Exposure to Benzo(a)pyrene Enhances Acetaminophen-Induced Liver Injury in Mice at Non-Hepatotoxic Doses

: Acetaminophen (APAP) is a widely used analgesic, especially for children. Its primary mechanism involves inhibiting cyclooxygenase enzymes and activating the endocannabinoid and TRPV1 systems. Though its toxicity is low, it can harm the liver in a dose-dependent manner. Low APAP doses can also increase pollutant-induced liver damage. Little is known about interactions be-tween APAP and benzo[a]pyrene (B[a]P). This study aimed to assess if co-exposure to non-hepatotoxic doses of B[a]P and APAP causes liver injury in mice, exploring the underlying mechanisms. Female ICR mice received 50 mg/kg B[a]P or a vehicle for three days, followed by 200 mg/kg APAP or a vehicle. Liver injury was assessed through histopathological examination, serum transaminase activity, and gene expression analysis. In the B[a]P/APAP group, several histology changes were observed, including ballooning injury, steatosis, necrosis, inflammation, and apoptosis. Transaminase levels correlated with histopathological scores, and there was an increase in hepatic cytochrome P450 family 1 subfamily a member 1 ( Cyp1a1 ) mRNA levels and a decrease in aryl hydrocarbon receptor ( Ahr ), cytochrome P450 family 2 subfamily e polypeptide 1 ( Cyp2e1 ), superoxide dismutase 1 ( Sod1 ), peroxisome proliferator activated receptor gamma ( Ppar-γ ), and caspase 3 ( Casp3 ). This suggests that prior exposure to B[a]P makes mice more susceptible to APAP-induced liver injury, involving changes in gene expression related to metabolism, redox balance, and cell proliferation. Therefore, using therapeutic APAP doses after exposure to B[a]P could lead to liver injury.


Introduction
Acetaminophen (APAP) is one of the most popular and widely used analgesic and antipyretic drugs globally [1,2], and it is included in the WHO model list of essential medicines [3].Although this chemical is safe for most individuals, there is a risk of developing drug-induced liver injury (DILI) in some patients with pre-existing pathological conditions such as liver damage [4], malnutrition [5,6], co-medication [7], age [8], genetics [9], or tobacco consumption [10], among others [1].
Once ingested, most APAP is excreted in bile and urine after combination with glucuronide and sulfate cofactors, and a fraction of APAP (5-10%) is metabolized to N-acetylp-benzoquinone imine (NAPQI) [1], which is detoxified through conjugation with glutathione [11,12].This last reaction can lead to cellular oxidative stress by glutathione depletion, reducing the ability of glutathione transferase to metabolize hydrogen peroxide [2,12].During an APAP overdose, large amounts of NAPQI are produced, depleting liver glutathione and causing liver injury [11,12].Acetaminophen overdose is the most frequent cause of acute liver failure (ALF) of any etiology in the United States (46%), and in Great Britain and Europe (40-70%) [13], APAP-induced liver damage represents the second most common cause of liver transplantation worldwide [14], being responsible for about 500 deaths per annum in the United States (U.S.) alone [15] and 150 to 200 deaths annually in England and Wales [16].
The molecular mechanisms underlying acetaminophen-induced liver damage have been extensively studied [11,17,18].One of the primary molecular pathways implicated in the progression of APAP-induced liver injury involves the induction of oxidative stress, triggered by the production of the reactive metabolite NAPQI through acetaminophen metabolism via cytochrome P450 (Cyp450), primarily CYP2E1 [19].NAPQI, capable of forming adducts through its interaction with cysteine groups of proteins [11,17,18], leads to the formation of mitochondrial permeability transition pores, resulting in disruptions in calcium homeostasis, uncoupling of oxidative phosphorylation, release of intramitochondrial ions and metabolic intermediates, mitochondrial inflammation, decreased ATP synthesis, and ultimately, hepatocyte viability loss [17].
In addition to the tissue damage induced by oxidative stress events resulting from APAP metabolites, several studies have also implicated APAP in potential genotoxic and carcinogenic effects.This is attributed to the formation of the quinone imine (NAPQI), which can chemically interact with DNA through a 1,4-Michael addition mechanism, following depletion of glutathione (GSH) [20].
The antioxidant defense mechanisms against APAP-induced oxidative stress processes have been extensively described [11,21,22].The formation of NAPQI-protein adducts in the mitochondrial respiratory chain (MRC) triggers the release of electrons to oxygen, thereby promoting the production of superoxide, which is then catalyzed into hydrogen peroxide and oxygen by superoxide dismutase (SOD), leading to oxidative/nitrosative stress [11,22,23].Hydrogen peroxide is either eliminated by catalase (CAT) or reacts with GSH.Consequently, when GSH levels decrease, and antioxidant activity is inhibited, reactive oxygen species combine with nitric oxide (NO) to form peroxynitrite, resulting in the formation of nitrotyrosine adducts that affect the function of intracellular proteins [11].
One of the exogenous factors recently studied concerning variations in drug effects and toxicity is related to individuals' exposome.This is because it can alter drug metabolism, change the bioavailability or excretion of the drug, or interfere with drug action and targets [24].
The exacerbation of APAP-induced liver injury has been demonstrated by studies involving pre-exposure or co-exposure of biomodels to various pollutants, including ozone [25], fenbendazole [26], and roxithromycin [27], among others [28,29].Within the reported toxicological interaction mechanisms from these investigations, several have been identified: increased oxidative stress, evidenced by significant expression of oxidative stress-responsive genes such as metallothionein-1 (Mt-1), heme oxygenase-1 (Hmox-1), and glutamate-cysteine ligase (Gclc) in the liver, and reduced cellular regeneration mechanisms due to overexpression of P21 [25]; depletion of tissue GSH levels due to the formation of conjugates [26]; and induction of oxidative stress through metabolic changes in each xenobiotic by inhibiting cytochrome P450 2D6 (CYP2D6) activity and increasing cytochrome P450 2E1 (CYP2E1) expression, leading to slower APAP elimination [27].These findings raise significant concerns, especially considering the multitude of environmental pollutants that are part of our daily exposome, which may be involved in the depletion of endogenous antioxidants.This is particularly worrisome as their mechanisms of action may coincide with that of APAP.Among these chemicals are the polycyclic aromatic hydrocarbons (PAHs), in particular benzo[a]pyrene (B[a]P), known for their capacity to induce oxidative stress [30][31][32].Benzo[a]pyrene is considered a high-priority substance of concern for human exposure (8th/275), according to the Agency for Toxic Substances and Disease Registry [33].B[a]P is generated through pyrolytic processes, particularly the incomplete combustion of organic materials during industrial and other human activities [34].This xenobiotic has been repeatedly found in the air, surface water, soil, and sediments [35][36][37].Human exposure to B[a]P is common [35], considering its sources of contamination in the environment, which include industrial and automobile emissions, hazardous waste sites, cigarette smoke, biomass burning, municipal incinerators, volcanic eruptions, home heating, and the consumption of charcoal-broiled and smoked foods [34,38].
This PAH has been categorized by the International Agency for Research on Cancer (IARC) as a human group 1 carcinogen because of its mutagenic and carcinogenic effects in animal models and its association with the development of several types of human cancer, including lung, breast, and liver [38,39].In addition to sufficient evidence of carcinogenicity in humans, the neurotoxicity, epigenotoxicity, alteration of various metabolic pathways, and reproductive toxicity of B[a]P have been demonstrated experimentally [31,35,[38][39][40][41][42].The molecular mechanisms associated with B[a]P toxicity involve the creation of stable and depurinating DNA adducts, repetitive redox cycling that generates reactive oxygen species (ROS), radical-cation mechanism, mechanism via formation of ortho-quinone, and interaction with the aryl hydrocarbon (AhR) receptor, among others [39,43].
The cellular antioxidant system, including key enzymes such as superoxide dismutase (SOD), glutathione peroxidase (GPx), catalase (CAT), and non-enzymatic components like glutathione, plays a crucial role in eliminating or regulating the elevated levels of ROS produced in the body [30].Despite this, some studies have demonstrated disturbances in antioxidant responses in the liver, lungs, kidneys, stomach, and brain induced by acute B[a]P treatment in a murine model [30,32].
APAP and B[a]P hepatotoxicity is associated with alterations in the activity of metabolic enzymes, including members of the Cytochrome P450 family [17,19,31,[43][44][45]; the generation of reactive byproducts, leading to the depletion of endogenous antioxidants produced during their metabolism [17,20,26,30,32]; as well as the activation or inactivation of transcription factors through their interaction with specific ligands.These factors, in turn, regulate the expression of genes involved in the metabolism and toxicity of these xenobiotics [50][51][52].
Despite the evident co-exposure to acetaminophen and B[a]P, especially in individuals who smoke cigarettes, are exposed to automobile emissions, and consume food contaminated with B[a]P, there is insufficient information regarding the effects of pre-exposure to B[a]P on non-hepatotoxic doses of acetaminophen-induced liver damage.Therefore, the objective of this research was to evaluate whether acute co-exposure to non-hepatotoxic doses of B[a]P and APAP could induce liver injury in mice and to explore the cellular and molecular mechanisms involved.

Body Weight and Liver-to-Body Ratio
Mice body weight did not show significant alterations following various treatments, including sesame oil (vehicle for B[a]P) and saline solution (vehicle for APAP), APAP alone, B[a]P alone, or B[a]P combined with APAP (Figure S1).However, a noteworthy increase (p < 0.05) in the liver-to-body weight ratio was observed in the group of mice treated with B[a]P and B[a]P combined with APAP compared to the control.No significant difference was observed between the control group and the group treated with APAP alone (Figure 1).

Body Weight and Liver-to-Body Ratio
Mice body weight did not show significant alterations following various treatments, including sesame oil (vehicle for B[a]P) and saline solution (vehicle for APAP), APAP alone, B[a]P alone, or B[a]P combined with APAP (Figure S1).However, a noteworthy increase (p < 0.05) in the liver-to-body weight ratio was observed in the group of mice treated with B[a]P and B[a]P combined with APAP compared to the control.No significant difference was observed between the control group and the group treated with APAP alone (Figure 1).

Serum Transaminase Activity
Alanine aminotransferase (ALT) and Aspartate aminotransferase (AST) activities were investigated as markers of hepatocellular injury in circulating blood.A significant increase (p < 0.05) in the activity of these transaminases was detected in the serum of mice in the group that received the co-treatment of B[a]P and APAP compared to the control group (Figure 2).

Serum Transaminase Activity
Alanine aminotransferase (ALT) and Aspartate aminotransferase (AST) activities were investigated as markers of hepatocellular injury in circulating blood.A significant increase (p < 0.05) in the activity of these transaminases was detected in the serum of mice in the group that received the co-treatment of B[a]P and APAP compared to the control group (Figure 2).

Body Weight and Liver-to-Body Ratio
Mice body weight did not show significant alterations following various treatments, including sesame oil (vehicle for B[a]P) and saline solution (vehicle for APAP), APAP alone, B[a]P alone, or B[a]P combined with APAP (Figure S1).However, a noteworthy increase (p < 0.05) in the liver-to-body weight ratio was observed in the group of mice treated with B[a]P and B[a]P combined with APAP compared to the control.No significant difference was observed between the control group and the group treated with APAP alone (Figure 1).

Serum Transaminase Activity
Alanine aminotransferase (ALT) and Aspartate aminotransferase (AST) activities were investigated as markers of hepatocellular injury in circulating blood.A significant increase (p < 0.05) in the activity of these transaminases was detected in the serum of mice in the group that received the co-treatment of B[a]P and APAP compared to the control group (Figure 2).

Relative Gene Expression
The results of the gene expression profile for all experimental groups are shown in Figures 3 and S2 and Table S2.B[a]P/APAP treatment caused changes in the relative expression of various genes coding for proteins involved in different biochemical processes.B[a]P/APAP co-exposure led to a significant increase in the expression of Cyp1a1

Relative Gene Expression
The results of the gene expression profile for all experimental groups are shown in Figures 3 and S2 and Table S2.B[a]P/APAP treatment caused changes in the relative expression of various genes coding for proteins involved in different biochemical processes.B[a]P/APAP co-exposure led to a significant increase in the expression of Cyp1a1 (Cytochrome P450 Family 1 Subfamily A Member 1), which is a gene encoding one of the enzymes responsible for the metabolism of xenobiotics, including B[a]P, and a decrease in the expression of the genes Ahr (Aryl Hydrocarbon Receptor), Cyp2e1 (Cytochrome P450 Family 2 Subfamily E Member 1), Sod1 (Superoxide Dismutase 1), Ppar-γ (Peroxisome Proliferator-Activated Receptor Gamma), Casp3 (Caspase 3), and Trp53 (Transformation-related Protein 53).These results suggest that B[a]P/APAP co-exposure promotes alterations in AhR activation, oxidative stress, lipid and xenobiotics metabolism, and apoptosis pathways.

Pathological Examination
Examination of liver tissue showed very few or no histological alterations in the liver of the control, APAP-, and B[a]P-treated mice.B[a]P/APAP treatment caused several hepatic changes found in the B[a]P/APAP group, including ballooning injury, steatosis, necrosis accompanied by lobular and portal inflammation, and apoptosis (Figure 4).The sum of hepatic histopathological scoring was significantly increased (p < 0.05) in mice treated with B[a]P/APAP (Table 1; Figure S3A).Additionally, a good correlation was found between transaminase values and the histopathological scores obtained (Figure S3B).
Examination of liver tissue showed very few or no histological alterations in the liver of the control, APAP-, and B[a]P-treated mice.B[a]P/APAP treatment caused several hepatic changes found in the B[a]P/APAP group, including ballooning injury, steatosis, necrosis accompanied by lobular and portal inflammation, and apoptosis (Figure 4).The sum of hepatic histopathological scoring was significantly increased (p < 0.05) in mice treated with B[a]P/APAP (Table 1; Figure S3A).Additionally, a good correlation was found between transaminase values and the histopathological scores obtained (Figure S3B).In severity, 0: absent or rare, 1: mild, 2: moderate, 3: marked.The internal numbers in each histopathological parameter correspond to the number of mice for each degree of severity (frequency).Total histopathological finding per severity was calculated as the sum of the product of the level of severity × number of mice with the corresponding level of severity for each histopathological parameter.

Discussion
The findings reported here indicate that previous exposure to polynuclear aromatic hydrocarbons can potentiate the hepatotoxicity of APAP, evidenced by changes in gene expression, increased transaminase levels, and histopathological markers of tissue damage.
The liver plays a crucial role in maintaining various physiological processes related to the immune and endocrine systems as well as in metabolism and the detoxification of xenobiotics, including polycyclic aromatic compounds and drugs [53].In this study, both B[a]P alone and B[a]P + APAP treatments induced an increase in the liver-to-body weight ratio.This increase in the relative weight of the liver, as observed in previous studies involving male and female F-344 rats exposed to B[a]P [54], may be indicative of an adaptive response within the liver aimed at maintaining the organism's homeostasis.Such adaptation could involve alterations in enzymatic activity [55], as demonstrated in this study (Figure 3A).
The treatment with B[a]P induced a pronounced increase of Cyp1a1 gene expression, which was exacerbated by APAP (Figure 3A).This gene encodes a member of the Cytochrome P450 superfamily of enzymes involved in both B[a]P detoxification and metabolic activation.The latter process results in DNA adduct formation and reactive oxygen species (ROS) production [60].It is well established that B[a]P binds to the aryl hydrocarbon receptor (Ahr), a ligand-dependent transcription factor, and induces the transcription of genes involved in various pathways, including xenobiotic and lipid metabolism, and immune responses, such as Cyp1a1 (Cytochrome P450, family 1, subfamily a, polypeptide 1), Cyp1b1 (Cytochrome P450, family 1, subfamily b, polypeptide 1), Fabp5 (fatty acid binding protein 5), Ugdh (UDP-glucose dehydrogenase), Car3 (Carbonic anhydrase 3), Nqo1 (NAD(P)H dehydrogenase, quinone 1), Myc (Myelocytomatosis oncogene), and Mt1 (Metallothionein 1), among others [61].Due to the involvement of the AHR protein in various biological processes, cells have developed different mechanisms to prevent excessive signaling.One of these mechanisms involves the overexpression of phase I and II enzymes of xenobiotic metabolism, facilitating their removal through ATP-dependent membrane transporters [62].
CYP1A1 exhibits a high capacity for AHR-mediated metabolism and activation of B[a]P [60], resulting in the generation of B[a]P 7,8-epoxide; B[a]P 7,8 diols; B[a]P 7,8dihydrodiol-9,10-epoxide (BPDE); and reactive oxygen species (ROS) as by-products of these reactions [30].In this study, exposure to B[a]P/APAP resulted in a decrease in Ahr gene expression compared to the control group (Figure 3A), likely due to negative feedback induced by the degradation of its ligand B[a]P through CYP1A1.This negative feedback mechanism serves to regulate AhR signaling and prevent adverse effects [63].
In addition to its role in B[a]P activation, detoxification, and ROS release through metabolic reactions [30], Cyp1a1 expression also appears to be involved in the regulation of other Cytochrome P450 enzymes [64].In this study, Cyp1a1 was overexpressed in mice treated with B[a]P and those exposed to B[a]P/APAP.However, Cyp2e1, which encodes one of the key enzymes involved in metabolizing APAP to its toxic metabolite, was downregulated (Figure 3A).This observation may be attributed to a potential repression of Cyp2E1 expression by the overexpression of Cyp1a1 induced by reactive oxygen species produced during its metabolic activity.This inference is supported by a previous study demonstrating the downregulation of the CYP2E1 gene following exogenous H 2 O 2 addition and glutathione depletion mediated by benzo[a]pyrene treatment [65,66].
The conversion of paracetamol to NAPQI, its primary reactive metabolite, is catalyzed by various proteins of the Cytochrome P450 superfamily, predominantly by CYP2E1.However, other isoforms, including CYP1A2 and CYP3A4, also contribute to the bioactivation processes of APAP, as observed by several authors [67][68][69].Lee and coworkers demonstrated that other P450 enzymes such as CYP1A2 may be responsible for the toxicity in Cyp2e1 knockout mice at high doses of APAP (600 mg/kg) [70].Therefore, the metabolic activation of APAP in the co-exposed group could be promoted by an upregulation of Cyp1a2 following prior exposure to B(a)P, considering that the expression of CYP1a2 can be induced by polycyclic aromatic hydrocarbons [71].
The metabolism of APAP leads to the production of N-acetyl-p-benzoquinone imine (NAPQI), a reactive metabolite detoxified by conjugation with glutathione [2,11,14].As this antioxidant molecule is depleted by APAP, the reactive oxygen species (ROS) production induced by the metabolism of B[a]P deepens the antioxidant arsenal in the cell.Together with the accumulation of NAPQI at low doses of APAP, extensive cell damage occurs, explaining features such as necrosis, apoptosis, inflammation, hemorrhage, and congestion of the liver parenchyma [1,11,72], causing increased levels of aminotransferases [15].
Oxidative stress is largely regulated by the antioxidant enzyme system, including SOD, CAT, GPx, peroxiredoxin (Prx), and glutathione reductase (GSR), among others.Therefore, the loss of expression of these enzymes could be used as biomarkers of oxidative stress [73].In this study, a downregulation in the expression of genes encoding oxidative stress-scavenging enzymes such as Sod1 (Figure 3B) and Hmox1 (Figure S2) was evidenced, suggesting disturbances in the antioxidant system due to the overproduction of epoxides and other B[a]P byproducts [30,32].It is also known that there are regulatory sequences in the promoter region of the Sod1 gene that correspond to binding sites for various transcription factors such as AhR, Nrf2, and NF-κB, among others [74].Thus, the downregulation of Sod1 could also be associated with the decreased expression of Ahr observed in B[a]P/APAP-treated mice, favoring an oxidative cellular environment.
In addition to the molecular alterations implicated in xenobiotic metabolism and the disruption of the antioxidant system resulting from the co-exposure to B[a]P/APAP, an aberration in lipid metabolism was evidenced through the analysis of Ppar-γ gene expression, a member of the peroxisome proliferator-activated receptor family.PPAR-γ plays a crucial role in reducing hepatic lipotoxicity and ameliorating steatosis [75] by regulating lipid and glucose metabolism, enhancing glycogen synthesis, and improving mitochondrial function and fat mobilization from muscle/liver tissues [76].Previous studies have demonstrated that B[a]P can reduce mRNA expression of Pparγ in mice, leading to dyslipidemia and abnormal glucose metabolism [77].Consistent with these findings, in the current study, Ppar-γ was downregulated in B[a]P-treated mice, resulting in Sci.Pharm.2024, 92, 30 9 of 15 alterations in liver morphology, including hepatic steatosis [78] (Figure 4).These metabolic changes could render the liver more susceptible to injury induced by APAP, as indicated by numerous studies involving cell lines, mouse models, and human populations [79][80][81].
PPAR-γ has been reported to play a role in cancer, acting either as a promoter or a suppressor in tumor cell growth.Its function depends on the signaling pathways activated in the cellular microenvironment through interaction with various ligands [76,82].Molecular mechanisms involved in its role as an antineoplastic regulator are based on the inhibition of cell proliferation, induction of apoptosis, and terminal differentiation [83,84].The findings in this study suggest that the downregulation of Casp3 (Figure 3B) in the B[a]P/APAP group might be mediated by reduced Ppar-γ activity, as demonstrated in mutant mice with follicular thyroid carcinoma (TRβ PV/PV ) [85].
The alterations in gene expression observed in the B[a]P/APAP group resulted in hepatic changes including ballooning injury, steatosis, and necrosis accompanied by lobular and portal inflammation (Figure 4).This suggests that prior exposure to B[a]P can elevate the risk of APAP hepatotoxicity even at non-toxic doses, possibly due to the depletion of enzymatic and non-enzymatic endogenous antioxidants that counteract the adverse effects of free radicals produced during benzo(a)pyrene metabolism [30,32].Additionally, there is a super-production of toxic APAP metabolites through the activation of CyP450 enzymes [52], as evidenced by changes in the expression of genes involved in xenobiotic metabolism, the antioxidant system, lipid metabolism, and the regulation of cell proliferation, such as Cyp1a1, Cyp2e1, Sod1, Ppar-γ, and Casp3.Consequently, liver tissue damage typical of acetaminophen toxicity is induced, characterized by alterations in liver morphology including vacuolization of centrilobular hepatocytes, steatosis, and necrosis, accompanied by inflammation [72].The manifestation of liver injury is reflected in the release of high concentrations of liver transaminases detected in the serum (Figure 5).Our results demonstrate a significant association between transaminase levels (ALT and AST) and hepatic histopathological scoring (p < 0.0001), with positive correlations (r = 0.77 and 0.71, respectively) (Figure S3).

Laboratory Animals
Pathogen-free ICR-CD1 female mice (6 weeks old, 18-20 g), obtained from the National Institute of Health (Colombia), were included.The mice were housed in a controlled environment, maintaining a temperature of 25 ± 2 °C, relative humidity of 70-85%, and a 12 h light/dark cycle and were provided with free access to standard rodent food (Laboratory Rodent Diet 5001, St. Louis, MO, USA) and water.Before the commencement of ex-

Laboratory Animals
Pathogen-free ICR-CD1 female mice (6 weeks old, 18-20 g), obtained from the National Institute of Health (Colombia), were included.The mice were housed in a controlled environment, maintaining a temperature of 25 ± 2 • C, relative humidity of 70-85%, and a 12 h light/dark cycle and were provided with free access to standard rodent food (Laboratory Rodent Diet 5001, St. Louis, MO, USA) and water.Before the commencement of experiments, the mice underwent a 2-week acclimatization period.All procedures were conducted in accordance with the recommendations of the European Union regarding animal experimentation (Directive of the European Council 2010/63/EU) and were approved by the Ethics Committee on Scientific Research at the University of Cartagena (Minute No. 106 of 15 March 2018).

Experimental Design
Thirty ICR female mice were randomly distributed using a blind method into four groups as follows: group I, the non-exposure group (n = 5); group II, animals treated with B[a]P (Sigma-Aldrich, St. Louis, MO, USA) (n = 7); group III, mice treated with APAP (Sigma-Aldrich, St. Louis, USA) (n = 6); and group IV, animals treated with B[a]P/APAP (n = 12).Mice body weight was measured daily.The experimental groups did not exhibit significant differences in body weight.
First, 50 mg/kg B[a]P or sesame oil (vehicle/control) was intraperitoneally (IP) administered to mice for three consecutive days.Twenty-four hours after B[a]P administration, mice were treated with 200 mg/kg APAP or saline (vehicle/control) by a single intraperitoneal injection (IP), followed by euthanization 24 h after co-exposure [26] (Figure 6).The doses of 50 mg/kg B[a]P and 200 mg/kg APAP and the number of mice for each experimental group were selected based on a pilot study, which demonstrated that mice treated with this dosage regimen did not exhibit elevations in serum ALT activity (assessed separately for each group) and considering the risk of loss of experimental units from the co-exposed group.IP administration was chosen to ensure the absorption of APAP and B(a)P from the peritoneal cavity by the portal system.The entire experiment was repeated with similar results.

Sample Collection
After treatment, mice were euthanized by exsanguination under sodium pentobarbital anesthesia administered by IP injection at a dose of 60 mg/kg [86].Whole blood, collected via the portal vein, was centrifuged at 2000× g for 15 min at 4 °C.Serum was stored at −80 °C for further analysis.A tissue section of the liver was immediately stored in RNALater ® (Qiagen, Germantown, MD, USA) for gene-expression analysis, and another portion was collected in tubes with 10% buffered formalin for histopathological analysis.

Enzyme Analysis
Serum transaminase activities were measured according to the International Federation of Clinical Chemistry (IFCC) reference method for alanine and aspartate aminotransferase, using commercially available kits (Biosystems S.A., Barcelona, Spain).Test reactions were conducted at 37 °C, with absorbance monitored at 340 nm using a semi-auto- The doses of 50 mg/kg B[a]P and 200 mg/kg APAP and the number of mice for each experimental group were selected based on a pilot study, which demonstrated that mice treated with this dosage regimen did not exhibit elevations in serum ALT activity (assessed separately for each group) and considering the risk of loss of experimental units from the co-exposed group.IP administration was chosen to ensure the absorption of APAP and B(a)P from the peritoneal cavity by the portal system.The entire experiment was repeated with similar results.

Sample Collection
After treatment, mice were euthanized by exsanguination under sodium pentobarbital anesthesia administered by IP injection at a dose of 60 mg/kg [86].Whole blood, collected via the portal vein, was centrifuged at 2000× g for 15 min at 4 • C. Serum was stored at −80 • C for further analysis.A tissue section of the liver was immediately stored in RNALater ® (Qiagen, Germantown, MD, USA) for gene-expression analysis, and another portion was collected in tubes with 10% buffered formalin for histopathological analysis.

Enzyme Analysis
Serum transaminase activities were measured according to the International Federation of Clinical Chemistry (IFCC) reference method for alanine and aspartate aminotransferase, using commercially available kits (Biosystems S.A., Barcelona, Spain).Test reactions were conducted at 37 • C, with absorbance monitored at 340 nm using a semi-automatic BTS 350 analyzer (Biosystems S.A., Barcelona, Spain).

Gene Expression Assays
Hepatic RNA was isolated using the RNeasy ® Mini Kit (Qiagen, Germantown, MD, USA) following the manufacturer's instructions.RNA concentration and purity were determined using a Thermo Scientific Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific, Wilmington, MA, USA).RNA was quantified by spectrophotometry (A260), purity was assessed by the A260/A280 ratio, and integrity was checked by visual inspection on an agarose gel after an electrophoresis run.RNA aliquots were stored at −80 • C for later use.
Extracted RNA (2 µg) was used as a template to synthesize cDNA utilizing a High-Capacity RNA-to-cDNA™ Kit (Applied Biosystems, Foster City, CA, USA).The cDNA was used as a template in a total volume of 20 µL, containing 10 pmol each of forward and reverse gene-specific primers.The amplification was carried out considering the appropriate annealing temperature for each pair of specific primers for the genes of interest.The Real-Time PCR reaction was performed on a StepOne ® Real-Time PCR System (Applied Biosystems, Foster City, CA, USA), using the PowerUp™ SYBR ® Green Master Mix kit (Applied Biosystems, Foster City, CA, USA).
Eleven genes coding for proteins involved in different biochemical pathways were analyzed, including markers of xenobiotic metabolism, inflammation, apoptosis, oxidative stress, and lipid metabolism.Accession numbers and primer sequences for the target genes are provided in Table S1.Changes in gene expression were determined using the relative standard curve method [87,88].The fold-change of the target gene expression is presented as the n-fold difference relative to the control group.β-Actin and Rps29 were employed as housekeeping genes.

Pathological Examination
Liver tissue sections were treated with 10% phosphate-buffered neutral formalin, dehydrated using a graded ethanol series (25-100%), and then embedded in paraffin.The tissue blocks were cut into 5 um thick slices, deparaffinized with xylene and ethanol, and then stained with hematoxylin and eosin (H&E) for the examination of histopathological changes [86].Images of different tissue sections were captured using a Nikon ECLIPSE E-100 microscope, coded, and examined in a blinded manner to avoid any type of bias.
The following parameters were examined in liver tissue: ballooning, steatosis, portal inflammation, lobular inflammation, and necrosis.For semi-quantitative effects, each histopathological finding was scored as follows: a score of 0 indicates severity absent or rare; 1, mild; 2, moderate; and 3, marked.Total scores for hepatic histopathology lesions in each mouse were calculated by the sum of scores from each of the parameters found.A minimum pathology was associated with a mouse liver score less than 5, moderate pathology if the total score was 5-10, and marked pathology if it was 10-15 [89,90].

Statistical Analysis
Results are expressed as the mean ± standard error of the mean (SEM).The normality and homogeneity of variance were verified using the Shapiro-Wilk and Bartlett tests, respectively.Differences between the means of the groups were evaluated by ANOVA, followed by Dunnett's post hoc test.In the absence of normality, the Kruskal-Wallis nonparametric test for independent variables followed by Dunn's post hoc test was used.
Spearman correlation analysis was employed to explore associations between histopathological scores, serum transaminase activities, and fold change of gene expression.
GraphPad Prism version 8.0.0 for Windows (San Diego, CA, USA) was utilized for statistical purposes, and a p-value < 0.05 was considered statistically significant.

Conclusions
The present study demonstrates that prior exposure to benzo[a]pyrene increases susceptibility to APAP-induced liver injury during treatment at non-hepatotoxic doses in mice.This susceptibility is attributed to changes in gene expression related to cellular metabolism, redox balance, and regulation of cell proliferation, leading to histopathological alterations in the liver, including ballooning injury, steatosis, inflammation, and necrosis (Figure 5).Consequently, therapeutic doses of APAP may result in liver injury in individuals exposed to B[a]P.Further studies are warranted to elucidate exposome-related factors contributing to acetaminophen-induced liver injury.

Figure 1 .
Figure 1.Effects of B[a]P/APAP co-exposure on the relative liver weight for the different experimental groups.The mice group exposed to B[a]P and B[a]P + APAP exhibited a significant increase in the liver-to-body weight ratio compared to the control group.Control, non-exposure group (n = 5); B(a)P, animals treated with B[a]P (n = 7); APAP, mice treated with APAP (n = 6); B(a)P + APAP, animals treated with B[a]P and APAP (n = 12).Data are expressed as mean ± SEM. * indicates significantly different control group.The level of significance was set at p < 0.05.

Figure 2 .
Figure 2. Serum ALT and AST levels in different treatment groups.Serum ALT activity was significantly elevated in B[a]P + APAP-treated mice.Control, non-exposure group (n = 5); B(a)P, animals treated with B[a]P (n = 7); APAP, mice treated with APAP (n = 6); B(a)P + APAP, animals treated

Figure 1 .
Figure 1.Effects of B[a]P/APAP co-exposure on the relative liver weight for the different experimental groups.The mice group exposed to B[a]P and B[a]P + APAP exhibited a significant increase in the liver-to-body weight ratio compared to the control group.Control, non-exposure group (n = 5); B(a)P, animals treated with B[a]P (n = 7); APAP, mice treated with APAP (n = 6); B(a)P + APAP, animals treated with B[a]P and APAP (n = 12).Data are expressed as mean ± SEM. * indicates significantly different control group.The level of significance was set at p < 0.05.

Figure 1 .
Figure 1.Effects of B[a]P/APAP co-exposure on the relative liver weight for the different experimental groups.The mice group exposed to B[a]P and B[a]P + APAP exhibited a significant increase in the liver-to-body weight ratio compared to the control group.Control, non-exposure group (n = 5); B(a)P, animals treated with B[a]P (n = 7); APAP, mice treated with APAP (n = 6); B(a)P + APAP, animals treated with B[a]P and APAP (n = 12).Data are expressed as mean ± SEM. * indicates significantly different control group.The level of significance was set at p < 0.05.

Figure 2 .
Figure 2. Serum ALT and AST levels in different treatment groups.Serum ALT activity was significantly elevated in B[a]P + APAP-treated mice.Control, non-exposure group (n = 5); B(a)P, animals treated with B[a]P (n = 7); APAP, mice treated with APAP (n = 6); B(a)P + APAP, animals treated

Figure 2 .
Figure 2. Serum ALT and AST levels in different treatment groups.Serum ALT activity was significantly elevated in B[a]P + APAP-treated mice.Control, non-exposure group (n = 5); B(a)P, animals treated with B[a]P (n = 7); APAP, mice treated with APAP (n = 6); B(a)P + APAP, animals treated with B[a]P and APAP (n = 12).Each bar represents the mean ± SEM. * indicates significantly different control group.The level of significance was set at p < 0.05.

Figure 3 .
Figure 3. Effects of B[a]P/APAP co-exposure on the mRNA levels of target genes in the liver of ICR mice.(A).Genes involved in xenobiotic metabolism (B).Genes related to oxidative stress and apoptosis.Expression was normalized against β-Actin and Rps29 (housekeeping genes).Control, nonexposure group (n = 5); B(a)P, animals treated with B[a]P (n = 7); APAP, mice treated with APAP (n = 6); B(a)P + APAP, animals treated with B[a]P and APAP (n = 12).Data are expressed as mean ± SEM. * indicates significantly different from control group; a indicates significantly different from B[a]P group.The level of significance was set at p < 0.05.

Figure 3 .
Figure 3. Effects of B[a]P/APAP co-exposure on the mRNA levels of target genes in the liver of ICR mice.(A).Genes involved in xenobiotic metabolism (B).Genes related to oxidative stress and apoptosis.Expression was normalized against β-Actin and Rps29 (housekeeping genes).Control, non-exposure group (n = 5); B(a)P, animals treated with B[a]P (n = 7); APAP, mice treated with APAP (n = 6); B(a)P + APAP, animals treated with B[a]P and APAP (n = 12).Data are expressed as mean ± SEM. * indicates significantly different from control group; a indicates significantly different from B[a]P group.The level of significance was set at p < 0.05.

Figure 4 .Table 1 .Figure 4 .Table 1 .
Figure 4. Effects of B[a]P/APAP on hepatic morphology of ICR mice.Light photomicrographs of liver sections from the control group, mice exposed to APAP alone, B[a]P alone, or a combination of B[a]P and APAP.Hematoxylin and eosin staining (H&E) (40× and 100×).He, hepatocytes with normal architecture; Si, sinusoids; PT, portal triad (portal vein, hepatic artery, and bile duct); Ba, ballooning injury; St, steatosis; Ne, necrosis; LI, lobular inflammation; PI, portal inflammation; Ap, apoptosis.Table 1. Scoring of hepatic histopathological examinations from different groups of mice exposed to APAP, B[a]P, and APAP + B[a]P.

Figure 5 .
Figure 5. Molecular mechanisms involved in the potentiation of acetaminophen hepatotoxicity by B[a]P.The overexpression of Cyp1a1 induced by B[a]P leads to the downregulation of Cyp2e1 and excessive production of reactive metabolites of B[a]P.This, along with the under-expression of the Sod1 and Ppar-γ genes, induces a dysregulation of the redox balance in liver tissue due to the depletion of antioxidants and the downregulation of apoptotic signals via Casp3.Thus, exposure to nonhepatotoxic doses of APAP results in the production of NAPQI (possibly mediated by CYP1A2), causing ballooning, steatosis, necrosis, and inflammation as well as the release of elevated levels of serum transaminases such as ALT and AST because of liver injury.

Figure 5 .
Figure 5. Molecular mechanisms involved in the potentiation of acetaminophen hepatotoxicity by B[a]P.The overexpression of Cyp1a1 induced by B[a]P leads to the downregulation of Cyp2e1 and excessive production of reactive metabolites of B[a]P.This, along with the under-expression of the Sod1 and Ppar-γ genes, induces a dysregulation of the redox balance in liver tissue due to the depletion of antioxidants and the downregulation of apoptotic signals via Casp3.Thus, exposure to non-hepatotoxic doses of APAP results in the production of NAPQI (possibly mediated by CYP1A2), causing ballooning, steatosis, necrosis, and inflammation as well as the release of elevated levels of serum transaminases such as ALT and AST because of liver injury.

16 Figure 6 .
Figure 6.Experimental Design.Animals were given 0 or 50 mg/kg B[a]P for three consecutive days and then exposed to 0 or 200 mg/kg APAP.Mice were euthanized at 24 h after APAP injection.

Figure 6 .
Figure 6.Experimental Design.Animals were given 0 or 50 mg/kg B[a]P for three consecutive days and then exposed to 0 or 200 mg/kg APAP.Mice were euthanized at 24 h after APAP injection.