Role Of Reactive Oxygen Species On Lipopolysaccharide-induced Teratogenesis In Mice

Background/ObjectLipopolysaccharide (LPS) is a toxic component of cell walls in gram-negative bacteria and is widely present in the digestive tracts of humans and animals. Gastrointestinal inflammatory diseases and excess alcohol intake are known to increase permeability of LPS from gastrointestinal tract into blood. Humans are constantly exposed to low levels of LPS through infection. High levels of LPS have also been detected in women with bacterial vaginosis. Lipopolysaccharide has been associated with adverse developmental outcome, including embryonic resorption, intra-uterine fetal death, intra-uterine growth retardation and preterm delivery. The present study aimed to determine whether maternal LPS exposure causes external and skeletal abnormalities and to assess the potential role of reactive oxygen species (ROS) in LPS-induced teratogenecity.MethodsLPS was administered in two different modes. In mode A, the pregnant mice were intraperitoneally (i.p.) injected with different doses of LPS (either 10μg/kg or 20μg/kg or 30μg/kg) daily from gestational (gd) 8 to gd 12. All dams were sacrificed on gd 18 for fetal examinations. The number of live fetuses, dead fetuses, resorption sites and implantation sites was counted. Live fetuses were weighed, and examined for external morphological malformations. Crown-rump and tail lengths were examined and skeletal development was evaluated. In mode B, to assess the potential role of reactive oxygen species (ROS) in LPS-induced teratogenecity, the pregnant mice were injected with two doses of LPS, one (20μg/kg, i.p.) administered on gd 8 and the other (20μg/kg, i.p.) administered eight hours latter. Some pregnant mice were pretreated with PBN (100 mg/kg, i.p.) 30 min before LPS. Twelve dams each group were sacrificed 6 h after the second LPS treatment. Maternal serum and amniotic fluid were collected for analysis of nitrite plus nitrate concentration. Maternal liver, placenta and embryo were excised for measurements of thiobarbituric acid-reactive substance (TBARS) and glutathione (GSH) content. Placentas were excised for measurement of nitrotyrosine residues. The remaining dams were sacrificed on gd 18 for fetal examinations.ResultsAn intraperitoneal (i.p.) injection with LPS daily from gestational day (gd) 8 to gd 12 resulted in the incidence of external malformations. The highest incidence of externally malformed fetuses was observed in fetuses from dams exposed to 20μg/kg LPS daily from gd 8 to gd 12, in which 34.9% of fetuses were externally deformed. In addition, 17.4% of fetuses per litter in 30μg/kg group and 12.5% of fetuses per litter in 10μg/kg group were externally malformed. Exencephaly and encephalomeningocele were the most common external malformations. In addition, more than 80% of fetuses had skeletal deformities in 30μg/kg group. External abnormalities were also observed in fetuses from dams exposed to only two doses of LPS (20μg/kg, i.p.) on gd 8, in which 76.5% of litters and 39.1% of fetuses were affected. LPS-induced teratogenecity seems to be associated with lipid peroxidation, nitrotyrosine residues and GSH depletion in maternal liver, placenta and embryo. Alpha-phenyl-N-t-butylnitrone (PBN), a free radical spin-trapping agent, abolished LPS-induced lipid peroxidation, nitrotyrosine residues and GSH depletion. Correspondingly, PBN pretreatment obviously decreased the incidence of external abnormalities, in which 37.5% of litters and 7.0% of fetuses were affected. ConclusionsMaternal lipopolysaccharide exposure results in external malformations and skeletal deformities. ROS might be, at least partially, involved in LPS-induced teratogenesis.