Xenobiotics metabolizing enzymes (XMES) are very important for metabolizing of exogenous and endogenous compounds . They are involved in the biotransformation of exogenous compounds like procarcinogen, drugs, solvents. They are categorized into parts: phase I and phase II enzymes. Cytochrome 450 family comes under phase I enzyme. Cytochrome P450 family of enzymes is mainly involved in synthesis and metabolism processes of exogenous and endogenous compounds. CYP genes are located mainly in the liver and within the cell are present in the endoplasmic reticulum and also in the mitochondria. CYP family is divided into four main families: CYP 1, CYP 2, CYP 3 and CYP4.
CYP has two main roles in the human body . First, CYP is used by the body to metabolize and transform a range of hydrophobic xenobiotics (exogenous, foreign compounds such as pesticides, carcinogens, and pollutants) to more polar metabolites so that they can be readily excreted in the urine. By biotransforming potentially toxic compounds to less potent forms, CYP works as a natural detoxifying agent of the body. Large populations of CYP are found bound to the endoplasmic reticulum in mammalian liver cells, the primary site of metabolism. The gene families most commonly found in humans and involved in metabolism include cytochrome P-450 1, 2, and 3 (known as CYP 1, CYP 2, and CYP 3). Second, CYP enzymes are used in the synthesis of important signaling molecules, such as steroid hormones in the endocrine glands, fat-soluble vitamins and Metabolism of fats (cholesterol and fatty acids).
CYP metabolisms have adverse effects as well as beneficial effects. CYP plays an important role in activating carcinogens, such as polyaromatic hydrocarbon (PAH), by an oxidation mechanism. They are also involved in the metabolism of certain medication that are ingested (endoplasmic reticulum) and also certain toxin/internal substances formed within the cells (mitochondria). These functions mainly depend upon iron present in it as a prosthetic group. Hence, they are called hemoproteins. Iron contains two 4s electrons and six 3d shell electrons in its valence shell. The function of this gene depends on the shifting oxidation state of iron between ferrous Fe2+ (loss of the 4s electrons) and ferric Fe3+ (loss of an additional 3d electron). As the ferric state results in a half-filled 3d shell, it is the more stable form of the two states. As a result, CYP can be readily reduced with the addition of an electron. It is a group of heme-thiolate monooxygenases and involved in an independent NADPH dependent electron transport pathway .
Fig1. Metal complex structure of CYP 450
5.1. CARCINOGEN IDENTIFICATION
The carcinogen from tobacco smoking is genotoxic carcinogens that are capable on inducing DNA lesion. This carcinogen is divided into two parts, first is direct carcinogens and second is procarcinogens. Direct carcinogens are highly reactive and having an electrophilic groups with surplus positive charge. They interact directly with DNA and form DNA adducts. The example of this carcinogens are N-nitrosoalkylurea, ethyl- and methylmethanesulfonate, N-methyl-N-nitronitrosoguanidine, sulfur mustard, diepoxybutane, beta-propiolactone, ethyleneimine, etc. Initially, procarcinogens are chemically in inactive form. When procarcinogens go inside the body, they interact with different types of enzyme that metabolise in the cell in two steps. In the first step, procarcinogen are activated and converted into electrophilic derivatives. In the second step, the metabolic products are neutralized by conjugation. The first step process is mainly done by CYTOCHROME 450 family and second step process is done by conjugating enzymes include acyltransferase, epoxidases, sulfotransferases, glutathione-S-transferases, UDP-glucuronyl transferases, and transaminases . They are activated by oxidation process that is mainly done by CYP gene.
The main procarcinogen in smoking that create negative effect on CYP1A1 genes are mainly polycyclic aromatic hydrocarbon group (Benzo(a)pyrene, Benz(a)anthracene, Benzo(b) fluoranthene, Benzo(c)phenanthrene) and N-nitrosamines (NNK) . CYP1A1 does not form directly tumour, it only makes procarcinogen to carcinogen that forms tumour formation. Cytochrome p450 enzyme comes under the phase1 enzymes which convertes the mutagens to epoxides in the K-region as well as the Bay region of Benzo (a) pyrene . CYP gene carry out oxidation and reduction of the hydrophobic ligands converting them into epoxides usually in the K-region which is then taken up by phase2 enzyme (GST) and metabolized into water soluble compounds (later excreted through urine). But if the epoxide is formed near the bay region then phase2 enzymes become idle without carrying out any metabolic process. The CYP gene metabolises the carcinogens to epoxides, which are highly reactive and binds to DNA and further activated to diol epoxides. They covert the carcinogen B[a] P into B[a]P-7,8-diol-9,10-epoxides which has got high DNA binding capacity. These mutagens are termed as Bay epoxides as they have tumourogenic effects on DNA. The presence of motif reason of CYP 450 oxidizes the PAH. The motif reason of CYP450 is Phe-X(6-9)-Cys-X-Gly, where X is a specific amino acid. Cysteine binds to heme iron and takes part in the transfer of one atmospheric oxygen atom to the substrate that contained in the pocket of the substrate-binding site in the enzyme active center. In few cases, the life span of metabolites is very short (several milliseconds) due to their high reactivity and fast conversion into stable hydroxylated derivatives. In other cases, due to slow decomposition of metabolites they easily enter into the nucleus, mitochondria, and other cell organelles. The reaction catalyzed by cytochromes P450 is a monooxygenase reaction, e.g., insertion of one atom of oxygen into an organic substrate (RH) while the other oxygen atom is reduced to water:
RH + O2 + 2H+ + 2e- → ROH + H2O
Fig2. Substrate oxidation in the cytochrome P450 system
Fig3. Bay and K region of Benzo(a)pyrene and epoxide formation of Bay region
Fig4. Epoxide formation of B(a)P
5.2. INDUCTION OF CYP GENE
The amount of CYP1A1 is responsible for tumour formation in organs. Basal expression of CYP1A1 is negligible. High amount of CYP1A1 increases the risk of tumour formation or cancer. It can create an imbalance condition between detoxification and activation that leads to adverse effects. It converts the procarcinogen to carcinogen by oxidation process. At high substrate concentrations detoxification becomes saturated and induction can increase the production of reactive metabolites compare to the capacity of cellular defenses, thereby producing toxicity or neoplasia .
Procarcinogen increases the amount of CYP1A1. CYP1A1 is induced by two pathways namely canonical and non-canonical signaling pathway of AhR. Canonical signaling pathway enhances the synthesis of CYP1A1 that causes tumour formation. There is no evidence to prove that Non-Canonical signaling pathway is the reason of tumour promoter. Our study show that the research is going on about non canonical signaling pathway. CYP1A1 is also called as AHH (Aryl Hydrocarbon hydroxylase). It is involved in the metabolic activation of aromatic hydrocarbon (polycyclic aromatic hydrocarbons). The studies on the mechanism revealed that ligand like B[a]P binds to the receptor AhR (aryl hydrocarbon receptor) (an intracellular protein that starts the induction process by biding inducer) in the cytosol. When B(a)P goes inside the body, it will pass through lipid membrane before reaching the cytosol. It is a hydrophobic as well as lipophilic compound that accumulates in the membrane of lipid bilayer, So that the membrane loses its reliability and an increase in permeability to protons and ions. Therefore, it creates imbalance the proton motive force and intracellular pH. In addition to the effects of lipophilic compounds on the lipid part of the membrane, proteins surrounded in the membrane are affected. The effects on the membrane-embedded proteins result to a large extent from changes in the lipid environment [7, 9]. So that PAH is able to go inside the cytosol and makes complex with Ahr receptor. Usually the AhR receptor is present as a part of the cytosolic protein complex which includes 2 hsp90, p23 and XAP2 or AIP (also known as co-chaperons). Once the ligand is bound to the receptor, the co-chaperons dissociate and the receptor is translolcated into the nucleus. This complex along with the ligand molecule heterodimerizes, with another molecule ARNT (AhR nuclear translocator). This heterodimer binds to the consensus regulatory sequences such as AhREs (Ah responsive elements), XREs (Xenobiotics responsive elements) or DREs (dioxin responsive elements) to be found in the promoter region of AhR target genes such as CYP1A1, thereby initiating the transcription by RNA-polyII . The transcription of CYP1A1 gene is inhibited by AhRR protein .The AhRR/ARNT heterodimer stops the transcription process initiated by XREs and also inhibits the heterodimer formation of AhR-ARNT [2,1].
Fig.5. Canonical signaling pathway of AhR
5.3. ALTERATION IN CYP1A1
Mainly four types of polymorphism happen in CYP1A1 that enhances the tumour formation in organs by increasing the oxidation process that makes procarcinogen to carcinogen. These four polymorphisms  are as follows:
M1 (Msp1), T→C substitution at nucleotide 3801 in the 3′-non-coding region.
M2 (Msp2), A→G substitution at nucleotide 2455 leading to an amino acid change of isoleucine to valine at codon 462.
M3, T→C substitution at nucleotide 3205 in the 3′-non-coding region.
M4, C→A substitution at nucleotide 2453 leading to an amino acid change of threonine to asparagine at codon 461.
These mutations do not happen due to smoking; it can lead to enhance the more AHH (aryl hydrocarbon hydroxylase) activity. Each mutation has different function and plays an important role in the tumour formation. For example, CYP1A1 Msp1 (M1) increases the catalytic activity and CYP1A1 Msp2 (M2) is directly related to other functional polymorphism and creates more PAH-DNA adducts in white blood cells . This mutations happen mainly in lung, larynx, pharynx, oral, breast, ovary, liver, colorectal and esophagus. The mechanism of CYP1A1 is same for all organisms and the formation of tumour in these organs depends upon geographical factor, demographic factor and number of cigarettes.
In lung cancer generally Msp1 (T→C) mutations occur, but in Brazil Msp2 (A→G) mutation associates with lung cancer [17, 19]. B(a)P and NNK carcinogen mainly involve with liver cancer.
Oral cancer, larynx and pharynx
Msp2 and Msp1 mutation also associates with oral cancer, larynx and pharynx [15, 16, 18]. And PAH and NNN are associated with these cancer. Benzo[a]pyrene, NNN and NNK are carcinogens that enhance the oral cancer . The studies of various articles show that CYP1A1 Msp1 (M1) mutation maximum associates with increasing the risk of oral squamous cell carcinoma.
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CYPIAI (M1) and CYP1A1 (M2) increase the risk of breast cancer [20, 21].PAH forms PAH-DNA adduct in breast tissue that associates with increased breast cancer. Generally PAH is used for the study of tumor formation in breast cancer because of three reasons, first it is a good model for understanding the chemical mechanism of tumour formation by chemicals. Second the geometric resemblance of PAH and endogenous estrogen is same. Third estrogens and PAH have aromatic ring. PAH are activated by two pathways, first by electron oxidation and second by CYP1A1. DNA adducts are formed by metabolically activated PAH binding with the nucleophilic groups of the two purine bases, adenine (A) and guanine (G). Both adducts may be either stable or depurinating (It is the loss of purine (adenine or guanine) from DNA backbone). The stable adducts is formed by covalently bonding to DNA unless removed during repair, where the depurinating adducts are those that are released from DNA by deterioration of the glycosidic bond .Stable DNA adducts are created when PAH binds with the exocyclic amino group of A or G, where depurinating adducts are formed by covalently bonding of PAH at the N-3 or N-7 position of Adenine or the N-7 or, sometimes, the C-8 position of Guanine [26, 27].
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