Cancer and Phase II Drug-Metabolizing Enzymes

Sheweita1, S.A and Tilmisany2, A.K. 1- Department of Bioscience & Technology, Institute of Graduate Studies & Research, Alexandria University, Egypt 2- Department of Pharmacology, Faculty of Medicine, King Abdulaziz University, Madinah, Saudi Arabia

Correspondence to: Dr. Salah A. Sheweita Department of Biochemistry Faculty of Medicine, PO Box 30001 King Abdulaziz University, Madinah, Saudi Arabia E-mail: [email protected] Fax: 966-04-8461407 Tel. 966-04-8461407 Key words: Cancer, tumor suppressor genes, alkylating agents, diet, glutathione, glutathioneS-transferase, epoxidase, sulfatase.

2 Summary: Cancer development results from the interaction between genetic factors, the environment, and dietary factors have been identified as modulators of carcinogenesis process. The formation of DNA adducts is recognized as the initial step in chemical carcinogenesis. Accordingly, blocking DNA adducts formation would be the first line of defense against cancer caused by carcinogens. Glutathione-S-transferases inactivate chemical carcinogens into less toxic or inactive metabolite through reduction of DNA adducts formation. There are many different types of glutathione S-transferase isozymes. For example, GSTπ serves as a marker for hepatotoxicity in rodent system, and also plays an important role in carcinogen detoxification. Therefore, inhibition of GST activity might potentiate the deleterious effects of many environmental toxicants and carcinogens. In addition, approximately half of the population lacks GST Mu expression. Epidemiological evidence showed that persons possessing this genotype are predisposed to a number of cancers including breast, prostate, liver and colon cancers. Also, lacking of GST Mu expression in human has been associated with increased predisposition to liver and colon cancers. In addition, individual risk of cancer depends on the frequency of mutational events in target oncogenes and tumor suppressor genes which could lead to loss of chromosomal materials and tumor progression. The most frequent genetic alteration in a variety of human malignant tumors is the mutation of the coding sequence of the p53 tumor suppressor gene. O6-alkylguanine in DNA leads to very high rates of G:C→A:T transitions in p53 gene. These alterations will modulate the expression of p53 gene and consequently change DNA repair, cell division, and cell death by apoptosis. Also, changes in the expression of BcI-2 gene results in extended viability of cells by over-riding programmed cell death (apoptosis) induced under various conditions. The prolonged life-span increases the risk of acquiring genetic changes resulting in malignant transformation. In addition, a huge variety of food ingredients have been shown to affect cell proliferation rates. They, therefore, may either reduce or increase the risk of cancer development and progression. For example, it has been found that a high intake of dietary fat accelerates the development of breast cancer in animal models. Certain diets have been suggested to act as tumor promoters also in

3 other types of cancer such as colon cancer, where high intake of fat and phosphate have been linked to colonic hyper-proliferation and colon cancer development. Different factors such as oncogenes, aromatic amines, alkylating agents, and diet have a significant role in cancer induction. Determination of glutathione S-transferase isozymes in plasma or serum could be used as a biomarker for cancer in different organs and could give an early detection.

4 1- Introduction Up to 90% of all cancers are possibly caused by environmental factors, such as tobacco smoke, diet and occupational exposures. The majority of the exogenous compounds such as chemical carcinogens require metabolic activation before they interact with cellular macromolecules and can cause cancer initiation through the formation of DNA damage. Exogenous genotoxic carcinogens encompass a wide range of chemically different compounds with the common property to form chemical bonds with DNA that result in the generation of "DNAadducts" [1]. In addition, the initial stage genetic damage based on endogenous processes that cause mutations or even gene deletions. Free radicals are one of the endogenous mediators of oxidative DNA damage and mutations. They are produced by normal oxidative metabolism in cells but their production is increased under pathological conditions [2,3]. Most of the genotoxic carcinogens such as polycyclic aromatic hydrocarbons, aromatic amines, alkyl and arylnitrosamines are activated by enzymes whose normal function is the metabolism, detoxification and elimination of potentially toxic compounds. The pro-carcinogens are activated by phase-I enzymes, most of which belong to the superfamily of cytochrome P450-dependent monooxygenases. Their normal function is to insert an oxygen into relatively inert and usually non-polar substrates and these intermediates formed by oxidative metabolism are then conjugated by phase-II enzymes with endogenous polar substrates such as glucuronides, glutathione or sulfate to enable the excretion of the now more polar products [4]. Phase-II enzymes therefore can reduce the cellular exposure to carcinogens whereas phase-I enzymes can increase it [5-8]. Some evidence suggests that genetic susceptibility to cancer may in part be determined by polymorphisms and altered functions of these enzymes. The xenobioticmetabolizing machinery contains two main types of enzymes: the phase-I cytochromes P-450 mediating oxidative metabolism which covered in our previous report [2], and phase II conjugating enzymes which are presented in this review. There are many different factors are involved in the causation of different types of cancers including alkylating agents, diet, aromatic amines, and tumor

5 suppressor gene. In addition to the role of phase-II drug metabolizing enzymes in the induction and/or suppression of different types of cancers.

1.1 Factors involved in cancer induction 1.1.1 Tumor suppressor genes. Tumor suppressor genes have been implicated in a variety of human cancers. It is suggested that their inactivation, due to point mutations within the gene or due to loss of chromosomal materials, can play an important role in differentiation and tumor progression. Recent studies have identified alterations of molecular events underlying urothelial neoplastic progression associated with specific genes along schistosomal bladder cancer. These include the activation of H-ras [9] and inactivation of p53 [10], and retinoblastoma gene [11].The protein products of oncogenes are known to directly participate in cell-cycle processes. Any alterations of these genes or their proteins can alter their functions, leading to uncontrolled cell growth and ultimately tumor formation. The most frequently activated oncogenes are the members of ras gene groups. They encode a low molecular weight (21 kDa) protein which mediates single transduction between tyrosine-kinase receptors and the nucleus. This process can be inhibited by different mutations in the various sites of ras genes. Several studies estimated the frequency of ras genes activation between 7 and 17% in human urinary bladder cancer [12]. Moreover, experimental studies on ras mutations in vitro showed a cumulative effect with other cellular alterations including c-erbB-2 and c-myc leading to more aggressive and invasive properties [13]. The p53 gene is one of the most studied tumor suppressor genes in human cancer. It is located on the short arm of chromosome 17 and encods a protein involved in the growth and regulation of cells [14]. it also regulates the multiple components of DNA damage control response and promotes cellular senescence

[15].

Habuchi

et

al.;(1993)[16]

reported

that

about

86%

schistosomiasis-associated bladder cancer had mutated p53 gene in exons 5, 6, 8 and 10. This is consistent with the frequencies of p53 mutations at different grades of the schistosomiasis-associated bladder cancer where, at the early

6 stage of the disease, p53 inactivation ranged between 0-38% as opposed to 3386% in the advanced tumor stage. Loss of p53 function has shown to allow cells to become permissive for genetic changes such as DNA amplification [17]. The most frequent genetic alterations in a variety of human malignant tumors is the mutation of the coding sequence of the p53 tumor supressor gene [10]. It has been recently reported that molecular changes in H-ras and p53 genes in bilharzial-bladder cancer are different from those reported in transitional cell carcinoma in Western countries [18]. Multiple mutations were found at the p53 locus in schistosomiasis-bladder cancer. These might be due to the involvement of specific etiological agents, such as N-nitrosamines [19,20], and abnormal tryptophan metabolites [21], that are responsible for the neoplastic progression in bilharzial-bladder cancer patients. About 89% of the N-nitrosoamines-induced p53 mutations have a high incidence (75-100) of G→A transitions [22]. It has been found that thirty patients out of ninety had tumors with mutations in exons 5-8 of the p53 gene: 17/53 squamous cell carcinoma (SCC), 8/23 transitional cell carcinoma, 4/13 adenocarcinoma and 1/3 other tumors. Out of 19 mutations in SCC, 16 were base pair substitutions (BPS), two were deletions and one an insertion. Of the BPS, nine were transitions at CpG dinucleotides and two were G→T transversions. All the mutations in TCC were BPS: four were transitions at CpG

dinucleotides

and

three

were

G→C

transversions.

Out

of

four

adenocarcinomas with mutations, two had transitions at CpG dinucleotides. It was suggested that the excess of transitions at CpG dinucelotides in schistosomiasis bladder cancer results from nitric oxide produced by the inflammatory response provoked by schistosomal eggs. Nitric oxide can produce such mutations either directly by deamination of 5-methylcytosine or indirectly through reduction of nitrate to nitrite, by nitrate reducing bacteria, leading to the formation of endogenous N-nitroso compounds which cause mutations in p53 gene and DNA alkylation [22]. O6 -alkylguanine in DNA leads to very high rates of G:C→A:T transitions. These alterations in p53 gene will consequently modulate the expression of genes that regulate DNA repair, cell division, and cell death by apoptosis [15].

7 Changes in cell-cycle control are thought to be critically associated with cancer development. A family of enzymes called the cyclin-dependent kinases (CDKs) controlled the progression from the G1 to the M phase during the cell cycle. CDK activity is dependent on positive regulators called cyclins and inhibited by a set of proteins termed CDK inhibitors. Cyclins are low molecular weight proteins whose function is markedly modulated during phases of the cell cycle. The role played by these proteins in human cancer has long remained unclear. However, in adenomas of parathyroid, the gene promoter of parathyroid hormone is fused to the gene encoding cyclin D1 and this could provide evidence that cyclins can be directly invovled in cancer development. Seven CDK inhibitors have been characterized including p57, p21, p27, p19, p18, p16INK4 and p15. It has been found that deletion was present in 23/47 samples and mutations in other 2 cases (53% of p16INK4 ) in schistosomiasis-associated bladder cancer patients [23]. They concluded that p16INK4

alterations are more frequent in

schistosomiasis-associated bladder cancer than in other bladder tumors [23]. p16INK4 binds specifically to CDK4 and CDK6 and inhibits these two kinases [24]. Interestingly, cyclin D1 activates CDK4 and CDK6. Thus, p16INK4 is a specific regulator of cyclin D1-dependent kinases. It is thus likely that p16INK4 alterations as a result of schistosomiasis can also be involved in cancer development. In another study, deletions in 9p, where CDKN2 resides, were found in 92% (10/11) of squamous cell carcinomas of Egyptian bladder cancer patients compared with only 10% (11 of 110) of transitional cell carcinoma of Sweden bladder cancer patients [25]. The authors suggested that a putative tumor supressor gene on 9p may contribute to squmaous cell carcinoma tumorigenesis [25]. The two bladder carcinomas, squamous cell carcinomas and transitional cell carcinoma, differ in their genetic alterations suggesting that distinct underlying genetic defects may explain, at least in part, the pathological differences between the two tumors of the bladder epithelium. BcI-2 gene was discovered in chromosomal translocations identified in B cell leukaemias and follicular lymphomas. The expression of this gene results in extended viability of cells by over-riding programmed cell death (apoptosis)

8 induced under various conditions. The prolonged life-span increases the risk of acquiring genetic changes resulting in malignant transformation [26]. BcI-2 can cooperate with viral or other cellular protooncogenes in the process of transformation and tumorigenesis, both in vivo and in vitro [27], and its expression is now reported in a variety of haematological and epithelial malignancies [28]. A positive correlation between BcI-2 expression and tumor progression has been described in prostate and gastrointestinal epithelial carcinomas [29]. Recently, it has been shown that BcI-2 was over expressed in schistosomiasis-associated bladder cancer [30]. The high level of BcI-2 expression in malignant cells, but not in the precancerous cells, suggests that they may be upregulated in the later stages of tumor progression.This could be of clinical significance, as BcI-2 may confer a resistance to cytotoxic agents on tumor cells. It has been shown that BcI-2 was only expressed at high levels in squamous carcinoma and adenocarcinoma, but not significantly in transitional cell carcinoma suggesting that the expression may also be related to tumor-cell linkage [30].

1.1.2 Role of aromatic amines. It is well known that human populations come into contact with a variety of chemical carcinogens [31]. Aromatic amines, among these chemicals, are a potent group of carcinogens widely present in the environment as consequence of human activities. Occupational exposure to aromatic amines most notably in the manufacture of dye-stuffs and in tire manufacture [32], are known to be the most important causes of

bladder cancer. Aromatic amines, after metabolic

activation, were shown to react with cellular DNA to form aromatic amine-DNA adduct and such adducts were linked with the mutagenic, toxic and carcinogenic effects of these agents [33-35]. Biochemical studies of aromatic amines show that several reactions are involved in the detoxification and/or activation of the parent compounds and their N-hydroxy and arylhydroxamic acid derivatives. Nhydroxylation is the primary pathway of most aromatic amines activation, mainly in the liver via a certain cytochrome P-450 isozyme [36]. The N-hydroxy derivatives can then enter the circulation, thus reacting with hemoglobin, or can

9 enter the bladder lumen and be reabsorbed into the bladder epithelium. Previous studies showed that acetyltransferases contained in urinary bladder, have a further bioactivation step to N-hydroxy derivative to form the highly electophilic derivative, N-acetoxy, that could bind covalently to urothelial DNA [37] and initiate bladder cancer. Supporting the role of aromatic amines in bladder cancer induction, epithelial hyperplasia and metaplasia were reported in the bladders of mice infected with S. haematobium and pretreated with acetylaminofluorene [38] and 2-naphthylamine in combination with E. coli

infection [39]. The wide

variation in carcinogen metabolism in humans has long been regarded as an important determinant of individual susceptibility to chemical carcinogenesis. In the case of aromatic amine carcinogens, it has become apparent that the biochemical basis for these differences may be the polymorphic distribution of specific carcinogen-metabolizing enzymes involved in their activation and/or detoxification. These polymorphisms can arise from both heritable and environmental factors, which can be assessed in epidemiological studies. The role of aromatic amines has been well established in cancer of the urinary bladder. Industrial exposures to 4-aminobiphenyl, 2-naphthylamine, and benzidine were clearly associated with a high incidence of transitional urothelialcell carcinomas. Occupational exposure to 4,4-methylenebis(2-chloroaniline) and o-toluidine have also been correlated with increased bladder cancer risk [40]. Likewise, cigarette smoking has often been implicated as a causative factor in urinary bladder carcinogenesis, and this association has been supported by findings that aromatic amines are present in nanogram quantities in cigarette smoke and that smokers have much higher level of aromatic amine-hemoglobin adducts than nonsmokers. It has been showed that several smoking-related DNA adducts present in human urothelium are characteristic of aromatic amine-C8deoxyguanosine, which identified as a major adduct. An increasing body of evidence indicates that aromatic amines may play a significant role in the etiology of human colorectal cancer. Greater susceptibility to this disease has long been associated with dietary factors, including consumption of well-done red meats and smoked meats [41].

10 1.1.3 Role of N-nitrosamines and DNA damage Various hypotheses have been proposed to explain the carcinogenic process of bladder cancer [42]. However, more concern was directed towards the possible role of the N-nitroso compounds, an important class of chemical carcinogens, in this process [19,20,43]. Experimental studies identified several compounds which are bladder carcinogens for rodents and/or dog, including N-nitrosomethylurea, N-butyl-N-(4-hydroxybutyl)nitrosamine and N-nitrosomethyldodecylamine [44,45]. The biogenesis of bladder cancer was studied in experimental animals, and there is now good evidence that it is a multistage process involving early and late stages which can be influenced respectively by genotoxic and nongenotoxic carcinogens acting sequentially on the target tissue and accelerating the development of bladder cancer [43]. The urinary excretion of N-nitrosamines (NNC) was studied in different populations from widely separate regions of the world in order to predict their possible exposure to this group of chemical carcinogens. Several of these studies showed that subjects with a high-risk of developing stomach, esophageal , colon

and urinary bladder cancers excreted higher levels of NNC and

precursors in their urine relative to low-risk groups [46]. Significant amounts of Nnitrosamines, nitrite and nitrate were detected in the urine of schistosomalinfested patients [19,20.] The presence of these compounds in the urine, therefore, could provide the initiating events critical for the formation of bladder cancer. In order to express their carcinogenic effects, their activation are necessary. There are two enzyme species responsible for the N-demethylation of dimethylnitrosamine (DMN) through an oxidative N-demethylation reaction, namely DMN-N-demethylases I & II ( which can operate at ~ 4 mM and ~200 mM of DMN respectively) [47,48]. It was found that following the N-demethylation of dimethylnitrosamine (DMN), a diazonium ion is produced leading ultimately to the formation of carbonium ion that methylate DNA. During schistosomiasis, the expression of hepatic carcinogen-metabolizing enzymes differs markedly from the pattern observed in uninfected control animals [49]. Therefore, the possibility of formation of carbonium ions could be high and could lead to a high incidence of liver damage and bladder cancer. It was suggested that the carcinogenic

11 effects of alkylating agents are proportional to the activities of their activating enzymes in the liver [50] since more of the active metabolites might be produced, when the demethylases are activated. Therefore, the carcinogenic effects of carbonium ion resulting from activation of N-nitrosodimethylamine might be increased toward the liver and probably other organs. Supporting our previous observations, schistosomiasis induced promutagenic methylation damage in tissue

DNA

of

S.

mansoni-infected

animals.

The

levels

of

O 6-

methyldeoxyguanisone (O6-MedG) detected in the liver DNA of infected animals were directly proportional with the levels of S. mansoni infection [51]. Methylation of DNA has been detected in liver tissue of experimental animals infected with S. mansoni and in various tissues of human populations especially in those of bladder cancer associated with schistosomiasis [51]. O6alkylguanine is thought to be formed in larger amounts than the other promutagenic base, O4-alkylthymine, especially in the case of alkylating agents [52]. These suggest a continuos and prolonged exposure to alkylating agents detected in large quantities in the urine of schistosomal patients. Such DNA damage in the bladder tissue of bilharzial patients might be responsible for the initiation of bladder cancer among bilharzial-suffering patients. The persistence of O6-alkylguanine in different tissues strongly depends on the capacity of the cellular DNA repair system, alkyltransferase, which is correlated with the mutagenic, and carcinogenic effects of chemical carcinogens [53]. The constitutive level of alkyltransferase (ATase) activity varies among the cells and tissues of different mammalian species [54,55]. Samples of bladder mucosa infected with schistosomiasis

from Egypt showed a relatively high

quantitative level of mutagenic lesions. This may be due to the increased efficiency of activating mechanisms. Bladder tissue has a lower capacity in repairing the damaged DNA, particularly when its cellular DNA contains higher amounts of alkylation, than other tissues in human and animal models [55]. The lower activity of alkyltransferase and unrepaired DNA damage could , therefore, enhance the incidence of bladder tumor.

1.1.4 Role of the diet.

12 A huge variety of food ingredients have been shown to affect cell proliferation rates. They, therefore, may either reduce or increase the risk of cancer development and progression. The significant differences in incidence of specific types of cancer in particular countries or regions of the world have directed attention to the possible influence of dietary components on the biological processes concerned with carcinogenesis. It is generally considered that the Western diet is deficient in fiber and it was originally suggested that fiber protected against colorectal cancer. This concept was based on the low incidence of the disease in East Africa where a high-fiber diet was consumed [56,57].

Evidence from both animal and experimental studies was used in

support of the concept that a high fiber content of diet protects not only against colorectal cancer [58], but also against other cancers, such as mammary cancer [59]. In a case-control study on the effect of diet on breast cancer risk in Singapore and China, a quantitative food-frequency questionnaire was used to evaluate the consumption of certain foodstuffs over a 12-month period prior to the interview of 200 Singaporean and Chinese women with histologically confirmed breast cancer and 420 matched controls. It was found that the red meat intake (but neither total meat nor saturated fat) was a predisposing factor with regard to breast cancer risk [60]. La Vecchia et al.; in 1988 indicated that green vegetable consumption was inversely related to the risk of breast cancer [61]. For many years, interest has centered on the potential dangers of a high fat content of the diet, especially with regards to breast cancer. Extensive information from experimental studies was offered in support of the concept that a high dietary fat intake is a causative factor for breast cancer but the evidence from epidemiological case-control studies appears to be to some extent equivocal and inconclusive [62]. Reports from many epidemiological studies concluded that there was little evidence to support the concept that high dietary fat intake is associated with breast cancer risk. [63]. There is growing evidence that dietary lipids may play a significant role in the induction of cancer. Various studies have demonstrated - mainly in animal models - that a high intake of dietary fat accelerates the development of breast cancer. Especially the content of n-6 fatty acids, such as linoleic acid, was shown to be associated with

13 increased rates of tumor formation, whereas n-3 fatty acids, such as eicosapentanoic acid and docosahexaenoic acid, did not affect tumor development but were able to inhibit the promoting effects of n-6 fatty acids [64]. Another potent suppressor of mammary tumors in animal studies is conjugated linoleic acid of which milk fat is a good source [65]. In humans, however, there are evidences that increased body weight is a more critical parameter with regard to cancer development in the mammary gland than fat intake per se or the fatty acid pattern [66]. Overnutrition in early life causes rapid growth that results in early menarche and consequently the mammary tissue is confronted with high levels of growth-stimulating estrogens for a longer time period. Overnutrition and high fat consumption in later life results in a breast cancer promoting hormonal imbalance by increasing the levels of free steroid hormones associated with visceral obesity [67]. Whereas certain dietary factors promote the proliferation of cells in mammary tissue by enhancing the circulating estrogen levels, there are also dietary ingredients that can suppress these effects. Phytoestrogens, found in vegetables such as soy, are weak estrogens but in competition with more potent endogenous estrogens display a net anti-estrogenic activity. Moreover, they may serve as aromatase-inhibitors thus reducing the synthesis of estrogens [68]. These actions of the phytoestrogens have been related to a reduced incidence of breast cancer associated with a diet rich in soy products [69]. Certain diets have been suggested to act as tumor promoters also in other types of cancer such as colon cancer [70, 71], where high intake of fat and phosphate have been linked to colonic hyperproliferation [70] and colon cancer development [71]. As there are dietary factors that increase the proliferation rate of colonic cells there are also compounds that block uncontrolled tissue growth. Butyrate, produced by the gut microflora from fermentable dietary fibres, is one of the key factors shown to suppress growth of cancer cells by increasing the expression of proteins that permit cell-cycle arrest. Especially the induction of the cyclindependent kinase inhibitor p21 seems crucial for the growth inhibiting effects of butyrate [72]. Regarding the role of proteins in the incidence of hepatocarcinogenesis, Lee et al. (1971) , found that the incidence of aflatoxin B1-induced hepatomas

14 was significantly higher in rainbow trout given a diet containing 49% fish protein than those given a 32% protein diet [73]. It has been showed that the enhancing effect of protein diet on aflatoxin-hepatocarcinogenesis was even more dramatic with higher protein concentration; the 9-month hepatoma incidences in rainbow trout fed 20 ppb AFB1 and a 40, 50, 60, or 70 % protein diet were 33, 48, 68 and 90%, respectively. In agreement with the previous studies, small amounts of Nnitrosamines

(N-nitrosodimethylamine,

N-nitrosopyrrolidine

and

N-

nitrosodiethylamine) with a range of 0.2-0.25 μg/kg sample, were detected in cheese samples stored for different periods of time. [3]. N-nitrosodimethylamine was also detected in some traditional Egyptian foods such as fava (fresh fava beans boiled in water until soft), fried fava (softented fava beans mixed with vegetables mainly onions, garlic, red peppers and spices), raw salted fish (Sardines), and raw salted fish (Mullet) with a range of 0.35-0.65 μg/kg sample [3].These foods are considered the main traditional meal of Egyptian farmers since they consumed

them almost daily. The presence of some N-nitroso

compounds in traditional Egyptian food, in addition to the fact that these compounds may be formed from endogenous sources in the stomach or bacterial synthesis [74] in the bladder, might play a significant role in the induction of bladder cancer in Egypt. Epidemiological studies suggest that the high rates of colorectal cancer in developed countries are potentially preventable by dietary means. National incidence rates for colon cancer are strongly correlated with average consumption levels of meat in 23 countries. In some prospective studies, individuals consuming higher amounts of red or processed meat, but not white meat or fish, experience a greater risk of developing colon cancer [75]. Vegetarians are known to be at low risk of cancer but it is not clear which aspects of vegetarianism are protective. Selected flavonoids found in a large variety of vegetables and fruits are also able to exert cell-cycle arrest. Their actions are linked to the increased expression of cell-cycle regulators, such as p21 or the reduced expression of different cyclins or of proteins also affecting proliferation, such as cyclooxygenase-2 (COX-2), [76, 77]. Especially the effects of these plant components on COX-2 expression could provide an important chemopreventive

15 mechanism in colon cancer development since COX-2 is overexpressed in about 90% of all human colorectal cancers and moreover, COX-2 inhibition by longterm treatment with non-steroidal anti-inflammatory drugs is considered as a chemopreventive strategy [78, 79]. The mechanism by which enhanced COX-2 expression promotes cancer development is most likely by an increase of cell proliferation due to increased production of growth promoting prostaglandins [80]. Vegetables, starch and non-starch polysaccharides, increased stool weight and reduced stool pH are also implicated in reduced risks of colon cancer [81,82]. G→A transitions at the second G of a GG pair at codon 12 or 13 of K-ras are common in colorectal cancer and are characteristic effects of alkylating agents such as N-nitroso compounds [83]. Human faecal specimens have been shown to contain N-nitroso compounds [84]. Alkylated DNA adducts of O6methylguanine have been detected in human colonic tissue and N-methyl-Nnitrosourea induces G→A transitions in codons 12 and 13 of K-ras in 30% of rat colon carcinomas [85]. 1.2 Enzymes of carcinogen inactivation 1.2.1 Glutathione-S-transferase (GST) The glutathione S-transferases (GSTs) are a group of inducible enzymes important in the detoxication of many different xenobiotics in mammals. The GSTs achieve detoxication by catalyzing the conjugation of reduced glutathione to various electrophilic substrates. The glutathione-S-transferase enzymes are soluble proteins predominantly found in the cytosol of hepatocytes and catalyze the conjugation of a variety of compounds with the endogenous tripeptide, glutathione. Cytosolic glutathione S-transferases can be divided into four families, termed alph (α), mu (µ), Pi(π), and theta (Ф), having different but sometimes overlapping substrate specificities [86-90]. Glutathione S-transferases are subjected to activation by endogenous disulfides and by various intermediates that form during the metabolism of drugs and other foreign compounds [91]. Individual variation in the expression of GST isozymes is well documented [92]. For example, the levels of GSTα class isozymes can vary markedly between individuals [93], and in the case of GSTµ class, as a consequence of genetic polymorphism, about 45% of individuals do not express

16 GSTµ subunits [94]. Such variation in expression of GST isozymes may predispose individuals to the toxic effects of environmental carcinogens. A number of studies demonstrating that high levels of GSTπ in neoplastic nodules in several rat models of hepatocarcinogenesis have prompted investigations of GSTπ levels in human tumors. The predominant form in most human tumors investigated was class GSTπ and comparison of matched pairs of normal and tumor tissues revealed high levels in stomach, colon, bladder, cervix and lung tumors [95-98]. GSTπ serves as a marker for hepatotoxicity in rodent system [99], and also plays an important role in carcinogen detoxification. Therefore, inhibition of GST activity and depletion of GSH levels might potentiate the deleterious

effects

of

many

environmental

toxicants

and

carcinogens.

Glutathione-S-transferases (GSTs) have the capacity to detoxify electrophilic xenobiotics by catalyzing the formation of glutathione (GSH) conjugates. GSTs are also engaged in the intracellular transport of variety of hormones, endogenous metabolites, and drugs, by virtue of their capacity to bind these substances [100]. A series of carcinogens drugs, metabolites, and related compounds were analyzed for binding to GST [101]. Broad specificity of binding is evident, but relative affinities are not determined by lipophilicity alone. This is exemplified by the striking differences between 2,3-benzo(a)anthracene, which was bound with high affinity, and 1,2-benzo(a)anthracene, which was classified as non-binding. Distinct structural requirements emerged from these results [102]. Steric factors appear to dictate binding; the polysubstituted anthracenes such as the dibenzanthracenes, pyrene, chrysene and other polycyclic aromatic compounds were bound. Similarly, dibenz(a,j)acridine was bound whereas acridine and acridine orange were not bound. It is well known that parasites and heavy metals caused marked changes in GST activity, and GSH levels [103105]. Inducers of GSTs are generally considered as protective compounds against cancer, acting blocking agents. The human diet contains many compounds that inhibit various steps of the carcinogenic process [106]. The coffee specific diterpenses cafestol and kahweol have been reported to be anticarcinogenic in several animal models. It has been postulated that this activity

17 may be related to their ability to induction of glutathione S-transferase π class [107]. GSTπ was induced in the stomach by coumarin and α-angelicalactone and in the pancrease by flavone [108]. Several dietary compounds have been demonstrated to reduce gastrointestinal cancer rates in both human and animals. For example, sulforaphane, indole-3-carbinol, D-limonene and relafen induced GSTα levels in small intestine and livers, GSTµ levels in stomach and small intestine, GSTπ levels in stomach and small and large intestine [109]. Aqueous extracts of either green or black tea were administered to rats as the sole drinking fluid for 4 weeks. Hepatic GST activity and UDP-Glucuronyl transferase were induced [109]. On the other hand, many different chemical compounds and dietary items were found to inhibit the expression and the activity of GST isozymes. Trivalent antimony was a potent inhibitor of glutathione S-transferases from human erythrocytes [110]. Based on this inhibition characteristics and the preferential accumulation of trivalent antimony in mammalian erythrocytes, for example, during therapeutic treatment of Leishmaniasis, antimony levels in erythrocytes may be high enough to depress GST activity, which might compromise the ability of erythrocytes to detoxify electrophilic xenobiotics [110]. Animals treated with acriflavine, a protein kinase c inhibitor, and allyl disulfide showed complete blockage of GST gene expression as early as 12 h of treatment [111]. Analogues of glutathione preferentially inhibit GSTα, have less effect on µ isozymes, and finally have little effect on rat Ф and π isozymes [112114]. Tissues highly expressing activities of GSTs are protected from cytotoxic damage elicited by electrophiles for which the conjugation to GSH is readily catalyzed. Purified GST Mu and Theta have high activities against polycyclic aromatic hydrocarbon epoxide metabolites that may be generated from constituents of tobacco smoke [115-117]. Additionally, over-expression of Alpha GSTs in cells enhances their protection from necrotic toxicity produced by cyclophosphamide or diethylnitrosamine [118,119]. Elevated levels of Alpha, Mu and Pi GSTs have been associated with protection of tissues from cytotoxicity produced by acetaminophen, carbon tetrachloride and aflatoxin B1 [120,121].

18 The GSTs also have been shown to detoxify cyclophosphamide [122], nitroglycerin [123], L-phenylalanine mustard [124], and chlorambucil [125]. Therefore, measurement of the concentrations and activities of GSTs, and their modulation in tissues that are subjected to physiological or environmental stimuli, may provide researchers with an important tool in monitoring the detoxication potential of cellular systems. For example, the GST isozymes are inducible to varying degrees by a number of xenobiotics, including phenobarbital, 3methylcholanthrene, phenolic antioxidants, azo dyes, flavonoids, TCDD, diphenols, thiocarbamates, 1,2-dithiol-3-thiones, isothiocyanates, cinnamates, coumarins, and -naphthoflavone in mice [126-130]. Thus, measurement of GST isozyme profiles in preclinical rodent model scan rapidly provide clues to potential drug interactions or modification in the detoxication of environmental contaminants. In addition, altered expression of the GSTs has been measured in mammalian tissues and in human serum, plasma and urine is observed. For example, increased levels of Alpha GST isozymes in plasma or serum have been related to acute or chronic liver disease [131]. Similarly, the levels of urinary Alpha GSTs relate to renal damage [132-134]. Increased GST plasma levels has been observed following hepatocellular damage elicited by acetaminophen [135], alcohol [136], or halothane [137]. The Alpha GST serum half life is short (less than 1 hr), and because of its high concentration in liver, its broad lobular distribution and rapid release from tissues, the Alpha GSTs are considered to be more sensitive indicators of hepatocellular injury than the standard serum transaminases. They may also give important information about the course of liver disease and their subsequent management. Measurement of Alpha GSTs in serum and urine were found to be better indicators than the serum transaminases in early prediction of rejection following liver or kidney transplantation, respectively [138]. The Alpha GSTs were also a better measure of successful intervention to alleviate rejection following transplantation, thereby demonstrating the important clinical use of monitoring GST levels in biological tissues [138]. Similarly, the plasma levels of Alpha GSTs were correlated with

19 onset and therapeutic suppression of chronic or acute hepatitis infection [139]. Elevated Alpha GSTs have been found in plasma of patients with hepatocellular damage caused by hypoglycemia [140], birth asphyxia [141], or autoimmune chronic hepatitis [142]. Approximately half of the population lacks GST Mu expression, termed the GST M1 null genotype [143-144]. Epidemiological evidence supports the conclusions that persons possessing this genotype are predisposed to a number of cancers including breast, prostate, liver and colon cancers [145-147]. Measurement of a lack of GST Mu class isozymes in human polymorphonuclear cells has been associated with increased predisposition to liver and colon cancers [144,147]. GST Pi is an acidic isozyme which is expressed in high concentrations in chemically-induced preneoplastic rat hepatocyte nodules [148-150] and in rat primary hepatomas [152]. The human ortholog, GST P1-1 [152], is also expressed in many human tumors, including colonic [153], hepatic [154], prostate [155], lung [156], breast, ovarian, gastric, and

renal

cell

[157,158]

carcinomas,

as

well

as

melanoma,

uterine

adenocarcinoma, and mesothelioma [157,159]. In contrast, GST P7-7 is present in only trace amounts in normal rat liver, and is expressed primarily in the bile duct epithelium. GST P7-7 has also been induced de novo in rat hepatic tissue by treatment with trans-stilbene oxide [160], lead nitrate [161], and pyrrole [162]. Elevated expression of GST P1-1 has been found in a number of human multidrug resistant cell lines and tumors [163-165] and GST P1-1 has been associated

with

the

resistance

of

these

cells

and

tumors

towards

chemotherapeutic agents. Therefore, measurement of GST Alpha, Mu and Pi class expression may have clinical benefit for monitoring therapeutic progression of

cancerous

disease

or

identification

of

populations

susceptible

to

chemotherapeutic interventions. 1.2.2 Glutathione and other amino acids conjugation Glutathione (GSH) is presented in most plant and animal tissues from which the human diet is derived. GSH can function as an antioxidant in the ingest, can maintain ascorbate in a reduced and functional form, can directly react with and inactivate toxic electrophiles in the diet, and can be broken down

20 to yield cysteine [166]. Studies have shown that dietary GSH enhances metabolic clearance of dietary peroxidized lipids and decreases their net absorption [167] and that consumption of food high in GSH content is associated with about a 50% reduction in risk of oral and pharyngeal cancer [168]. Therefore, an improved understanding of the distribution of GSH in foods and the factors affecting GSH absorption and distribution can be expected to provide the basis for improved food selection and preparation to reduce risk of chronic diseases. Glutathione (GSH) one of the most abundant intracellular thiols, aids in protection of cells from the lethal effects of toxic and carcinogenic compounds [169,170]. It has shown to be an important determinant of cellular sensitivity to a wide variety of drugs and other cytotoxic compounds [171-173]. Both oxidationreduction reactions may eventually result in GSH depletion even though the former type of reaction primarily leads to glutathione oxidation and should be effectively compensated by glutathione reductase activity [174]. GSH depletion is often associated with cytotoxicity, and there are some indications that conjugation reactions can be more detrimental to the cell than redox cycles [175, 176]. It thus seems possible that GSH depletion may promote tumor development through a mechanism that involves cytotoxicity and other different ways [177, 178]. In addition, preventing of cellular glutathione induction by buthionine sulfoximine increased the cytotoxicity, the frequency of sister chromatid exchange and prolonged the cell cycle. The cytosolic glutathione-Stransferase and GSH play an important role in the detoxification of many environmental chemicals including mutagens and carcinogens [179-183]. Their main function is the conjugation of GSH to a variety of electrophilic compounds. Previous studies have demonstrated that GSH and GST reduced the covalent binding of epoxides of well known chemical carcinogens, e.g. aflatoxin B1 and benzo [a]pyrene, with DNA [184-188]. It has been reported that such DNAbinding was found to be effective in decreasing the hepatocarcinogenesis caused by these compounds [189,190]. Glutathione conjugates are not excreted per se but rather undergo further enzymatic modification of the peptide moiety resulting in the urinary or biliary excretion of cysteinyl-sulfur substituted, n-acylcysteines, more commonly

21 referred to mercapturic acids [178]. Mercapturic acid formation is initiated by glutathionase conjugation followed by removal of the glutamate moiety by glutathionase and subsequent removal of glycine by a peptidase enzyme, the latter two enzymes being present in both liver, gastrointestinal tract and kidney. In the final step, the amino group of cysteine is acetylated by a hepatic nacetylase resulting in formation of the mercapturic acid derivative. This latter acetylation reaction is reversible and deacetylases can reform the amino metabolite [191]. It is clear that glutathione conjugation serves as a protective mechanism whereby potentially toxic, electrophilic metabolites, are detoxified either as glutathione conjugates or mercapturic acids [189-191]. However, it is becoming increasingly recognized that glutathione conjugation is not exclusively a detoxification reaction and that certain xenobiotics are toxicologically activated by this conjugation route, either as such or as a result of further processing of the glutathione conjugate. Therefore, cellular levels of glutathione are an important determinant of xenobiotic toxicity [175,176,190]. 1.2.3 UDP-Glucuronsyltransferase The human UDP-glucuronosyltransferase (UGTs) appear to consist of two multigene superfamilies, designated UGT1A and UGT2A/UGT2B [192]. These transferases exhibit distinct but overlapping substrate specificity and are known to catalyze the glucuronidation of a variety of phenols, arylamines, steroids, and bilrubin.

Only a few years ago it was generally accepted, that phase II

metabolites of drugs, such as O- or N-glucuronides, are rapidly excreted following their formation in the body and that these metabolites are not active or reactive [193,194]. The resulting acyl glucuronides were found to be electrophiles, reacting with sulfhydryl group. Conjugation with D-glucuronic acid represents the major route for elimination and detoxification of drugs and endogenous compounds possessing a carboxylic acid function [195,196]. Carcinogens and their reactive metabolites may also be metabolized by alternative routes (e.g.; by conjugation or by hydrolyze activities) to relatively harmless intermediates that can be eliminated from the body, although in some

22 cases these may be further transformed into highly reactive chemical species [197,198]. As with the enzymes of carcinogen activation those responsible for inactivation may play pivotal roles in the determination of the target tissue specificity of a particular carcinogen. It has also been suggested that the sulfation of certain chemical carcinogens could lead to more toxic conjugates, which can cause cell necrosis [199-201]. A major fraction of the N-hydroxy derivatives of aromatic amines is converted to the glucuronide, which is then excreted in the bile and urine [199]. However, the glucuronide also may be hydrolyzed to release the free N-hydroxy arylamine, which is a potent electrophile [202]. 1.2.4 Sulfate conjugation Many drugs are oxidized to a variety of phenols, alcohols or hydroxylamines which can then serve as excellent substrates for subsequence sulfates conjugation, forming the readily excretable sulfate esters. However, in organic sulfate is relatively inert and must first be activated by ATP [203]. For phenolic metabolites, the key enzyme in this sequence is sulfotransferase. The sulfotransferase enzymes are soluble enzymes found in many tissues including liver, kidney, gut and platelets and catalyze the sulfation of drugs such as paracetamol, isoprenaline and salicylamide and many steroids. It appears that the sulphotransferase exist in multiple enzyme forms with the steroid sulfating enzymes being distinct from the sulfotransferase responsible for drug conjugation reaction [204-206]. It should be emphasized that sulfation conjugation reactions are not as widespread or as of quantitative importance as glucuronide conjugation reactions, due in part to the limited bioavailability of inorganic sulfate and hence PAPS. This is particularly true when a drug is actively metabolized to phenolic products or when high body burdens of phenolic drugs are reached (for example, in overdose), resulting in effective saturation of this metabolic pathway [207]. 1.2.5 N-Acetyltransferase Xenobiotic-metabolizing enzymes constitute an important line of defense against a variety of carcinogens. The wide variation in carcinogen metabolism in human has long been regarded as an important determinant of individual susceptibility to chemical carcinogenesis. The carcinogenicity of several

23 arylamines and amides for the liver and the urinary bladder has prompted studies on the metabolism of these carcinogens and on their reactive metabolites, which catalyzed by mixed-function oxidases in the hepatic endoplasmic reticulum [208]. Distinct genes, designated NAT1 and NAT2 code N-acetyltransferase activities in human. NAT1 activity appears to be monomorphically distributed in human tissues, while the latter exhibits a polymorphism that allows the detection of phenotypically slow and rapid metabolizers [209]. The NAT2 polymorphism can have a significant effect on individual susceptibility to aromatic amine-induced cancers. N-Acetylation of arylamines represents a competing pathway for Noxidation, a necessary metabolic activation-step occurring in the liver. The unconjugated N-hydroxy metabolites can then enter the circulation, and be transported to the urinary bladder lumen, where reabsorption and covalent binding to urothelial DNA can occur [210]. Low activity of arylamine Nacetyltransferase 2 (slow NAT2 acetylator) was consistently associated with urinary bladder cancer risk [211]. Also, NAT2 plays a significant role in risk of lung cancer among non-smokers Chinese women [212]. Some studies have shown that meat consumption is associated with breast cancer risk. Several heterocyclic amines, formed in cooking of meats, are mammary carcinogens in laboratory models [213,214]. Heterocyclic amines are activated and rapid NAT2 activity may increase risk associated with these compounds [213,214], Since NAT2 increased the binding of quinoline, a heterocyclic amine derivative, with DNA by 12 fold, while NAT1 increased such binding by 4 fold [214].

1.2.6 Epoxide hydrolase Epoxide hydrolase is widely distributed throughout the animal kingdom, including human. In the rat, it almost occurs with highest activity being found in the liver and smaller amount being found in kidney, lung, and adrenal gland. In liver, epoxide hydrolase occurs predominantly in the endoplasmic reticulum fraction, nuclear membranes, and the cytosol [215]. The soluble epoxide hydrolase (sEH) plays a significant role in the biosynthesis of inflammation mediators as well as xenobiotic transformations [216]. Epoxide hydrolase is induced by most of the

24 xenobiotic

inducers

of

the

mixed-function

oxidase

system.

Decamethylcyclopentasiloxane (D5) is a cyclic siloxane with a wide range of commercial applications. Liver microsomal epoxide hydrolase activity and immunoreactive protein increased 1.7- and 1.4-fold, respectively, in the D5exposed group [217]. The naturally occurring organosulfur compounds (OSCs) diallyl sulfide (DAS), diallyl disulfide (DADS) and dipropyl sulfide (DPS) were studied with respect to their effects on microsomal epoxide hydrolase (mEH). DADS increased the mEH levels in the liver, intestine, and kidney, while DAS and DPS moderately induced mEH level in the liver [218]. A series of organosulfur compounds were developed as chemopreventive active compounds against hepatotoxicity and carcinogenisity of aflatoxin B1 [219]. The mechanism of chemoprotection involved inhibition of the P450-mediated metabolic activation of chemical carcinogens and enhancement of electrophilic detoxification through induction of epoxide hydrolase, which would facilitate the clearance of activated metabolites through conjugation reaction [219]. Although hydrolysis in most of the cases result in detoxication, in some cases hydrolysis may lead to activated molecules that may attack macromolecules (proteins, RNAs, DNAs), resulting in toxicity [220]. On the other hand, the discovery of substituted ureas and carbamates as potent inhibitors were found to enhance cytotoxicity of transstilbene oxide, which is active as the epoxide, but reduce cytotoxicity of leukotoxin, which is activated by epoxide hydrolase to its toxic diol [216]. They also reduce toxicity of leukotoxin in vivo in mice and prevent symptoms suggestive of acute respiratory distress syndrome [216]. Genetic polymorphisms of biotransformation enzymes are in a number of cases a major factor involved in the interindividual variability in xenobiotic metabolism and toxicity [221]. This may lead to interindividual variability in efficacy of drugs and disease susceptibility. Polymorphisms in exons 3 and 4 of microsomal epoxide hydrolase in 101 patients with colon cancer and compared the results with 203 control samples. The frequency of the exon 3 T to C mutation was higher in cancer patients than in control [221]. This sequence alteration changes tyrosine residue 113 to histidine and is associated with lower enzyme activity when expressed in vitro.

25 Therefore, slow epoxide hydrolase activity may be a risk factor for colon cancer [221]. Conclusions: The GSTs constitute a primary pathway for the detoxication of cellular electrophiles generated endogenously or form xenobiotic administration or exposure. Preclinical studies have correlated enhanced metabolism of electrophiles with increased levels of GST isozymes within various tissues. Expression of GSTs within individuals can then provide an indicator the metabolic potential of their tissues and possible deficiencies in their susceptibility to dietary or environmental carcinogens. GSTs are over-expressed in certain tumor types, and measurement of GSTs in serum or in pathological specimens can be used to follow the course of disease and the success of intervention. Some isozymes of glutathione S-transferase could be used as a biomarker for detection of cancer in different organs.

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