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.