Bilirubin and Heme as Growth Inhibitors of Chicken Embryos in Ovo

ABSTRACT: The increased morbidity during pregnancies complicated by hematologic or liver disease has generally been attributed to the metabolic abnormalities of the illness itself. Because tetrapyrrole concentrations are elevated in these conditions, we introduced bilirubin or heme (prepared as 10 mM solutions) into the air sac of fertilized chicken eggs to study their effect on the growth of normal chicken embryos. In 9-d eggs, the injection of 0.06 mL heme resulted in significant embryo growth inhibition as indicated by overall wt (91 ± 3% versus control), tibia length (84 ± 2%), tibia wt (81 ± 3%), femur length (88 ± 1%), and femur wt (78 ± 3%); doses greater than 0.10 mL resulted in substantial fetal losses. The injection of 0.06 mL bilirubin into the same-age eggs also resulted in less than normal tibia length (87 ± 2% versus control), tibia wt (75 ± 4%), femur length (91 ± 2%), and femur wt (81 ± 3%); larger doses resulted in more pronounced growth inhibition, but fetal losses were less common than with heme. Older chick embryos (12-d) appeared more resistant to the effects of bilirubin: 0.15 mL bilirubin inhibited only tibia and femur wt; larger doses were required to significantly suppress the other growth parameters. The sameage chicken embryos, however, remained exquisitely sensitive to heme; 0.05 mL resulted in less than normal whole embryo wt (88 ± 2% versus control), tibia length (80 ± 1%), tibia wt (76 ± 1%), femur length (78 ± 1%), and femur wt (77 ± 1%). Substantial fetal loss occurred with heme doses above 0.10 mL as well as among the less mature chicks. Significant inhibition of the growth of long bones (tibia and femur) also occurred when we added 0.05 mM bilirubin or 0.025 mM Hb to bones maintained in organ culture in vitro for 5 d. We conclude that several heme compounds and bilirubin inhibit the growth and survival of normal chick embryos in ovo and in vitro.We suggest that prenatal exposure to high levels of these compounds may have a previously underestimated negative influence on fetal development.