MATERIALS and METHODS

A. Chemicals

Solutol HS15 (Kolliphor HS15) (CAS: 70142-34-6), obtained from Sigma-Aldrich (St Louis, MO, USA) prepared as a 20 % aqueous solution (20% HS15) was used as the vehicle. Solutol was used as a vehicle for Benzo(a)pyrene studies. Deionized water was used as a vehicle for acridine orange study. Benzo(a)pyrene (CAS: 50-32-8; ≥96% pure) purchased from Sigma-Aldrich (St Louis, MO, USA) was used in the metabolomics as well as gene expression studies. Acridine orange (CAS: 10127-02-3; pure, ≥55% dye content), purchased from Acros Organics (Bridgewater, NJ, USA), was used for two photon microscopy study.

B. Egg Handling

The protocol of the study is described in detail in Williams et al. (2014) and Thakkar et al. (2024). Briefly, fertilized eggs (SPF Premium) of white leghorn chicken (Gallus gallus ) were purchased from Charles River Laboratories (North Franklin, CT). Eggs were weighed, numbered, and randomly divided into control and dosed groups (at least 10 eggs per group). On day 0 incubation day, eggs were placed in automatic egg turners and incubated in GQF Manufacturing Company Hova Bator Model 2362N Styrofoam incubators (Murray McMurray Hatchery, Webster City, IA, USA) at 37 ± 0.5 °C and 60 ± 5% humidity. Viability was assessed by transillumination on incubation day 8, and eggs that did not develop were discarded. Separate incubators were used for control and dosed eggs to avoid any possible cross contamination. Doses of compounds were selected based on available acute toxicity data (oral LD50 in rodents, extrapolated on ~60g egg). For imaging studies, acridine orange was administered at 10 μg/egg. For analysis of metabolites and genomic changes, B(a)P was injected at 250 µg/egg. Test compounds and respective vehicles were administered in total volume of 0.15 ml/egg via 3 daily injections into the air sac on incubation days 9 through 11. For metabolite and gene expression analyses, a group of naïve (non-dosed) eggs that did not receive any injections was also included. The eggs were terminated two to three hours after the last injection. The eggshells were opened, the fetuses removed and decapitated. Fetal weights, including the head, were recorded after removal of the surrounding excess yolk. Viability percentage was calculated based on the ratio of embryo-fetuses alive upon termination to the total number of embryo-fetuses in the group. The abdominal cavity was opened, and the livers were removed, weighed, and processed for further analyses.

C. Two-Photon Microscopy

Instrument Setup: Two-photon imaging of tissue samples was performed using Leica Stellaris 8 DIVE system (Leica Microsystems, Wetzlar, Germany). The microscope is equipped with a mode-locked titanium: sapphire laser for excitation, capable of delivering femtosecond pulses at the desired wavelength. The laser power and wavelength were optimized based on the fluorophores for acridine orange (460/650). The microscope was configured for both two-photon excitation and detection, allowing for deep tissue imaging with high spatial resolution.Sample Mounting: Prior to imaging, tissue samples were mounted on to a slide and a drop of water was added with coverslip mounted on top. Care was taken to ensure that the sample was securely positioned and oriented for optimal imaging.Imaging Parameters: The imaging parameters, including laser power, wavelength, scanning speed, and image resolution, were carefully optimized. Laser power was adjusted to achieve sufficient signal intensity while minimizing photobleaching and phototoxicity. The scanning speed was optimized to balance imaging speed with signal-to-noise ratio and resolution requirements. Z-stack imaging was performed to capture three-dimensional information about the tissue structure, with the step size adjusted based on the desired axial resolution.Image Acquisition: Two-photon imaging was performed using optimized parameters, with image acquisition conducted in both x, y and z dimensions. Z-stack images were acquired by scanning through the tissue volume at consecutive focal planes. Care was taken to minimize exposure to laser light and phototoxic effects on the sample during image acquisition.

D. LC-HRMS

Frozen liver samples were sent to Frontage Laboratories (Exton, PA) for the analysis using LC-HRMS with Xcalibur and Freestyle Compound Discoverer software.Sample Preparation: Liver samples were weighed in the non-skirted homogenizing tube containing 0.5 mm Zirconium and mixed with 9-fold of IPA/H2O=70:30 (weight: volume = 1g: 9 mL) followed by 45 seconds homogenization at 4000 cycles per minute. The homogenized liver samples were volume proportional pooled into three separate mixtures by treatment (untreated, solvent treated, BP treated). 100 µL of pooled sample were mixed with 200 µL organic solvent (ACN with 0.1µg/mL ISD). Then vortexed and centrifuged for 5min at 13000 rpm. Take 250 µL of supernatant and dry it down to 100 µL under N2 prior to LC/HRMS.Instrumentation: The analytical instrumentation utilized in this study consisted of a Thermo Scientific Vanquish Ultra-performance liquid chromatography (UPLC) system equipped with multiple units identified by serial numbers: 8315629, 8315641, 8315545, and 6504418. Coupled to the UPLC system was a Thermo Scientific Q Exactive mass spectrometer identified by the serial number 10374L.UPLC Conditions: For chromatographic separation, a mobile phase comprising 0.1% formic acid in water (Mobile Phase A) and 0.1% formic acid in acetonitrile (Mobile Phase B) was employed. The separation was achieved on a Phenomenex Kinetex BP column (2.6 x 100 mm) using a gradient elution program with varying percentages of Mobile Phase B over time: 5% at 0 min, 5% at 1 min, 75% at 7 min, 95% at 10 min, maintaining 95% until 12 min, returning to 5% at 12.5 min, and equilibrating at 5% until 15 min. The flow rate was set at 0.4 mL/min, and injection volumes ranged from 2 to 10 µL.Mass Spec Conditions: The mass spectrometer was operated in positive ionization mode with a spray voltage of 3.50 kV. Additional parameters included an S-lens RF level of 55, probe heater temperature set at 375°C, and capillary temperature maintained at 325°C. The sheath gas flow rate was set to 45 units, with auxiliary gas at 15 units and sweep gas at 1 unit. Mass spectra were acquired over a range of m/z 150-850 with a full MS resolution of 35,000 and an automatic gain control (AGC) target of 3e6. MS/MS experiments were conducted at a resolution of 17,500, with an AGC target of 1e5, using collision energies (CE) of 30, 40, and 55.Reagents: Reagents used in the analysis included Fisher Optima LC/MS grade solvents: water, acetonitrile, methanol, and formic acid. These reagents were chosen to ensure high purity and compatibility with the analytical instrumentation employed in this study.

E. RNA Sequencing

RNA extraction and sequencing from the liver samples were performed at Azenta Life Sciences (South Plainfield, NJ).RNA extraction: Total RNA was extracted using Qiagen Rneasy Plus Mini kit following manufacturer’s instructions (Qiagen, Hilden, Germany).Library Preparation with PolyA selection and Illumina Sequencing: Quantification RNA samples was done using Qubit 2.0 Fluorometer (Life Technologies, Carlsbad, CA, USA) and RNA integrity assessment was done using Agilent TapeStation 4200 (Agilent Technologies, Palo Alto, CA, USA). Prior to library preparation, ERCC RNA Spike-In Mix (Cat: 4456740) from ThermoFisher Scientific, was added to normalized total RNA following manufacturer’s protocol. NEBNext Ultra II RNA Library Prep Kit was used to prepare RNA sequencing libraries (NEB, Ipswich, MA, USA). Enrichment of mRNAs with Oligod(T) beads for a brief time. In the next step at 94 °C enriched mRNAs were fragmented for 15 minutes. Both first and second strand cDNA were synthesized. The cDNA fragments were then end-repaired and adenylated at the 3’ ends. Universal adapters were ligated to the cDNA fragments, followed by the addition of indexes and library enrichment through PCR with a limited number of cycles. The validation of the sequencing library was done on Agilent TapeStation (Agilent Technologies, Palo Alto, CA, USA), and quantification was done by using Qubit 2.0 Fluorometer (Invitrogen, Carlsbad, CA) along with quantitative PCR (KAPA Biosystems, Wilmington, MA, USA). On a flowcell the sequencing libraries were clustered. The flowcell was loaded on the Illumina NovaSeq instrument post clustering. Using a 2x150bp Paired End (PE) configuration, the samples were sequenced as a next step. Control software was used to do image analysis and base calling. Raw sequence data (bcl files) generated by the sequencer were converted into fastq files and de-multiplexed using Illumina’s bcl2fastq 2.17 software. One mismatch was allowed for index sequence identification.Data Analysis: After investigating the quality of the raw data, Trimmomatic v.0.36 was used to trim sequence reads remove possible adapter sequences and poor-quality nucleotides. The trimmed reads were aligned to the human reference genome available on ENSEMBL using the STAR aligner v.2.5.2b, resulting in the generation of BAM files. Unique gene hit counts were calculated using featureCounts from the Subread package v.1.5.2, counting only unique reads that fell within exon regions. The gene hit counts table was then used for downstream differential expression analysis. DESeq2 was employed to compare gene expression between the sample groups, using the Wald test to generate p-values and log2 fold changes. Genes with adjusted p-values < 0.05 and absolute log2 fold changes > 1 were identified as differentially expressed for each comparison. Gene ontology analysis was performed on the statistically significant genes using the GeneSCF software, clustering the genes based on their biological processes and determining their statistical significance using the human GO list. The gene code was converted to gene symbol using Biotools.fr (https://www.biotools.fr/mouse/ensembl_symbol_converter). STRING v. 12.0 and Cytoscape v. 3.1 databases were used for gene mapping, functional enrichment analysis, and network visualization.