SERS and SEIRA Detection of Polycyclic Aromatic Hydrocarbons and their Derivatives in Murine Tissues: Bioaccumulation and Clearance

Abstract

The toxicological effects of polycyclic aromatic hydrocarbons (PAHs) and polycyclic aromatic compounds (PACs) have attracted considerable research interest due to their known bioaccumulation. However, time-resolved and longitudinal studies that examine in vivo bioaccumulation and clearance remain limited. Here we use Surface-Enhanced Raman Scattering (SERS) and Surface-Enhanced Infrared Absorption (SEIRA) spectroscopies to detect and track the presence of two PAHs: pyrene, benzo[a]pyrene, and one PAC: 5,12-naphthacenequinone, in murine organ tissues. Mice were exposed to a three-component mixture of these three chemicals, then their organs were collected at 24-, 48-, and 72- hours post-exposure. The accumulation of each of these chemicals in the brain, liver, kidneys, heart, and spleen over a 72-hour post-exposure period was determined by SERS and SEIRA and independently quantified by gas chromatography-mass spectrometry (GC-MS) analysis. The liver and kidneys displayed the highest level of accumulation, reflecting their important roles in detoxification, while significant levels were detected in the brain, indicating permeability of the blood-brain barrier and the potential for neurotoxic effects. Benzo[a]pyrene showed the highest retention and slowest clearance, while 5,12-naphthacenequinone showed surprisingly minimal bioaccumulation. The independent detection of benzo[a]pyrene-induced DNA adducts in multiple organs also highlights its mutagenic and carcinogenic risk.

Graphical Abstract

Introduction

Polycyclic aromatic hydrocarbons (PAHs) are hazardous environmental contaminants known for their toxicity and carcinogenicity in humans.^1–3 The connection between PAH exposure and carcinogenicity was first documented in 1775, and increased ambient levels of these environmental toxins remain a global threat to human health today.⁴ Primarily generated through the incomplete combustion of organic matter such as diesel fuels and wildfire-fueling vegetation, PAHs can enter the body through inhalation, ingestion, or dermal absorption. Due to their hydrophobic nature, these compounds easily diffuse across cell membranes, where they bind to receptors and activate signal pathways that influence gene expression.^{3, 5} PAHs can be chemically modified into Polycyclic Aromatic Compounds (PACs) by a variety of ways, which can result in their increased solubility and intracellular uptake, leading to increased toxicity relative to their unfunctionalized PAH counterparts.⁶ Numerous studies have shown that PAHs can be metabolized and converted into specific PACs through a multi-step process: (i) Cytochrome P450 (CYP)-mediated oxidation, facilitated by enzymes such as CYP1A1 and CYP1B1, which transform PAHs into reactive epoxide intermediates capable of forming DNA adducts and initiating carcinogenic processes.^7–12 (ii) Conjugation reactions catalyzed by phase II enzymes, including glutathione S-transferases (GSTs), UDP-glucuronosyltransferases (UGTs), and sulfotransferases (SULTs), which attach polar groups to PAHs or their metabolites, enhancing their solubility and promoting excretion.^13–15 (iii) Redox cycling of PAH metabolites, which leads to multiple redox reactions that generate reactive oxygen species, contributing to oxidative stress, lipid peroxidation, and cellular damage.^16–19 Given their complex biological effects, the health risks associated with PAH and PAC exposure highlight the critical need for accurate detection and quantification within biological matrices and living systems.

Various analytical methods, including high-performance liquid chromatography (HPLC) coupled with fluorescence detection, gas chromatography-mass spectrometry (GC-MS), and time-of-flight secondary ion mass spectrometry (ToF-SIMS), have been developed for PAH detection.^20–25 While each method offers unique advantages and contributes to the comprehensive study and management of PAH/PAC pollution, they often require extensive preparation and significant operational time, which can be a drawback for rapid analysis needs. Recent advancements in surface-enhanced vibrational spectroscopies have highlighted the synergistic potential of combining SERS with SEIRA for the enhanced detection of PAHs/PACs.^{21, 26–27} This approach utilizes the complementary vibrational information provided by both SERS and SEIRA, facilitating the direct identification of PAHs/PACs at sub-parts-per-billion concentrations without the need for extensive sample preparation or separation processes.^{21, 28}

In this study, we utilize SERS and SEIRA to map the bioaccumulation of specific PAH/PACs in murine tissues and track the clearance of each chemical over a 72-hour period following exposure. We temporally quantify the accumulation of each contaminant within the various organs to follow their biodistribution and elimination. We observe that PAHs and PACs undergo both chemically- and organ-specific bioaccumulation and clearance dynamics. This type of study may shed light on the individual role of specific PAHs or PACs in the origin and pathology of diseases, such as the various cancers known to have origins in exposure to environmental toxins.

Results and Discussions

Murine models were utilized to investigate the presence of PAHs and PACs, providing insight into their distribution and potential health impacts on vital organs, specifically, the liver, lungs, brain, heart, and spleen. The experimental workflow is illustrated in Fig. 1A. Animals were subjected to an intraperitoneal (IP) injection of either corn oil (CO) as a vehicle control or a mixture of PAHs and were euthanized at various time points of 24, 48, and 72 hours post-exposure. Liver, kidneys, lungs, brain, heart, and spleen were collected for analysis. PAHs/PACs were extracted from tissues, reconstituted in either acetone or hexane, then applied to an engineered substrate composed of gold nanoshells (NS), which were specifically designed to enhance SERS and SEIRA spectroscopic signals.^29–32 Fig. 1B illustrates a substrate specifically engineered for the detection of PAHs/PACs utilizing both Surface-enhanced Raman Spectroscopy (SERS) and surface-enhanced infrared adsorption spectroscopy (SEIRA).²⁶ The SERS substrates were composed of NS with radii [r₁, r₂] = [63, 75] nm, which corresponds to a plasmon resonance of 785 nm in H₂O (the wavelength of the Raman pump laser used in all SERS measurements). NSs with radii [r₁, r₂] = [100, 115] nm, resulting in dipole plasmon resonances at 965 nm and quadrupole plasmon resonances at 685 nm in H₂O, were constructed for SEIRA measurements. The optical properties of both NSs used to constitute the SERS and SEIRA substrates are shown in Fig. S1 of the Supporting Information. When closely packed on a substrate, the hybridization of the NS dipolar plasmons gives rise to a broadband superradiant plasmon mode in the infrared, which enhances the local electromagnetic field across the mid-infrared chemical fingerprint region of the spectrum, crucial for SEIRA signal enhancement.^33–34 The plasmonic response of closely-packed NS with their plasmon resonance tuned to the pump laser wavelength supports their SERS signal enhancement.^34–35 A scanning electron microscopy (SEM) image of the dispersed NSs used for SERS is shown in Fig. 1C.

Figure 1. Schematic overview of the experimental workflow for detecting PAH/PACs in murine tissue samples.

(A) Schematic diagram illustrating the extraction process. Following PAH/PAC exposure, organ tissues (e.g., liver, kidney, lung, brain, heart, and spleen) are collected. Tissues are homogenized and the PAH/PAC are extracted, followed by resuspension in acetone. (B) Illustration of SERS and SEIRA substrate preparation and data analysis. The resulting extract is drop-cast on a Au NS-based substrate for detection. Subsequent analysis is performed using SERS and SEIRA spectroscopies. (C) Scanning electron microscopy (SEM) image of dispersed Au NS.

Animals received one-time intraperitoneal injections of a mixture of pyrene (PYR), benzo(a)pyrene (B[a]P), and 5,12-Naphthacenequinone (TQE), or corn oil (CO) as a vehicle control, and were studied at 24, 48, and 72 hours post-treatment. Freshly prepared Au NSs were deposited onto quartz substrates coated with poly-L-lysine, followed by the drop-dry deposition of 20 μL of the tissue extracts in acetone. SERS data were collected using a Renishaw inVia Raman microscope employing a 785 nm laser wavelength, with an acquisition time of 20 seconds and a laser intensity of 55 μW. Approximately 25–30 spectra were collected from each sample from various areas of the substrate, then averaged to minimize variability in the SERS spectra. Representative SERS spectra of murine liver, kidney, brain, and lung tissues collected at 24, 48, and 72 hours post-exposure are presented in Fig. 2, while SERS spectra from heart and spleen tissues are provided in Fig. S2 (Supporting Information). The Raman spectra of PYR and B[a]P standards (shown in black and dark grey, respectively) are shown as references. Additionally, Fig. S3 presents all SERS spectra plotted on the same scale to emphasize the time-dependent decrease in Raman peak intensity. When excited with a 785 nm laser, B[a]P exhibits a stronger fluorescence than PYR. This fluorescence from B[a]P increases the baseline of the Raman spectrum, resulting in a lower SNR relative to PYR.

Figure 2. Spectral Analysis of Pyrene and Benzo(a)Pyrene in Murine Tissues using SERS.

SERS spectra of acetone-extracted PYR and B[a]P from contaminated murine liver (A), brain (B) kidney (C), and lung (D) tissues collected at 24, 48, and 72 hours post-exposure (shown in gradient colors). Spectra from control (corn oil (CO)) tissues are shown in a darker solid color. For reference, Raman spectra of solid PYR and B[a]P powders are displayed in black and dark grey, respectively.

SERS spectra identifying PYR and B[a]P were detected across all examined tissues (lung, liver, brain, kidney, heart, and spleen) of the exposed mice post-injection, confirming the broad distribution of these contaminants. PYR was identified by the characteristic vibrational peaks at 593, 806, 1243, and 1408 cm⁻¹, corresponding to the CC skeletal stretch mode, CH out-of-plane bending, CC stretching/CH in-plane bending, and CC stretching/ring stretching, respectively.^36–38 Similarly, B[a]P exhibited distinct peaks at 334 cm⁻¹ (CC and CH in-plane bending), 527 cm⁻¹ (CC, CH in-plane bending, and CCCC torsion), 613 cm⁻¹ (CC and CH in-plane bending), 637 cm⁻¹ (CC, CH in-plane bending, and CC torsion), 1025 cm⁻¹ (CC stretching and CH in-plane bending), 1239 cm⁻¹ (CH in-plane bending and CCCH torsion), 1347 cm⁻¹ (CH in-plane bending, CC stretching, CCCC torsion), and 1390 cm⁻¹ (CCC stretching).³⁹ All vibrational modes of PYR and B[a]P are indicated by the yellow dashed lines in all panels of Fig. 2. The absence of these spectral features in control samples emphasizes the specificity of this method for identifying the contaminants in biological tissues. Similar findings were observed when the mice were exposed to individual PAHs or PACs. The spectroscopic data presented in Figs. S3 and S4, Supporting Information, confirm the successful detection of PYR, B[a]P, and TQE in murine liver and kidney tissues using SERS and SEIRA. Reference spectra of the pure compounds further validate the identity of the observed peaks, with light blue, green, and orange lines indicating the characteristic signals of PYR, B[a]P, and TQE, respectively.

SEIRA spectroscopy was utilized (Fig. 3) to investigate the molecular signatures of TQE in murine samples. Additionally, Fig. S4 shows SEIRA spectra plotted on the same scale to emphasize the time-dependent decrease in IR band intensity. The SEIRA sensitivity of TQE arises from its polar functional groups, which exhibit a high dipole moment and efficiently interact with enhanced IR fields. In contrast, PYR and B[a]P molecules are better detected by SERS, as their symmetric, π-conjugated aromatic structures lead to Raman scattering through changes in polarizability rather than dipole moment. Fourier-transform infrared spectroscopy (FTIR) of the pure compounds was performed as a reference. All spectral data were baseline-corrected prior to analysis. For SEIRA, freshly prepared Au NS were deposited on Si substrates, followed by the drop-dry deposition of 50 μL of biosamples in acetone. The array of SEIRA data was collected using a Bruker Vertex 80V spectrometer with a SiC globar lamp source, a liquid nitrogen-cooled HgCdTe (MCT) detector, and 256 scans with a resolution of 4 cm⁻¹. Samples were measured in a normal transmission geometry with an 8 mm window.

Figure 3. Spectral Analysis of 5,12-Naphthacenequinone in Murine Tissues using SEIRA.

SEIRA spectra of hexane-extracted TQE from treated murine liver (A), brain (B), kidney (C), and lung (D) tissues collected at 24, 48, and 72 hours post-exposure (shown in gradient colors). The spectra from control (corn oil (CO)) tissues are shown in a darker color. The FTIR spectrum of TQE is shown as a reference spectrum at the bottom of each panel. Dashed lines highlight the characteristic SEIRA peaks of TQE, indicating its presence in the tissues.

SEIRA analysis of murine samples revealed distinct absorption bands at 1165 cm⁻¹ (CH bending), 714 cm⁻¹, and 754 cm⁻¹ (CH wagging), indicative of the presence of TQE in the contaminated tissues.²¹ The contaminated liver and brain samples exhibit a strong band at 1107 cm⁻¹, which does not correspond to the vibrational modes of TQE. This band likely arises from C–O stretching modes formed through interaction between PAC and biomolecules within the tissue matrix. These characteristic bands were absent in the untreated controls. A strong, narrow band at 1680 cm⁻¹ corresponding to the C=O stretch of the quinone was observed in the contaminated samples, but appears to shift by 20 cm⁻¹ relative to the pure compound, potentially due to interactions with molecular components of the tissue extract and with the NS surface. This quinone-related band is distinct from the broader absorption at 1760 cm⁻¹, most likely attributed to fatty acids. Additionally, a prominent band at 1540 cm⁻¹, associated with the CC stretch in the aromatic rings, further supports the presence of TQE. These spectral signatures were absent in the control group of mice, indicating a clear differentiation between treated and untreated samples.

The time-dependent accumulation and clearance of B[a]P, PYR, and TQE in brain, kidney, liver, and lung tissues after injection were evaluated using SERS spectra, by analyzing the Raman peak area of selected signals in the spectra (Fig. 4A). Error bars represent the standard deviation calculated from Raman spectral data collected from three individual mice, with 25 to 30 spectra acquired per animal. Error bars are smaller than point markers and therefore not visible. Characteristic vibrational bands, 611 cm⁻¹ for B[a]P, 589 cm⁻¹ for PYR, and 1657 cm⁻¹ for TQE, were analyzed using a Lorentzian and pseudo-Voigt peak-fitting procedure, followed by integration of the main peak area. A representative SERS spectrum and its corresponding spectral fit, used for the time-dependent calculation of PAH/PAC distributions, are shown in Fig. S5. The time-dependent profile of PAHs/PAC across different murine tissues was assessed by plotting the integrated peak areas as a function of time post-injection (Fig. 4A). A consistent decline in PAH signal intensity was observed in all tissue types, likely indicating progressive clearance from the tissues. It can be clearly seen that the rate of reduction varied among tissues, with some exhibiting more rapid decreases than others. None of the tissues demonstrated complete elimination of PAHs within the 72-hour period, suggesting a degree of persistence for these chemicals in the analyzed organs. Although SERS and SEIRA responses provided a time-dependent qualitative trend, accurate quantification of PAHs/PAC concentrations is limited by factors such as surface saturation, analyte binding affinity, and substrate-dependent spectral variability. Therefore, to overcome these limitations, GC–MS/MS was employed to obtain absolute concentration measurements and monitor time-dependent changes across tissues. This method enabled the detailed mapping of PAH/PAC biodistributions across different organs, as shown in Fig. 4B. Error bars represent the standard error of the mean, calculated from the average concentrations measured in tissues from three individual mice at each post-exposure time point. Error bars are not visible where their size is smaller than the data point markers.

Figure 4. Detection and distribution of PAHs in murine tissues at 24, 48, and 72 hours post-treatment.

(A) Time profiles of the characteristic area Raman peak of the 611 cm⁻¹ band for B[a]P, 589 cm⁻¹ band for PYR, and 1657 cm⁻¹ band for TQE measured at 24, 48, and 72 hrs. post-injection, demonstrating dynamic changes in compound distribution. (B) GC-MS/MS quantification of B[a]P, PYR, and TQE in tissues collected at corresponding times. PYR and TQE concentrations were statistically significant across all tissues and time points (t(1) < 12.71, p > 0.05) . B[a]P concentrations were statistically significant except in lung at 72 hours post-injection (t(1) < 12.71, p > 0.05).

Among the examined tissues, the liver exhibited the highest accumulation of PAHs, reflecting its central role as the body’s primary organ for detoxification. Similarly, the kidneys, vital organs in the filtration and xenobiotic detoxification process, also demonstrated significant retention of PAHs.^{14 40–41} Both organs serve as critical targets in the metabolism and elimination of PAHs. Such accumulation can disrupt renal function and lead to nephrotoxicity, compromising the organ’s ability to filter waste effectively. A significant accumulation of PAHs/PAC was observed in brain tissue, indicating potential permeability of the blood-brain barrier (BBB) to these compounds. Once in the brain, PAHs and their metabolites can contribute to oxidative stress, neuro-inflammation, and potential neurotoxicity, raising concerns about their impact on central nervous system function.^42–44 Among the analyzed compounds, B[a]P exhibited the highest tissue absorption and slowest clearance rate, likely due to its five-ring structure, which enhances lipophilicity and persistence.^45–47 The higher persistence of B[a]P may also explain the apparent increase in liver tissue concentration observed between 48 and 72 hours post-exposure. Elimination of this PAH from the liver may be influenced by metabolic factors that vary from organism to organism, leading to higher variability in the data. This is further reflected in the Raman peak intensities, which also show relatively large error bars, indicating significantly higher variability compared to the other PAH/PAC’s and tissues. In contrast, the more water-soluble TQE, with its smaller ring system, exhibited the lowest accumulation, suggesting limited bioavailability and/or faster elimination.

Exposure to PAHs can lead to the formation of mutagenic, bulky DNA adducts. To investigate DNA adduct formation, we utilized B[a]P and benzo[b]fluorenthene (B[b]F) as representative PAHs. Pregnant dams (C57BL/6J) mice were orally administered a mixture of B[a]P and B[b]F (7.5 mg/kg each) during the gestational days (GD) 16–19. The offspring were euthanized on postnatal day 15 for further analysis. The DNA extracted from the lung and brain was subjected to ³²P-postlabeling to determine the presence of PAH-DNA adduct formation. Additionally, we conducted DNA adduct studies in the livers and lungs of unrelated adult mice. Twelve-week-old A/J mice received a single dose of B[a]P (40 µmol/kg) and were euthanized seven days later. Adducts were detected in all examined tissues of the treated animals, while none were found in the corn oil-treated control groups. (Fig. 5).

Figure 5. DNA Adduct Formation of PAHs in Mice.

Representative ³²P-postlabeled PAH–DNA adducts profiles in: (i) lung and (ii) brain of neonates exposed in utero to B[a]P via maternal exposure; (iii) lung and (iv) liver of (unrelated) adult A/J mice (8 weeks old) treated with 40 µmol/kg B[a]P. Samples were collected 21 days post-treatment. Based on our previous publication, Spot 2 corresponds to the benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide deoxy-guanosine (BPDE-dG) adduct.^{12, 48}

Chromatographic analysis of ³²P-postlabeled DNA adducts revealed remarkably similar profiles in the lung and brain tissues of newborn mice exposed to B[a]P in utero, suggesting comparable transplacental distribution and DNA reactivity of B[a]P in these organs. Transplacental formation of DNA adducts in different organs after administration of individual PAHs including B[a]P has been previously demonstrated by Lu et al.⁴⁹ However, to our knowledge, this is the first report documenting DNA adduct formation after in utero exposure to a mixture of B[a]P and B[b]F (Fig. 5 i and ii). The ability of these compounds to cross the fetal blood-brain barrier (BBB) is of particular concern, as it may directly influence the development and function of the immature brain. In contrast, distinct DNA adduct patterns were detected in the lung and liver tissues of adult mice treated with B[a]P, reflecting tissue-specific metabolic activation and DNA interaction in mature animals. Notably, spot 2 corresponds to the benzo(a) pyrene-7,8-dihydrodiol-9,10-epoxide deoxy-guanosine (BPDE-dG) adduct, a well-characterized biomarker of PAH-induced DNA damage.^{12, 48}

Conclusions

This study demonstrates the toxicological effects of PAHs/PACs on murine organ tissue utilizing SERS and SEIRA for sensitive detection. Qualitative spectral analysis confirmed the presence of B[a]P, PYR, and TQE in the exposed tissues, while GC-MS/MS provided quantitative validation of their widespread accumulation in critical organs, including the brain, liver, lungs, heart, spleen, and kidneys. Additionally, ultrasensitive assays revealed elevated levels of PAH-induced DNA adducts, highlighting the genotoxic impact of these compounds. SERS and SEIRA spectroscopies provide rapid, cost-effective, and non-destructive tools for environmental toxicology and biomedical research, which could revolutionize our approach to monitoring, assessing, and mitigating chemical exposure risks in both clinical and ecological settings.

Materials and Methods

Materials.

(3-aminopropyl) triethoxysilane (ES, 99%), tetra chloroauric acid (HAuCl₄·3H₂O), tetrakis hydroxymethyl phosphonium chloride (THPC), poly-L-lysine hydrobromide (MW 150,000−300,000) poly-L-Lysine (PLL) pyrene, and anthracene, were purchased from Sigma-Aldrich. Formaldehyde (37%), sulfuric acid (H₂SO₄, 100%), hydrogen peroxide (H₂O₂, 30%), potassium dihydrogen phosphate (KH₂PO₄), and 200-proof ethanol were obtained from Fisher Scientific. All chemicals were used as received without further purification. The water was deionized and filtered using a Milli-Q water system (18.2 MΩ cm at 25 °C, Millipore). Quartz slides were obtained for substrate fabrication (Fisher Scientific). The 120 nm diameter aminated SiO₂ cores in ethanol (10 mg/mL) were purchased from nanoComposix, Inc., San Diego, CA.

Synthesis Au Nanoshells (Au NS).

The Au NSs were synthesized using a previously reported procedure.⁵⁰ Small Au colloids (2–5 nm diameters) were synthesized by reducing HAuCl₄ with THPC reducing agent. Then, 300 μL aminated SiO₂ cores were incubated with 20 mL of a THPC (2–5 nm diameter Au colloids) solution for 24 hours to enable the attachment of the Au colloids onto the SiO₂ surface, followed by multiple cleaning cycles to remove excess reaction chemicals. Following this step, an electroless plating process reduced Au onto the SiO₂ surface, forming a continuous Au shell. The electroless deposition was achieved by reducing Au³⁺ from a 3 mL solution (1 day aged) of 1.8 mM KH₂PO₄ and 0.4 μM HAuCl₄, with the addition of 15 μL of formaldehyde. The Au NSs were fabricated with the core and shell radii of [r₁, r₂] = [63, 75] nm, which corresponds to a plasmon resonance of 785 nm (the wavelength of Raman laser) in H₂O. For the SEIRA measurements, the radii were [r₁, r₂] = [100, 115] nm.

SERS Substrate Preparation.

Cleaned quartz slides (from Thermo Scientific^™ Quartz microscope slides, fused, 76.2 x 25.4 mm) were immersed in a 0.01% w/v aqueous solution of PLL (MW 150,000 to 300,000) for 5 hours to facilitate the subsequent attachment of nanoparticles to the quartz surface. The surfaces were cleaned with ethanol and dried in a flow of N₂. Cut silicone isolators (Grace Bio-Labs Press-To-Seal silicone isolator, No PSA, 9 mm diameter) were placed on the quartz-PLL substrate, followed by a dry drop deposition of 100 μL 10¹⁰ part./mL Au NS or NPs aqueous solution into the isolator well. Then, 50 μL of bio-samples were directly dried on the Au NSs SERS substrates. Before acquiring the SERS spectra, the substrates were fully immersed in Milli-Q water.

SEIRA Substrate Preparation.

A 525 μm-thick silicon wafer (undoped,<100>, FZ, University Wafers) was cut into small square supports and cleaned through sonication, first in acetone and then in isopropanol. Subsequently, the silicon supports were blow-dried with dry N₂. Next, 100 μL of the NSs was drop-cast onto a silicon support and allowed to dry under ambient conditions. Following this, 50 μL of the bio-sample was directly drop-cast onto the substrate and left to dry. The NSs utilized for the SEIRA experiments were fabricated with core and shell radii [r₁, r₂] = [100, 115] nm, resulting in dipole plasmon resonances at 965 nm and quadrupole plasmon resonances at 685 nm in H₂O.

Murine model.

Female C57BL6J mice, aged 8–10 weeks, were purchased from Jackson Laboratories. All experimental protocols were approved by the Institutional Animal Care and Use Committee of Baylor College of Medicine (Protocol No. AN-907). The animals were housed under controlled conditions at the animal facility of Texas Children’s Hospital, Baylor College of Medicine, Houston, Texas. Mice were randomly assigned to treatment groups receiving intraperitoneal injections (IP) of a mixture of B[a]P (50 mg/kg), PYR (50 mg/kg), and TQE (40 mg/kg) dissolved in corn oil (CO) or CO alone as vehicle control (10 mL/kg). Treatments were administered once, and animals were monitored for 24 hours post-administration.

Euthanasia and Tissue Collection.

Following the treatment period, mice were euthanized (at 24, 48, and 72 hrs.) under deep anesthesia to minimize suffering. Tissues from the lungs, liver, brain, spleen, heart, and kidneys were collected, immediately frozen, and stored at −80°C until use.

Extraction procedure.

The tissue sample (see Table S1 for a summary of the tissue weight for extraction) was homogenized in 1000 µL of hexane using a Janke & Kunkel Ultra-Turrax T25 mixer at 20,000 RPM for 30 seconds. Following initial homogenization, an additional 1000 µL of chloroform was added, and the mixture was vortexed. The homogenate mixture was then allowed to stand at room temperature for 30 minutes to facilitate phase separation. Subsequently, the samples were centrifuged at 5000 rpm for 10 minutes. The supernatant was carefully transferred to a new container and dried under a stream of nitrogen at room temperature to ensure complete removal of solvent. Finally, the dried residues were reconstituted in 200 µL of acetone, vortexed to ensure homogeneity, and used for further assay analysis.

DNA Adduct Analyses.

Genomic DNA was isolated using a combination of organic solvent extraction and enzymatic digestion of protein and RNA,⁵¹ and stored at −80°C until use. DNA adducts were detected using the nuclease P1-enhanced bisphosphate version of the ³²P-postlabeling assay following established protocols.⁵² Briefly, 10 µg of DNA was enzymatically degraded to normal (Np) and modified (Xp) deoxyribonucleoside 3’-monophosphates and their base-modified derivatives with a mixture of micrococcal endonuclease and spleen phosphodiesterase. After 3’-dephosphorylation of the normal nucleotides with nuclease P1, the enriched nuclease P1-resistant modified 3’-nucleotides were converted to 5’-32P-labeled deoxyribonucleoside 3’,5’-bisphosphate derivatives by incubation with carrier-free [γ-³²P]ATP and T4 polynucleotide kinase. The radioactively labeled digestion products were applied to modified PEI cellulose thin layers and chromatographed overnight (15–16 h) with solvent 1 (D1, 2.3 M sodium phosphate, pH 5.70) to purify bulky adducts. Labeled PAH adducts retained in the lower (L, 2.8 × 1.0 cm) part of the D1 chromatogram were each, after brief autoradiography on Cronex 4 X-ray film, contact-transferred to individual acceptor sheets and resolved by two-dimensional TLC. The bulky DNA adducts were separated with solvents 2 (3.82 M lithium formate, 6.75 M urea, pH 3.35) and solvent 3 (0.72 M sodium phosphate, 0.45 M Tris-HCl, 7.65 M urea, pH 8.20) in the first and second dimensions, respectively. ³²P-labeled DNA adducts were visualized by screen-enhanced autoradiography at −80⁰C using Kodak XAR-5 film and were detected with the aid of Amersham Typhoon Biomolecular Phosphorimager (GE Healthcare, Chicago, IL, USA). The instrument automatically subtracted appropriate blank count rates from sample values.

GC-MS Sample preparation and analysis. Sample Preparation.

Tissue samples were homogenized in 1 mL of hexane, followed by the addition of 1 mL of chloroform to facilitate the extraction of the target analyte. The resulting lysate was vortexed at room temperature for 30 minutes to ensure thorough mixing and solubilization of the analyte and centrifuged at 5000 rpm for 15 minutes to separate the organic phase. The supernatant was collected and subjected to solvent evaporation under reduced pressure using a Genevac vacuum concentrator. The dried extract was subsequently reconstituted in 50 µL of chloroform for analysis. Quantification of B[α]P, TQE, and PYR was performed using a gas chromatography-tandem mass spectrometry (GC-MS/MS) system consisting of an Agilent 7890A GC coupled with an Agilent 7000 Triple Quadrupole Mass Spectrometer. The instrument operated in split injection mode with a split ratio of 50:1, and analytes were separated on a DB-5MS capillary column (30 m in length, 250 µm in internal diameter, and 0.25 µm in film thickness). Helium was used as the carrier gas at a constant flow rate of 1.7 mL/min. The inlet temperature was maintained at 250 °C, while the oven temperature was initially set at 60 °C, held for 1 minute, and then ramped at 10 °C/min to 325 °C and held at the final temperature for 10 minutes, resulting in a total run time of 37.5 minutes. The transfer line to the mass spectrometer was set at 290 °C. Detection was carried out in electron ionization (EI) mode at 70 eV, with an ion source temperature of 250 °C. Analytes were monitored using Selected Ion Monitoring (SIM) mode with target m/z values of 258.3 (TQE), 202.25 (PYR), and 250.0 (B[α]P). Data acquisition and quantitative analysis were performed using Agilent MassHunter Quantitative Analysis software.

Measurements.

SERS measurements were acquired with a Renishaw inVia Raman microscope (Renishaw) with a 785-nm excitation wavelength and 55-μW laser power at the samples. Backscattered light was collected using a 63X water immersion objective lens (Leica), with a numerical aperture (NA) of 0.9 and a 20-second exposure time. SEIRA spectra were recorded in a Bruker VERTEX 80v FTIR spectrophotometer in transmission mode at normal incidence. The instrument has a mercury cadmium telluride (MCT) detector, a KBr beam splitter, and a SiC Globar light source. The mirror velocity was 20 kHz, and the spectral resolution was 4 cm−1. Spectra were measured under a rough vacuum (∼1.0 hPa) with 256 scans and the background of the empty chambers. For baseline subtraction, a standard infrared (IR) spectrum of the silicone substrate was employed. All SEIRA data were processed using baseline subtraction. Extinction measurements were performed on a Cary 5000 UV/Vis/NIR Varian spectrophotometer. SEM measurements were performed using an FEI Quanta 400 field emission SEM at an acceleration voltage of 20 kV. Homogenization of the bio-samples was achieved with a T25 ULTRA-TURRAX^® instrument.