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Abstract
Summary
During the last three decades, there has been a steady increase in the use of perfluorinated compounds such as perfluorooctane sulfonate (PFOS), perfluorooctanoic acid (PFOA), perfluorooctane sulfonamide (PFOSA), perfluorohexane sulfonate (PFHS), perfluorobutane sulfonate (PFBS), and perfluorononanoic acid (PFNA). These compounds have wide commercial applications and are wide spread pollutants of toxicological importance. They are environmentally persistent, bioaccumulative and toxic. The goal of this study was to evaluate the reproductive, genetic and biochemical effects of PFOS and PFOA in pregnant ICR mice and the reflection of these effects on their offspring.
A total number of 160 Adult pregnant ICR mice at 7 weeks of age were used for this study. 60 pregnant dams out of them were treated with PFOS and 20 were left as control and received the solvent only. 60 dams were treated with PFOA and 20 dams were left as control and received deionized water. Both the PFOS and PFOA groups were subdivided according to the dose of the chemical given to the pregnant dams into 3 equal sub groups (20 pregnant dams each). The PFOS sub groups received 1, 10 and 20 mg/kg body weight PFOS. On the other hand, the PFOA sub groups received 1, 5 and 10 mg/kg body weight PFOA.
Maternal body weight, food consumption and water intake were monitored daily throughout gestation period. Ten dams per group were exposed to the chemicals from GD0 to GD17 then euthanized on GD18 (24 hours after the last treatment) under diethyl ether anesthesia. Blood was collected from the descending aorta and serum samples were obtained for determination of blood serum levels of Lactate dehydrogenase (LDH), Gamma glutamyl transferase (GGT), Aspartate aminotransferase (AST), Alanine aminotransferase (ALT), alkaline phosphatase (ALP), creatinine, blood urea nitrogen, total bilirubin, total protein, albumin, globulins, calcium, inorganic phosphorus, glucose, triglycerides, phospholipids, total cholesterol, non esterified fatty acids, hydroxyl butyric acid and serum leptin concentration.
Maternal liver, kidneys, lungs and brain were dissected and weighed; the organ/body weight ratio was calculated to obtain the relative organ weight. A portion of the liver was dissected and used immediately for comet assay. The previous organs were kept in neutral buffered formalin 10% for histopathological examination. The gravid uterus of the same dams was removed and examined for prenatal evaluation of fetuses. The liver of the fetuses were dissected and used immediately for comet assay. Individual live fetuses were prepared for teratological evaluation.
The remaining 10 pregnant mice per group were exposed to the chemicals from GD0 to GD18 and allowed to give birth; the neonates of 5 dams were monitored for 4 days for postnatal survival. All the neonates of the remaining 5 dams were kept in Bouin’s fixative for three days then kept in 70% ethanol till histopathological examination. Statistical significance was determined by the analysis of variance (ANOVA). When a significant treatment effect was detected, each treatment group was tested for difference from the control group using Dunnett’s t-test.
Results of the present study revealed significant reduction in maternal weight gain and daily feed consumption after exposure to 20 mg/kg PFOS and 10 mg/kg PFOA. Daily water intake was significantly increased after exposure to 20 mg/kg PFOS and after exposure to 5 mg/kg PFOA in late gestation.
There were significant increases in the absolute and relative weight of the maternal liver in a dose dependent manner associated with hypertrophy of hepatic cells after exposure to both of PFOS and PFOA, and significant increase in the relative lung and brain weight after exposure to PFOS at 20 mg/kg group. Relative kidney weight was significantly increased after exposure to PFOA.
Serum lipids (triglycerides, phospholipids, total cholesterol, and non-esterified free fatty acids), protein and leptin levels were significantly decreased after exposure to PFOS and PFOA at 20 mg/kg and 10 mg/kg respectively. In addition, exposure to PFOA resulted in significant increases in serum GGT, AST, ALP activities.
PFOS treatment induced DNA damage in maternal and fetal liver at 10 and 20 mg/kg groups. However, exposure to PFOA at 10 mg/kg induced DNA damage in maternal liver only.
PFOS treatment reduced the number of live fetuses accompanied with increased fetal resorption only at 20 mg/kg group, while reduced the fetal body weight in a dose dependent manner. Gross examination of fetuses (GD18) showed presence of an abnormal swelling in the back of the neck region in all fetuses of the 20 mg/kg and some fetuses of 10 mg/kg groups.
Teratological evaluation revealed presence of several skeletal abnormalities such as, cleft palate, delayed eruption of incisors, spina bifida occulta (delayed closure of the vertebral spine), delayed ossification of phalanges and sternum, wavy ribs, curved fetus (curved vertebral column), and abnormal (kinked) tail mostly at 10 and 20 mg/kg PFOS groups. Maternal exposure to PFOA reduced the fetal body weight at 5 and 10 mg/kg groups. There were no significant effects on the prenatal survival or resorbed fetuses, in addition to few skeletal abnormalities as delayed ossification of the sternum and phalanges accompanied by delayed eruption of incisors in the 10 mg/kg group.
Both of PFOS and PFOA reduced the survival rate of the neonates, as there was a 100% neonatal mortality (all neonates died immediately after birth) at 20 mg/kg and 10 mg/kg for PFOS and PFOA respectively.
The abnormal swelling observed in fetuses of mice received 20 mg/kg PFOS was observed in neonates after birth. Histopathologically, it appeared as a dilatation of vessels with blood in the Dura matter and discontinuously trimmed by the endothelial-like spindle shaped cells. Histopathological examination of neonatal lungs revealed that all neonates of the 20 mg/kg PFOS group showed severe lung atelectasis.
At 10 mg/kg PFOA group, some neonates showed whole body edema. Histopathological examination of neonatal head and lungs revealed that some of the neonates showed slight to moderate degree of dilatation of the brain blood vessel accompanied by slight lung atelectasis.
One of the probable explanations for the neonatal death in mice exposed to PFOS might be that the intracranial blood vessel dilatation press the respiratory center of the brain and prevent the lungs to start respiration after birth.
PFOS and PFOA induced neonatal mortality for offspring but the cause of death may be different. Further investigations are required to explore the exact mechanisms of neonatal death after maternal exposure to these compounds.