This post was written by Katie Jones.
Bisphenol A (BPA) is a chemical compound found in many plastics. It has been identified as an environmental pollutant and can be a potential danger to human health as a disruptor of the endocrine system. When buying plastic baby bottles, cups, and food containers, many will have a BPA-free stamp somewhere on the container for this reason. As someone who worked in a dental office as a sterilization technician for two years, BPA has continued to fascinate me because many of the instruments I worked with were made of plastic. My father has also been diagosed with non-Hodgkins lymphoma within the last year, which his oncologists have said may have a BPA link.
With the increasing prevalence of childhood obesity, scientists at the University of Geneva in Switzerland decided to research the effects of BPA exposure on perigonadal adipose tissue in rats from early in gestation to three weeks after birth. Their research is summarized in a paper titled Perinatal Exposure to Bisphenol A Alters Early Adipogenesis in the Rat. It was published by Emmanuel Somm et al. and appeared in the October 2009 issue of Environmental Health Perspectives from pages 1549-1555.
Introduction: Based on previous studies, it has been found that BPA is in the serum of pregnant women and umbilical cord serum collected at birth.1 It has also been indicated that BPA has a presence in amniotic fluid and placenta.1 When rats were exposed to BPA in utero, females showed accelerated puberty and increased body weight,2 changes to the mammary glands,3 and abnormal development of the genitals.1 Males showed abnormal changes in structure and function of the prostate.4,5
In the body, BPA has been known to disrupt normal endocrine functioning by binding to estrogen receptors alpha and beta.6,7 It also acts as a thyroid hormone antagonist and targets protein disulfide isomerase, which is involved in the folding, assembly, and shedding of many proteins in the cells.8,9 BPA also interferes with glucose homeostasis10 and increases the presence of adipose tissue cells in the body with increased enzyme levels and fatty acid binding proteins.11
At the time, there was a lack of research concerning BPA exposure in vivo, which also accounted for the unexplained trends of obesity in industrialized countries. Because of this, Somm et al. studied the effects of BPA on adipose tissue and its gene expression in Sprague-Dawley rats three weeks after birth. They also monitored the body weight and food intake of these same rats if they were fed a standard diet or high-fat diet after the three-week mark.
Methods: Animal Care, Diets, and BPA Exposure before Weaning: The male and female rats were given a rodent experimental diet low in phytoestrogens 10 days before mating until the end of the gestation period. Pregnant female rats were exposed to BPA at a concentration of 1 milligram per liter in the water supply from day six of the gestation period until three weeks after the birth of the offspring. Pregnant females in the control group were given 1% ethanol instead of BPA. All plastics used during the research were BPA-free to avoid contamination. Daily food intake, volume of water consumed, and body weight were measured daily in the mother rats before the three-week post-birth mark.
Animal Care and Diets after Weaning: At the three-week mark, a group of rats that were from both the BPA-exposed group and the ethanol control group were anesthetized and decapitated. Epididymal white adipose tissue (eWAT) was removed from males and parametrial white adipose tissue (pWAT) was removed from females. Brown adipose tissue was also collected near the shoulder region from both males and females. These fat pads were weighed and then frozen in liquid nitrogen for further analysis.
After the three-week mark, a second group of rats that were from both BPA-exposed and ethanol control groups started to get normal drinking water and were fed with either a standard rat diet or a high-fat diet. Their body weight was measured from ages four weeks to fourteen weeks. Their food intake was also measured by weighing the solid pellets on grids atop the cages.
Histologic Examination of Adipose Tissue: The pWAT that was removed from the first group of rats was fixed in a paraformaldehyde solution for 24 hours and then set in paraffin. Small sections (5 micrometers) were cut and stained.
RNA Preparation and mRNA Quantification: RNA was extracted from the adipose tissue and liver samples from the rats using Trizol reagent. 5 micrograms total of RNA were reverse transcribed using a leukemia virus reverse transcriptase, RNAsin ribonuclease inhibitor, primers, dNTP (deoxyribonucleotide triphosphate) and DTT (dithiothreitol). Polymerase chain reaction (PCR) determined the coding of CAAT enhancer binding protein alpha (C/EBP-α), peroxisome proliferator-activated receptor gamma (PPAR-ɣ), sterol regulatory element binding protein 1-C (SREBP-1C), GATA binding protein 2 (GATA-2), preadipocyte factor 1 (Pref-1), lipoprotein lipase (LPL), acetyl-CoA carboxylase (ACC), fatty acid synthase (FAS), peroxisome proliferator-activated receptor alpha (PPAR-α), peroxisome proliferator-activated receptor-gamma coactivator (PGC-1α), stearoyl-CoA desaturase 1 (SCD-1) and glucose transporter 4 (GLUT4). The primer sequences for each were listed in this same section.
Plasma Measurements: Plasma glucose, noesterified fatty acid and triglyceride levels, and cholesterol levels were measured.
Glucose Tolerance Test: 14-week-old male rats from both the BPA and control groups had been fed a high-fat diet and fasted from the previous day. They were injected with 1.5 milligrams of glucose per gram of body weight. Blood samples were then collected through the tail at certain time intervals (0, 15, 30, 60, and 120 minutes) following the administration of glucose.
Statistics: The results were expressed as the mean plus or minus the standard error of measurement (SEM). The analysis of variance (ANOVA) method was used. When taking into account birth weight and the weight at three weeks between the BPA and control groups, a regression method was used.
Results: Maternal Physiology during Gestation: During the pregnancies of the female rats, the weight gain throughout the gestational period was not significantly different in those exposed to BPA (135 ± 7 g) versus the control group (140 ± 6 g). Food intake was also similar between the two. The daily amount of water consumed was not changed by the presence or absence of BPA. By the end of gestation, the scientists estimated that the total exposure of BPA to each rat was approximately 70 micrograms of BPA per kilogram of body weight each day once gestation had ended. Bottle leakage, spills, and evaporation were not taken into account. I saw this as problematic because leakage, spills, and evaporation could have accounted for a large portion of the measured intake and dramatically changed the results.
Body Weight of Offspring at Birth, Sex Ratio, and Litter Size: At birth, the body weight of male rats born to BPA-exposed mothers was significantly higher (7.33 ± 0.12 g) than those of the control group (6.91 ± 0.15 g). The same applied to the female rats born to BPA-exposed mothers (7.03 ± 0.11 g) versus the control group (6.47 ± 0.12 g). This trend is seen in Figure 1A-B. Litter size was determined to not affect the results. The sex ratio of the newborn rats was not affected by prenatal BPA exposure.
Body Weight and Fat Pad Weights at Weaning: Body weight measurements were repeated at age three weeks, which can be seen in Figure 1C-D. The body weight of BPA-exposed male rats (53.36 ± 1.02 g) was not much different from the control group (52.24 ± 1.11 g). However, the BPA-exposed females still had a higher body weight on day 21 (53.73 ± 0.65 g) versus the control group (47.79 ± 1.44 g).
To study the fat pads, a few of these rats were sacrificed in the same manner as those mentioned earlier. The BAT and eWAT/pWAT were removed and weighed. In males, the BAT weight remained unchanged but the eWAT mass was higher in the BPA group (56 ± 6 mg) compared to the control group (39 ± 6 mg). In the pWAT of females, the mass was significantly higher in the BPA group (95 ± 9 mg) than the control group (33 ± 6 mg). In contrast to the males, the BAT was also higher in the BPA group (178 ± 13 mg) than the control group (116 ± 11 mg). These results can be seen in Figure 2A-B. The results also suggest a positive correlation between the increase in body weight of the BPA-exposed female rats and the increase in fat pad weight. This is shown in Figure 2C.
Overall, the results showed that females are more sensitive to weight gain when exposed to BPA. With BPA binding to estrogen receptors, which are seen more in females, this seems to make sense.
Expression of Genes Involved in Adipogenesis and Lipogenesis in Adipose Tissue At Weaning: Histologic sections of the females’ pWAT tissue were taken. In the control group, the cells appeared normal, but in the BPA group, the adipose cells were much larger. This is seen in Figure 2D.
The mRNA levels of the preadipogenic transcription factors C/EBP-α, PPAR-ɣ, and SREBP-1C were significantly increased by about double each in the BPA female rats’ pWAT. The levels of the inhibitors GATA-2 and Pref-1 remained unchanged. The levels of lipogenic enzymes LPL, FAS, and SCD-1 also increased in the pWAT of the BPA females. Levels of GLUT4 also increased. These results are shown in Figure 2E-F. These results compared with the histologic examinations of the pWAT tissue show that the exposure to BPA enhances adipogenesis by increasing gene transcription. This explains why the adipose cells are much larger and the pWATs were heavier.
Expression of Genes Involved in Lipogenesis in Liver at Weaning: In BPA-exposed females, there were significant increases in SREBP-1C, ACC, and FAS, but no changes to PPAR-α or PPAR-ɣ (Figure 3A). There were no significant differences in circulating triglycerides, which could have altered gene expression (Figure 3B-E).
Postweaning Body Weight: The BPA-exposed rats were weighed weekly from ages four to fourteen weeks. There was no statistical difference in the body weight of BPA-exposed males versus the control males on the standard rat diet, but the BPA-exposed males on a high-fat diet were much heavier than the controls (Figure 4A-B) In females, the BPA-exposed were heavier than the controls on both standard rat and high-fat diets (Figure 4C-D). These results show a sex-specific difference in weight gain in BPA-exposed rats.
Postweaning Food Intake and Glucose Tolerance at Adulthood: There was no impact of perinatal BPA exposure on daily food intake or energy intake in males or females on either diet. Glucose tolerance tests were performed in the same manner in both BPA-exposed males and control males. No disturbances in glucose tolerance were found.
Discussion: Based on this study, it is evident that exposure to bisphenol A perinatally affects the generation of fatty tissue. The body weight of the rats exposed to BPA was higher than that of the control groups. The genes, proteins, and enzymes responsible for formation of adipose cells were also overexpressed, resulting in hypertrophied adipose cells in rats exposed to BPA. It was also indicated that being exposed to BPA as well as a high-calorie diet makes one more susceptible to obesity.
Some explanations for the increased weight gain in the BPA-exposed rats can be examined by looking at the mechanisms of BPA. By binding to estrogen receptors, BPA can directly act on the hypothalamus and control appetite. This happens because BPA can cross the blood-brain barrier. This could explain why those rats exposed to BPA had an increased food intake. It could also explain why the females consistently had higher body weights than the controls, as there is a higher presence of estrogen in females than in males.
In conclusion, the direct exposure to BPA through the parent (placenta and milk) in vivo increases adipose tissue creation and storage in a sex-specific manner. Using these results, BPA exposure could be related to the increase in childhood obesity, but further studies are needed to confirm this.
Because this study is one of very few of its kind, I would say it is too soon to accept or dismiss these results. Given the circumstances, I do believe this is an interesting and exciting idea in the application to childhood obesity. Knowing the dangers of BPA and our exposure to plastics on a normal basis, this experiment could be very valid in determining the relation. On another note, I did feel this experiment could have had completely different results in a few areas. For example, the amount of BPA consumed by the pregnant rat during the gestational period was 70 micrograms of BPA per gram of body weight each day. This BPA was in the water bottle, and the scientists did not take into account spills, leaks, or evaporation, and these were stated as an error. I understand that those variables were very hard to measure, but they could have dramatically altered the results had they been attainable.
This study also had multiple variables that were tested, including body weight, RNA analysis, histological analysis, glucose testing, and the effect on the liver. These variables may have been necessary to fully explain the results, but at times the article became muddled and confusing to me because there were so many different variables. If I had performed this same experiment, I would have focused on less variables, probably excluding the glucose tolerance and effect on the liver since they did not yield the significant results that the others did.
Overall, I enjoyed this aspect of relating BPA to obesity. It will be interesting to see the grey areas be answered in the future, such as, if BPA and a high-calorie diet increases susceptibility to obesity, would the same result be seen if plastics were regulated to be BPA-free and exposure was much lower? That experiment would require a longitudinal study (and probably several decades), but this study opened up opportunities for questions such as that one to be answered.
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