Regulation of Human Cytochrome P4501A1 (hCYP1A1): A Plausible Target for Chemoprevention?

Human cytochrome P450 1A1 (hCYP1A1) has been an object of study due to its role in precarcinogen metabolism; for this reason it is relevant to know more in depth the mechanisms that rule out its expression and activity, which make this enzyme a target for the development of novel chemiopreventive agents. The aim of this work is to review the origin, regulation, and structural and functional characteristics of CYP1A1 letting us understand its role in the bioactivation of precarcinogen and the consequences of its modulation in other physiological processes, as well as guide us in the study of this important protein.


Introduction
Cytochrome P450 (CYP) is a superfamily of hemoproteins, with monooxygenase activity, which are spread into the three domains of life. They are biological catalysts that metabolize endogenous compounds such as hormones, bile acids, cholesterol, and xenobiotics like environmental pollutants and drugs. The hCYP1A1 is an enzyme of biomedical and toxicological interest, which catalyzes the biotransformation of polycyclic aryl hydrocarbons (PAHs), aromatic amines, and polychlorinated biphenyls into polar compounds, which can be conjugated to soluble compounds suitable for excretion by urine or bile. Nevertheless, under specific circumstances, this enzyme catalyzes the bioactivation of compounds capable of reacting with macromolecules, such as DNA, leading to the start of mutagenic process.
Every day, we are exposed to compounds that are substrates of CYP1A1, through environmental pollution, food, and, particularly, cigarette smoke. The importance of this protein in chemical carcinogenesis induced by PAHs has been demonstrated in CYP1 knockout mice, in which the lack of this protein shows less formation of adducts PAH-DNA [1,2]. In addition, rodent exposition to CYP1A1 inhibitors diminished the number of tumors induced by PAHs [3,4].
Epidemiologic studies focused on the relationship among PAH exposition, PAH-DNA adducts level, and cancer incidence in humans demonstrate an increased risk in colon adenocarcinoma [5], breast cancer [6], and lung cancer [7] in those individuals with higher levels of adducts.
This data suggests that imbalance between detoxification and bioactivation of carcinogens, independence of enzyme catalysis, regulation of gene expression of CYP1A1, and cellular environment are crucial factors at the beginning of chemical carcinogenesis process. Because of this, several questions are still to answer; we propose that a global view of the function and regulation of this enzyme would help to answer these questions; thus, the aim of this work is to integrate the knowledge that has been generated until now about the origin, regulation, and structural characteristics of hCYP1A1.
2 BioMed Research International endogenous and exogenous substrates in bacteria, archaea, eukaryotes, and viruses [8,9]; therefore these proteins must descend from a prokaryotic common ancestor ∼3 billion years ago, before the oxygenation of the atmosphere and emergence of eukaryotic cells [10,11].
The first CYP proteins were involved in the biosynthesis of compounds required for the formation and maintenance of cell structures and then following CYP proteins coevolved as defense mechanisms in plants and insects and more recently a set of these enzymes evolved in response to xenobiotics [12,13].
CYPs belonging to families 1-4 are the main mediators of exogenous metabolism; however, cytochromes from family 1 are of particular biomedical and toxicological interest because of their affinity to halogenated polycyclic, aromatic amines, aromatic hydrocarbons, and endogenous compounds, whose metabolites can be toxic, mutagenic, or carcinogenic [14][15][16].
CYP genes of family 1 are grouped into six subfamilies: CYP1A, CYP1B, CYP1C, CYP1D, CYP1E, and CYP1F, from these 1E and 1F are found in urochordates; 1A, 1B, 1C, and 1D are found in fish and amphibians; in mammals the subfamilies that are mainly distributed are 1A and 1B and in some cases 1D [9,17].
CYP1A and CYP1B diverged from a common ancestor ∼450 million years ago (Ma); thus, CYP1A appears early in aquatic vertebrates, as a single copy, which has been identified in teleost fish, while mammals and birds have paralogous genes of CYP1A: CYP1A1, CYP1A2, in mammals, and CYP1A4, CYP1A5 in birds, which emerged ∼250 Ma from a duplication event and one inversion, common for both lineages [15,18,19] (Figure 1).
In humans, the CYP1A1 gene consists of 6069 bp and is located at the CYP1A1 CYP1A2 locus on chromosome 15q24.1, sharing a regulatory region of 23306 bp with the CYP1A2 gene that is oriented in opposite direction. The 5 flanking region is shared by both genes and contains a bidirectional promoter and DNA motifs, known as response elements, that activate and regulate the expression of these genes [20,21].
The participation of multiple signaling pathways in the regulation of the hCYP1A1 transcription has been reported. Next, an overview about the pathways involved in this regulation is reviewed.

Upregulation of CYP1A1
The constitutive hCYP1A1 gene has low level of expression in extrahepatic tissues of adult humans. However, liver and extrahepatic expression of this enzyme can be induced by many substrates through multiple pathways. The aryl hydrocarbon receptor (AHR) pathway has been widely studied and it appears to be the main protein receptor that influences CYP1A1 induction. The AHR is a cytosolic ligand-activated transcription factor associated with two heat shock proteins of 90 kDa (Hsp90), a hepatitis B virus X-associated protein (XAP2), and a chaperone of 23 kDa (p23). This receptor is activated by endogenous ligands and several xenobiotics such as polycyclic aromatic hydrocarbons (PAHs), heterocyclic amines, and halogenated biphenyls [22]. After ligand activation, AHR undergoes conformational changes that promote its translocation into the nucleus, via importin, where it is dissociated from the chaperone proteins (Hsp90, XAP2, and p23), and binds to the nuclear translocator AHR (ARNT) [23,24]; then the formed AHR-ARNT complex (AHRC) binds to xenobiotic responsive elements (XRE) (5 -TNGCGTG-3 ) located at the enhancer element [25].
Thirteen XRE have been identified in the regulatory region of human CYP1A1 [25]. It has been speculated that they are located at the major grooves of the DNA and they would be exposed during nucleosomal movements, allowing the AHRC binding. In turn, this promotes the recruitment of chromatin remodeling proteins such as p300, SRC1/2, and BRG1 [26], subsequent hyperacetylation of lysines 9 and 14 in histone 3 (H3K9ac and H3K14ac), and methylation of lysine 4 in histone 3 (H3K4me) (from dimethylation to trimethylation) at the promoter; meanwhile hyperacetylation of lysine 16 in histone 4 (AcH4K16) and increased phosphorylation of serine 10 in histone 3 (pH3S10) take place at the enhancer element. The increase of acetylation marks at the promoter region of mouse CYP1A1 (mCYP1A1) is consistent with the releasing of a basal repressive complex, which is composed of histone deacetylase 1 (HDAC1) and DNA methyltransferase 1 (DNMT1). It has been suggested that marks at the enhancer could stabilize the open chromatin state to allow the AHRC-mediated transcriptional loop [27][28][29]. Finally, this AHR-dependent pathway has target genes such as CYP1A1, CYP1A2, and CYP1B1 and aldehyde dehydrogenase 3A1 (ALDH3A1) [30,31]. Figure 2 shows some regulatory mechanisms involved in CYP1A1 regulation.
A number of pathways also modulate CYP1A1 transcription through binding to the promoter, interactions with AHR, or both mechanisms. Next, we briefly describe some of them.
The canonical Wnt/ -catenin signaling pathway is involved in the adult tissue homeostasis regulation, embryonic development, and tumorigenesis. It has also been implicated in the induction of some CYPs, including mCyp1a1. In mice, this was demonstrated by the specific loss of CTNNB1 that encodes -catenin and leads to a decrease of mCyp1a1 induction by AHR agonists such as 3-methylcholanthrene (3-MC), -naphthoflavone ( -NF), and butylated hydroxyanisole. Additionally, it has been observed that maximum mCyp1a1 induction was obtained when -catenin acted as coactivator of AHR, although this protein also binds to the transcription factor TCF, which has a binding site in mCyp1a1 promoter, suggesting a different mode of action [32][33][34]. Similarly, in rat hepatoma, it has been observed that the interaction between AHR and hypophosphorylated retinoblastoma protein (pRb) aids maximum induction of rat CYP1A1 by 2.3, 7.8 tetrachlorodibenzo-p-dioxin (TCDD); pRb plays an important role in cell cycle control and it has been proposed that it could also act as a coactivator of AHR [35,36].
Furthermore, several nuclear receptors are involved in the upregulation of hCYP1A1; for example, the constitutive androstane receptor (CAR) [37] which is also a regulator of the expression of the CYP2A, 2B, 2C, and 3A subfamilies is activated by drugs; the liver X receptor (LXR ) that   is involved in lipid homeostasis is activated by oxysterols [38,39]; and the peroxisome proliferator-activated receptor (PPAR ), is activated by fibrates, phthalates, arachidonic acid, and its derivatives [40,41]. These receptors bind to their specific responsive elements located in the gene promoter, activate the transcription, and potentiate the induction of hCYP1A1. The crosstalk amongst signaling pathways involved in regulating the expression of CYP1A1 could have implications for drug-drug, drug-toxic, and drug-food interactions.

Downregulation of CYP1A1
The tight regulation of CYP1A1 is highly necessary due to the known harmful effects of electrophilic compounds produced by the enzymatic activity of CYP1A1; a number of CYP1A1 downregulation mechanisms have been described; for example, the AHR repressor protein (AHRR) is a target gen of the transcriptional activity of AHR and competes with AHR for binding to XREs. AHRR has been described as a negative tissue-specific regulator of mCYP1A1 expression [43,44]. Its overexpression in transgenic mice suppresses the mCYP1A1 induction in lung, spleen, and adipose tissue [45]. Moreover, it has been suggested that rat CYP1A1 regulates its own expression because it catalyzes the removal of AHR agonists and thus decreases the activation of this pathway [46,47].
Hypoxia inducible factor participates as a negative regulator of hCYP1A1 expression through the competition with AHR for the binding to ARNT. Under hypoxia conditions,  basal hCYP1A1 expression decreases [48] and induction by AHR ligands is inhibited [49,50].
Moreover, the retinoic acid receptor pathway (RAR) is also implicated in the regulation of hCYP1A1 expression through two mechanisms. In the first one, RAR modulates the transcriptional expression of this protein through its binding to a retinoic acid responsive element (RARE) located in the hCYP1A1 promoter [51,52]. In the second one, the corepressor SMRT (silencing mediator for retinoid and thyroid receptors), which is attached to RAR, is released upon activation of RAR by retinoic acid; subsequently released SMRT can interact with AHR and reduce hCYP1A1 induction [53].
Another protein involved in the downregulation of hCYP1A1 induction is the nuclear factor I (NFI). NFI activates the expression by binding to promoter of hCYP1A1 and it is sensitive to oxidative stress [54]. It has been demonstrated that increased activity of hCYP1A1 generates reactive oxygen species, which in turn can lead to the oxidation of the single cysteine residue on NFI and then it is released from the hCYP1A1 promoter, thus decreasing the expression of this gene [55,56].
The presence of a glucocorticoid responsive element in the intron one of the CYP1A1 gene in several species has been reported. The activity of the glucocorticoid receptor potentiates the effect of activated AHR in rat hepatocytes unlike human hepatocytes where dexamethasone (glucocorticoid analog) decreases the hCYP1A1 protein but not mRNA induced by 3-MC [57,58]. However, additional studies are needed to clarify the effect of glucocorticoids on CYP1A1 gene and protein levels.
Gut-enriched Kruppel like factor (KLFG or KLF4) is a regulator of cell proliferation, differentiation, apoptosis, and cellular reprogramming and has been identified as a negative regulator of rat CYP1A1 transcription in a dependent way of its binding to the basic transcription element (BTE); moreover, this effect might also be part of the interaction between KLFG and Sp1, an CYP1A1 transcriptional activator [59].

Epigenetic, Posttranscriptional, and Posttranslational Regulation of CYP1A1
Until now, several modes of action have been reported for the regulation of human CYP1A1. In essence, transcriptional expression has been reviewed, but there is another kind of gene regulation that involves epigenetic mechanisms BioMed Research International 5 such as methylation, acetylation, histone ubiquitination, or DNA methylation and hydroxylation. In this regard, to explore the role of these mechanisms on the regulation of hCYP1A1 expression studies were conducted using the DNMTs inhibitor, 5-aza-2-deoxycytidine (5AzadC), and HDACs inhibitors, trichostatin A (TSA) and sodium butyrate. Table 1 summarizes the effects of these inhibitors on CYP1A1 expression. Such effects are species-specific and depend on whether the tissue is derived from healthy or cancerous donations. This review focuses mainly on hCYP1A1 regulation and just on enriching the data presented; Table 1 shows results from studies conducted in human, mouse, or rat cell lines primary cultures. According to the results it is not possible to conclude whether hCYP1A1 has a DNA methylation dependence regulation or not. It seems that tissue and temporal issues might have been involved in this regard as well as the tumor state. We cannot rule this, but tumor or cancer state allows an increased DNA methylation in hCYP1A1 regulatory region, at least in prostate [27] and lung [65,69]; thus, in these models this gene has no constitutive expression which is activated by exposition to 5AzadC.
There is another type of hCYP1A1 regulation, which is through posttranscriptional modulation. Some in silico studies have been conducted in order to determine a possible regulation of CYP1A through noncoding RNAs. Based on web databases analyses, six putative micro RNAs (miRNAs), hsa-miR-125b-2, hsa-miR-488, hsa-miR-657, hsa-miR-892a, hsa-miR-511, and hsa-miR-626, with one or more binding sites to the 3 UTR region of hCYP1A1 were identified [21]. Following the same strategy, an additional study used five different bioinformatics programs and predicted 332 miRNAs to target hCYP1A1 UTRs, from which 12% were predicted in at least 2 programs [110].
Interestingly, in a study performed in human breast cancer cell line MCF-7 exposed to BaP leads to diminish miR-892a expression and function. This miRNA binds to 515-535 nucleotides of 3 -UTR of human CYP1A1 and acts as translational repressor of this transcript. The putative effect of miR-892a was previously predicted by an in silico study [111]. Another study conducted in normal human liver tissues ( = 92) searched for a correlation between the protein level of CYP1A1 and the expression of miRs and a negative correlation was found for miR-200a ( = −0.36), miR-142-3p ( = −0.36), and miR-200b ( = −0.36) [112]. Nevertheless, another study with healthy human liver tissues from individuals of different ages determined that upregulation of miR-125b-5p was related to downregulation of CYP1A1 from fetal and pediatric samples. The effect of this miRNA was also previously predicted [113].
At this point we realize that the protein expression of CYP1A1 is tissue-, health-and age-specific; thus, it is not strange to expect that also the mechanisms and factors involved in its expression would be specific as we can observe from the previous data where two miRNAs were predicted in silico and confirmed in vivo, but none of them were found repeatedly among the studies reviewed here. It would be obvious that if there are differences in miRNAs found among results with human CYP1A1, there could be much more differences between human and other species models. This assumption is supported by a report conducted in mice fetal thymocytes where miR-31 was found as a negative regulator of mCyp1a1 translation after exposition of cells to TCDD. Furthermore, miR-31 has matched with 3 -UTR of the transcript of this protein [114].
There are some studies reporting indirect regulation of CYP1A1 through the regulation of AHR by small noncoding RNAs, as in the case of the Sprague-Dawley rats treated during 2 weeks with an antagonist of the corticotrophin releasing factor I. Results show that rat liver CYP1A1 expression was increased through an atypical pathway different from AHR ligand and suggest the involvement of miR-29a-5p, miR-680, and miR-700 which were negatively expressed 10-, 6-and 8.6fold, respectively. Whether these miRNAs could act through rCYP1A1 direct binding or not is still unknown because the first two had binding sites in the 3 -UTR region of both rCYP1A1 and AHR [115]. More information about hCYP1A1 regulation through its 3 UTR region shall be discovered in the near future to achieve this objective; also more tissues and health conditions are needed to be studied.
Until this point we covered evolutionary origin of CYP1A1 and its transcriptional and posttranscriptional regulation, but once the CYP1A1 protein is formed its cellular lifetime is regulated too. The half-life time of this protein is of ∼2.8 hours; this suggests a mechanism of protein degradation and the studies prompted to proteasomal degradation pathway. In fact, treatment with ubiquitin-proteasome inhibitor MG132 keeps the levels of CYP1A1, while lysosomal inhibitors do not [116][117][118]. In spite of these experiments, there are no reports that could help us figure out the mechanism of degradation of CYP1A1.
Another possible regulation of CYP1A1 is through the degradation of its heme group, which has been explored in human hepatoma cell line HepG2 exposed to different heavy metals. Here an increase in hemooxygenase 1 was found; this enzyme is involved in the metabolism of the heme group. Its increased levels found after heavy metals exposition correlate with diminished activity of CYP1A1, while protein level and gene expression remain unchanged [117,119,120].

Structural Characteristics of Human CYP1A1 and Its Ligands
Human CYP1A1 has a molecular weight of 58.16 kDa and consists of 512 amino acids of which the first thirty of the Nterminal region allow the association of the protein with the mitochondrial membrane and the disordered region of the smooth endoplasmic reticulum rich in unsaturated fatty acids, unlike the human CYP1A2 which is located in the sorted regions rich in cholesterol, sphingomyelin, and saturated fatty acids. Moreover, these thirty residues would also be mediating the interaction with NADPH-CYP reductase [121][122][123][124]. Directed mutagenesis in the residues of the human protein showed altered kinetic parameters and demonstrates the importance of certain amino acids like Phe123, Phe224, Glu256, Asp313, Gly316, Ala317, Thr321, Val382, and Ile386 (Table 2) in the recognition, binding, and affinity for the AzadC increased hCYP1A1 expression in HLAC NHBE and HBEC were low methylated than HLAC at enhancer region.
[69] substrates. However, the spatial orientation of these residues was known until the three-dimensional structure of human CYP1A1 was resolved by X-ray crystallography at a resolution of 2.6Å [125].
The protein crystallization of human CYP1A1 allowed us to know that this protein is comprised by twelve -helices (A-L), three -sheets ( 1-3), and four helical short regions (A , B , F , and G ) forming six sequences as putative substrate   [77] recognition sites (SRS) important for ligand selectivity of this enzyme [125,126], which are shown in Figure 3 and listed as follows.
(i) SRS1 corresponds to the amino acid region 106-124 of loop between helix B and helix B and portion of loop between helix B and helix C. In turn, it forms part of the wall of the active site and it is proposed as a site for the input and output of ligands that influence the regioselectivity for the oxidation of substrates [127,128]. (ii) SRS2 is part of the helices E and F, as well as of the residues 217-228, in the loop that connects these regions. Its role is similar to SRS1 participating in the ligand orientation [129,130]. (iii) SRS3 is found in helix G from amino acid 251 to amino acid 262 [126].
(v) SRS5 goes from residue 381 to residue 386 and connects helix J to the beta sheet. In other CYPs this region has been associated with the entry of the ligand due to its high flexibility [130]. (vi) SRS6 is the shortest region and is located in the loop near the 3 sheet [126].
The human CYP1A1 structure allows binding planar molecule with ∼12.3Å in length and ∼4.6Å in width, conformed by aromatic, polyaromatic, and heterocyclic rings which are essential for the formation of -stacking in the protein active site, mainly with Phe-224 at helix F, conferring stability to the enzyme-substrate complex [43,80,81,[131][132][133][134][135]. Nevertheless, for specific substrate redox reaction to be produced (Table 3), ligand also requires to be oriented with its reactive group facing the heme group [136,137].

CYP1A1 through Development
Besides its importance in the metabolism of xenobiotics, CYP1A1 is also involved in the metabolism of endogenous compounds, such as arachidonic acid, eicosapentaenoic acid [93], 17 -estradiol [95], and melatonin [94].
Arachidonic acid and eicosapentaenoic acid are biotransformed by this enzyme to products such as 14, 15-epoxyeicosatrienoic acid and 17, 18-epoxyeicosatetraenoic acid, which influence cardiovascular pressure [93]. This attribute highlighted the importance of the association between heart diseases and CYP1A1 polymorphisms [138][139][140].
Treatment with the CYP1A inhibitor, -naphthoflavone, shows that the activity of CYP1A1 is important for the proper development of the embryo's cardiovascular system [141][142][143]. However, so far there is not enough information about the impact of this isoform in the endogenous metabolism, so it is essential to conduct more studies that can help us to understand the mechanisms of these processes and their impact on the human health.
The use of different animal models has proved that activity and basal expression of CYP1A1 during embryonic development are organ-stage-specific (Table 4), where the liver and cardiovascular tissues have the highest expression. In the chicken, exposure to CYP1A1 inducers causes an increase in heart size and weight, while, in fish, edema in pericardium as well as modifications in the normal shape of the organ has been reported [141,142,[144][145][146][147][148].
Searching whether the function of CYP1A1 is crucial for life, a line of knockout mice for this gene was produced [149]. These animals show decreased liver, kidney, and heart weight, as well as increased blood pressure and lower heart rate compared to wild type mice, thus demonstrating the importance of CYP1A1 in the cardiovascular system [150].
In adulthood, the human CYP1A1 expression is low and is found particularly in tissues of the respiratory system such 50-245 hpf Gallbladder EROD in vivo (arbitrary units) [109] as trachea and lungs, but after induction, it is also detected in other organs such as liver, adrenal gland, bladder, heart, kidney, ovary, placenta, prostate, testis, thyroid, salivary gland, and spleen [96,151]. Among these organs, different levels of the protein are detected [152].

Concluding Remarks
CYP1A1 is a relevant enzyme for biotransformation of environmental compounds into mutagenic metabolites; this fact has a strong effect on worldwide population; therefore, the knowledge of its tridimensional structure as well as its ligands allows us to the rationale search and development of inhibitors that would become chemopreventive agents for diseases related to exposure to CYP1A1 activated carcinogens.
On the other hand, the presence of CYP1A1 among several species forces us to choose biological models that share with humans similar CYP1A1 characteristics in order to obtain results able to be extrapolated. The animals frequently used for this purpose are rats and mice, in which some of the regulatory mechanisms and other data, reported here, have been described. Moreover, as already mentioned in the "upregulation of CYP1A1" Section, several pathways could be involved like the recently reported WNT-catenin, RAR, or CAR pathways that regulate CYP1A1 expression by direct interaction with its gene promoter or with that of AHR or both. However, these alternative pathways are poorly described and more studies in this regard are required to know how and what are the factors involved as well as the specific conditions necessary for their action on CYP1A1 expression, like the tissue and its microenvironment or culture cell type used just to mention two of them. The discoveries of pathways that converge in CYP1A1 regulation are opportunities for the selection of new therapeutic targets that allow drug development for chemoprevention.
For the study of CYP1A1, we need to take into account that impairment of gene expression or enzyme activity could lead to adverse effects because it is involved in endogenous metabolism, an issue discussed in "CYP1A1 through development," with particular interest in cardiotoxicity.
The integration of data generated about CYP1A1, factors, and mechanisms that play a role in carcinogen bioactivation will help us to rise up strategies that improve our life quality. In this context, some key questions that need to be addressed are written below.
It will be worth to continue the searching for chemopreventive agents that inhibit CYP1A1 even if it seems to be involved in the normal development of the heart. It is a good strategy to improve chemopreventive agents acting on different regulating CYP1A1 pathways at the same time; meanwhile they have fewer side effects. What is the real contribution of CYP1A1 in the process of carcinogen bioactivation knowing that it shares regulatory elements with additional CYPs of the same family? Do the cardiotoxicity effects produced in the lack of CYP1A1 activity be a window for searching new therapeutic targets for cardiovascular diseases? What is the biological relevance of reactive oxygen species production by CYP1A1? Why do tissues have differences on CYP1A1 expression? Is the tissue-specific, or even cell-specific, expression of CYP1A1 explained by differences in endogenous metabolism requirements or by alternative modulation of a particular set of AHR co-activators? Do the specific CYP1A1 expression and induction play a role in the development of a particular cancer ligand related?