control and experimental fetuses on the twentieth day of pregnancy. Serum was obtained from pooled blood samples of two to three fetuses. Paper electrophoresis was carried out in a Spinco model R system using barbital buffer at pH 8.6. The paper strips were subsequently stained with bromphenol blue and their density measured in a Spinco Analytrol to determine the relative concentration of each component. Total protein was measured in a Bausch and Lomb Serum Protein Meter. The predominant malformations seen in abnormal fetuses were apparent anophthalmia, 59% ; hydrocephalus, 37% ; and exencephaly, 19%. Other malformations were noted but with less frequency. Twentyday fetuses from trypan blue treated mothers had a significantly lower total protein concentration (1.50 g/100 ml) than control fetuses (1.95 g/100 ml). A comparison of the absolute protein concentration of the serum fractions revealed significant differences between control and experimental fetuses in beta globulin, alpha-1 globulin, and albumin. In each instance the absolute concentration was less in the fraction from fetuses of trypan blue treated mothers. Fetal gamma, alpha-2, and alpha-3 globulins were not affected by the maternal treatment. The disazo dye, trypan blue is known to cause alterations in the serum proteins of the pregnant rabbit when administered either as subcutaneous injections prior to and during pregnancy (Langman and van Drunen, ’59) or, as a single subcutaneous injection on the second day of gestation (Beaudoin and Ferm, ’61). Both papers report a significant increase in the alpha and beta globulin concentration. This increase occurred during the first ten days of pregnancy, a time critical to embryogenesis. Langman and van Drunen reported 39% resorptions and 17% of the survivors malformed at autopsy, proof that a teratogenic dose of trypan blue was employed. Beaudoin and Ferm did not carry their experiments to the end of gestation, however, their dose was based on a teratogenic concentration previously reported by Ferm (’56). Alterations of serum protein composition, following trypan blue treatment, is not limited to the rabbit. Yamada (’59) and Paoletti et al. (’62) reported serum protein changes in male rats treated with heavy doses of trypan blue. In each paper reference is made to an “abnormal” protein band migrating in close association with the alpha-2 globulin during paper electrophoresis. No marked changes were reported for any other globulin. Dijkstra and Gillman, (’60) reported a faint band that may or may not be related to the “abnormal” protein reported by Yamada. The band observed by Dijkstra and Gillman, however, was not present in every sample. It has also been demonstrated that teratogenic doses of trypan blue administered to the rabbit causes alterations in the protein composition of the yolk sac fluid of nine-day old embryos which mimic those of the maternal serum protein alteration (Hommes, ’59; Beaudoin and Ferm, ’61). The relationship between the maternal serum proteins and the proteins found in the yolk sac and fetus in the rabbit have been reviewed by Hemmings and Brambell (’61 ). Apparently free passage of maternal proteins across the yolk sac is possible, certainly albumin and gamma globulins can do so. In addition, it is probable that maternal albumin and most maternal globulins are able to pass to the fetal circula1 Supported by research grant AM-05603-03 from the National Institute of Metabolic Diseases, Public Health Service, U. S. Department of Health, Education, and Welfare. a Medical Student Research Fellow.