Rat UGT2B1 enzyme-linked immunosorbent assay (ELISA) kit was purchased from Abbexa LLC (cat# abx552486) (Sugar Land, TX, USA). The customized primers for real-time quantitative polymerase chain reaction (RT-qPCR) targeting the specific gene sequences were purchased from Integrated DNA Technologies, Inc. (Coralville, IA, USA). Acetic acid (cat#320099), acetonitrile (cat#34998-4L), alamethicin from Trichodermia viride (cat#A4665), anhydrous sodium carbonate (cat# 791768-1 kg), anhydrous sodium chloride (cat#746398), bovine serum albumin (BSA, cat#A7906), cupric sulphate (cat# C1297-500g), Folin Ciocalteu’s phenol reagent (cat#F9252-500 mL), formic Acid (cat#F0507), high performance liquid chromatography (HPLC) grade methanol (cat#34860-4L-R), HPLC grade water (cat#270733), magnesium chloride (MgCl₂; cat#M8266), MPA (cat#M5255), MPA-d₃ solution (cat# M-137-1ML), protease inhibitor cocktail (PIC; mixture of 6 inhibitors i.e., AEBSF [4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride], aprotinin, bestatin hydrochloride, E-64 [N-(trans-epoxysuccinyl)-L-leucine 4-guanidinobutylamide], leupeptin hemisulfate salt, and pepstatin A; (cat# P8340)), sodium hydroxide (cat#S5881-500g), trisma hydrochloride (tris-HCL; cat#T5941), and uridine 5′-diphosphate glucuronic acid (UDPGA; cat#U6751) were acquired from Sigma-Aldrich (Oakville, ON, Canada). High-capacity complementary deoxyribonucleic acid (cDNA) reverse transcription kit (cat#4368814) and SYBR™ green polymerase chain reaction master mix (cat#4309155) were obtained from Applied Biosystems. D-sucrose (cat#BP220-1), TRIzol reagent (cat#15596018), phosphate-buffered saline (pH 7.4, cat#10010023), sodium potassium tartrate tetrahydrate (cat#S387-500), and ultrapure DNase/RNase-free distilled water (cat#10977-015) were purchased from Thermo Fisher Scientific (Waltham, MA, USA). MPA beta-D-glucuronide (MPAG; cat#TRC-M831520), MPA d3-beta-D-glucuronide (MPAG-d₃; cat#TRC-M831522), and MPA acyl-beta-D-glucuronide (AcMPAG; cat#TRC-M831525) were obtained from Toronto Research Chemicals (Vaughan, ON, Canada).

Adult Sprague Dawley (SD) rats (males: 130–150g; approximately 6 weeks old on arrival, females: 155–175 g; approximately 8 weeks old on arrival; with target weight range for both sex after acclimatization to be ∼200–300 g) [28] were obtained from Charles River Laboratories (Québec, QC, Canada). Prior to experiments, all animals were acclimatized for 14 days in the Health Sciences Laboratory Animal Services at the University of Alberta. A 12-h light/dark cycle was maintained, with ad libitum access to food (PicoLab rodent diet 20, Cat#5053, North American lab supply, Toronto, Canada) and water throughout acclimatization and experimental periods. Animals were housed in same-sex pairs in standard static cages without restraint. Music exposure studies were conducted in groups of eight rats (n = 4/sex). Following acclimatization, animals were placed in a room with monitored sound levels and continuously exposed in a continuous loop (for 24 h) to digital recordings of music delivered via digital speaker (JBL Charge 5, Harman International Industries, Northridge, CA, USA). Each group was exposed to combinations of two or three musical elements (i.e., tempo, rhythm, and harmony). Sound levels were monitored with digital decibel meters (axGear, Richmond, BC, Canada) positioned adjacent to cages, and maintained in the range from soft (60 ± 5 dB) to loud (80 ± 5 dB) [23]. At the conclusion of the music exposure period, animals were euthanized under isoflurane anesthesia (4% for induction, 2.5% for maintenance during organ collection) [29]. Livers were excised and stored at −80 °C until further analysis. The animal experiment was approved by the University of Alberta Research Ethics Board (animal use protocol no: AUP00004477; under the Animal Care and Use Committee; Health Sciences 1). For positive controls, SD rats (n = 1 for each sex) were treated with an intraperitoneal injection of 20 μg/kg 2,3,7,8-tetra chlorodibenzo-p-dioxin (TCDD) in corn oil (a known inducer for UGT enzymes [30]).

To avoid inter-composer variability, we used music elements from a single student composer (Callum Drysdale; see Supplementary material for full bibliography) arranged in four tempo-rhythm combinations: fast tempo with regular rhythm (FT RR), slow tempo with regular rhythm (ST RR), slow tempo with irregular rhythm (ST IR), and fast tempo with irregular rhythm (FT IR). These musical pieces were utilized to assess their effects on MPA metabolism. Based on initial findings, two additional music combinations were created (by Callum Drysdale) by adding either atonal harmony (AH) or tonal harmony (TH) to the FT IR combination, which produced the strongest effects on MPA glucuronidation (see Results). Next, we compared music produced by three student composers (Callum Drysdale [original composer], Jasmine Hourahine, and Drew Aarssen; see Supplementary material for full bibliography) using the combination that showed the greatest effects (i.e., FT IR AH; see Results). We also tested two published works incorporating the same FT IR AH elements (i.e., First Construction (In Metal), by John Cage [31] and Violin Concerto Op. 36 - 3. Allegro, by Arnold Schoenberg [32]. The lawful copy of publicly available music files for these two published music pieces were played and exposed to animals for the purpose of academic publications, and therefore no copyright was required based on the assessments of University of Alberta Copyright Services Specialist. Digital audio files for our synthesized music can be provided upon request. Finally, to enable comparison across student-composed and published music with FT IR AH elements, we quantified the percentage of continuous (i.e., legato; [from Italian, meaning “tied” or “bound”] is a musical articulation indicating that notes should be played in a smooth, flowing, and connected manner—each note transitions seamlessly into the next with no silence between them [33]) and intermittent (i.e., staccato; [from Italian, meaning “detached”] is a musical articulation indicating that notes should be played in a short, sharp, and disconnected manner—each note is cut shorter than its written value, with a brief silence between notes [33]) frequencies from their individual spectrograms. To avoid potential confounding effects, the percentages of staccato and legato were kept constant (i.e., 99.67% and 0.33% respectively) in the initial screening between the FT IR, FT RR, ST IR, and ST RR groups.

Microsomal protein was isolated as previously described [34]. Briefly, 0.6 g of liver tissue was homogenized in 1.9 mL of homogenization buffer (5 μL PIC per mL of sucrose solution). The homogenate was centrifuged (Eppendorf centrifuge 5810 R 15-amp version, Hamburg, Germany) at 14,000 rpm for 20 min at 4 °C, and the resulting supernatant (i.e., S9 fraction) was ultracentrifuged (Beckman Coulter Optima MAX-XP ultracentrifuge, Mississauga, ON, Canada) at 40,000 rpm for 1 h at 4 °C. The microsomal pellet was resuspended in homogenization buffer by vortexing. Protein concentration was determined using the Lowry assay [35] with bovine serum albumin as the standard.

Microsomal incubations were performed as previously described by our group [36]. Initial velocity conditions were established by varying microsomal protein concentration (0.05–1.05 mg/mL), incubation time (0–60 min), and MPA concentration (0.1–8 μg/mL [0.31–25 μM, MPA molecular weight: 320.34 g/mol]). Briefly, microsomal protein was preincubated with alamethicin (10 μg/mg protein) for 30 min. MPA was then added to a final reaction volume of 100 μL in incubation buffer (100 mM Tris-HCl, 10 mM MgCl₂, and 1% BSA). The MPA stock solution (1,000 μg/mL, except for enzyme kinetic experiments where the high concentration stock was prepared as 50,000 μg/mL) was prepared in methanol, and the final methanol concentration in all incubations was maintained at <1% (vehicle concentrations in controls were matched to the exact methanol concentrations in the experimental groups). Reaction mixtures were equilibrated at 37 °C for 5 min in a shaking water bath (Precision shake bath model 25, Jouan Inc., Winchester, VA, USA). Reactions were initiated by adding UDPGA to a final concentration of 5 mM, and terminated by adding 152 μL of ice-cold protein precipitation solution containing internal standards (MPA-d₃ [20 μg/mL, 1 μL] and MPAG-d₃ [100 μg/mL, 1 μL]), together with 123 μL methanol, 25 μL acetonitrile, and 2 μL 10% acetic acid. MPAG and AcMPAG formation was quantified by liquid chromatography–tandem mass spectrometry (LC–MS/MS) as previously described [37] (see section LC-MS/MS analysis for the quantification of MPA metabolites). Specific incubation conditions for each experiment are provided in the corresponding figure and table legends.

The enzyme kinetic study was conducted in rat liver microsomes under initial velocity conditions using increasing concentrations of MPA (0–320 μg/mL, [0–998.9 μM, MPA molecular weight: 320.34 g/mol]) to determine kinetic parameters, including V _max (maximum reaction rate), K _m (substrate concentration at half V _max ), and intrinsic clearance (CL _int ; Equation 1). To determine the model of best fit, the concentration-velocity data (i.e., combined, male and female) were fitted in Graphpad Prism (version 10.5.0; GraphPad Software LLC, Boston, USA) to multiple kinetic models, including Michaelis-Menten, substrate inhibition, allosteric sigmoidal, and specific binding with hill slope. Model selection was based on visual inspection and goodness-of-fit statistics, as previously described [38], including R² (coefficient of determination), sum of squares, root mean square, root mean square error, and the corrected Akaike information criterion [38]. Because only the Michaelis-Menten model (Equation 2) provided acceptable and best fits across all individual animals using these graphical and statistical criteria, it was used for subsequent analyses. Incubation conditions and the resulting V _max , K _m , and CL _int values are provided in the corresponding figure and/or table legends.

CL _int was calculated using Equation 1 [38]: C L int = V max K m (1)

Michaelis-Menten with Equation 2 [38, 39]: v = V max × S K m + S (2)

Where, v is velocity, [S] is substrate (MPA) concentrations, V _max , and K _m as defined above.

MPAG and AcMPAG formation in rat liver microsomal incubations was quantified using an LC-MS/MS assay developed in our laboratory [37] and partial-validated in this matrix in accordance with the current United States Food and Drug Administration bioanalytical method validation guidelines [40] (see supplementary material, Table 1). Briefly, after the incubation, 100 μL of reaction mixture (sample or calibrators) was mixed with 152 μL ice-cold protein precipitation solution containing 1 μL of MPA-d₃ (20 μg/mL), 1 μL MPAG-d₃ (100 μg/mL), 123 μL methanol, 25 μL acetonitrile, and 2 μL of 10% acetic acid. This mixture was vortexed-mixed for 30 s on a fixed speed vortex mixer (Fisher Scientific (Ottawa, Ontario, Canada), and centrifuged twice (Eppendorf centrifuge 5424 R, Hamburg, Germany) at 18,600 g at 4 °C for 10 min. A 10 μL aliquot of the resultant supernatant was injected for LC-MS/MS analysis. Chromatographic separation was performed on an Agilent Eclipse XDB C18 column (5 µm particle size, 4.6 × 250 mm diameter column) using mobile phases A and B (phase A: water with 2 mM ammonium acetate and 0.1% v/v formic acid, phase B: methanol with 2 mM ammonium acetate and 0.1% v/v formic acid). Gradient elution was used to achieve chromatographic separation (0–2 min: 30% phase B, 2–6 min: 30% → 100% phase B, 6–8 min: 100% phase B, 8–8.5 min 100% → 30% phase B, 8.5–15 min: 30% phase B). A 1 mL/min flowrate was maintained throughout the run time. Detection was achieved using a triple quadrupole mass spectrometer (LCMS-8050 Triple Quad LC-MS/MS; Shimadzu, Kyoto, Japan) [37].

Total RNA was isolated from frozen liver tissue using TRIzol reagent (Invitrogen) as per manufacturer’s instructions (Invitrogen, Waltham, MA, USA). RNA extraction, quantification, and cDNA synthesis was performed as described in an earlier study [42, 43]. cDNA was amplified by RT-qPCR as described previously [44] using 96-well optical plates and the comparative delta delta cycle threshold (ΔΔCT) method [45] on a QuantStudio 3 system (Applied Biosystems, Thermo Fisher Scientific, Waltham, MA, USA). Forward and reverse primer nucleotide sequences (Table 1) for each gene were verified for the Rattus norvegicus using National Center for Biotechnology Information’s basic local alignment search tools (BLAST) through BLASTN 2.17.0+ web program version [46]. Fold changes in mRNA expression were considered statistically significant when the p-value was less than the Bonferroni-corrected alpha (α/k, where k = number of genes tested) [47]. Relative mRNA expression was calculated using β-actin as the housekeeping gene. mRNA fold change was calculated as 2^−Δ(ΔCT) using Equations 3, 4 [34]: Δ CT = CT target gene   ‐  CT β ‐ actin (3) Δ Δ CT = Δ CT FT IR AH   ‐   Δ CT control (4)

Protein expression of UGT2B1, an enzyme potentially involved in AcMPAG formation [11], was measured in liver microsomal protein from rats exposed to FT IR AH (composed by Callum) and from control rats. Sample dilutions were conducted as needed to ensure the samples were within the calibration range of the assay kit. Microsomal samples were processed according to the manufacturer’s instructions using a commercial enzyme-linked immunosorbent assay (ELISA) kit (Abbexa LLC; cat# abx552486; Sugar Land, TX, USA) to detect and quantify UGT2B1. Briefly, a 96-well plate pre-coated with an anti-UGT2B1 antibody was used. Microsomal protein (0.2 mg/mL; selected based on preliminary experiments) and standards (100 μL, 0.16–10 ng/mL, prepared from lyophilized protein) were incubated with pre-coated ELISA plate for 2 h at 37 °C, followed by incubation with detection reagent A (100 μL, detection antibody 100X (1:8,000) in 50% Glycerol) and B (100 μL, Horseradish peroxidase-conjugated Avidin 100X [1:30,000] in 50% Glycerol) for 1 h at 37 °C. After appropriate washing steps as per manufacturer’s instructions (wash buffer, tris buffer saline with 1% tween-20 and 0.33% Thymol), 0.05% 3,3′,5,5′-tetramethylbenzidine (90 μL, 10–20 min incubation) was used for colorimetric detection. Wells containing UGT2B1 produced a blue signal, which turned to yellow upon the addition of stop solution (50 μL, 1 M Sulfuric acid). Absorbance was measured immediately at 450 nm using a SpectraMax iD series multimode microplate reader (Molecular Devices LLC, San Jose, CA, USA). Relative absorbance was calculated by subtracting the matrix optical density, and UGT2B1 concentrations were determined from the standard curve.

All results are presented as mean ± standard error of the mean (SEM). Statistical significance was set as p < 0.05; when multiple statistical tests were considered together, a Bonferroni-corrected alpha value was used. All analyses were performed in GraphPad Prism (version 10.5.0; GraphPad Software LLC, Boston, USA). Parametric tests (unpaired t-test or one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test) were used when data met assumptions of normality and equal variance. When these assumptions were not met, nonparametric tests were applied (i.e., Mann–Whitney or Kruskal–Wallis ANOVA followed by Dunn’s test).

First experiments characterized the initial velocity conditions (i.e., linear reaction rates with MPAG or AcMPAG formations that are <10% of the initial incubated MPA concentrations [38] for microsomal protein (i.e., 0.2 mg/mL), incubation time (i.e., 15 min), and MPA concentration (i.e., 1.2 μg/mL) using select female and male rodents. Negative control incubations (i.e., MPA-free, UDPGA-free, and protein-free) showed no detectable MPA metabolites (data not shown). MPAG and AcMPAG formation (Figure 1) was quantified for the first four music conditions (FT RR, ST RR, ST IR and FT IR; composed by Callum). In the combined sample (i.e., male and females, n = 8), none of the music combinations affected MPAG formation. In contrast, FT IR significantly reduced AcMPAG formation by 42.5 ± 14.4% (mean ± SEM, p < 0.05, n = 8) compared with the no-music control group, and by 31.2 ± 5.5% (p < 0.05, n = 8) compared to FT RR (Figures 1A,B). When analyzed by sex, AcMPAG formation in female rodents was reduced in FT IR group by 52.6 ± 17.6% (p < 0.005, n = 4) versus control and by 33.6 ± 4.6% (p < 0.05, n = 4) versus FT RR (Figure 1D). In male rodents, AcMPAG formation only showed a trend toward a reduction (24.9 ± 12.1%, p > 0.05, n = 4) (Figure 1F). Additionally, ST RR decreased AcMPAG formation in females by 39.1 ± 8.3% (p < 0.05, n = 4) versus the control (Figure 1D), an effect not observed in males. In contrast, none of the music elements affected MPAG formation in female or male rodents (Figures 1C,E). To confirm that the in-vitro microsomal incubation conditions were suitable for MPAG attenuation (a negative finding in our experiments), MPAG formation was also evaluated in the presence of 100 μM niflumic acid, an inhibitor of human microsomal UGT1A9 [48] which is the primary enzyme responsible for MPAG formation [36] (as a potential inhibitor toward the rodent orthologs for MPA glucuronidation [11, 49, 50]). Niflumic acid reduced MPAG formation demonstrating positive inhibitory control (Supplementary material, Figure 1).

Because the FT IR group showed a reduction in AcMPAG formation, we investigated this combination further by adding harmony (tonal or atonal) as an additional music element. We tested fast tempo and irregular rhythm with tonal (FT IR TH) or atonal harmony (FT IR AH) composed by Callum to assess their effects on MPAG and AcMPAG formations (Figure 2). MPAG formation was comparable to that in control animals in both the FT IR TH and FT IR AH groups (Figures 2A,C,E). In contrast, AcMPAG formation was reduced in the FT IR AH group by 74.4 ± 10.8 (p < 0.005, n = 8 combined sample), 71.2 ± 14.3% (p < 0.005, n = 4 [females]), and 80.1 ± 8.0% (p < 0.0005, n = 4 [males]) versus the control; and by 56.2 ± 11.7% (p < 0.05, n = 8), 64.5 ± 7.6% (p < 0.01, n = 4 [females]), and 41.5 ± 13.5% (p < 0.05, n = 4 [males]) versus the FT IR TH group (Figures 2B,D,F). Only the male rodents had a reduction in AcMPAG formation in the FT IR TH by 48.4 ± 13.4% (p < 0.05, n = 4) compared to the control (Figure 2F).

The effects of FT IR AH exposure on the enzyme kinetics of MPAG and AcMPAG formation are shown in Figure 3 for the combined sample (n = 8), while Table 2 summarizes sex-stratified kinetic parameters. Across all animals, enzyme kinetics were best described by the Michaelis-Menten model compared with alternative models. For MPAG formation, FT IR AH exposure was associated with a modest increase in K _m from 35.0 ± 2.84 μg/mL (controls, n = 8) to 49.9 ± 3.5 μg/mL (FT IR AH, p < 0.01, n = 8). Sex-stratified analyses showed only an increase in females (32.3 ± 10.7%, p < 0.05, n = 4, Table 2) in FT IR AH group versus the control group. In contrast, V _max and CL _int values for MPAG formation remained comparable to controls. For AcMPAG formation, K _m did not differ from controls; however, both V _max and CL _int were markedly reduced following FT IR AH exposure. V _max decreased by 89.7 ± 46.4% (p < 0.05, n = 7 [3 males, 4 females]), with only trends toward decreases observed in females (90.1 ± 72.3%, p > 0.05, n = 4) and males (89.1 ± 32.3%, p > 0.05, n = 3; Table 2). Similarly, CL _int decreased by 91.5 ± 29.7% (p < 0.05, n = 7 [3 males, 4 females]), including trends toward a reduction observed in females (94.0 ± 39.4%, p > 0.05, n = 4) and males (85.4 ± 24.8%, p < 0.05, n = 3, Table 2). In addition, there were no significant differences between male and female animals for V _max , K _m , and CL _int for MPAG and AcMPAG within the FT IR AH group. The lack of statistical significance in sex-stratified analyses was likely due to the small samples (i.e., secondary objectives).

The effects of different composers’ FT IR AH music on MPAG and AcMPAG formations were further evaluated using music from Callum [C], Jasmine [J], and Drew [D] (Figure 4). MPAG formation was not affected by music from any composer (Figures 4A,C,E). In contrast, AcMPAG formation was reduced in rats exposed to music composed by Callum (by 72.8 ± 7.5%, p < 0.005, n = 8; 68.6 ± 13.0%, p < 0.005, n = 4 [females]; 77.3 ± 9.4%, p = 0.07, n = 4 [males]) and Jasmine (by 77.8 ± 8.4%, p < 0.0001, n = 8; 74.1 ± 13.6%, p < 0.005, n = 4 [females]; 81.8 ± 11.7%, p < 0.005, n = 4 [males]) compared with the control group. Music composed by Drew produced a smaller, non-significant reduction in AcMPAG formation (Figure 4). When directly compared with Drew’s music, AcMPAG formation was lower (by trend or statistical significance) in rats exposed to Callum’s music (by 35.3 ± 9.2%, p > 0.05, n = 8; 50.4 ± 14.1%, p < 0.05, n = 4 [females]; 19.4 ± 2.8%, p > 0.05, n = 4 [males]) and Jasmine’s music (by 40.3 ± 9.3%, p < 0.01, n = 8; 55.9 ± 13.8%, p < 0.01, n = 4 [females]; 23.9 ± 3.8%, p > 0.05, n = 4 [males]). The lack of statistical significance in sex-stratified analyses for male rodents was likely due to the small samples (i.e., a secondary objective).

Rodents were also exposed to two published, pre-composed musical pieces [31, 32] with FT IR AH components to evaluate their effects on MPAG and AcMPAG formation (Figure 5). Consistent with all other mentioned music testing, MPAG formation was not affected by either of the music when compared to the controls. Moreover, AcMPAG formation in rats exposed to John cage music was also comparable to controls. In contrast, Arnold Schoenberg music reduced AcMPAG formation by 32.0 ± 10.7% (p < 0.05, n = 7 [4 males, 3 females]) versus the control. When analyzed by sex, a trend toward a reduction was observed in females (15.1 ± 13.2%, p > 0.05, n = 4) with a significant effect evident in males (45.6 ± 12.9%, p < 0.05, n = 4).

In order to quantitatively delineate differences in the three student composers and the two published composers with FT IR AH elements (Figures 4, 5), the individual music pieces were analyzed for percentage of legato and staccato based on spectrograms (Supplementary material, Figure 3). The frequency range for all five music pieces were comparable within 20 Hz to 20 kHz (except Drew’s piece which had negligible frequency above 16 kHz and the whole piece represents intermittent breaks between the music frequencies). For Callum’s music, 68% of music exhibited tempo between 110 and 160 bpm, with the remaining 32% at ∼80 bpm, and the majority of the music was legato (i.e., 92.80% legato, 7.20% staccato). Jasmine maintained the tempo between 65 and 70 bpm also with high percentage of legato (92.52% legato, 7.48% staccato). In contrast, Drew maintained tempo at ∼105 bpm with intermittent presence of frequencies (i.e., 60.98% presence of audible frequencies and 39.02% gaps, where virtually the whole piece is composed as staccato above 900 Hz [and legato is present below 900 Hz, where SD rats hearing capacity starts at 250 Hz]) throughout the entire piece. John cage and Arnold Schoenberg maintained tempo between 90 and 126 bpm with reduced percentage of legato (i.e., John cage [18.24% legato, 81.76% staccato] and Arnold Schoenberg [22.16% legato, 77.84% staccato]).

mRNA expressions of rodent hepatic Ugts were measured in rats exposed to FT IR AH music (composed by Callum) and in control rats with no music exposure (Figure 6) using RT-qPCR. For each of the 6 Ugt genes examined, FT IR AH exposure produced changes of < 3-fold, and none was significant (i.e., using Bonferroni-corrected alpha value of 0.0083 based on 6 compared genes). For the Ugt enzymes potentially involved in MPA glucuronidation in rats (i.e., Ugt1a1, 1a6, 1a7, and 2b1) [11, 49, 50], the overall fold changes were 1.24 ± 0.14, 1.60 ± 0.19, 1.37 ± 0.29, and 1.65 ± 0.36, respectively (p > 0.0083, n = 8 [4 males, 4 females]). When analyzed by sex, females showed fold changes of 1.18 ± 0.25, 1.59 ± 0.38, 0.95 ± 0.16, and 1.12 ± 0.36, respectively (p > 0.0083, n = 4), while males showed 1.31 ± 0.15, 1.62 ± 0.17, 1.79 ± 0.50, and 2.18 ± 0.53 fold increase respectively (p > 0.0083, n = 4). TCDD-exposed rats (i.e., positive control) showed substantial increases in Ugt1a6 and Ugt1a7 expression (∼34-fold and 14-fold, respectively n = 1 per sex) compared with controls (Supplementary material, Figure 2).

Notably, no differences in UGT2B1 protein expression, the putative enzyme for AcMPAG formation in rodents [11] were observed in the liver tissue of rodents exposed to control and the FT IR AH music (composed by Callum) (Figure 7). UGT2B1 protein concentrations in the FT IR AH group were 377.7 ± 29.6 ng/mL (p > 0.05, n = 8 [4 males, 4 females]), with similar values in females (387.8 ± 34.0 ng/mL, p > 0.05, n = 4) and males (367.7 ± 53.4 ng/mL, p > 0.05, n = 4) compared to the controls (368.8 ± 26.9 ng/mL [n = 8; 4 males, 4 females], 418.6 ± 36.2 ng/mL, [females, n = 4] and 319.1 ± 20.2 ng/mL [males, n = 4]).