Food-Borne Polycyclic Aromatic Hydrocarbons and Circadian Disruption

Circadian disruption has been found to increase the risk of metabolic diseases, brain disorders, and cancer. The aryl hydrocarbon receptor (AhR), responsible for xenobiotic metabolism, is known to be activated by certain environmental stimuli, including polycyclic aromatic hydrocarbons (PAHs). Exposure to these stimuli may lead to diseases related to circadian disruption, with AhR activation suggested as a leading cause. Both the aryl hydrocarbon receptor nuclear translocator (ARNT) and aryl hydrocarbon receptor nuclear translocator-like (BMAL1) are class II basic helix–loop–helix/Per-ARNT-SIM (bHLH-PAS) proteins. These proteins form heterodimers with stimulated class I bHLH-PAS proteins, including circadian locomotor output cycles kaput (CLOCK) and AhR. Due to their sequential similarity, the overactivation of AhR by toxicants, such as PAHs, may lead to the formation of heterodimers with BMAL1, potentially causing circadian disruption. Dysregulation of BMAL1 can affect a wide range of metabolic genes, emphasizing its crucial roles. However, this issue has not been adequately addressed. Previous studies have reported that the inhibitory effects of phytochemicals on AhR activation can ameliorate diseases induced by environmental toxicants. Additionally, some phytochemicals have shown preventive effects on circadian misalignment. Therefore, this Review aims to explore potential strategies to prevent circadian disruption induced by food-borne toxicants, such as benzo[a]pyrene; to generate new ideas for future studies; and to highlight the importance of investigating these preventive strategies.


CIRCADIAN CLOCK
Research on the mechanisms controlling circadian rhythms was awarded the 2017 Nobel Prize in Physiology and Medicine; this provided the concept that rhythmic outputs of expression of some genes are regulated by clock genes, including aryl hydrocarbon receptor nuclear translocator-like (BMAL1), circadian locomotor output cycles kaput (Clock), cryptochrome (Crys), and period (Pers), which form a regulatory feedback loop controlled by signals in a cellautonomous and self-sustained manner. 1,2Thousands of genes are driven by these core circadian regulators and result in tissue-specific functions under a certain rhythmicity. 3.1.Circadian Clock, Master Clock, and Peripheral Clock.The circadian clock serves as the internal timing machine that ensures organisms maintain the rhythmic consistency to adapt to the Earth's rotational period of around 24 h. 4 The rhythmic biological clock can autonomously regulate the above-mentioned physiological functions of organisms periodically in the short-term absence of environmental cues, such as light.5 Most mammals present regular rhythmic oscillation, known as circadian rhythm, in their regular activities such as the sleep−wake cycle and dietary stress, which regulate essential physiological functions including body temperature maintenance, metabolism processes, energy homeostasis, and hormone secretion.4 The endogenous biological rhythm, approximately 24 h in duration, controls many biological functions, such as gene expression, and is preserved in most organisms across evolution.6 The suprachiasmatic nucleus (SCN), the central pacemaker of the circadian clock known as the master clock of the mammalian hypothalamus, is responsible for modifying the rhythmic output signals with light stimuli.7 It is recognized as the dominant controller of behavioral rhythm.However, circadian genes are capable of expressing in isolated peripheral organs with or without signals from the SCN. 4 Light is known as the only exogenous photic entrainment to the mammalian circadian system, but there are other nonphotic entrainments that hold biological significance.8 Notably, light and nutrient consumption have a large impact on biological rhythms, and the latter could directly affect peripherals (Figure 1). 9 "There is no organ and no function in the body which does not exhibit a similar daily rhythmicity", according to Gierse (1842) and restated by Dr. Aschoff (1965).The concept that circadian rhythm affects the body's functionality and peripheral liver has been proposed for at least nine decades.10 Nonphotic entrainment could be partially responsible for circadian misalignment.Unlike the master clock SCN, the circadian rhythm of peripheral clocks in other organs or tissues can be potentially disrupted by a variety of environmental stimulative factors including but not limited to temperature, jet lag, sleep disorder, dietary stress, artificial light, and stress causing allostatic load accumulation.11,12 The circadian activities in different tissues can be specifically modulated by certain signaling molecules produced by neuroendocrine tissues and their interaction with their cognate receptor counterparts located and functioning in those tissues. 9Briefly, via endocrine outflow, the SCN clock could affect organ function dependently/independently through peripheral clock synchronization (Figure 2).13 Furthermore, exposure to environmental toxicants and drugs has been shown to have a disruptive impact on circadian rhythm, which can result in metabolic disease development.14 This is indicated in the growing epidemiological evidence that shows shift work has become a risk factor for hypertension, stroke, and coronary heart disease, while social jetlag may be associated with an elevation in triglyceride levels, a reduction in high-density lipoprotein (HDL), and insulin sensitivity.15 Cardiovascular disease, nonalcoholic fatty liver disease (NAFLD), 16 obesity, and leptin resistance 17 are the suggested consequences of metabolic dysfunction due to circadian disruption.These results are supported by established findings that provide evidence that energy homeostasis, especially glucose and lipid metabolism, could be regulated by circadian machinery.14 Moreover, disruptive effects on circadian rhythm may also lead to cognitive deficits and neurodegenerative diseases, such as Alzheimer's disease and Parkinson's disease. 1.2. Mlecular Mechanism.It is clearly understood that the molecular clock of most mammals constitutes a transcriptional−translational feedback loop (TTFL) that consists of core clock genes including initiators, Clock, brain-and-muscle Figure 1.Overview of the principal circadian clock of mammals.Ambient light is one of the entrainment factors that affect the master clock and hormone output, and these hormone signals will control the peripheral clocks in other organs, thereby impacting fasting/feeding times. Fting/ feeding times and diet composition are also entrainment factors that could regulate peripheral clocks and the compositions of gut microbiota.Consequently, gut microbial metabolites affect peripheral clocks in organs such as the liver.
ARNT-like protein (BMAL1) that transcribes "period" genes (PER1, PER2, and PER3), cryptochrome genes (CRY1 and CRY2) that function as inhibitors in the transcriptional feedback loop, and Rev-Erb-α and ROR-α that can positively and negatively regulate BMAL1. 11In addition to core clock genes, the binding of heterodimer CLOCK-BMAL1 onto the E-box located in the promoter region also promotes the transcriptome.It is suggested that 7−13% of genes are regulated by the circadian system, so-called clock-controlled genes (CCGs).Although the core components of molecular clocks are mostly the same in different tissues, the tissuespecific expression patterns of these circadian genes lead to minorly overlapped cycling transcripts. 2As listed by Zhang and Kay in 2010, some CCGs have been successfully identified in which their clock function and their oscillations controlled by the intrinsic clock are responsible for the efficiency of energy use and environmental change anticipation. 18These CCGs encode proteins involved in transcription, translation, signal transduction, the cell cycle, and metabolic processes that have been widely discussed in recent years.
In the following section, the role of BMAL1, which has been identified as a factor in the incidence of diseases or malfunction of organs, will be introduced.

Brain and Muscle ARNT-like Protein (BMAL1).
Brain and muscle ARNT-like protein-1 (BMAL1) is a protein that is expressed in some brain regions at a high level and is responsible for the synchronization of peripheral oscillations in muscle and other organs. 19As a major and essential component of the circadian clock, BMAL1 is one of the most widely studied genes because of its impact on disease incidence.In 2008, Lamia et al. found significant physiological changes in liver-specific deleted BMAL1 −/− mice in both normal light−dark cycles and constant dark. 20Under the conditions of a standard light−dark cycle, BMAL1 −/− mice showed significant increases in body weight and total fat content, slower restoration of blood glucose, and hypersensitivity trends in serum insulin.At the molecular level, the absence of BMAL1 led to a loss of the rhythmic expression of glucose transporter 2 (Glut2) in both mRNA and protein levels.Deletion of BMAL1 in the liver was found to be more relevant to metabolic dysregulation, while mutation of BMAL1 (Mop3) nonspecifically in certain organs may increase the progression of aging and mortality.In 2005, Burger et al. found the development of joint ankyloses in Mop3 null mice, which severely decreased their activity levels. 21It was concluded that the absence of the MOP3 protein was the major factor causing mice to exhibit reduced locomotor activity and fail to thrive during aging due to abnormal ectopic mineralization and ossification at the insertion site of tissues and bones.Therefore, Mop3 was suggested to be a potential inhibitor of ossification in ligaments and tendons (     induce hepatomegaly, nonalcoholic fatty liver disease (NAFLD), and even spontaneous hepatocellular carcinoma (HCC) with a sufficient induction period. 16The authors revealed that chronic circadian disruption could cause dysregulation of the sympathetic nervous system (SNS), which results in activation of the constitutive androstane receptor (CAR) and inhibition of the Farnesoid X receptor (FXR).The combined effect of intrahepatic bile acid accumulation and progression to nonalcoholic steatohepatitis (NASH) and advanced liver fibrosis activated by circadian disruption was the major cause of spontaneous HCC.In addition to hepatic diseases, the concept of circadian dysfunctions inducing obesity and leptin resistance were demonstrated by the same research team in the previous year.It has been revealed that both the central clock and the peripheral clock control leptin endocrine homeostasis.BMAL1/CLOCK in adipose tissue controls leptin transcription, driving the rhythmicity of serum leptin. 17Briefly, BMAL1/CLOCK could stimulate C/EBPα for leptin transcription at a particular phase, but chronic jetlag abolished the rhythmic bindings.Although the serum leptin levels were higher, failure to activate the pSTAT3/POMC pathway indicated leptin resistance induced by chronic jetlag.Adiponectin and leptin are both recognized as family members of adipokines, and the secretion of leptin was elucidated to be controlled by the circadian clock.Similarly, in 2015, Barnea et al. suggested that the expression of adiponectin occurred in a circadian manner and its expression mediators, peroxisome proliferator-activated receptor γ (PPARγ) and PPARγ coactivator 1α (PGC1α), should be known as CCGs. 22t was found that expression of AdipoQ (encoding gene of adiponectin) is positively controlled by PPARγ+RXR and CLOCK+BMAL1 but negatively related to the addition of CRY1.Furthermore, the expression of adipoQ significantly reduced when the core clock gene Clock was silenced.However, the underlying molecular mechanism required further clarification.Clock is the first clock gene identified in vertebrates that is similar to BMAL1, which encodes the basic helix−loop−helix (bHLH)-PAS transcription factor.Early in the year 2000, Oishi et al. proved that BMAL1 loses its rhythmicity and is sharply expressed at all phases in the brain, liver, heart, and kidney when the Clock is mutated, while at the same time expression of Per2 and Dbp dramatically decreases without significant oscillation. 23.2.2.Role of BMAL1 in Cancer Progression.It has been found that the effects of circadian disruption are not limited to metabolic disease but also contribute to cancer progression (Table 1).In 2016, Papagianakopoulos et al. demonstrated that lung cancer progression was promoted and accelerated by jetlag. 24Both Kras and Kras +p53 mutations contributed to spontaneous tumorigenesis in mice, but it was found that jetlag induction during the tumor progression period significantly increased the tumor burden.To dissect the role of clock genes in tumorigenesis, Per2 and BMAL1 mutations were assessed.The finding showed that the loss of BMAL1 and Per2 further increased tumor burden and decreased survival, which comparatively had a greater impact in the BMAL1-mutated group.Furthermore, it was revealed that the BMAL1 mutation could increase the tumor burden, possibly via a p53-dependent mechanism, while the Per2 mutation was related to an increase in c-Myc.Lastly, an increase in lung cancer proliferation was observed to be associated with the consumption of glucose and glutamine, implying that cancer progression could be highly related to metabolism controlled by circadian genes.
One study suggested that human pancreatic ductal adenocarcinoma could be predicted by BMAL1 expression.According to Li et al. (2016), up to 70.1% of patients with pancreatic ductal adenocarcinoma exhibited low expression of BMAL1 in tumor samples.Among them, the cases could be further classified into clinical stages I, II, III, and IV with percentages of 30.8%, 61.3%, 84.6%, and 94.1%, respectively, which indicated the ratio increased with advancing stages and positively correlated to overall survival and disease-free survival times. 25Another clinical case study by Jiang et al. confirmed the finding.The mRNA and protein levels of BMAL1 were significantly lower in the pancreatic tumor as compared to the noncancerous tissue from the individual in up to 45 cases, and the expression was negatively correlated to the TNM stage. 26o determine the role of BMAL1 in pancreatic cancer, PC cell line BxPC-3 with a higher BMAL1 protein level was silenced with shBMAL1, while AsPC-1 with lower BMAL1 expression was transfected to overexpress BMAL1.Surprisingly, upregulated genes of BxPC-3/shBMAL1 cells were primarily involved in survival and cell proliferation, while those related to the apoptotic process were dramatically downregulated in the GO analysis.Furthermore, it was revealed that genes associated with the p53 pathway, NF-κB pathway, and PI3K-Akt pathways were downregulated, indicating the suppressive role of BMAL1 in pancreatic cancer.Conversely, AsPC-1 that stably overexpressed BMAL1 markedly decreased proliferation and arrested in the G2/M phase compared to its controlled counterpart.The tumor suppressive effect was maintained in xenograft models, resulting in a smaller tumor size and reduction in invasiveness when BMAL1 was overexpressed.Most importantly, BMAL1 was suggested to directly bind on the promoter region for transcription of the p53 gene.
In line with the above findings, Kiessling et al. suggested tumor growth could be inhibited by enhancing the function of the circadian clock.It was found that by treating cancer cells with dexamethasone (DEX), an agonist of the glucocorticoid receptor, the rhythmicity of cells could be observed, accompanied by cell cycle regulators. 27Interestingly, after being treated with DEX, fewer cells entered in the S phase, while more were in the G0/G1 phase, which indicated a decrement in the number of cells undergoing DNA replication.DEX-induced activation of the circadian clock was assumed to be responsible for slowing tumor cell proliferation and upregulation of rhythmic clock genes, including Per1, Per2, Cry1, and Nr1d1, along with the protein level of BMAL1.To further confirm that the inhibitory effect of DEX on tumor growth was due to activation of the clock, the BMAL1 gene was knocked down; as expected, the relative tumor volume could no longer be suppressed and the rhythmicity of most BMAL1 target genes was disrupted.
The importance of BMAL1 was also noted in ferroptotic cancer cell-associated inflammation. 28Nuclear protein HMGB1 is the mediator triggering the inflammatory response, and it was demonstrated that HMGB1 expression induced by ferroptosis in acute pancreatitis could be regulated by BMAL1.BMAL1 could reverse the downregulation of antioxidant enzyme expression and proteins involved in the membrane repair system, resulting in a protective effect against tissue injury and a reduction in the HMGB1 level that is positively correlated with cancer-related inflammation.
Collectively, it has been corroborated that BMAL1 exhibits conspicuous and causal roles in the incidence of metabolic diseases and cancer progression.Moreover, the above findings suggest the potential to involve BMAL1 in tumor inhibition strategies.However, it could not be elucidated if circadian disruption induced by jetlag, social jetlag, or shift work could lead to severe consequences, such as clock gene mutation or deletion.Nevertheless, circadian misalignment or abnormal expression might deteriorate disease conditions or progression.On the other hand, the evidence showed that exposure to air pollution could lead to circadian toxicity. 29Some of the proteins involved in the xenobiotic detoxification process are also found to have a molecular link with the circadian clock.For instance, the mRNA level of aryl hydrocarbon receptor (AhR) and its downstream detoxification enzymes were found to be regulated by the Clock gene. 30Another study demonstrated that the inhibition or disruption of Per1 and Per2 might result in greater expression of cyp1a1 and cyp1b1 induced by TCDD in both in vivo and in vitro studies, which suggests an important role for Per1 in AhR-regulated gene modulation. 31Neuronal PAS domain protein 2 (NPAS2), as the paralog of CLOCK, was identified as a regulator of hepatic CYP1A2 in both cell and animal models. 32Both AhR and CLOCK are class I basic helix�loop−helix/Per-ARNT-SIM (bHLH-PAS) proteins that sense environmental signals and form heterodimers with class II proteins, resulting in target gene regulation. 33Huang et al. demonstrated high coordination of CYP1A1 and PER2 in the posterior pituitaries and livers of rats, which suggests the possible interaction or similar responses of AhR/ARNT and CLOCK/BMAL1 systems toward environmental changes. 34These studies hint at a causal loop between xenobiotic detoxification and circadian regulation.Therefore, this Review will be focused on the effect of food-borne activators of xenobiotic detoxification and their possible effect on circadian disruption.

AHR ACTIVATION INTERFERES WITH CIRCADIAN RHYTHM
Several studies mentioned the interference in the circadian rhythm caused by environmental toxicants.For example, benzo[a]pyrene (B[a]P) may cause lung inflammation via its alteration of the circadian pattern of blood pressure. 35It has been reviewed that exposure to air pollutants may affect rhythmic pulmonary and cardiovascular functions and consequently increase the risk of vascular and cardiometabolic disorders. 29Another study suggested that parental DNA damage and hypermethylation of the Per1 promoter caused by B[a]P could pass to offspring. 36B[a]P is a potent substrate for CYP1A1 and 1B1, and AhR is a ligand-activated transcription factor that can be activated by B[a]P and is responsible for the transcription of the genes involved in xenobiotic metabolism. 37However, further clarification is needed to understand the underlying mechanisms of these toxicants disrupting or destroying the circadian rhythm and causing disease.

ARNT and BMAL1
. ARNT forms a heterodimer with activated AhR and subsequently regulates transcription. 38MAL1 is a protein that has a sequence homology similar to the ARNT protein, which includes similar splice patterns of intron/exon and five conserved exons that compose the Per-ARNT-Sim (PAS) domain. 39Core clock genes, such as BMAL1, CLOCK, and the family members of Cry and Per, are known as PAS-domain-containing proteins, and the domain facilitates the formation of heterodimers for protein−protein interactions and self-regulation.Due to the similarity of PAS domains, activated AhR can form a heterodimer with BMAL1 instead of ARNT, which may consequently disrupt CLOCK/BMAL1 activation and dampen the transcriptional rhythm.
The ligands of AhR vary from environmental toxicants to pharmaceuticals and phytochemicals.Environmental toxicants include tetrachlorodibenzo-p-dioxin (TCDD), which has a high affinity to AhR, halogenated aromatics, and polyaromatci hydrocarbons (PAHs). 40Pharmaceuticals and phytochemicals that can act as AhR ligands can be classified by their sources, which may include dietary ligands such as quercetin and resveratrol, metabolized food components such as indole[3,2b] carbazoles, and gut microbial metabolites such as butyrate and indole-3-acetic. 41In 2008, it was demonstrated that exposure to TCDD, an AhR agonist, could lead to changes in the gene expression of BMAL1 and Per1. 42It was further proven by immunoprecipitation that heterodimerization of BMAL1 with AhR increased in mouse ovarian extract following TCDD intervention. 43This finding was also supported by a study that demonstrated the enhancement or alteration of circadian and clock-controlled metabolic genes in AhR depletion mice. 44Exposure to TCDD has also been found to abolish the oscillating expression of up to 5636 clockcontrolled genes in the liver.The finding suggested that activation of AhR could severely disrupt the circadian regulation of hepatic metabolism. 45Supportingly, the binding of activated AhR to the E-Box of the promoters of the clock gene and lipolysis gene was found to increase and consequently lead to an alteration in adipose tissue rhythmicity and impairment of lipolysis in mouse adipose tissue. 46Transcription of Per1 is driven by the binding of the CLOCK/ BMAL1 heterodimer to the E-box of the Per1 promoter, and it was found that AhR activation could lead to dysregulation of rhythmic Per1 and contribute to metabolic issues. 47he findings of these studies suggest that activation of AhR can have a circadian influence that may subsequently affect the host metabolism.On the other hand, it was also reported that the timing of exposure to the pollutants or toxicants known as AhR ligands can be critical because of the responsive expression of the xenobiotic enzymes that are controlled by circadian regulation. 48Therefore, it should be understood that environment toxicants, including food-borne toxicants, could be potential nonphotic entrainments causing circadian disruption.

POLYCYCLIC AROMATIC HYDROCARBONS (PAHS)
Polycyclic aromatic hydrocarbons (PAHs) are persistent environmental pollutants.Due to their ubiquity, actions for remedying the health issues caused by PAHs are a global concern. 49PAHs consist of multiple aromatic rings that can present in different molecular arrangements (linear, cluster, or angular) and are classified into different groups according to the number of rings present in the compounds, where those with lower molecular weight (2−3 rings) possess higher solubility in water (Figures 3 and 4). 50More than 200 PAHs can be generated via pyrolysis, a process that involves three main factors: organic matter, reduced oxygen levels, and hightemperature conditions.Human exposure to PAHs can occur through dietary and nondietary sources.The latter includes skin contact and inhalation, while the former can occur via the consumption of environmentally contaminated foods or foods that have undergone high-temperature processes, such as roasting, toasting, and frying. 51revious studies have been conducted to determine the health effects of PAH exposure.A variety of adverse effects and health risks have been discovered, including carcinogenicity, teratogenicity, genotoxicity, neurotoxicity, and immunotoxicity.Short-term but high-concentration exposure can lead to skin and eye irritation, confusion, nausea, and impairment in lung function causing asthma, while long-term lower-dose exposure is implicated in the incidence of cancers, such as lung cancer, skin cancer, and cancers of the digestive tract. 52In addition, the association between long-term exposure to PAHs and an increase in the prevalence of hypertension has been reported in several studies due to the overactivation of AhR. 53herefore, reducing opportunities for PAH exposure has become a major concern to protect public health and safety.Some strategies have been suggested to minimize the exposure risk of humans to PAHs, such as regulation of cooking or food processing practices. 54Nevertheless, once PAH contamination has occurred it is difficult remove 55 and therefore methods for the detection of PAHs are required.
Besides inhaled air, dietary intake serves as a major route of human exposure to PAHs, 56 and because it is controllable the detection of PAH in food has gained its place in the research field.For the detection of PAH contamination in food, common analytical targets include naphthalene (NA), anthracene (ANT), benzo[a]anthracene (BAA), dibenzopyr-ene (DBP), and benzo[k]fluoranthene (BKF). 57Among a list of PAHs that are commonly found in foods, 15 have confirmed genotoxicity. 57Until 2008, benzo[a]pyrene (B[a]P) was the only marker recognized for the identification of the occurrence of PAH contamination in food, however, several markers are currently used. 58The regulations were changed for the following reasons: (1) even if the concentration of B[a]P is low in food, contamination with other PAHs may still be present and (2) B[a]P is a minor contributor to total cancer risk at around 11%. 59 Before introducing PAH2, PAH4, and PAH8 as more accurate markers, it is important to note that according to regulations, smoked molluscs, muscle meat of smoked fish, heat-treated meat products, cocoa butter, and chocolates are food commodities with high potential to be contaminated by B[a]P.The maximum allowable level of B[a]P is 5 μg/kg.Due to the contamination of PAHs in water, direct consumption of aquatic organisms could contribute to the intake of PAHs.Additionally, PAH contamination has been found in other foods, including wheat flour (total PAHs 0.71−1.66μg/kg), toasted bread (total PAHs 7.38−18.0μg/kg), cabbage (total PAHs 9 μg/kg), lettuce (total PAHs 14 μg/kg), and parsley (total PAHs 15−120 μg/kg).It should be noted that the contamination levels could be higher in industrial cities. 57 Furthermore, meat products, oils, and water-based food products are also highly susceptible to PAH contamination.According to a review by Domingo and Nadal, the estimated intake of PAHs could reach up to 59.2 μg/day, although most studies have reported much lower intake amounts of PAHs. 60AH8 includes benzo  and B[a]P.Among them, BAA and chrysene are considered light compounds that can be easily inhaled. 62It was previously reported that up to 60% of lung cancers were caused by B[a]P and other PAHs found in cigarettes, with each cigarette potentially containing 20−40 ng of B[a]P.The carcinogenic response could also occur in the bladder, breast, cervix, and prostate. 63According to a recent clinical study investigating the correlation between smoking and oxidative stress in chronic obstructive pulmonary disease patients, B[a]P was the most abundant carcinogenic PAH in smokers and was positively correlated with higher serum uric acid levels. 64lthough lower molecular weight PAHs are predominant in commercial cigarettes, B[a]P is used as a representative marker for total PAHs based on the strong positive correlation between them. 65However, B[a]P might not be an appropriate surrogate for all 14 PAHs in cigarettes. 66A multiyear study of PAHs in cigarettes suggested that the B[a]P level is representative of 4-and 5-ring PAHs, while fluorene is representative of 3-ring PAHs, with a high observed correlation. 67he toxicity of these PAHs was tested by Brunstrom et al., and the results showed that benzo[k]fluoranthene had the highest potency (LD 50 of 56 nmol/kg egg), followed by dibenzo[a,h]anthracene (LD 50 of 140 nmol/kg egg). 68In addition, dibenzo[a,h]anthracene and indeno [1,2,3-c,d]pyrene were found to have the highest potency in inducing EROD enzyme activities.Indeno[1,2,3-c,d]pyrene was found to be a prominent PAH in ambient PM2.5, which could be responsible  for enhancing antigen-induced allergic inflammatory responses. 69In 2005, it was demonstrated that exposure to benzo[k]fluoranthene could reduce splenic and thymic cellularity in mice. 70Moreover, exposure to benzo[k]fluoranthene could lead to genotoxic damage by inducing oxidative stress, as evidenced in an in vivo study. 71lthough B[a]P was declared inappropriate as the marker for the occurrence of PAH contamination in food, it remains the most extensively studied PAH due to its high toxic equivalency factor (TEF) value and genotoxicity. 57Because B[a]P is the most well-studied food-borne PAH and serves as one of the most famous ligands of AhR, we focus on it in the following section.

Benzo[a]pyrene (B[a]P).
As previously described, B[a]P is a highly toxic carcinogenic PAH.Up to 95% of nonsmokers' B[a]P exposure is from diet. 72The International Agency for Research on Cancer (IARC) has designated B[a]P as a group I human carcinogen, and this classification is supported by evidence from preclinical and epidemiological studies. 73B[a]P exerts its genotoxic effects following metabolization by cytochrome P450 (CYP) enzymes.In detail, B[a]P is converted into BaP-7,8-oxide by CYPs, then converted into dihydrodiol by microsomal epoxide hydrolase (mEH), and consequently becomes B[a]P-7,8-dihyrodiol-9,10epoxide (BPDE) within this three-stage formation pathway. 74PDE is the active metabolite that reacts with DNA directly and forms an adduct, preferentially a premutagenic guanine adduct, which finalizes in G to T transversion. 75.1.1.Phase I Enzymes.CYPs were first discovered by Klingenberg in 1954, and their function and importance were determined nearly a decade later.Numerous CYPs are involved in drug metabolism or xenobiotic detoxification metabolism. 76The involvement of the CYP450 enzyme is compulsory in the detoxification process to mediate phase I reactions, including oxidation, reduction, or hydrolysis reactions that result in metabolized molecules with an introduced functional group (−OH, − SH, − NH 2 , or − COOH). 77In the metabolism of B[a]P, the isoforms CYP1A1 and CYP1B1 are responsible for the production of epoxide intermediates, including B[a]P-7,8-oxide, B[a]P-9,10-oxide, or 3-hydroxy-B[a]P, and the conversions by CYP450 enzymes are known to be microsomal NADPH-dependent. 78Although there are 18 mammalian CYP families, families CYP1−4 are responsive and inducible by environmental factors, such as diet, drugs, and chemical inducers. 79It was previously reported that PAHs can induce several different members of CYP families, and B[a]P is a potent substrate for CYP1A1 and 1B1. 80The efficiency of CYP1B1 to oxidize B[a]P is around half that of CYP1A1, and it is notable that other family members, including CYP1A2, CYP2C8, CYP2C9, and CYP3A4, have also been found to be involved in B[a]P oxidation. 81xidation of B[a]P by CYP enzymes is followed by the catalysis of epoxide hydrolases (EHs) to promote the biotransformation of highly reactive epoxides into diols. 82hese hydrated products include B[a]P-9,10-diol from B[a]P-9,10-oxide and B[a]P-7,8-diol from B[a]P-7,8-oxide.At this stage, metabolic activation by phase I enzymes leads to the formation of carcinogenic metabolites, while the involvement of phase II enzymes leads to a detoxification pathway. 83B[a]P-7,8-diol is mono-oxygenated by CYP1A1 and CYP1B1 and finally yields B[a]P-7,8-diol-9,10-epoxide (BPDE), the electrophilic carcinogen that can covalently bind to DNA guanine residues to form adducts. 78 As a reactive electrophile, the formation of BPDE can cause DNA damage, DNA mutations, and carcinogenesis.High levels of BPDE-DNA adducts could even increase the risk of abortion during early pregnancy. 84ccording to previous studies, 0.01−0.1 μM BPDE could form 800−9600 bulky DNA adducts.The unrepaired BPDE-DNA adducts could lead to toxicity caused by replicative stress and genomic instability. 85In the in vitro study, a significant increase in the level of DNA lesions was detected upon treatment with BPDE at a lower concentration of 10 nM. 86otably, BPDE can form adducts not only by binding to nucleic acids but also by binding to proteins and lipids. 87lthough CYP450 enzymes are responsible for the formation of BPDE, their primary role and ultimate purpose are detoxification and protection of the host.
3.1.2.Phase II and Phase III of the Detoxification Process.The first phase of the detoxification process by CYP450 enzymes is followed by phase II detoxification: the conjugation of endogenous hydrophilic substances at the reactive sites of phase I metabolites. 88The corresponding enzymes for the conjugation are known as phase II enzymes and include glucuronyl transferase, sulfotransferase (SULT), glutathione transferase, N-acetyl transferase, and methyltransferase for the transfer of glucuronic acid, sulfate, glutathione, an acetyl group, and a methyl group onto a phase I metabolite, respectively. 88DP-glucuronosyltransferase (UGT), SULT, and glutathione S-transferase (GST) catalyze most conjugation reactions among these phase II enzymes. 77The conjugation of these anionic groups increases the hydrophilicity of the parent compound and stops these compounds from diffusing across the phospholipid membrane barrier. 89In each stage of the B[a]P detoxification process, different phase II enzymes are responsible for the formation of detoxified products.For instance, GSTs can catalyze the conjugation with B[a]P-7,8epoxide to form glutathione conjugates, while UGTs and SULTs are involved in the conjugation with B[a]P-7,8dihydrodiol. 90 In the final stage, also called phase III, the metabolites with hydrophilic conjugates can be excreted or eliminated via membrane carriers, primarily ATP-binding cassette (ABC) and solute carrier (SLC) transporters. 91.2.Aryl Hydrocarbon Receptor (AhR) Activation.In addition to environmental toxicants, several AhR ligands have been identified previously, including endogenous ligands such as tryptophan metabolites and gut microbiota-derived compounds (Figure 5). 40On the AhR protein, there is a PAS-B domain that allows binding of ligand,s while the bHLH-PAS-A domains on both AhR and ARNT proteins allow their dimerization. 92For instance, the binding of B[a]P to AhR leads to the dimerization of AhR and aryl hydrocarbon receptor nuclear translocator (ARNT), followed by nuclear translocation and binding of the heterodimer onto DNA, such as xenobiotic responsive elements (XREs), resulting in the transcription of regulated genes, including CYP enzymes. 92,93lthough xenobiotic metabolism is the first discovered function of AhR, its activation also controls several mechanisms including cell cycle regulation, protein interaction, and epigenetic mechanisms. 92t has been reported that oxidative stress via AhR activation by B[a]P could lead to mitochondria-mediated intrinsic apoptosis. 94Additionally, under conditions with abundant endogenous AhR levels, the pro-apoptogenic effect of B[a]P via CYP1A1 activation is enhanced. 95Another study suggested that oral gavage of B[a]P disrupts fat synthesis and glucose homeostasis and induces inflammation, possibly via AhR activation. 96Activation of AhR by B[a]P also led to proinflammatory responses in respiratory allergy via increased IL-33 expression and eosinophil infiltration in the lung. 97These studies suggest the deleterious effect on human health caused by B[a]P-activated AhR.

INTERVENTION OR SUPPLEMENTATION OF PHYTOCHEMICALS POTENTIALLY IMPROVES CIRCADIAN DISRUPTION
In medicine, phytochemicals have renowned advantages associated with treatment costs and alleviation of side effects and, at the same time, exhibit preventive and therapeutic effects.Several studies show the effectiveness of phytochemicals in disease attenuation or gut microbial dysbiosis improvement.In comparison, the effect of phytochemicals on circadian disruption is not as commonly recognized.In this section, some recent studies demonstrating the effect of phytochemicals on circadian rhythm disorders will be discussed.
In 2020, Song et al. reported that flavonoids from Cyclocarya paliurus showed modulatory effects on constant-darknessinduced mice in terms of their hepatic clock gene misalignment and intestinal microbiota dysbiosis. 98The extract was rich in kaempferol and quercetin derivatives, and the major outcomes of the supplementation were reflected in the body weight, the decrement of the F/B value, gut microbial diurnal oscillation, and the corresponding functionality of gut microbes.In the same year, it was found that polymethoxyflavones containing 3,5,7,3′,4′-pentamethoxyflavone, 5,7,4′-trimethoxyflavone, and 5,7-dimethoxyflavonein black ginger (Kaempferia parviflora) were regulative, reflected in the expression of CCGs including Bmal1, Cry1, and Per2. 99Moreover, the extract also ameliorated some negative effects induced by jetlag in mouse models.Qi et al. revealed that dietary tea polyphenols composed of gallic acid, epigallocatechin, epigallocatechin-3gallate, and epicatechin-3-gallate could ameliorate memory impairment and metabolic disorder, including insulin resistance and glucose/lipid metabolism dysfunction induced by constant darkness in a mouse model. 100The same research team also found that tea polyphenols could mitigate circadian clock dysregulation induced by oxidative stress, particularly the mitochondria impairment elicited by hydrogen peroxide via Bmal1-dependent pathways, such as Nrf2/ARE/HO-1 and Akt/CREB/BDNF signaling pathways. 101Further studies have determined that dietary phytochemicals, such as dietary natural cocoa, 102 nobiletin, 103 myricetin, 104 EGCG, 105 resveratrol, 106 pterostilbene, 107 and caffeine, 108 could potentially ameliorate circadian rhythm disruption or overcome health issues induced by circadian dysregulation.Therefore, dietary phytochemicals are potential modulatory agents for circadian rhythm disruption or sleep disorders.
On the other hand, phytochemicals or plant compounds with antioxidant properties can also protect the circadian clock by directly reducing the formation of PAHs in food.The pyrolytic formation of PAHs is a crucial process that generates a significant amount of free radicals.These radicals may ultimately form aromatic hydrocarbon rings.For instance, methyl radicals and acetylene radicals can form benzene rings.Additionally, small radical molecules can form aromatic hydrocarbon rings through polymerization, addition reactions, and cyclization reactions. 109Additionally, it was reported that edible oils account for approximately 50% of dietary exposure to PAHs, possibly due to the high oil solubility of these PAHs. 110Several factors might contribute to the occurrence of PAHs, such as the types of raw materials used for edible oils, refining processes, storage conditions, and the types of cooking oils. 111The production of cyclohexane or hydroperoxides occurs during the thermal treatment of food lipids through oxidation and breakdown.This process ultimately forms naphthalene or naphthalene-like compounds through further oxidation or cyclization and may result in the synthesis of PAHs through polymerization.In another context, exposure of B[a]P to UVA radiation can cause ROS accumulation in lipids, and these concentrated ROS can induce lipid peroxidation. 112lthough the formation of PAHs in oil is not well-verified, it is believed that they could form due to free radicals generated from fatty acid oxidation, which could be evidenced by the finding showing that catechin in oil can effectively inhibit the generation of PAH4 at levels above 0.02% under heating conditions. 113Additionally, more studies have demonstrated that nonlipid substances with antioxidant properties in oil can significantly inhibit PAH formation during heating processes. 114The inhibition of PAH formation, accompanied by the reduction in the peroxide value (POV) and thiobarbituric acid reactive substances (TBARs) by apple polyphenols in barbecued pork, suggested the contribution of lipid oxidation to PAH formation. 115The use of rapeseed oil, sesame oil, or sunflower oil could help reduce PAH formation. 116herefore, eliminating radicals with antioxidants could be a potential strategy.It has been suggested that supplementing phytochemicals with antioxidant properties could achieve this goal.For instance, the formation of PAHs during the roasting process of sunflower seeds could be significantly reduced when the seeds were flavored with hogweed, which contains several phenolic compounds with strong antioxidant properties, such as rutin, coumarin, and quercetin. 117Another study suggested that a marinade containing dietary antioxidants, including quercetin or the volatile organosulfur phytochemical in garlic, diallyl disulfide, could effectively decrease PAH levels in grilled meat. 118As mentioned above, PAH8 has been introduced as a more accurate marker to indicate the contamination of PAHs in food.As demonstrated by Wang et al., naringenin and quinic acid exhibited a compelling inhibitory effect on PAH8 formation in charcoal-grilled chicken wings. 119Although the authors suggested that there was no correlation between the reduction of phenolic compounds and PAH8 production, more antioxidant analysis should be conducted before a conclusion is reached.In another study, it was suggested that ABTS could effectively demonstrate the correlation between the antioxidant properties of flavonoids and PAH formation. 120This indicates that the antioxidant properties of phytochemicals could be indicative of their capability to inhibit PAH formation.
There are several mechanisms that have been pointed out to ameliorate circadian misalignment, but the involvement of AhR regulation was not mentioned.

PERSPECTIVE: EFFECT OF PHYTOCHEMICALS ON AHR ACTIVATION
Due to the crucial role of AhR in circadian regulation, modulating the activation of AhR represents a strategy to prevent consequential disruption.Some phytochemicals may act as potential contributors.For instance, resveratrol has been reported to possess antagonistic activity on AhR to compete with TCDD, as resveratrol has a close structural homology to AhR antagonistic ligands. 121Although resveratrol was not as efficient as an AhR inhibitor compared to α-naphthoflavone, it has the advantage of lower toxicity.Human paraoxonase 1 (PON-1) is a hepatic-secreted enzyme that is transcriptionally regulated by AhR.Interestingly, compared to quercetin, TCDD is a poor inducer in regulating the gene expression of PON-1. 122These results suggested that the binding of different AhR ligands may lead to distinct gene regulation depending on the target sequence.Therefore, the intervention of phytochemicals to compete with environmental toxicants upon AhR activation could be a possible strategy to avoid circadian disruption while at the same time maintaining xenobiotic metabolism.5.1.From Flavonoids to Stilbenoids.The role of phytochemicals in AhR activation can be agonistic or antagonistic.It has been reviewed that flavonoids that share a similar structural backbone with quercetin, such as daidzein, genistein, and apigenin, may act as AhR agonists, but at the same time some may also exhibit antagonistic activity to inhibit the activation of AhR induced by environmental pollutants. 123or example, one flavonoid that can be naturally found in citrus peel was reported to reduce the nuclear translocation of AhR induced by 7,12-dimethylbenz[a]anthracene (DMBA) or TCDD, as well as its downstream gene expression. 124A molecular docking study screened a list of flavonoids and showed that some could possess blocking capacities for AhR-ARNT heterodimer formation up to 50−60%, while most of the studied flavonoids interact with AhR and ARNT to some extent. 125Notably, fisetin showed the greatest blocking capability among the studied flavonoids, and more importantly resveratrol, a member of the stilbene family, also showed a probability of blockage of 30%, suggesting the potential of stilbenoids to suppress the transcriptional ability of AhR. 125hronic activation of AhR has been found to promote the expression of resistant genes to the inhibitor of cutaneous melanoma, which may subsequently lead to aggressive tumors.The addition of resveratrol in treatment with the inhibitor could effectively reduce the number of resistant cells by inhibiting AhR activation, delaying tumor growth. 126In 1999, resveratrol was revealed as a competitive antagonist of dioxin and other AhR ligands. 121Resveratrol intervention has also been reported to rescue osteoblastogenesis from deterioration induced by indoxyl sulfate, a gut microbial tryptophan metabolite, by acting as an AhR antagonist. 127Moreover, resveratrol could also prevent epigenetic silencing of the BRCA-1 gene, which is AhR-dependent, and lead to the repair of DNA damage caused by xenobiotics. 128t was reviewed that the binding energies of resveratrol and its derived metabolite, lunularin, against the PAS-A domain of AhR were −6.64 and −6.76 kcal/mol, respectively. 129In 2010, piceatannol, hydroxylated resveratrol, was found to exhibit a similar antagonistic effect on AhR activation 130 which indicates that the stilbenoids that share the same backbone are potential candidates as AhR antagonists.The inhibitory effect of pterostilbene, known as methoxylated resveratrol, on the nuclear translocation of AhR was reported to protect keratinocytes against the damage induced by particulate matter. 131nvironmental pollutants and food-borne pollutants have become unavoidable issues to be faced in this age.In addition to cancers and metabolic diseases, circadian disruption has also been known to be influenced by these pollutants via AhR activation.More importantly, circadian disruption has recently been reported to adversely affect the host metabolic function.Inhibition of excessive AhR activation has been identified as a strategy to alleviate the progression of the disease.On the other hand, some phytochemicals such as flavonoids and stilbenoids have also been found to exhibit an antagonistic effect on AhR activation (Figure 6).However, there are fewer studies on the suppressive effect of AhR activation against food-borne pollutants in circadian disruption.Hopefully, this review will provide new insights into preventing pollutantinduced circadian disruption via AhR overactivation through phytochemical intervention.

Figure 2 .
Figure 2. The principal circadian clock of mammals via hormone secretion.The peripheral clocks of organs affected by hormones controlled by the circadian clock include the lung, heart, muscle, liver, stomach, kidney, adipose tissue, and pancreas.Hormones secreted by these tissues could provide feedback to the master clock.

Figure 5 .
Figure 5.Some microbial metabolites are known AhR activators.Modulation of gut microbiota leads to the production of distinct gut microbial metabolites, including short-chain fatty acids and indoles.These metabolites subsequently affect the activation of certain receptors, such as AhR.

Figure 6 .
Figure 6.AhR activation by environmental toxicants may be responsible for phase I enzyme transcription and circadian disruption.Some phytochemicals may exhibit an antagonistic effect on AhR activation.