Effect of Perinatal Bisphenol A Exposure on Serum Lipids and Lipid Enzymes in Offspring Rats of Different Sex*

GAO Liang， WANG Han Ning， ZHANG Ling， PENG Fang Yuan，JIA Yue， WEI Wei， and JIA Li Hong，#

Letter to the Editor

Effect of Perinatal Bisphenol A Exposure on Serum Lipids and Lipid Enzymes in Offspring Rats of Different Sex*

GAO Liang1， WANG Han Ning2， ZHANG Ling1， PENG Fang Yuan1，JIA Yue1， WEI Wei1， and JIA Li Hong1，#

Rats were exposed to 1 or 10 μg/mL bisphenol A （BPA） in water during pregnancy and lactation. Offspring rats were given normal water and a standard diet from weaning to postnatal day （PND）50. Perinatal exposure to BPA resulted in significantly increased body weight， visceral adipose tissue， abnormal serum lipids， and lower adiponectin （ADP） levels in both female and male offspring rats. Liver adipose triglyceride lipase （Atgl）mRNA levels and ADP protein in visceral adipose tissue were significantly decreased in BPA-exposed offspring rats. In both female or male offspring rats，obesity and dyslipidemia induced by perinatal exposure to BPA were associated with down regulation of Atgl mRNA in liver and ADP protein in visceral adipose tissue.

Emerging data suggest that Bisphenol A （BPA）may be an important contributing factor to the obesity epidemic； especially early life exposure to BPA can alter developmental programming，increasing the risk of metabolic disorders［1-2］. However， the exact mechanism of BPA-mediated effects is not fully understood. Based on several studies showing BPA-mediated changes in serum lipids and lipid-related metabolic enzymes in rats of different sex， we explored the effect of perinatal BPA exposure on serum lipids. Specifically， we evaluated changes in fat synthetase （Fas） and adipose triglyceride lipase （Atgl） mRNA in liver tissue and measured adiponectin （ADP） in serum as well as in visceral adipose tissue in male and female offspring rats that were exposed to BPA in utero.

Male （250-300 g） and female （200-220 g）Sprague-Dawley （SD） rats were purchased from the animal center of China Medical University ［license number， SYXK （Liao） 2008-0005］. All animals were handled in accordance with the Guidelines for Animal Experimentation issued by the Chinese Association for Laboratory Animal Science. After a 1-week adaptation period in a room with standard temperature （22±2 °C） and illumination on a 12-h light-dark cycle， females were mated with males. A sperm-positive vaginal smear indicated the first day of pregnancy. Physiologically normal pregnant rats were individually housed and randomly allocated into three groups （n=7/group）. Two groups were exposed to BPA （Sigma Aldrich， St. Louis， MO） with free access to water containing 1 μg/mL （low dose）or 10 μg/mL （high dose） BPA from gestation day 6 until the end of lactation. The third group comprised of control animals given water containing 1% ethanol， the vehicle for BPA solution. Pups were weaned on postnatal day （PND） 21 and given access normal drinking water and fed with a standard diet until PND 50. At end of the experiment， blood samples were collected from abdominal aorta under aether anesthesia， and serum was separated by centrifugation and stored at -80 °C until subsequent analysis. Liver， perigonadal and perirenal adipose tissue were dissected， weighed， and stored at -80 °C until subsequent analysis of gene or protein expression to assess lipid metabolism.

Serum triglyceride （TC）， cholesterol （TG）， high density lipoprotein （HDL）， and low density lipoprotein （LDL） levels were determined by commercially available reagent kits （Biosino Biotechnology and Science Beijing， China）. Fas and Atgl gene expression levels in liver specimens were measured by real-time （RT） polymerase chain reaction （PCR）. Briefly， total RNA was isolated from frozen liver tissue using TRIzol? （Takara Biotechnology， Dalian， China）. All samples with anabsorbance ratio at 260/280 nm approaching 2 were considered to contain pure RNA. First-strand DNA was reverse-transcribed from 0.5 μg total RNA using SuperScript first-strand synthesis system （Takara，Dalian， China）， according to the manufacturer’s instructions. RT-PCR reaction mixtures （25 μL）contained 100 ng template cDNA， 400 nmol/L each of forward and reverse primers， and 12.5 μL SYBR Premix Ex TaqTM11 （Takara）， and reaction was performed on a PTC-100TMprogrammable thermal controller （MJ Research， Waltham， MA）. PCR was performed on a thermal cycler （MJ Research） with an initial denaturation at 94 °C for 2 min， followed by 31 cycles of denaturation at 94 °C for 30 s， annealing at 55 °C for 30 s， and extension at 72 °C for 35 s； final extension was at 72 °C for 10 min. PCR products were detected by electrophoresis on a 1.5% agarose gel containing GeneFinder （Bio-V， China）. Products in gel were quantified using densitometry with the Bioimage software （Millipore， Bedford， MA）. Signals for target mRNAs were normalized to the signal of the housekeeping gene β-actin， and results were expressed as target/β-actin mRNA ratio. Predesigned primers， which were purchased from Takara Biotechnology， were as follows： rat Fas： forward，5’-AGGTGCTAGAGGCCCTGCTA-3’， reverse， 5’-GTGC ACAGACACCTTCCCAT-3’； Atgl： forward， 5’-ACCTGTG CCTTACCGTTCAC-3’， reverse， 5’-GGCAAGAGTGACAT GCAGAA-3’； β-actin： forward， 5’-GCCCATCTATGAG GGTTACGC-3’， reverse， 5’-GAGCCACCAATCCACACAG AG-3’.

ADP protein expression was measured by western immunoblotting. Protein samples were prepared from visceral adipose tissues， and concentrations were determined by Pierce BCA? protein assay kit （Thermo Scientific， Rockford， I）. Samples containing 50 μg total protein were mixed with equal volumes of 5 × sodium dodecyl sulfate（SDS）-acrylamide gel loading buffer and incubated at 95 °C for 5 min. Twenty μL of each sample was loaded into the wells of 14% SDS-acrylamide gels. Proteins were separated for 1.5 h at 100 V and transferred to membranes. For immunodetection of ADP， membranes were blocked overnight at 4 °C with TBS containing 0.1% Tween-20 and 5% nonfat milk （TBSTM）， followed by incubation with a mouse monoclonal anti-human ADP （1：1000； sc-26496；Santa Cruz Biotechnology， USA） in TBSTM at 4 °C overnight. Membranes were then incubated for 1 h with a rabbit anti-mouse IgG secondary antibody conjugated with horseradish peroxidase in TBSTM. An antibody against glyceraldehyde-3-phosphate dehydrogenase （GAPDH） was used as loading control. Autoradiographs were scanned for densitometric analysis to quantify changes in ADP expression using Phoretix 1D advanced software. The signal intensities of ADP bands were normalized to corresponding GAPDH bands. Five samples from each group were analyzed.

For statistical analysis， SPSS software （version 20.0 for Windows； Chicago， IL） was used. All data were presented as means±standard deviation. Differences between groups were analyzed by analysis of variance （ANOVA） followed by appropriate post-hoc tests， and P values of ＜0.05 were considered as statistically significant.

Our results showed that body weights of both female （Figure S1A see the www.besjournal.com）and male （Figure S1B see the www.besjournal.com）offspring rats born to dams exposed to 1 or 10 μg/mL BPA from PND 1 to PND 50 were significantly higher than those of offspring rats born to control dams （n=7 belonging to seven diffrent litters in each group， P＜0.05）， as well as there was more weight perigonadal adipose tissure in BPA-exposed female offspring rats， and more weight perirenal adipose tissue in BPA-exposed male offspring rats compared controls （Figure S3 see the www.besjournal.com）. However， the effect of perinatal BPA exposure on body weight was no statistically different between the low-dose （1 μg/mL BPA） and high-dose （10 μg/mL BPA） groups，suggesting that the effect of BPA on body weight was not dose-dependent. The ubiquity of BPA in environment necessitates careful assessment of exposure to low-dose BPA［3］. Numerous studies showed that BPA exposure during early development could result in higher body weight in female rats［2，4］. However， very few studies examined the effect of BPA exposure on body weight in male rats. Furthermore， conflicting results of epidemiologic studies investigating the effect of BPA exposure on human body weight might be associated with the dose， time and route of BPA exposure［5-6］. Our study also indicated that perinatal exposure to BPA resulted in the different distribution of visceral adipose tissue， such as more fat around uterus for female rats and around kidney for male rats. This effect of BPA on distribution of visceral adipose tissue was worth study further.

Serum lipid and ADP levels in female and male offspring are shown in Table 1. In female offspring rats， both low- and high-dose BPA exposure during perinatal period led to significant increases in serumTG and TC levels （P＜0.05 and P＜0.01， respectively）and significant decreases in serum HDL and ADP levels （P＜0.05 and P＜0.01， respectively）， compared to controls. Conversely， compared to control males，significantly higher levels of serum TC and significantly lower levels of serum ADP were observed only in the high-dose male offspring rats（P＜0.05 and P＜0.01， respectively）； no significant differences were observed between the low-dose and the control male offspring rats. These findings suggested that dyslipidemia was more severe in female offspring rats than in male offspring rats following exposure to BPA during gestation and lactation. Previously， perinatal BPA exposure was shown to result in higher serum TG levels in adult offspring rats that were fed a high-fat diet after weaning［7］. Our results indicated that perinatal BPA exposure precipitated abnormal lipid levels in both female and male offspring rats despite being fed a standard diet after weaning.

ADP protein expression in visceral adipose tissue of female offspring rats was significantly lower in both low- and high-dose groups than in control offspring rats， whereas a similar change was only observed in male offspring rats in the high-dose group （P＜0.05 and P＜0.01， Figure S2 see the www.besjournal.com）. As a vital adipokine， ADP is primarily expressed in and released from adipose tissue. It is important to note that ADP was demonstrated to positively correlate with serum HDL and negatively correlate with serum TG in mice； ADP was also found to confer protection from metabolic syndrome in humans［8］. A recent study found that BPA at environmentally relevant doses inhibited ADP release from human adipose tissue explants and adipocytes［9］. In the present study， serum ADP levels in both BPA-exposed female and male offspring rats were significantly lower； in addition， ADP protein expression was also lower in visceral adipose tissue. This decrease in ADP protein expression in tissue coincided with the increased body weight of offspring rats. These findings provide evidence to support our hypothesis that decreased ADP might be involved in abnormal lipid metabolism induced by BPA.

We also assessed changes in the expression of Fas and Atgl， two rate-limiting enzymes catalyzing fat synthesis and lipolysis， respectively， in liver. As shown in Figure 1， the levels of liver Fas mRNA were not significantly different between female and male offspring rats perinatally exposed to BPA and control rats （P＞0.05）. In contrast， liver Atgl mRNA expression was significantly lower in female offspring rats in both the low-dose and high-dose groups （P＜0.05）. Importantly， liver Atgl mRNA levels were decreased only in male offspring rats perinatally exposed to 10 μg/mL BPA （P＜0.05）. ATGL is a major hepatic lipase that regulates TG turnover and initiates the breakdown of intracellular TGs into fatty acid monomers； thus， lower Atgl expression indicates TC accumulation in hepatic cells［10］. Our results suggested that hyperlipidemia induced by perinatal BPA exposure in offspring rats was associated with down regulation of Atgl mRNA expression in liver. Furthermore， sex-specific changes in Atgl mRNA suggested that female offspring rats were more susceptible than male offspring rats to perinatal exposure to BPA.

In conclusion， perinatal exposure to low- as well as high-dose BPA induced obesity and dyslipidemia in both female and male offspring rats， which was likely due to the downregulation of Atgl in liver and ADP protein in visceral adipose tissue.

Table 1. The Effect of Perinatal Exposure to BPA on Serum Lipids and ADP Levels in Female and Male Offspring Rats

#Correspondence should be addressed to Professor JIA Li Hong， Tel： 86-24-31939080； E-mail： lhjia@mail. cmu.edu.cn

Biographical note of the first author： GAO Liang， male，born in 1986， an assistant/master's degree， studying PhD，majoring in cause and mechanism of children obesity and related diseases.

Received： March 10， 2016；

Accepted： September 1， 2016

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A total of 48 articles were acquired after the literature searches using Wanfang Data， 69 articles were acquired by CQVIP database， and only 1 article was acquired by PubMed. Then the iterative literatures were eliminated， and last a total of 71 articles were remained， among which 26 articles are related with epidemic hemorrhagic fever infections in laboratories. And 20 of 26 articles are about staff infections， 6 of 26 articles refer to laboratory animal infections （Figure 1）. There were eleven incidences that occurred in seven different regions of China［4，8-20］. It has been well-documented that humans can become infected with Hantaviruses and develop epidemic hemorrhagic fever through contact with the urine， feces and saliva of rodents. Before the 1990s， epidemic hemorrhagic fever infections in humans were mainly caused by and transmitted through wild rodents （e.g. Apodemus and Rattus norvegicus）， whereas after 1990s，hantavirus-infected laboratory mice and rats were considered the major source of infection to humans［21］. In this study， we reviewed the events that occurred in China from 1995 to 2015， and found that infected laboratory mice and rats were the dominant cause of epidemic hemorrhagic fever among laboratory personnel， which was consistent with previous reports.

Fault Tree Construction FTA is an important failure analytical tool. FTA was originally developed and introduced in 1962 by Watson at Bell Laboratory under the US Air Force Ballistics Systems Division to evaluate a missile launch control system［22］， and extended to a broad range of fields such as aviation，rail transportation， chemical industry and medicine［23-25］. In FTA， an undesired top event is analyzed by using Boolean logic in order to combine a series of lower-level faults to determine not only the direct cause of the undesired event， but also indirect causes leading to the top event.

A fault tree， which is a logical diagram that allows the logical prioritization of contributors leading to the undesired top event， was constructed to identify basic faults or lowest level factors that may lead to epidemic hemorrhagic fever among laboratory personnel working with animals either directly or indirectly. We aimed to unveil the logical relationships of infection and to determine the most effective approach to preventing infection （Table 1）. In this study， EasyDraw software was utilized to construct a fault tree diagram with event， gate and transfer symbols， from which the causes of infection were noted according to a review of the literature，and further revised and confirmed by two biosafety experts.

Upon the completion of the comprehensive literature review， we constructed a fault tree diagram. Laboratory epidemic hemorrhagic fever served as the top failure event that was first noted. Additionally， the possible reasons for infection，including pathogens harbored by experimental animals， exposure to animals （e.g. feeding or holding）， unsuitable personal protective equipment（PPE）， and lack of proper vaccinations， fell just below the top level of the fault tree as the second level of the intermediate events. With logic graphic symbols，the fault tree was broken down to lower levels and finalized by a total of 12 lower or basic elements or faults （X） which may lead to the undesired event，and probabilities were calculated （Table 2 and Figure 2）.

Table 1. Relative Terms

Table 2. Basic Events in the Fault Tree

Fault Tree Analysis： Minimal Cut Sets After the fault tree was assembled， we conducted FTA. The premise was that if all the basic events occurred，then the top failure was inevitable. In most cases，however， a unique combination of basic events are necessary and sufficient to cause the undesired event， and the combination was defined as a minimal cut set and the number of minimal cut sets indicate the probability of the top failure event［26-27］. Boolean algebra algorithms were used to calculate the number of the minimal cut sets， and a total of 32 minimal cut sets were obtained as follows：

｛X1， X4， X5， X9｝｛X2， X3， X4， X5， X9｝｛X1， X4， X6，X9｝｛X2， X3， X4， X6， X9｝｛X1， X4， X7， X9｝｛X1， X4， X8，X9｝｛X2， X3， X4， X7， X9｝｛X2， X3， X4， X8， X9｝｛X1， X4， X5，X10｝｛X1， X4， X5， X11｝｛X2， X3， X4， X5， X10｝｛X2， X3， X4，X5， X11｝｛X1， X4， X6， X10｝｛X1， X4， X6， X11｝｛X2， X3， X4，X6， X10｝｛X2， X3， X4， X6， X11｝｛X1， X4， X7， X10｝｛X1， X4，X7， X11｝｛X1， X4， X8， X10｝｛X1， X4， X8， X11｝｛X2， X3， X4，X7， X10｝｛X2， X3， X4， X7， X11｝｛X2， X3， X4， X8， X10｝｛X2，X3， X4， X8， X11｝｛X1， X4， X5， X12｝｛X2， X3， X4， X5，X12｝｛X1， X4， X6， X12｝｛X2， X3， X4， X6， X12｝｛X1， X4， X7，X12｝｛X1， X4， X8， X12｝｛X2， X3， X4， X7， X12｝｛X2， X3， X4，X8， X12｝.

As listed above， each minimal cut set contained 4-5 basic events. For instance， the cut set ｛X1， X4， X5，X9｝ was composed of 4 basic events： X1， animals purchased without complying with established standards； X4， lack of vaccination； X5， without PPE；and X9，injury by scratch， bite or edge tools. However，for laboratory workers who were not vaccinated against epidemic hemorrhagic fever nor wore PPE，and were injured by scratches and bites by infected animals， their risk of infection was much greater. Another minimal cut set ｛X2， X3， X4， X8， X12｝consisted of five basic faults. If laboratory personnel worked in animal facilities within disease outbreak regions where no appropriate equipment to block wild rodents from entering animal facilities， and get exposed to wastes from the infected animals， but did not wear PPE or wore inappropriate PPE， they were likely to be infected by aerosols. In this study， there were 32 minimal cut sets， indicating at least 32 basic fault events that could occur and lead to epidemic hemorrhagic fever if the feasible precautions were not properly undertaken.

Fault Tree Analysis： Minimal Path Sets If none of the basic events occurred， then the top event would not occur. Meanwhile some of the basic events could occur separately or non-simultaneously. The combinations of these basic events were not sufficient to cause the top event， and they are defined as path sets. The minimal path sets referred to the safe modes in which the top event would not occur if these basic events would be prevented simultaneously. Therefore， the fault tree was transformed into the successful tree and produced five minimal path sets which were as follows： ｛X1， X2｝｛X5， X6， X7， X8｝ ｛X9， X10， X11， X12｝ ｛X4｝ ｛X1， X3｝.

As revealed， there were 1-4 basic events in a minimal path set， in which the undesired event would not occur if all the basic events in the minimal path sets would not happen. Each preventive measure， in turn， was identified from the minimal path sets that could be used to prevent epidemic hemorrhagic fever. In this study， a total of five minimal path sets were identified， representing at least five approaches that could prevent the undesired event （Table 3）.

Table 3. Measures to Prevent Epidemic Hemorrhagic Fever Accidents

The five different preventive measures in terms of feasibility and efficiency were prioritized. As a result， being vaccinated was considered as the most effective approach to preventing epidemic hemorrhagic fever， and other preventive measures included improving training for laboratory personnel，compliance with standards and regulations，installation of adequate equipment in animal facilities and adornment of PPE. Among the preventive measures in the minimal path sets，however， avoiding the production of biological aerosols was the least feasible.

Structural Importance Analysis Structural importance analysis was performed to prioritize each basic fault for its importance to the undesired top event， and the impact on the failure rates in the fault tree. According to the minimal cut sets， the order of basic faults for their importance to the top event was as follows： I ［X4］＞ I ［X1］＞ I ［X2］ = I ［X3］＞ I［X5］ = I ［X6］ = I ［X7］ = I ［X8］ = I ［X9］ = I ［X10］ = I ［X11］= I ［X12］. As displayed above， the basic fault， not being vaccinated against epidemic hemorrhagic fever affected the occurrence of the top event most，followed by the two basic faults： purchase of animals that did not comply with standards and regulations，and location of facilities within areas of disease outbreaks， which were more important to the top event than the following basic faults of wearing unsuitable PPE and exposure to the infected animals.

Main Findings and Suggestion

In this study， 12 basic elements were analyzed and the possibilities of each element were assessed using a fault tree diagram. Next， FTA was conducted，and 32 minimal cut sets and 5 minimal path sets were found to be associated with the epidemic hemorrhagic fever. Finally， the structural importance was analyzed， and results showed that not receiving a vaccination affected the occurrence of the top event more than any other factor did.

To the best of our knowledge， this is the first report that utilizes FTA to analyze the undesired infection in the field of biomedicine. Similar to FTA for safety issues occurring in different industries， all factors leading to the infection， such as facility，animals， handling procedures， skills and management were investigated and analyzed. Our main findings in this study are summarized and discussed below.

Firstly， laboratory management should be strengthened to ensure no pathogens are transmitted by laboratory animals. Having built upon our analysis in this study， we suggest the following preventive measures： （1） Laboratory animals should be purchased in compliance with the national standards and regulations as described in GB14922.2-2011［28］. In particular， experimental mice and rats should not carry hantavirus. Although the strict regulations and standards have been issued and implemented［29］， unfortunately， in 2004 an infection event was reported as a direct consequence of a Wistar rat， that was believed to be infected at the time of purchase［4］； （2） Animal facilities and laboratory management should be improved and preventive measures should be taken to block wild rodents within the disease-outbreak regions from entering animal facilities thus transmitting infection to laboratory animals. While it may seem impossible not to have any animal facilities in the areas of disease outbreaks， the improvement and regular inspection of animal facilities， along with self-inspection， can be made to effectively prevent infection； （3） Screening and detection of viruses should be regularly conducted in order to identify infected animals as early as possible.

Secondly， operating procedures should be optimized and direct contact with animals and their wastes should be avoided. We must strengthen the training programs for laboratory personnel to improve their experimental skills to minimize exposure. In the mean time， standard operating procedures for disposal of animals and their wastes should be implemented strictly in order to reduce risk of infection as much as possible.

Thirdly， the present study showed that being vaccinated against epidemic hemorrhagic fever was the most effective measure in preventing infections for laboratory personnel. Therefore， laboratory workers who handle experimental animals should be offered vaccines against epidemic hemorrhagic fever infection. In fact， the administration of vaccines against epidemic hemorrhagic fever provides the laboratory personnel with important advantage by greatly reducing the risk of epidemic hemorrhagic fever. Bivalent inactivated vaccines， which are available to individuals in disease-outbreak areas nationwide at no cost， function by stimulating the immune system to produce a specific antibody against both type I and type II epidemic hemorrhagic fever. In 2005， an infectious incidence of epidemic hemorrhagic fever resulted in four out of five individuals being infected， with the one who was not infected having been vaccinated against epidemichemorrhagic fever［14］. Until now， there has been no information about vaccination rates among laboratory personnel working with animals， but it is estimated that the rate may remain low across the nations. The incidence noted above， along with our analysis in this study， demonstrated that the administration of the vaccines may effectively prevent infection. Of course， vaccination is the last defense against infections. Thus， we cannot completely rely on vaccination ignoring other protections.

Finally， awareness of biosafety should be raised and personal protection should be enhanced. A wealth of previous studies have demonstrated that the aerosol has been the most common way to cause infection， but aerosol generation would be difficult to avoid. Since the proper use of PPE is very important， funds to purchase adequate PPE should be provided， and the training program for laboratory personnel on proper use of PPE should be offered.

#Correspondence should be address to ZHAO Chi Hong. Tel： 86-10-58900336， E-mail： zhaoch@chinacdc.cn

Biographical note of the first author： LIU Xiao Yu，female， majoring in pathogen biology.

Received： July 1， 2016；

Accepted： September 2， 2016

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*This research was supported by Liaoning Nature Science Foundation of China （project number 2015020466）； and by the National Natural Science Foundation of China （project number 81072311）.

1. Department of Child and Adolescent Health， School of Public Health， China Medical University， Shenyang 110122，Liaoning， China； 2. Department of Sports Medicine and Joint Surgery， the First Affiliated Hospital of China Medical University， Shenyang 110001， Liaoning， China