This dataset includes data from a study investigating how developmental exposure to an environmental PCB mixture, the MARBLES mix, affects post-weaning dam mice bladder. The MARBLES mix mimics the relative composition of the 12 most abundant PCB congeners identified in the serum of pregnant women at higher risk of having a child with a neurodevelopmental disorder. Female dam mice were dosed with MARBLES mix (vehicle, 0.1, 1, or 6 mg/kg/day) via diet two weeks before mating with a male, and dosing continued through mating, gestation, and lactation until collection (67 ± 10 days dosed). PCBs and hydroxylated PCBs (OH-PCBs) levels were quantified in bladder, liver, blood, adipose, and urine. Cytochrome P450 (CYP) expression was measured in bladder and liver. This dataset provides each PCB and OH-PCB concentration in different tissue types, as well as the quality assurance and quality control (QA/QC) results. CYP expressions in bladder and liver were also reported in the dataset.

Animal exposure

All animal procedures were conducted in accordance with the NIH Guide for the Care and Use of Laboratory Animals and were approved by the University of Wisconsin-Madison Animal Care and Use Committee (protocol #V006099). The study design and conduct of the animal study have been reported following the ARRIVE guidelines in earlier publications. Briefly, C57BL/6J mice (RRID: IMSR_JAX:000664, Jackson Labs, Bar Harbor, ME, USA) were housed in clear plastic cages with corn cob bedding. Feed (Diet #2019 for breeders and 2020x for maintenance; Teklad, Indianapolis, IN, USA) and water were available ad libitum. Housing was maintained on a 12 h light and dark cycle at 22 ± 2 °C.

Female nulliparous mice (n=11-13), aged approximately 50 ± 8 days, were exposed to 0 (vehicle control), 0.1 (low dose), 1.0 (medium dose), or 6.0 mg/kg (high dose) MARBLES mix per day. These doses have been shown to produce biological effects in both dams and their offspring, without causing overt toxicity or adversely affecting reproductive outcomes. Body mass was also not altered following exposure to the MARBLES mix. For PCB administration, a 20 mg/mL PCB mix stock in organic peanut oil (Spectrum Organic Products, LLC, Melville, NY) was diluted in peanut butter (Trader Joe’s, Monrovia, CA) to make working stocks of 0.025, 0.25, and 1.5 mg PCB/g peanut butter to give to mice for oral daily consumption. Mice were dosed for two weeks, were then paired with a male, and dosing continued through mating, gestation, and lactation until collection (67 ± 10 days dosed).

To refine and reduce the number of animals, the tissues used here were generated as part of a larger study aimed at producing offspring. Additionally, to add relevance to voiding function, some dams underwent voiding assays (void spot assay, uroflow, and anesthetized cystometry) before being euthanized. Bladder and liver were immediately collected following euthanasia, weighed, snap frozen, and stored at -80°C. Blood was collected via cardiac puncture and placed in glass vials containing 80 μL of 7.5% EDTA, then stored at -80°C. Urine was collected as free catch urine upon scuffing animals one day prior to voiding testing/collection, or was collected from a plexiglass plate after mice freely urinated through a grid during uroflow testing. Urine was stored in glass vials at -80°C. For the bladder, samples from 2-3 animals were pooled (n=2-4 pools per exposure group) to generate enough tissue for PCB and metabolite quantification. Some of the pooled samples contain 1 tissue from a dam who failed to take care of their litter to weaning, but continued to be dosed to the wean date.  All other tissues were not pooled, with n=5-6 mice per experimental group.

PCB and OH-PCB extraction from tissues.

A liquid-liquid extraction method was used to simultaneously extract and analyze PCBs and OH-PCBs. Briefly, liver (28±4 mg, n=5-6 mice/exposure group), adipose tissue (143±18 mg, n=5-6 mice/exposure group), or pooled bladders (45±8 mg, n=2-4 pools/exposure group) were homogenized with 3 mL 2-propanol using a TissueRuptor (QIAGEN, Hilden, Germany). After spiking 10 ng PCB15 and PCB117 and 10 ng OH-PCB (4'-OH-PCB9, 4-OH-PCB91, and 4'-OH-PCB159) as surrogate standards to all samples, PCBs and OH-PCBs were extracted with diethyl ether and hexane (1:9, v/v), followed by washing the organic extracts with 5 mL of 0.1 M phosphoric acid in 0.9% sodium chloride solution. The extracts were concentrated under a gentle stream of nitrogen and derivatized with diazomethane at 4°C overnight.(Kania-Korwel et al., 2008) The extracts were passed through a sulfuric acid and silica gel (1:2, w/w) cartridge for further cleanup. Finally, the extracts were concentrated under gentle nitrogen flow and spiked with the internal standard (d-PCB30 and PCB204) for gas chromatography with tandem mass spectrometry (GC-MS/MS) analysis.

Blood samples (415±15 mg, n=5-6 mice/exposure group) were extracted similarly, but with slight modifications. Briefly, 1 mL of 6 M HCl was added to the serum. After spiking the surrogate standard, PCB and OH-PCB were extracted using a 2-propanol and hexane: methyl tert-butyl ether (MTBE) (1:1, v/v) mixture. After washing with 3 mL 1% KCl, the extracts were concentrated and derivatized as described above. After further cleanup with 2-propanol and tetrabutylammonium hydrogen sulfate (TBA), the extracts were subjected to the same cleanup steps as described for the extraction of bladder tissue.

Twenty µL of sulfatase (type H-2 from Helix pomatia, Sigma-Aldrich, St. Louis, MO, USA) was added to pooled urine samples (282±146 mg, n=5-6 mice/exposure group) to convert PCB conjugates to OH-PCBs in a shaking water bath for 16 h at 37 °C. The extraction of PCBs and OH-PCBs in urine samples after deconjugation followed the same procedure as described above for blood extraction.

GC-MS/MS analysis

PCBs and OH-PCBs were quantified by a GC-MS/MS system (Agilent 7890B GC system, Agilent 7000D Triple Quad, Agilent 7693 autosampler) equipped with an SPB-Octyl capillary column (50% n-octyl/50% methyl siloxane, 30 m length, 0.25 mm inner diameter, 0.25 µm film thickness; Supelco, Bellefonte, PA). Helium was used as the carrier gas (0.8 mL/min), and nitrogen was used as the collision gas. The gas chromatograph was operated in solvent vent injection mode with the following setup: initial temperature, 45 °C; initial time, 0.06 min; ramp, 600 °C/min to inlet temperature of 325 °C at 5 psi. The oven temperature program of the gas chromatograph was 45 °C for 2 min, 45 to 75 °C at 100 °C/min, hold for 5 min, 75 to 150 °C at 15 °C/min, hold for 1 min, 150 to 280 at 2.5 °C/min, and hold 5 min. The triple quadrupole electron ionization source was set to 230 °C. Congener mass was quantified by applying a relative response factor obtained from the calibration standard for each congener.

Quality assurance/quality control (QA/QC) for PCB extraction.

Method blanks, tissues from vehicle-only exposed animals, and ongoing precision and recovery (OPR) standards were extracted in parallel to the samples with each sample batch to ensure the rigor and reproducibility of the analyses. Analyte masses were corrected based on the recoveries of their corresponding surrogate standards (SS). PCB15 and PCB117 were used as SS for PCBs, while 4′-OH-PCB9, 4-OH-PCB91, and 4′-OH-PCB159 served as SS for OH-PCBs. Deuterium-labeled d-PCB30 (CDN Isotopes, Quebec, Canada) and PCB204 were used as internal standards (volume correctors).

RT-qPCR in the bladder and liver.

The Cytiva RNAspin isolation kit was used according to the manufacturer’s instructions (Fisher Scientific, Waltham, MA, USA) to isolate RNA from frozen bladder and liver tissue. Briefly, tissues were homogenized in lysis buffer containing 10 µL of molecular grinding resin (G Biosciences, St Louis, MO, USA). cDNA was generated from 350 ng of RNA using GoScript Reverse Transcription Kit according to the manufacturer’s instructions (Promega, Madison, WI, USA). Real-time PCR was performed in 12 µL reactions consisting of 6 µL of SsoFast EvaGreen Supermix (Bio-Rad, Hercules, CA, USA), 0.5-1 µL of a combined 10 µM stock of forward and reverse primers, 3-3.5 µL of water, and 2 µL of cDNA. Primer3 or NCBI Primer Design and Primer BLAST were used to design and confirm primer targets. Primers were ordered from IDT (Coralville, IA, USA).

Primer efficiency was assessed through serial dilutions of cDNA to determine the optimal annealing temperature. Efficiencies of 90 to 111% were obtained for all primers used. Table S8 lists primer sequence and annealing temperatures. Samples were run in triplicate on a CFX Maestro real-time system with Bio-Rad CFX software. The PCR protocol consisted of 95 °C for 2 minutes, then 39 cycles of 95 °C for 5 s, annealing temperature specified in Table S8 for 30 s, followed by a final 95 °C for 5 s, 65 °C for 5 s, and ramp to 95 °C for 5 s to generate a melt curve. Relative mRNA abundance was determined using the delta CT method as described (Livak and Schmittgen, 2001), and normalized to Ppia (bladder) or Pgk1 (liver) abundance. Some low-abundance transcripts did not amplify in all samples, or outliers were detected as described in the statistics section. In the final analysis, a total of n=3-5 mice per exposure group were used.

Statistical Analysis.

Statistics were performed using GraphPad Prism (version 10.0.3, RRID:SCR_002798). PCB levels were normalized by tissue wet weight. To compare the PCB and OH-PCB profiles of two groups, the similarity coefficient cos θ (ranging from 0-1). For qPCR, data were assessed for normality using the Shapiro-Wilk and Kolmogorov-Smirnov tests. If the data passed normality tests, a one-way ANOVA with Tukey’s post hoc test was performed; if the data failed to pass normality tests, a Kruskal-Wallis test with Dunn’s multiple comparisons test was used. If the data passed the normality test but did not pass Bartlett’s test for assumptions of equal standard deviation, Welch’s ANOVA was performed. Prism’s ROUT method was used to identify and remove outliers. P values < 0.05 were considered significant and are indicated by an asterisk in the figures.