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The enzyme serves as a quinone reductase in connection with the conjugation reactions of hydroquinones involved in the detoxification pathways as well as in the biosynthetic processes such as vitamin K-dependent gamma-carboxylation of glutamate residues in the synthesis of the prothrombin .. It also involved in the nitric oxide biosynthesis, response to toxin, synaptic transmission, cholinergic, xenobiotic metabolism. . A wide variety of substrates such as quinones reduces two electrons which are catalyzed by NQO1. The DT-diaphorase enzyme reduces the quinone-imines, azo and nitro compounds but the most efficient substrate is quinone. The enzyme functions through the hydride transfer mechanism and requires a pyridine nucleotide cofactor. Both the NADPH and NADH proceeds with the reduction of electrons in the same pattern. After the attack of the free radical, it can generate antioxidant forms of both ubiquinone and vitamin E. The capability to protect the cells from oxidative challenge and the ability to decrease the quinones through an obligate two electron mechanism that precludes generation of reactive oxygen radicals and considered that NQO1 is an enzyme of chemoprotective. NQO1 stabilizes tumor suppressor gene p53 .Its interaction with the p53 in protein-protein interaction has been shown. Certain compounds such as antitumor quinones are bioactivated by the two electron reduction .So NQO1 serves like an activating enzyme .
Why NQO1 enzyme is described as an anticancer agent?
NQO1enzyme is described as anticancer agent because formation of B[a] P DNA adduct which is generated by CYP450 reductase reduced by NQO1enzyme. .
Quinone metabolism pathway
The two electron reduction of quinoid compounds into hydroquinone is catalyzed by NQO1 which is a cytosolic flavor enzyme.Phase-1enzyme such as Cytochrome p450 catalyzes one electron reduction from quinone. This process formed alkylating species and free radicals as the metabolite undergo auto oxidation under aerobic condition. NQO1 preventing quinones from entering the one electron reduction and two electron reduction is catalyzed by NQO1 which causes the formation of the less toxic hydroquinones that are excreted readily when conjugated. In this way cell protection from oxidative damage occurs by NQO1 .
The AhR repressor and the phase II xenobiotic metabolizing enzyme NAD (P) H: quinone oxidoreductase (NQO1) are induced by polyaromatic hydrocarbons. It is believed that the AhR and AhR repressor constitute a xenobiotic signal transduction negative feedback loop. AhR repressor transcription induced by the liganded AhR that expressed the AhR repressor. It also inhibits the AhR function. .Recent studies suggest that the AhR also plays a role in tumor progression, i.e. from a benign to a malignant tumor, in animal models and very likely also in humans, beside its function in tumor initiation and promotion. There was found that AhR not only play a role in PAH-dependent tumor initiation but also it is involved in the PAH-dependent tumor promotion and progression. It was currently indicated that the AhR induces gene transcription beyond metabolism. In several cell lines, the activation of the AhR leads to cell cycle arrest in G1-phase, which is due to a direct association of the hypophosphorylated retinoblastoma protein (pRB) with the AhR. Thus progression into S-phase is inhibited. (36). The transcription factor, nuclear factor kappa B modulates AhR signaling was found. For cellular proliferation, differentiation and motility, cell-cell contact is known to be a critical regulator. Proliferation inhibition by cell-cell contact is generally referred to as contact inhibition .It is also known as contact-dependent inhibition of growth. Contact inhibition is thought to be continuously active, playing a critical role in the repression of somatic cell proliferation in adult tissues. When contact inhibition is released, it was found that cell is proliferated abnormally. Either due to increased proliferation or decreased level of apoptosis, tumor promotion is characterized by unbalanced proliferation. It is likely that contact inhibition loss is a possible event during promotion of the tumor. .
Fig-2-AhR signaling pathway
The Aryl hydrocarbon Receptor (AhR) is a transcription factor. It is activated through a ligand. It was originally discovered because of its stimulation by a variety of the aromatic hydrocarbons with the benzo[a]pyrene as prototype. The AhR is activated by the PAH. Here, PAH is acted as a ligand. The above fig shows that the unliganded receptor AhR forms a complex with two heat shock protein
90 (Hsp90) molecules, XAP2 and co-chaperones p23 in the cytosol. Whenever PAH (ligand) binds to the AhR, the expression of the gene is induced. The ligand binding results in AhR nuclear translocation, the chaperone proteins dissociation, heterodimerization with ARNTand subsequent binding of the AhR-ARNT heterodimer to dioxin-responsive elements (DREs) with the consensus core recognition sequence 5'-TNGCGTG-3'. 5'-TNGCGTG-3' is also known as xenobiotic-responsive elements (XREs). Several genes encoding phase I and II xenobiotic metabolizing enzymes, such as NQO1 leads to transactivation. AhR pathway activation occur by PAHs that's why leads to their detoxication and excretion and at the same time, to their metabolic activation to genotoxic compounds. 
5.1- Carcinogen identification
The tobacco smoke carcinogens affecting the NQO1gene are benzo[a] pyrene which is a PAH, responsible for bladder cancer  , PAHs for esophageal cancer  and benzene for leukemia .
What is bladder?
Bladder is a muscular, balloon like organ that collects the urine from each kidney to bladder through a narrow tube, ureter. The urine from bladder is eliminated through another narrow tube called urethra .
Interaction mechanism between bladder and carcinogen (B[a] P)
Tumor in the bladder develops due to its interactions with the carcinogenic substances like polyaromatic hydrocarbons (PAH) in the tobacco smoke rather than a direct exposure to them. PAH targets the bladder through blood . The carcinogens from the tobacco smoke are directed from lungs into the blood stream and reach all parts of the body. The blood is filtered by kidneys and concentrated into the urine. The urine is stored and discharged from the bladder, hence the carcinogens in the urine mount up and targets the linings of the bladder walls made up of the urothelial cells. This damage to the cell lining makes the smoker more likely to develop bladder cancer [17, 18]. Genetic polymorphisms of enzymes that catalyze the exogenous or the endogenous carcinogens may determine the individual susceptibility to development of cancer. Most of the chemical carcinogens in cigarettes require metabolic activation by phase I enzymes and detoxification by phase II enzymes such as NAD (P) H: quinone oxidoreductase 1(NQO1). Metabolic activation of the PAHs by phase I enzymes lead to oxidized products, including quinones, resulting in the reactive oxygen species (ROS). In contrast, detoxification of certain carcinogens lead to the less-toxic and more hydrophilic derivatives, which are readily excreted. NQO1, an essential two-electron reductase and protect the cells from oxidative damage by decreasing ROS and reducing formation of DNA adducts by benzo[a]pyrene 3, 6-quinone, one of the carcinogen identified in the cigarette smoke. However, NQO1 can also catalyze the activation of some procarcinogens found in cigarette smoke. The activity of the NQO1 enzyme depends on the polymorphisms at the NQO1 locus. The major polymorphism of the NQO1 involves a single nucleotide C to T transition at nucleotide 609 (codon 187) of exon 6, which codes for a proline (Pro) to serine (Ser) amino acid substitution in protein. Lack of the protein as a result of NQO1 T/T genotype is associated with the accelerated degradation of the mutant NQO1 protein. Therefore, the variant allele (T) of NQO1 is associated with reduced enzymatic activity, and may lead to diverse abilities of the metabolic activation and detoxification. Cigarette smoking is thought to be a major risk factor for the bladder cancer, and smoke contains many carcinogens including the benzo[a]pyrene. NQO1 can convert benzo[a]pyrene intermediates to the less-toxic metabolites. The NQO1 variant genotypes that result in the reduced enzyme activity may lead to development of the cancer .
B[a] P metabolism
Fig-3-Interaction mechanism between phase-1enzyme cytochrome p450s and phase-2 enzyme NQO1in the metabolism of B[a] P .
Multiple reactions are catalyzed by phase-1enzyme such as CYP450. The enzyme play a major role in the activation of carcinogen such as B[a] P which is a PAH.
Phase-2 enzymes catalyze conjugation of the reactive intermediates and resulting urine formation through the process of the excretion. It was found that higher levels of reactive carcinogen metabolites are produced due to higher activation of carcinogen by phase-1 enzyme and lower detoxification by phase-II enzymes. Then higher level of reactive carcinogen metabolites bind to the DNA. After that binding, DNA adducts are formed. If the DNA repair enzymes are unable to remove the adducts, it replicate errors that initiate the carcinogenic casecade and causes mutation . It was found that oxidative metabolite of benzo (a) pyrene [benzo (a) pyrene 3, 6-quinones] causes mutation in NQO1which is present in tobacco smoke .
Fig-4-chemical structure of benzo[a]pyrene 
Fig-5- Benzo[a] pyrene metabolism showing DNA damage ,.
Quinones produces ROS by redox cycle between quinone and diphenol. B[a]P
is eliminated by excretion of glutathione and glucorunides conjugates. The
primary step for B[a]P elimination is oxidation by CYP1A1 and CYP1B1.
B[a] P oxidation by CYP1A1and CYP1B1 is also responsible for the bioactivation of the carcinogenic metabolites. For the detoxification of the primary metabolites, conjugation with glutathione and glucorunic acid are essential. B[a] P-3, 6-quinones forms B[a] P diphenoles and the reaction is catalyzed by NQO1 enzyme. Then it prevents the formation of semiquinones and redox cycle. Later it generates ROS .
Relation of benzene with bladder cancer
In chromosome 16q22, the NQO1 gene is located. In this gene many SNPs have been discovered.At nucleotide position 609, a nonsynonymous SNP was found.
At codon 187 in the protein (dbSNP ID: rs1800566), the variant is a C-to-T transition and results in a proline to serine amino acid substitution. According to the in vitro studies, the variant allele results in reduced enzymatic activity. It was found that C-T transition at codon 187results pro-ser substitution associated with benzene toxicity .
NQO1 enzyme depends on the polymorphisms at the NQO1 locus. The major polymorphism of the NQO1 involves a single nucleotide C to T transition at nucleotide 609 (codon 187) of exon 6, which codes for a proline (Pro) to serine (Ser) amino acid substitution in protein. Lack of the protein as a result of NQO1 T/T genotype is associated with the accelerated degradation of the mutant NQO1 protein. Therefore, the variant allele (T) of NQO1 is associated with reduced enzymatic activity, and may lead to diverse abilities of the metabolic activation and detoxification. Cigarette smoking is thought to be a major risk factor for the bladder cancer, and smoke contains many carcinogens including the benzo[a]pyrene. NQO1 can convert benzo[a]pyrene intermediates to the less-toxic metabolites. The NQO1 variant genotypes that result in the reduced enzyme activity may lead to development of the cancer .
What is oesophagus?
Oesophagus is otherwise known as food pipe. It is a hollow tube which carries liquids and foods from the throat to the stomach .
How smoking causes DNA damage in oesophageal cancer?
Fig-6-Inflammation and oesophageal adeno carcinoma
It was found that Cigarette smoking causes inflammation systemically and in the oesophageal epithelium by swallowing smoking products. A chronic state of systemic and localized inflammation and oxidative stress promotes the DNAdamage, cell proliferation and telomere shortening that increase the risk of developing clones containing the small-scale and large-scale genomic alterations, eventually leading to widespread chromosomal instability and oesophageal adenocarcinoma. 
How the carcinogens affect the function of the DNA in oesophagus?
Genetic polymorphisms of enzymes that catalyze the exogenous or the endogenous carcinogens may determine the individual susceptibility to development of cancer. Most of the chemical carcinogens in cigarettes require metabolic activation by phase I enzymes and detoxification by phase II enzymes such as NAD (P) H: quinone oxidoreductase 1(NQO1). Metabolic activation of the PAHs by phase I enzymes lead to oxidized products, including quinones, resulting in the reactive oxygen species (ROS). In contrast, detoxification of certain carcinogens lead to the less-toxic and more hydrophilic derivatives, which are readily excreted. NQO1, an essential two-electron reductase and protect the cells from oxidative damage by decreasing ROS and reducing formation of DNA adducts by benzo[a]pyrene 3, 6-quinone, one of the carcinogen identified in the cigarette smoke. However, NQO1 can also catalyze the activation of some procarcinogens found in cigarette smoke. Cigarette smoking is thought to be a major risk factor for the bladder cancer, and smoke contains many carcinogens including the benzo[a]pyrene. NQO1 can convert benzo[a]pyrene intermediates to the less-toxic metabolites. The NQO1 variant genotypes that result in the reduced enzyme activity may lead to development of the cancer. .
Same as bladder.
NADP (H): Quinone oxidoreductase 1(NQO1) is one of the most important enzyme. Some unstable PAH metabolites are highly reactive and may attack the DNA molecule in the process of the NQO1 enzyme metabolic reaction. Then PAH DNA adducts are formed. NQO1 detoxify a large no. of synthetic and environmental chemicals. In the 609th codon of NQO1DNA, a polymorphism of C-T leads to the formation of the NQO1*2 allele. The NQO1*2 allele is weak in its biochemical activity. There are only 2-4% enzymatic activity found in the NQO1 T allele in comparison to its wild type form. NQO1 m-RNA is expressd by the polymorphic NQO1allele (T/T). But the expressed m-RNA has no detectable protein because the proteosomal system rapidly degraded the mutant NQO1 protein. A proline to serine change in the 187th amino acid location of the NQO1 protein sequence caused by C-T substitution. It was found that oxidative compounds such as PAH stimulates the NQO1 gene expression.There was found a relation between esophageal tissue expression and their underlying NQO1*2 allele (C609T) genotypes .
What is leukemia?
Leukemia is the cancer of blood and bone marrow because of an abnormal proliferation of blood cells, generally leukocytes (WBCs) [24, 25]
How cigarette smoke causes DNA damage in blood?
Leukemia-causing chemicals like benzene present in cigarette smoke. .
It is a known human carcinogen.30% increase risk of leukemia getting through smoking. 14% of all adult leukemia cases causes through smoking. It is also regarded as a primary cause of leukemia. ,. It was found and considered that the main cause of leukemia is cigarette smoking. This is a major risk factor also. Benzene, polonium-210, and polycyclic aromatic hydrocarbons (PAHs), these chemicals are present in tobacco smoke. Lungs absorbed these carcinogens .These carcinogens are spread through the bloodstream. These carcinogens damage the DNA within blood cells.. Hence, normal bone marrow cells are transformed to leukemic cells due to DNA damage. . There are found that some pieces are lost from WBC from their DNA.It was shown that abnormal leukemic cells proliferate over by over through replication. An immature blast is produced that will not mature properly like normal blood cells. Those leukemic cells will not die and multiplication occurred until production of abnormal cells than normal healthy blood cells. Due to translocation, the mutation also occurs where one part of chromosome displaces and attached to other part of the chromosome. Normal DNA sequences will disrupt due to the translocation. It leads stimulates the growth of the tumor and cause prevention of tumor suppression .One in four cases of acute myelogenous leukemia (AML) is the result of cigarette smoking is estimated. .Another major source of public exposure to benzene is cigarette smoking. Approximately 2 mg/day was inhaled by a pack-a-day smoker. In NQO1gene, inherited Polymorphism encodes carcinogen activation and detoxification. It contributed indirectly to leukemia. The NQO1 enzyme is involved in the metabolism of benzene .
DI AND TRI HYDROXY BENZENE (HQ, CAT, BT)
Figure-7- benzene metabolism pathway leading to toxicity and detoxification
The diagram above shows that the hepatic enzyme CYP2E1 metabolized benzene
Benzene forms benzene oxide through metabolisation that immediately forms phenol. Again phenol is metabolized by CYP2E1 which forms di and tri hydroxyl benzene such as catechol (CAT), hydroquinone (HQ), and 1, 2, 4-benzenetriol (BT). Thus it plays an essential role in toxicity of benzene by activating it to toxic metabolites. Hydroquinone is converted to benzoquinone in the bone marrow by Myeloperoxidase.Benzoquinones are regarded as hematotoxin and genotoxin compounds.. NQO1 converted benzo quinones back to less toxic hydroxybenzenes. People lack significant activity of NQO1 making them potentially susceptible to benzene toxicity. A homozygous mutation (C -T) at position 609 in the NQO1 gene is caused by this variation. It was found thatNQ01609 mutation would be susceptible to benzene hematotoxicity with high CYP2E1 activity.NQO1 is also a risk factor for leukemia. Benzene poising risk increased at a7.6 fold by homozygous NQO1 mutant allele . Those who are homozygous for the point mutation, the NQO1 protein and activity are absent in them .
Fig-8-St of benzene-C6H6 
Benzene is a chemical compound. The chemical formula of benzene is C6H6.It
is a colorless aromatic hydrocarbon which is a known carcinogen .
A critical role in benzene toxicity was played by benzene metabolism.
It was also found that hepatic metabolism play a vital role in benzene metabolism.
Secondary metabolism of benzene metabolites contributed to toxicity in the bone marrow .
Fig-9-Benzene metabolic pathway
First step-Phenol formation
CYP450+NADPH+H+ +O2 CYP450+NADPH
The above reaction shows that H2O2 generated by cytochrome p450 when acting as oxidases of NADPH.
HOOH 2 *OH
This reaction indicates the generation of hydroxyl radicals.
Benzene + * OH phenol
It indicates that hydroxyl radicals formed from H2O2 hydroxylated to benzene to form phenol.
The first product is benzene oxide. Phenol is produced from benzene oxide by nonenzymatically rearrangement.
Second step-formation of benzenedihydrodiol
Epoxide hydrolase hydrated benzene oxide to form 1, 2-benzene dihydrodiol.
Third step- catechol formation from benzenedihydrodiol
Benzene dihydrodiol forms catechol through oxidation.The oxidation is carried out by the enzyme dihydrodiol dehydrogenase.
Fourth step-Formation of hydroquinone or catechol from phenol
The further hydroxylation of phenol gives rise hydroquinone or catechol .
Fifth step-Formation of 1, 2, 4-trihydroxy benzene
The other names of 1,2,4-Trihydroxybenzene are Hydroxyhydroquinone; Benzene-1,2,4-triol, 1,2,4-Benzenetriol .Either hydroxylation of hydroquinone or catechol produces 1,2,4-trihydroxy benzene.
Sixth step-Formation of benzo quinone
Hydroquinone is converted to benzo quinone in bone marrow by Myeloperoxidase. Benzo quinones are regarded as hematotoxin and genotoxin compounds. 
NQO1 converted benzo quinones back to less toxic hydroxybenzenes. People lack significant activity of NQO1 making them potentially susceptible to benzene toxicity .
Potential mechanism for benzene toxicity
Benzene metabolites produced in the liver. It is then transported to the bone marrow. Benzene damaged the DNA in the bone marrow.DNA damage result the depression of bone marrow which causes aplastic anemia.Then it leads to marrow dysplasia and ultimately it leads to acute leukemia.
Fig-Pathway of damage of DNA by benzene in bone marrow cells
The pathway shows two mechanisms.
In first mechanism the pathway shows that benzene metabolites cause the damage of the DNA.Benzene metabolic activation bind to the DNA covalently and after binding mutagenic events are produced that are expressed as leukemia.
In second mechanism the pathway shows that metabolite production which causes oxidative stress. Through oxidative stress oxidative damage to DNA occurred .
Oxidative stress and benzene toxicity
Oxidative stress caused by benzene toxicity through benzene metabolites. Hydroquinones are oxidized to form p-benzo quinone.p-benzoquinone bind covalently to the cellular macromolecules or glutathione. It is highly reactive. Benzene metabolites are involved in the redox cycling. Benzene metabolites participate in auto oxidation of a reduced form of the metabolite .Thus reactive oxygen and an oxidized species are produced from benzene metabolites. Bone marrow is one of the richly oxygenated organ.It has the ability to produce reactive oxygen species. From the benzene metabolites four electrons reduced from oxygen and generate hydrogen peroxide, hydroxyl radical and superoxide anion radicals. The starting materials are produced through the oxidized metabolite by undergoing their reduction of flavor protein. Then the starting material reenters the redox cycle. It was found that hydroquinone-p-benzo quinone reenter redox cycle.
Fig-10-benzo quinone reduction by FPH2 reductase
The figure shows that p-benzo quinone reduction proceeds through CYP450 reductase.
Semiquinone anion radical is the first product.
Semiquinone is reduced again by FPH2.
In this step protonation occurs. In the second reduction hydroquinone is formed.
The next step in redox cycling is reoxidation to p-benzo quinone would be inhibited because hydroquinone and is the substrate for auto oxidation leading to the formation of superoxide anion. Thus at physiological pH it is unlikely that hydroquinone-p-benzo quinone undergoes redox cycling.
Fig-11-Interaction of p-benzo quinone with glutathione (slow auto oxidation)
If p-benzo quinone is not reduced. It reacts with GSH and premercapturic acid of hydroquinone is produced from this reaction, which again produces mercapturic acid, and leading to the production of reactive oxygen species through slow auto oxidation.
Fig- 14-The formation of p-benzo quinone 2,3-oxide from p-benzo quinone and its metabolic fate
By the addition of the HOOH, p-Benzo quinone is converted to its epoxides.
Either by one-electron reduction (1) catalyzed by P450 reductase or by two-electron reduction (2) catalyzed by DT diaphorase, p-Benzoquinone is reduced to 1,2,4-benzenetriol.
Reaction of GSH with p-benzo quinone 2, 3-oxide leads to the formation of glutathionyl 1, 2, 4-benzenetriol .
Due to a C-T substitution at nucleotide 609 in the cDNA, a polymorphism exist which giving rise to a missense mutation in codon 187 (proline-serine). The frequency of an inactivating polymorphism in NQO1 appears to be increased in myeloid leukemia patients with abnormalities of chromosomes 5 and/or 7 but not in those with balanced translocations. Thus completely lack enzyme activity
Susceptible to leukemogenic changes induced by carcinogens in individuals who are homozygous for the inactivating allele of NQO1 [30, 31]. It was also found that reduced enzyme activity associated with individuals with the NQO1*2 variant . It was observed that statistically significant association was found between NQO1 C609T polymorphism and risk of chronic myelocytic leukemia .It was
Suggested that NQO1C609T polymorphism caused leukemia in childrens. So NQO1 C609T polymorphism is a major risk factor for childhood leukemia..