Evaluation of low doses BPA-induced perturbation of glycemia by toxicogenomics points to a primary role of pancreatic islets and to the mechanism of toxicity

Abstract

Epidemiologic and experimental studies have associated changes of blood glucose homeostasis to Bisphenol A (BPA) exposure. We took a toxicogenomic approach to investigate the mechanisms of low-dose (1 × 10(-9 )M) BPA toxicity in ex vivo cultures of primary murine pancreatic islets and hepatocytes. Twenty-nine inhibited genes were identified in islets and none in exposed hepatocytes. Although their expression was slightly altered, their impaired cellular level, as a whole, resulted in specific phenotypic changes. Damage of mitochondrial function and metabolism, as predicted by bioinformatics analyses, was observed: BPA exposure led to a time-dependent decrease in mitochondrial membrane potential, to an increase of ROS cellular levels and, finally, to an induction of apoptosis, attributable to the bigger Bax/Bcl-2 ratio owing to activation of NF-κB pathway. Our data suggest a multifactorial mechanism for BPA toxicity in pancreatic islets with emphasis to mitochondria dysfunction and NF-κB activation. Finally, we assessed in vitro the viability of BPA-treated islets in stressing condition, as exposure to high glucose, evidencing a reduced ability of the exposed islets to respond to further damages. The result was confirmed in vivo evaluating the reduction of glycemia in hyperglycemic mice transplanted with control and BPA-treated pancreatic islets. The reported findings identify the pancreatic islet as the main target of BPA toxicity in impairing the glycemia. They suggest that the BPA exposure can weaken the response of the pancreatic islets to damages. The last observation could represent a broader concept whose consideration should lead to the development of experimental plans better reproducing the multiple exposure conditions.

Figure 1

Differentially expressed genes between vehicle and 1x10⁻⁹ M BPA-treated samples for 48 h. Volcano plots of microarray data of hepatocytes (a) and islets (b). The y-axis value is the negative logarithm base 10 of the corrected P-value. A green horizontal line on the plot represents the user-defined significant threshold for P-value. The x-axis is shown as the logarithm base 2 of the fold change in expression level between treated and control. The green lines on the plot represents the user-defined significant threshold for P-value (horizontal) and fold change (vertical). Green dots are downregulated genes. (c) HeatMap showing the gene expression profile in the pancreatic islets microarray data. The expression value of each gene is mapped to a color-intensity value, as indicated by the color bar; (d) qRT-PCR validation of some inhibited genes in treated islets (I) and hepatocytes (H) compared with microarray data. Data are reported as the negative inverse of fold change value. The qRT-PCR results are expressed as the mean±SD of three independent experiments (N=3). *P-value <0.05; **P-value <0.01; ***P-value <0.001 compared with vehicle-only-treated cells

Figure 2

Time-dependent effects of BPA on transcript inhibition. Level of transcripts was determined by qRT-PCR after exposure to 1 × 10⁻⁹ M BPA for 24 h and 48 h. The selected genes were grouped by functional categories: respiratory chain subunits, Uqcrb and Ndufs4 (a); ATP-dependent pump subunits, ATP1b1 and ATP6v1f (b); ROS detoxification, Gpx3 and Sod2 (c); protein synthesis and degradation, Vapa, Iars, Zfand2a (d). Data are reported as fold change values. The results are expressed as the mean±SD of three independent experiments (N=3). *P-value <0.05; **P-value <0.01; ***P-value <0.001 compared with vehicle-only-treated islets

Figure 3

BPA impairs ROS intracellular level, mitochondrial membrane potential and islet viability. (a) ROS intracellular level as determined by CellROX reagent and (b) measurement of mitochondrial potential as determined by JC-1 reagent. For both assays, equal number of dispersed islets was cultured in the presence of 1x10⁻⁴ M, 1x10⁻⁹ M BPA or vehicle only for different times. Menadione 3x10⁻⁵ M for 30 min (in a) and 2x10⁻⁶ M CCCP (in b) were used as positive controls. Fluorescence emission was normalized on protein amount and data are reported as fold change values. (c) Murine islets cell viability determined by MTT assay. Dispersed islet cells, in equal number, were cultured in the presence of 1x10⁻⁴ M, 1x10⁻⁶ M, 1x10⁻⁹ M BPA, 25 mM glucose or vehicle only for 24 h or 48 h. Data are reported as fold change values. (d) Quantification of apoptotic islet cells by IF-TUNEL staining. Dispersed islet cells were cultured in chamber slides 1x10⁻⁴ M, 1x10⁻⁶ M, 1x10⁻⁹ M BPA, 25 mM glucose, 3x10⁻⁵ M menadione or vehicle only (DMSO) for 48 h and then visualized under a fluorescence microscope. Data are reported as percentage of TUNEL-positive cells/total cell number (DAPI staining). (e) qRT-PCR of Bax and Bcl-2 transcripts in murine pancreatic islets. Level of transcripts was determined by qRT-PCR after exposure to 1x10⁻⁹ M BPA for 24 h and 48 h. Data are reported as fold change values. All the results are expressed as the mean±SD of three independent experiments (N=3). *P-value <0.05; **P-value <0.01; ***P-value <0.001 compared with vehicle-only-treated islets

Figure 4

Pathways involved in BPA apoptosis triggering. (a) Schematic representation of transcription binding sites for fork head domain factors (FKHD), nuclear factor kappa B (NF-κB) and estrogen responsive elements (ERE) predicted by Genomatix software in Bax gene promoter fragment −1000/+50 bp; (b) qRT-PCR analyses of Bax transcript in murine pancreatic islets cultured in the presence or absence of 1 × 10⁻⁹ M BPA, 3 mM NAC, 2 × 10⁻⁵ M BMS-345541, 1 × 10⁻⁵ M LY-294002 or 1 × 10⁻⁵ M ICI-182,780, for 48 h. Data are reported as fold change values. *P-value <0.05; **P-value <0.01; ***P-value <0.001 compared with BPA-only-treated islets; (c) IF-TUNEL staining of cells co-exposed to BPA and ROS and NF-κB inhibitor. Dispersed islet cells were cultured in chamber slides in the presence or absence of 1 × 10⁻⁴ M BPA, 1 × 10⁻⁹M BPA, 3 mM NAC, 2 × 10⁻⁵ M BMS-345541 for 48 h and then visualized under a fluorescence microscope. Data are reported as percentage of TUNEL-positive cells/total cell number (DAPI staining). *P-value <0.05; **P-value <0.01; ***P-value <0.001 compared with BPA-only-treated islets; (d) IkB-α protein was determined in whole cellular extracts prepared from intact murine pancreatic islets cultured in the presence or absence of 1 × 10⁻⁴ M and 1 × 10⁻⁹ M BPA for 12 h and 18 h by western blot; (e) protein quantification of IkB-α in murine pancreatic islets cultured as already reported (d and Supplementary Figure S3). The signal intensity was determined with imageJ software and normalized on β-actin protein signal intensity. Data are reported as fold change values. *P-value <0.05; **P-value <0.01; ***P-value <0.001 compared with vehicle-only-treated islets; (f) qRT-PCR quantification of IkB-α transcript in murine pancreatic islets. Level of transcripts was determined by qRT-PCR after exposure to 1 × 10⁻⁹ M BPA for 12 h. Data are reported as fold change values. *P-value <0.05; **P-value <0.01; ***P-value <0.001 compared with vehicle-only-treated islets. All the results are expressed as the mean±SD of three independent experiments (N=3)

Figure 5

Effects of BPA on islets functionality. (a) Insulin secretion after 1 h of 16 mM glucose stimulation of islets exposed to 1 × 10⁻⁴ M, or 1 × 10⁻⁹ M BPA. Data are reported as fold change values. The results are expressed as the mean±SD of three independent experiments (N=3). *P-value <0.05; **P-value <0.01; ***P-value <0.001 compared with vehicle-only-treated islets; (b) glycemia measurements in hyperglycemic mice transplanted with pancreatic islets. The animals were intravenously injected (day −3) with STZ for hyperglycemia induction and transplanted with pancreatic islets from healthy syngenic donors, three days later (day 0). Transplanted islets were cultured for 48 h with (Gr.2, N=5 and Gr.3, N=4) or without (control group, Gr.1, N=5) 1 × 10⁻⁹ M BPA. Gr.2 animals were watered with 50 μg/kg bw/day BPA from the day of STZ injection until the day of killing. *P-value <0.05; **P-value <0.01; ***P-value <0.001 compared with control animals (Gr.1)

Figure 6

Mechanisms of BPA mitochondrial dysfunction and apoptosis in pancreatic islets. Genes deregulated upon BPA exposure as determined by microarray analysis (in red) and qRT-PCR (in green)