Lactobacillus crispatus 7-4 Mitigates Salmonella typhimurium-Induced Enteritis via the γ­Glutamylcysteine-Mediated Nrf2 Pathway.

Salmonella typhimurium (S. typhimurium) constitutes a major public health concern. We have previously proven that Lactobacillus crispatus 7-4 (L. crispatus 7-4) can inhibit the growth of S. typhimurium and thus can be used as a biocontrol strategy to suppress foodborne S. typhimurium infections. However, the inhibitory effect and in-depth mechanism of L. crispatus 7-4 remain to be elucidated. In this study, we found that L. crispatus 7-4 can protect against S. typhimurium-induced ileum injury by promoting intestinal barrier integrity, maintaining intestinal mucosal barrier homeostasis, and reducing intestinal inflammatory response. Furthermore, we demonstrated that this probiotic strain can increase the abundance of Lactobacillus spp. to maintain microbial homeostasis and simultaneously increase the amount of γ­glutamylcysteine (γ-GC) by activating the glutathione metabolic pathway. The increased γ-GC promoted the transcription of Nrf2 target genes, thereby improving the host antioxidant level, reducing reactive oxygen species (ROS) accumulation, and removing pro-inflammatory cytokines. In other words, L. crispatus 7-4 could activate the enterocyte Nrf2 pathway by improving γ-GC to protect against S. typhimurium-induced intestinal inflammation and oxidative damage.

Perinatal bisphenol S exposure exacerbates the oxidative burden and apoptosis in neonatal ovaries by suppressing the mTOR/autophagy axis.

Bisphenol S (BPS) is an emerging environmental endocrine disruptor capable of crossing the placental barrier, resulting in widespread exposure to pregnant women due to its extensive usage. However, the impact of perinatal maternal exposure to BPS on reproductive health in offspring and the underlying molecular mechanism remain underexplored. In this study, gestational ICR mice were provided with drinking water containing 3.33 mg/L BPS to mimic possible human exposure in some countries. Results demonstrated that BPS accelerated the breakdown of germ-cell cysts and the assembly of primordial follicles in neonates, leading to oocyte over-loss. Furthermore, the expression levels of folliculogenesis-related genes (Kit, Nobox, Gdf9, Sohlh2, Kitl, Bmp15, Lhx8, Figla, and Tgfb1) decreased, thus compromising oocyte quality and disrupting early folliculogenesis dynamics. BPS also disrupted other aspects of offspring reproduction, including advancing puberty onset, disrupting the estrus cycle, and impairing fertility. Further investigation found that BPS exposure inhibited the activities and expression levels of antioxidant-related enzymes in neonatal ovaries, leading to the substantial accumulation of MDA and ROS. The increased oxidative burden exacerbated the intracellular apoptotic signaling, manifested by increased expression levels of pro-apoptotic markers (Bax, Caspase 3, and Caspase 9) and decreased expression levels of anti-apoptotic marker (Bcl2). Concurrently, BPS inhibited autophagy by increasing p-mTOR/mTOR and decreasing p-ULK1/ULK1, subsequently down-regulating autophagy flux-related biomarkers (LC3b/LC3a and Beclin-1) and impeding the degradation of autophagy substrate p62. However, the imbalanced crosstalk between autophagy, apoptosis and oxidative stress homeostasis was restored after rapamycin treatment. Collectively, the findings demonstrated that BPS exposure induced reproductive disorders in offspring by perturbing the mTOR/autophagy axis, and such autophagic dysfunction exacerbated redox imbalance and promoted excessive apoptosis. These results provide novel mechanistic insights into the role of autophagy in mitigating BPS-induced intergenerational reproductive dysfunction.

Microencapsulate Probiotics (MP) Promote Growth Performance and Inhibit Inflammatory Response in Broilers Challenged with Salmonella typhimurium.

Antibiotic-resistant bacteria are prevalent in husbandry around the world due to the abuse of antibiotic growth promoters (AGPs); therefore, it is necessary to find alternatives to AGPs in animal feed. Among all the candidates, probiotics are promising alternatives to AGPs against Salmonella infection. The anti-Salmonella effects of three probiotic strains, namely, Lactobacillus crispatus 7-4, Lactobacillus johnsonii 3-1, and Pediococcus acidilactici 20-1, have been demonstrated in our previous study. In this study, we further obtained the alginate beads containing compound probiotics, namely, microencapsulate probiotics (MP), and evaluated its regulatory effect on the health of broilers. We incubated free and microencapsulate probiotics in simulated gastric and intestinal juice for 2 h, and the results showed that compared to free probiotics, encapsulation increased tolerance of compound probiotics in the simulated gastrointestinal condition. We observed that the application of probiotics, especially MP, conferred protective effects against Salmonella typhimurium (S.Tm) infection in broilers. Compared to the S.Tm group, the MP could promote the growth performance (p < 0.05) and reduce the S.Tm load in intestine and liver (p < 0.05). In detail, MP pretreatment could modulate the cecal microflora and upregulate the relative abundance of Lactobacillus and Enterobacteriaceae. Besides, MP could reduce the inflammation injury of the intestine and liver, reduce the pro-inflammatory cytokines (IL-6, TNF-α, IL-1ß) expression, and induce of anti-inflammatory cytokine (IL-10) expression. Furthermore, MP could inhibit NLRP3 pathway in ileum, thereby attenuating S.Tm-induced inflammation. In conclusion, MP could be a new feeding supplementation strategy to substitute AGPs in poultry feeding.

G protein-coupled estrogen receptor activation by bisphenol-A disrupts lipid metabolism and induces ferroptosis in the liver.

As a metabolic disruptor, bisphenol A (BPA) has been widely reported to disrupt lipid balance. Moreover, BPA has gained significant attention due to its estrogenic activity. While both ferroptosis and the G-protein-coupled estrogen receptor (GPER) have been implicated in lipid metabolism, their link to BPA-induced lipid accumulation remains unclear. In this study, chickens were randomly assigned to three groups and housed them for 4 weeks: a control group (0 µg/L BPA), a low dose group (50 µg/L BPA) and a high dose group (5000 µg/L BPA) to investigate the underlying mechanism of BPA-induced hepatotoxicity. Our results showed that BPA exposure significantly increased the contents of TG, TC, and LDL-C while decreasing HDL-C levels. We also found that BPA treatment altered the levels of genes involved in fatty acid ß-oxidation (ampkα, cpt-1, and ppaα), synthesis (acc, fas, scd-1, and srebp-1) and absorption (lpl and cd36). Moreover, the results showed that the BPA group had higher levels of IL-1ß, IL-18 and TNF-α. These results indicated that BPA exposure disrupted lipid metabolism and induced inflammation in the liver. We also demonstrated that BPA caused hepatic ferroptosis by raising iron content and the expression of genes related to lipid peroxidation (lpcat3, acsl4 and alox15), while reducing the expression of antioxidant system-associated genes (gpx4, slc7a11 and slc3a2). Importantly, BPA remarkably activated GPER expression in the liver. Interestingly, inhibition of GPER remarkably ameliorated BPA-induced lipid metabolism disorder, inflammatory response, and ferroptosis, indicating the crucial role of GPER in BPA-induced liver abnormalities. These findings highlight the link between GPER and ferroptosis in BPA-induced hepatotoxicity, providing new insights into the potential hazard of BPA.

The gut microbiota metabolite glycochenodeoxycholate activates TFR-ACSL4-mediated ferroptosis to promote the development of environmental toxin-linked MAFLD.

Metabolic dysfunction-associated fatty liver disease (MAFLD) has become the most common chronic liver disorders in the world, and yet has no approved pharmacotherapy due to the etiology is complex. In the last ten years, increasing evidence have identified the environmental pollutants as risk factors for MAFLD. However, the underlying mechanism remains unclear. Our study found that bromoacetic acid (BAA, a typical kind of environmental toxin) increased triglycerides and total cholesterol levels as well as induced obvious hepatic steatosis and inflammation. The lipidomics showed that ferroptosis was implicated in the environmental toxin-linked MAFLD. Besides, the analysis of microbial metabolomics showed significant change of gut microbiome in BAA groups and the content of gut microbiota metabolite (glycochenodeoxycholate, GCDCA) increased sharply. In vitro study, we observed features of ferroptotic cells by transmission electron microscopy after BAA/GCDCA treatment. Besides, we demonstrated that BAA/GCDCA significantly increased iron contents, with upregulating transferrin receptor (TFR) and acyl-CoA synthetase long-chain family 4 (ACSL4) expression levels. By contrast, iron chelator or silencing TFR relieved BAA/GCDCA-induced lipid metabolism disorder and inflammation. What's more, the interaction between TFR and ACSL4 was also identified. Taken together, we found that, in response to environmental toxin, gut microbiota metabolite GCDCA activates TFR-ACSL4-mediated ferroptosis, which triggered subsequent lipid metabolism disorder and inflammation. Moreover, these findings firstly highlighted the functional relevance among ferroptosis, lipid metabolism and gut microbiota metabolite during environmental pollutant exposure, which shed light on the deep mechanism of environmental toxin-related MAFLD, providing potential targets for the prevention of MAFLD.

Estrogen receptor ß activation inhibits colitis by promoting NLRP6-mediated autophagy.

Estrogen receptor ß (ERß) and NOD-like receptor family pyrin domain containing 6 (NLRP6) are highly expressed in intestinal tissues. Loss of ERß and NLRP6 exacerbate colitis in mouse models; however, the underlying mechanisms are incompletely understood. Here, we report that ERß directly activates the NLRP6 gene expression via binding to estrogen responsive element of Nlrp6 gene promoter. ERß also physically interacts with the NLRP6 nucleotide-binding domain and promotes NLRP6 inflammasome assembly. The ERß-NLRP6 axis then interacts with multiple autophagy-related proteins, including ULK1, BECN1, ATG16L1, LC3B, and p62, and affects the autophagosome biogenesis and autophagic flux. Finally, NLRP6-mediated autophagy suppresses the inflammatory response by promoting the K48-linked polyubiquitination of ASC, Casp-1 p20, IL-1ß, TNF-α, and prohibitin-2. Thus, ERß-NLRP6 direct an anti-inflammatory response by promoting autophagy. Our work uncovers an ERß-NLRP6-autophagy pathway as a regulatory mechanism that maintains intestinal epithelial cell homeostasis and facilitates tissue repair in colitis.

Lactobacillus crispatus-derived exopolysaccharides with antibacterial activity limit Salmonella typhimurium invasion by inhibiting inflammasome-mediated pyroptosis.

In this study, a novel heteropolysaccharide (EPS 7-4) with a molecular weight of 53 387 Da was isolated from Lactobacillus crispatus, and it was mainly composed of mannose (36.9%) and glucose (30.8%). EPS 7-4 showed excellent inhibitory effects on the proliferation, biofilm formation, and virulence factor gene expression of Salmonella typhimurium (S. typhimurium) by disrupting the integrity of the bacterial wall. Furthermore, EPS 7-4 can effectively restrict bacterial translocation, upregulate the abundance of Lactobacillus spp. and Bifidobacterium spp., and alleviate the S. typhimurium induced severe inflammatory response in the intestinal tract of mice. Besides, we demonstrated that EPS 7-4 can protect mice by inhibiting S. typhimurium induced pyroptosis, with the mechanism that EPS 7-4 affects ASC oligomerization during inflammasome-mediated pyroptosis. Therefore, due to its excellent anti-bacterial and anti-inflammatory abilities, EPS 7-4 is a promising health regulator owing to its excellent antibacterial and anti-inflammatory abilities.

Bromoacetic acid causes oxidative stress and uric acid metabolism dysfunction via disturbing mitochondrial function and Nrf2 pathway in chicken kidney.

Since the outbreak of COVID-19, widespread utilization of disinfectants has led to a tremendous increase in the generation of disinfection byproducts worldwide. Bromoacetic acid (BAA), one of the common disinfection byproducts in the environment, has triggered public concern because of its adverse effects on urinary system in mammals. Nevertheless, the BAA-induced nephrotoxicity and potential mechanism in birds still remains obscure. According to the detected content in the Taihu Lake Basin, the model of BAA exposure in chicken was established at doses of 0, 3, 300, 3000 µg/L for 4 weeks. Our results indicated that BAA exposure caused kidney swelling and structural disarrangement. BAA led to disorder in renal function (CRE, BUN, UA) and increased apoptosis (Bax, Bcl-2, caspase3). BAA suppressed the expression of mitochondrial biogenesis genes (PGC-1α, Nrf1, TFAM) and OXPHOS complex I genes (ND1, ND2, ND3, ND4, ND4L, ND5, ND6). Subsequently, BAA destroyed the expression of Nrf2 antioxidant reaction genes (Nrf2, Keap1, HO-1, NQO1, GCLM, GCLC). Furthermore, renal oxidative damage led to disorder in uric acid metabolism genes (Mrp2, Mrp4, Bcrp, OAT1, OAT2, OAT3) and exacerbated destruction in renal function. Overall, our study provided insights into the potential mechanism of BAA-induced nephrotoxicity, which were important for the clinical monitoring and prevention of BAA.

Bromoacetic acid induces neurogenic injury in the chicken brain by activating oxidative stress and NF-κB inflammatory pathway.

The bromoacetic acid (BAA) is one of the most teratogenic and neurotoxic disinfection byproducts. Birds take environmental water as their habitat and are inevitably affected by BAA in the environment. However, the neurotoxicity caused by BAA in birds has not been reported and the mechanism remains unclear. In this study, we chose chickens as the avian model to explore the effects of different concentrations of BAA on the brain tissues. Here, we selected the 3 µg/L dose of BAA detected in Tai Lake basin as a reference, and designed 1-, 100-, and 1000-fold of the environmental exposure dose as the experimental doses to explore the neurotoxicity of BAA in birds. Results showed that BAA increased the number of pyknotic nuclear neurons, deformed vascular sheaths, and glial cells in the brain. BAA inhibited the activity of antioxidant enzymes and the expression of antioxidant genes. With the increase of BAA concentration, the oxidative stress-responsive transcription factor NF-κB was activated. Furthermore, BAA remarkably changed the expression of lipid metabolism related genes (i.e., acc, gpat, hmgr, pparα, cpt1, and ampkα). Importantly, BAA decreased the mRNA and protein expression levels of autophagy-related genes (i.e., atg5, ulk1, beclin1, and lc3). Meantime, BAA increased the mRNA and protein levels of apoptotic and pro-apoptotic genes, such as p53, bax, cytochrome c, caspase-9, and caspase-3. Overall, our study provided new insights into the potential neurotoxic effects of BAA in birds, which was important for the clinical monitoring and prevention of BAA.

Fumonisin B1 induced intestinal epithelial barrier damage through endoplasmic reticulum stress triggered by the ceramide synthase 2 depletion.

Fumonisin B1 (FB1) contamination in feed is of great concern nowadays. The intestine would be the first line when FB1-contaminated food or feed was ingested. However, the intestinal toxicity and mechanism of FB1 have rarely been studied. In this study, we found that FB1 inhibited cell viability, and promoted the severe release of lactate dehydrogenase. Meantime, FB1 destroyed the intestinal physical barrier by reducing the expressions of tight junctions. And FB1 induced excessive production of cytokines like tumor necrosis factor-α, resulting in damage to the intestinal immunological barrier. Furthermore, we observed that FB1 preferentially inhibited the expressions of ceramide synthase 2 (CerS2) and upregulated the expression of endoplasmic reticulum (ER) stress markers. The siRNA-mediated knockdown of CerS2 and CerS2 overexpression proved that CerS2 depletion induced by FB1 triggered ER stress, which then destructed the intestinal barrier. FB1-induced intestinal impairment could be restored by CerS2 over-expression or 4-Phenylbutyric acid (ER stress inhibitor). Overall, our findings demonstrated intestinal toxicity and potential mechanism of FB1, and the intestinal impairment risk posed by FB1 must be taken seriously.

Testicular toxicity of bisphenol compounds: Homeostasis disruption of cholesterol/testosterone via PPARα activation.

The widespread application of bisphenols (BPs) has made them ubiquitous in the environment. Although the side effects of bisphenol A (BPA) substitutes have received increasing attention, studies on their reproductive toxicity remain lacking. In this research, the effects of BPA and its substitutes, including bisphenol S (BPS), bisphenol F (BPF), and bisphenol AF (BPAF), on the male reproductive system were evaluated. Results proved that these BPs disturbed germ cell proliferation, induced germ cell apoptosis, and perturbed sperm physiologies and spermatogenesis, which resulted from the disruption of testosterone (T) biosynthesis in Leydig cells (LCs). Importantly, in vitro and in vivo studies indicated that the exhausted cholesterol in LCs accounted for the reduced T production. Furthermore, the knockdown of peroxisome proliferator-activated receptor alpha (PPARα) remarkably ameliorated the downregulation of cholesterogenesis-related genes (i.e., Hmgcs1, Hmgcr, and Srebf2), indicating that PPARα played a critical role in BPs-induced testicular dysfunction. Overall, our studies indicated that BPS, BPF, and BPAF could induce testicular toxic effects similar to that of BPA, which were associated with the PPARα pathway.

Pathogenicity and virus shedding ability of fowl adenovirus serotype 4 to ducks.

Fowl adenovirus serotype 4 (FAdV-4) is the pathogen causing hepatitis-hydropericardium syndrome (HHS) in broilers. Since June 2015, it has emerged as one of the leading causes of economic losses in the poultry industry in China. Although most studies on FAdV-4 have focused on its pathogenicity to broilers, limited studies have been performed on other natural hosts such as ducks and geese. In this study, we assessed the pathogenicity of FAdV-4 to ducks of different ages through intramuscular injection and found that infected ducks showed severe growth depression. The infected ducks also suffered from extensive organ damage and had histopathological changes in the liver, spleen, and kidney. Although the virus infection caused lymphocyte necrosis of immune organs and the development of the bursa of Fabricius (bursa) was inhibited, the humoral immune response of infected ducks to FAdV-4 remained strong. The infected ducks also had high viral load in tissues and shed virus after the challenge. Overall, our research demonstrates that FAdV-4 can infect ducks and adversely affect the productivity of animals. And the viruses shed by infected ducks can pose a potential risk to the same or other poultry flocks.

Estrogen Receptor α Regulates Metabolic-Associated Fatty Liver Disease by Targeting NLRP3-GSDMD Axis-Mediated Hepatocyte Pyroptosis.

Metabolic-associated fatty liver disease (MAFLD) is currently one of the main causes of chronic liver disease, but its potential mechanism remains unclear. This study proved that estrogen receptor α (ERα) could negatively control hepatocyte pyroptosis by inhibiting NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome activation, gasdermin D (GSDMD)-N generation, propidium iodide (PI) uptake, lactate dehydrogenase (LDH) release, and pro-inflammatory cytokine (IL-1ß and IL-18) release. Furthermore, inhibition of pyroptosis ameliorated ERα deletion-induced metabolic dysfunction, insulin resistance, and liver injury. Mechanistically, ERα was confirmed to inhibit pyroptosis by directly interacting with GSDMD, and GSDMD blockade reversed the ERα inhibition-induced pyroptosis and improved lipid accumulation in hepatocytes. Notably, the treatment of wild-type (WT) mice with genistein, a phytoestrogen, could attenuate high-fat diet (HFD)-induced liver lipid steatosis and inhibit NLRP3-GSDMD-mediated pyroptosis. Results provide new insights into the underlying mechanism of pyroptosis regulation and uncover the potential treatment target of MAFLD.

Lactobacillus johnsonii 3-1 and Lactobacillus crispatus 7-4 promote the growth performance and ileum development and participate in lipid metabolism of broilers.

Long-term use of antibiotic growth promoter (AGP) in animal production is the main cause of antimicrobial resistance of pathogenic bacteria. Therefore, seeking alternatives to AGP is crucial for animal husbandry. Among all AGP alternatives, probiotics are promising candidates. In this study, two strains of lactic acid bacteria, L. johnsonii 3-1 and L. crispatus 7-4, were isolated from the feces of wild Gallus gallus, which exhibited obvious anti-pathogenic activity and improved the growth performance of broilers. Furthermore, we found that these two strains participated in the lipid metabolism of broilers by reducing the content of TC and TG in ileal epithelial cells and up-regulating the liver AMPKα/PPARα/CPT-1 pathway, which affects abdominal fat deposition. In summary, L. johnsonii 3-1 and L. crispatus 7-4 have the potential to be used as AGP substitutes and participate in the lipid metabolism of broilers to reduce abdominal fat deposition. Importantly, our study reveals for the first time that L. crispatus participates in liver lipid metabolism to reduce abdominal fat deposition in broilers.

Bisphenol A and genistein have opposite effects on adult chicken ovary by acting on ERα/Nrf2-Keap1-signaling pathway.

The reproductive toxicity of endocrine-disrupting chemicals has become a matter of great concern. However, the potential toxicological mechanism of typical environmental estrogens, bisphenol A (BPA) and genistein (GEN), on adult ovary remains ambiguous. In this study, we used laying hens as the experimental model and aimed to clarify the effect of long-term exposure to safe reference doses of BPA and GEN on adult ovary. Results demonstrated that 1/10 no-observable-adverse effect-level dose (1/10 NOAEL, 500 µg/kg body weight [bw]/day) of BPA significantly reduced the production performance and caused the degeneration of follicles and stromal cells and the increase of atretic follicles. Moreover, 1/10 NOAEL dose of BPA undermined the redox homeostasis of the ovary through activating Keap1 and suppressing the Nrf2-signaling pathway (Nrf2, NQO1, and HO-1). On the contrary, GEN (20, 40 mg/kg bw/day) dramatically improved the antioxidant capacity of the ovary by regulating the Nrf2-Keap1 pathway, enhancing the activities of antioxidant-related enzymes (CAT, GSH-Px, and T-SOD), and inhibiting the excessive accumulation of lipid peroxidation products (MDA). Parallel in vitro studies confirmed that the differential role of BPA and GEN on ovarian redox balance was directly mediated by Nrf2-Keap1 antioxidant system. And GEN could ameliorate BPA-induced oxidative stress. Importantly, our research found that exposure to BPA and GEN altered estrogen receptor alpha (ERα) expression in the ovary. And the use of specific ERα agonist/antagonist confirmed that BPA and GEN have opposite regulatory effects on the Nrf2-Keap1 pathway by targeting ERα.

Comparative effects of genistein and bisphenol A on non-alcoholic fatty liver disease in laying hens.

Bisphenol A (BPA) and genistein (GEN) are selective estrogen receptor modulators, which are involved in the occurrence and development of metabolic syndrome. However, their roles in non-alcoholic fatty liver disease (NAFLD) of laying hens have not been reported. Here, we investigated the effects of different concentrations of GEN and BPA on the NAFLD of laying hens. Results showed that GEN ameliorated the high-energy and low-protein diet (HELP)-induced NAFLD by improving pathological damage, hepatic steatosis, and insulin resistance and blocking the expression of NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome-related factors. By contrast, high dose of BPA could aggravate these changes with serious symptom of NAFLD and suppress the level of ERα in the liver considerably, while GEN could reverse this phenomenon in a dose-dependent manner. In general, our research shows that the protective effect of GEN on NAFLD aims to improve the metabolic disorders and inflammation closely connected to ERα, while BPA can inhibit the expression of ERα and exacerbate the symptom of NAFLD. In conclusion, we elucidate the opposing effects of GEN and BPA in NAFLD of laying hens, thus providing a potential mechanism related to ERα and inflammation.

Effects of bisphenol A at the safe reference dose on abdominal fat deposition in aged hens.

Bisphenol A (BPA) is an endocrine-disrupting chemical. Its influence on lipid homeostasis remains to be proven. In this study, the obese model of laying hens were induced using high-fat diet (HFD) to determine the lipid metabolism interference of BPA, especially its influence on estrogen receptors (ERs) and oxidative damage, at the dose of tolerable daily intake (TDI, 50 µg/kg body weight [BW]/day) and no observable adverse effect level (NOAEL, 5000 µg/kg BW/day). The results demonstrated that the TDI dose of BPA interacted with ERα more effectively than the NOAEL dose of BPA. The TDI dose of BPA increased the expression of ERα (esr1), which further changed the expression of lipid metabolism-related genes, such as cpt-1, lpl, creb1, and apov1. Furthermore, the abdominal fat rate, hematoxylin-eosin staining of adipocytes, and the average area of the hens were reduced. Therefore, the TDI dose of BPA played an estrogen-compensating role and weakened the effect of HFD on obesity in aged hens. By contrast, BPA at NOAEL dose exhibited great oxidative stress, which remarkably inhibited the activities of antioxidant-related enzymes (total superoxide dismutase and glutathione peroxidase) and promoted the excessive accumulation of lipid peroxidation products (malondialdehyde). Moreover, the increase in oxidative stress corresponded well with the increase in the expression of fat-forming genes (srebp-1, fas, acc, and ppar γ). That is, BPA at NOAEL may accelerate the process of fat formation.