Activity Of Carcinogen-metabolizing Enzymes Research Articles

N-nitrosodimethylamine induced changes in the activities of carcinogen-metabolizing enzymes in the liver of male mice: role of glutathione and gossypol as antioxidants

The importance of nutrition in protecting living organisms from the toxic effects of environmental carcinogens has attracted the attention of many researchers. In order to exert their toxic and/or carcinogenic effects, most carcinogens need to be activated primarily by phase I drug-metabolizing enzymes including; cytochrome P450, cytochrome b5, arylhydrocarbon hydroxylase (AHH), N-nitroso-dimethylamine N-demethylase I (NDMA-N-dI), NADPH-cytochrome c reductase and the expression of cytochrome P450 2E1. Changes in the activity of these enzymes were determined in livers of male mice pretreated with either glutathione (GSH) or gossypol as antioxidants, for seven consecutive days before administration of NDMNA as single dose for two hours before decapitation of mice. The total hepatic content of cytochrome P450 and AHH activity were induced, whereas the activity of NDMA-N-dI and expression of cytochrome P450 2E1 were reduced after treatment of mice with NDMA as a single dose. On the other hand, preteatments of mice with either gossypol or GSH for seven consecutive days decreased the total cytochrome P450 content as well as AHH activity whereas the activity of NDMA-N-dI and the expression of cytochrome P450 2E1 increased. Treatment of mice with NDMA only induced the total hepatic content of cytochrome P450 and AHH activity. Interestingly pretreatment of mice with GSH for seven consecutive days before injection of NDMA was found to restore the induced cytochrome P450 and AHH activity caused by NDMA to their normal levels. However, pretreatment of mice with gossypol prior to administration of NDMA did not restore such activities to their normal levels. Treatment of mice with either GSH or gossypol induced the expression of cytochrome P450 2E1. Interestingly, gossypol/NDMA treated mice restored the induced cytochrome P450 2E1 expression, caused by gossypol, to its normal level. However, GSH/NDMA-treated mice did not restore the induced level of cytochrome P450 2E1 caused by GSH, to its normal level. It is concluded that, pretreatment of mice with GSH and gossypol prior to administration of NDMA induced the expression of cytochrome P450 2E1. Therefore, GSH and gossypol, could induce the toxicity and/or carcinogenicity of N-nitrosamines whereas they might protect the liver and probably other organs from the toxic and carcinogenic effects of other carcinogens such as polycyclic aromatic hydrocarbons, e.g. benzo[a]pyrene, through inhibition of total cytochrome P450 content and AHH activity. Key words: Aryl hydrocarbon hydroxylase, Cytochrome P450 2E1, N-nitrosodimethylamine N- demethylase, glutathione, antioxidants.

Anthocyanin-Rich Grape Extract Blocks Breast Cell DNA Damage

Anthocyanins, belonging to the flavonoid family of phytochemicals, have received attention as agents that may have potential in preventing chronic diseases such as cardiovascular diseases and certain cancers. In the present study, an anthocyanin-rich extract from Concord grapes [referred to as Concord grape extract (CGE)] and the anthocyanin delphinidin were evaluated for their capacity to inhibit DNA adduct formation due to the environmental carcinogen benzo[a]pyrene (BP) in MCF-10F cells, a noncancerous, immortalized human breast epithelial cell line. CGE at 10 and 20 microg/mL and delphinidin at 0.6 microM concentrations significantly inhibited BP-DNA adduct formation. This was associated with a significant increase in activities of the phase II detoxification enzymes glutathione S-transferase and NAD(P)H:quinone reductase 1. In addition, these grape components also suppressed reactive oxygen species (ROS) formation, but did not induce antioxidant response element-dependent transcription. Taken together, these data suggest that CGE and a component grape anthocyanin have breast cancer chemopreventive potential due in part to their capacity to block carcinogen-DNA adduct formation, modulate activities of carcinogen-metabolizing enzymes, and suppress ROS in these noncancerous human breast cells.

Modulation of xenobiotic-metabolizing enzymes and redox status during chemoprevention of hamster buccal carcinogenesis by bovine lactoferrin

Objective Chemoprevention by dietary constituents has emerged as a novel approach to control oral cancer incidence. We therefore evaluated the chemopreventive efficacy of bovine milk lactoferrin (bLF) on 7,12-dimethylbenz[a]anthracene (DMBA)-induced hamster buccal pouch (HBP) carcinogenesis. Methods Hamsters were divided into four groups. The right buccal pouches of animals in groups 1 and 2 were painted with 0.5% DMBA three times a week for 14 wk. Animals in group 2, received in addition, basal diet containing 0.2% bLF. Group 3 animals were given 0.2% bLF alone. Group 4 animals served as control. The status of carcinogen-metabolizing enzymes, the extent of lipid peroxidation and glutathione-dependent antioxidants in the buccal pouch and liver as well as bone marrow micronuclei incidence were used as biomarkers. Results All the hamsters painted with DMBA alone for 14 wk, developed HBP carcinomas that showed diminished lipid peroxidation and increased activities of carcinogen-metabolizing enzymes and antioxidants with enhanced bone marrow micronuclei. In the liver of tumor bearing animals, the increase in phase I enzymes and lipid peroxidation was accompanied by reduced activities of antioxidant and phase II detoxification enzymes. Administration of bLF decreased the incidence of DMBA-induced micronuclei and HBP carcinomas by decreasing phase I enzymes, modulating lipid peroxidation and enhancing antioxidant and phase II enzyme activities. Conclusion The chemopreventive effects of bLF is mediated by reducing DMBA-induced genotoxicity and modulating carcinogen-metabolizing enzymes and oxidant-antioxidant profile in the target organ as well as in the liver.

Glutathione Alleviates the Influence of N-Nitrosamines on the Activity of Carcinogen-Metabolizing Enzymes in the Liver of Male Mice

Most xenobiotics and carcinogens are metabolized primarily by the hepatic drug-metabolizing enzymes including cytochrome P450, cytochrome b5, arylhydrocarbon hydroxylase (AHH), and dimethylnitrosamine N-demethylase I (DMN-dI) as phase I drug-metabolizing enzymes. Therefore, changes in the activities of the above mentioned enzymes as well as free radical levels measured as thiobarbituric acid reactive substances [TBARS] were determined in the livers of sixty mice pretreated with glutathione [100 mg/kg], as antioxidant, before intraperitoneal administration of dimethylnitrosamine [DMN] [10 mg/kg], diethylnitosamine [DEN] [10 mg/kg], or methylpropylnitrosamine [MPN] [10 mg/kg] for seven consecutive days. Ten control animals received distilled water as vehicle for comparison with treated animals. The hepatic content of cytochrome P450 induced after treatment of mice with DMN, DEN and MPN while DMN, DEN, or MPN increased AHH activity. Also, cytochrome b5 content and DMN-N-dI induced after treatment with DMN or DEN. Interestingly, glutathione pretreatment restored the activities of the previously mentioned enzymes caused by DMN or DEN. On the other hand, DMN, DEN, and MPN depleted the levels of glutathione, and increased the levels of free radicals [TBARS]. DMN and DEN increased the activity of GST. Interestingly, pretreatment of mice with glutathione before administration of DMN, or DEN was found to restore the levels of TBARS and GSH to their normal levels. In addition, GSH pretreatments induced the activity of GST above the normal levels after administration of DMN or MPN except DEN did not change such activity. It was concluded that N-nitrosamines enhanced the activity of phase I & II carcinogen-metabolizing enzymes as well as increased the production of free radicals. The mechanism by which GSH restores the activity of carcinogen-metabolizing enzymes or free radical production to their normal levels is possibly due to induction of GST activity and/or a direct binding to the xenobiotic molecules.

Narcotic drugs change the expression of cytochrome P450 2E1 and 2C6 and other activities of carcinogen-metabolizing enzymes in the liver of male mice.

Drug-metabolizing enzymes play a great role in the bioactivation and also detoxification of zenobiotics and carcinogens such as N-nitrosamines and polycyclic aromatic hydrocarbons (PAHs). Therefore, the present study was undertaken to investigate the effect of narcotic drugs such as cannabis (hashish) and diacetylmorphine (heroin) on the activity of N-nitrosodimethylamine N-demethylase I [NDMA-dI], arylhydrocarbon [benzo(a)pyerne] hydroxylase [AHH], cytochrome P450 (CYP), cytochrome b(5), NADPH-cytochrome c reductase, glutathione-S-transferase, and levels of glutathione and thiobarbituric acid-reactive substances (TBARS). In addition, the present study showed the influence of hashish and heroin after single (24 h) and repeated-dose treatments (4 consecutive days) on the expression of cytochrome P450 2E1 (CYP 2E1) and cytochrome P450 2C6 (CYP 2C6). The expression of CYP 2E1 was slightly induced after single-dose and markedly induced after repeated dose-treatments of mice with hashish (10 mg kg(-1) body weight). Contrarily, heroin markedly induced the expression of CYP 2C6 after single-dose and potentially reduced this expression after repeated-dose treatments. It is believed that N-nitrosamines are activated principally by CYP 2E1 and in support of this, the activity of NDMA-dI was found to be increased after single- and repeated-dose treatments of mice with hashish by 23 and 41%, respectively. In addition, single- and repeated-dose treatments of mice with hashish increased: (1) the total hepatic content of CYP by 112 and 206%, respectively; (2) AHH activity by 110 and 165%, respectively; (3) NADPH-cytochrome c reductase activity by 21 and 98%, respectively; (4) and glutathione level by 81 and 173%, respectively. Also, single-dose treatments of mice with heroin increased the total hepatic content of CYP, AHH, NADPH-cytochrome c reductase, and glutathione level by 126, 72, 39, 205%, respectively. However, repeated dose-treatments of mice with heroin did not change such activities except cytochrome c reductase activity increased by 20%. Interestingly, the level of free radicals, TBARS, was potentially decreased after single or repeated-dose treatments with either hashish or heroin. It is clear from this study that the effects of hashish are different from those of heroin on the above mentioned enzymes particularly after repeated dose treatments. It is concluded that hashish induced the expression of CYP 2E1 and other carcinogen-metabolizing enzymes activities, and this induction could potentiate the deleterious effects of N-nitrosamines and aromatic hydrocarbons, e.g. benzo(a)pyrene, upon the liver and probably other organs. Such alterations may also change the therapeutic actions of other drugs, which are primarily metabolized by the P450 system, when administered to peoples using hashish or heroin.

Changes in the expression of cytochrome P450 2E1 and the activity of carcinogen -metabolizing enzymes in Schistosoma haematobium-infected human bladder tissues

The bioactivation of N-nitrosamines and polycyclic aromatic hydrocarbons (PAHs) is mediated primarily by the mixed-function oxidase system, which includes dimethylnitrosamine N-demethylase I, arylhydrocarbon [benzo( a)pyerne] hydroxylase, cytochrome P450, cytochrome b 5, and ethoxycoumarin deethylase. Most of carcinogens and xenobiotics are conjugated and detoxified by phase II drug-metabolizing enzymes such as glutathione S-transferase. The present study showed the influence of Schistosoma haematobium on the activity of the above-mentioned enzymes in 13 schistosome-infected human bladder tissues compared with those of 15 schistosome-free samples. The contents of cytochrome P450 and cytochrome b 5 were increased in the bladder tissues by 48 and 69%, respectively. Moreover, the activities of dimethylnitrosamine N-demethylase I and arylhydrocarbon hydroxylase, ethoxycoumarin O-deethylase, ethoxyresourfin O-deethylase, and pentoxyresorufin O-pentoxyresorufin were increased by 75, 159, 49, 63 and 44%, respectively. The signal intensity for cytochrome P450 2E1 was greatly increased over the control. Also, the activity of glutathione S-transferase was increased by 89%. On the other hand, the activity of glutathione reductase and the level of reduced glutathione were decreased by 40 and 57%, respectively. Interestingly, the level of free radical, thiobarbituric acid reactive substance, was increased in the schistosome-infected human bladder tissues by 125%. The present study clearly demonstrated that S. haematobium changes the activity of carcinogen-metabolizing enzymes. We conclude that S. haematobium could enhance the carcinogenicity of polycyclic aromatic hydrocarbons (e.g. benzo(a)pyrene) and N-nitrosamines (e.g. dimethylnitrosamine) through induction of their corresponding bioactivating enzymes in human bladder tissues.

Alcohol and prostate cancer.

This paper reviews epidemiologic evidence that alcohol consumption is related to risk for prostate cancer. Significantly increased risks of prostrate cancer have been noted among alcohol abusers. A pooled analysis based on linear dose-response model suggests that daily consumption of four alcoholic drinks per day is associated with about a 21% increased risk for prostate cancer among heavy drinkers. In addition 17.4% of prostate cancer among heavy drinkers is attributable to alcohol consumption. Acute and chronic alcohol consumption tends to result in greater serum estrogen and lower androgens in both men and women. Moreover alcohol tends to improve the permeability of cell membranes to carcinogens alter carcinogen metabolizing enzyme activity and inhibit deoxyribonucleic acid repair. Overall the studies have noted that risks of prostate cancer are associated with the levels of exposure to alcohol irrespective of the type of beverage consumed. However there are limitations noted in the studies that could affect the findings. Hence cohort studies with quantitative assessment and re-evaluation of findings are needed to address such limitations.

Relationship between schistosomiasis and bladder cancer.

Carcinoma of the urinary bladder is the most common malignancy in the Middle East and parts of Africa where schistosomiasis is a widespread problem. Much evidence supports the association between schistosomiasis and bladder cancer: this includes the geographical correlation between the two conditions, the distinctive patterns of gender and age at diagnosis, the clinicopathological identity of schistosome-associated bladder cancer, and extensive evidence in experimentally infected animals. Multiple factors have been suggested as causative agents in schistosome-associated bladder carcinogenesis. Of these, N-nitroso compounds appear to be of particular importance since they were found at high levels in the urine of patients with schistosomiasis-associated bladder cancer. Various strains of bacteria that can mediate nitrosation reactions leading to the formation of N-nitrosamines have been identified in the urine of subjects with schistosomiasis at higher intensities of infection than in normal subjects. In experimental schistosomiasis, the activities of carcinogen-metabolizing enzymes are increased soon after infection but are reduced again during the later chronic stages of the disease. Not only could this prolong the period of exposure to activated N-nitrosamines, but also inflammatory cells, stimulated as a result of the infection, may induce the endogenous synthesis of N-nitrosamines as well as generating oxygen radicals. Higher than normal levels of host cell DNA damage are therefore anticipated, and they have indeed been observed in the case of alkylation damage, together with an inefficiency in the capacity of relevant enzymes to repair this damaged DNA. In experimental schistosomiasis, it was also found that endogenous levels of host cell DNA damage were related to the intensity of infection. All of these factors could contribute to an increased risk of bladder cancer in patients with schistosomiasis, and in particular, the gene changes observed may have potential for use as biomarkers in the early detection of bladder cancer that may assist in alleviating the problem.

β-Carotene and Aryl Hydrocarbon Hydroxylase in the Rat: An Effect of β-Carotene Independent of Vitamin A Activity

Animal models demonstrate a cancer-protective effect of vitamin A. However, human epidemiologic studies correlate the intake of the precursor, beta-carotene, rather than active vitamin A, to a reduced risk for certain cancers. This suggests that beta-carotene may have cancer-protective properties independent of its vitamin A activity. In the present rat study, effects of beta-carotene or active vitamin A on carcinogen metabolizing enzyme activity were evaluated. The activity of intestinal aryl hydrocarbon hydroxylase (AHH, EC 1.14.14.1) was higher in rats fed a purified diet supplemented with beta-carotene than in rats fed the control diet containing adequate vitamin A as retinyl palmitate (165 +/- 30 vs. 90 +/- 18 pmol/min x mg), P less than (0.05). Supplementing the control diet with retinyl acetate had no effect. This AHH-enhancing effect of beta-carotene on the activity of the intestinal mucosal enzyme was not seen on the hepatic enzyme, which is consistent with the nearly complete conversion of beta-carotene to vitamin A prior to reaching the liver. These results demonstrate an effect of beta-carotene on carcinogen metabolism which is independent of its vitamin A activity. This may help explain human epidemiologic data, and may lead to further work which would allow for prudent dietary recommendations toward a reduction in cancer risk.

Fetal and adult human liver differ markedly in the fluidity and lipid composition of their microsomal membranes.

The fluidity and lipid composition of the human hepatic microsomal membrane were studied in 11 livers from 16- to 21-week-old fetuses and in 5 adult livers, and compared with those of fetal and adult rat liver microsomes. Membrane fluidity was analyzed by measurement of fluorescence polarization using the fluorophore 1,6-diphenyl-1,3,5-hexatriene. The lipid apparent microviscosity (eta) of human fetal liver microsomes was 2.17 +/- 0.13 poise, as compared with 1.08 +/- 0.08 poise in adult liver (p less than 0.001). Similar differences in fluidity were found between fetal and adult rat liver microsomes. The more "fluid" adult microsomes had higher phospholipid/cholesterol and phosphatidylcholine/sphingomyelin molar ratios than those of the more "rigid" fetal microsomes. The degree of unsaturation of the adult microsomal lipids was much higher than that of the fetal lipids. The ratios of unsaturated/saturated fatty acids in microsomal lipids highly correlated with the eta values obtained for the combined group of fetal and adult human livers, suggesting that the developmental increase in degree of unsaturation of the microsomal lipids is a major determinant of the increased fluidity of adult as compared with fetal liver microsomes. These differences in fluidity and lipid composition between fetal and adult human liver microsomes may be a critical factor in the regulation of hepatic microsomal drug and carcinogen metabolizing enzyme activity, and could so determine the extent of toxicity and teratogenicity of drugs and/or their metabolites in the developing human fetus.

Effects of Dietary Lipid Saturation on Prolactin Secretion, Carcinogen Metabolism and Mammary Carcinogenesis in Rats

Isoenergetic diets containing 20% corn oil, 20% beef tallow, or an equal mixture of 10% corn oil and 10% beef tallow (mixed fat) were fed to 30 rats per diet for 28 weeks following weaning. DMBA [7,12-dimethylbenz(a)anthracene] was administered (1.75 mg/100 g body weight) in a single oral dose after 4 weeks of feeding. After 28 weeks, 70% of the rats fed corn oil had mammary tumors versus 47% for mixed fat and 30% for tallow. Diet had no effect on the number of tumors per tumor-bearing rat or the proportion of tumors that were adenocarcinomas. Other rats assigned to each of the three diets were killed at the time corresponding to DMBA administration for examination of hepatic mixed-function oxidase activity. NADPH cytochrome c reductase activity and cytochrome P-450 content were higher in rats fed corn oil or mixed fat rather than tallow. However, no significant differences in aryl hydrocarbon hydroxylase, glutathione transferase, and uridine-diphosphoglucuronide transferase activities were observed. The effects of dietary fat saturation on enzyme activity failed to show a clear association with DMBA carcinogenesis. In other rats assigned to the three dietary treatments for 4 or 16 weeks, lipid saturation did not change serum prolactin (PRL) concentrations during diestrus or proestrus. PRL secretion was examined following a provocative stimulus (perphenazine) in rats fed the experimental diets for 4 or 10–22 weeks. Although perphenazine increased serum PRL and depleted the pituitary of PRL, differences in dietary lipid saturation caused no significant changes in these indices. These data show that the incidence of mammary tumors in rats fed high fat diets (20% by weight) was greater in those fed corn oil compared to beef tallow. The effect of dietary lipid source on tumorigenesis was not associated with changes in carcinogen-metabolizing enzyme activity or PRL secretion.

Carcinogenesis and aging: III. The role of age in initiation and promotion of carcinogenesis

The problem of an interrelation between carcinogenesis and aging is discussed from the point of view of the two-stage model of carcinogenesis. Data on the carcinogenic effect of subcutaneously administered benzo(a)pyrene (BP), intravaginally applied 7, 12-dimethylbenz(a)anthracene (DMBA), and intravenously injected N-nitrosomethylurea (NMU) to young and old animals are presented. Both the obtained results and data from available literature show the absence of a uniform effect of age on carcinogenesis. Age-associated dynamics of carcinogen-metabolizing enzyme activity, accuracy in DNA repair, and proliferative activity of target and non-target tissues have been discussed as the determinant factors for observed differences. Data on the growth rate of some transplantable tumors in young and old animals are also presented. As a rule, the above tumors grow more rapidly in old animals in comparison with young ones. The role of age-associated hormonal, metabolic, and immunological changes in tumor promotion is discussed. The similarity is stressed of a number of changes occurring at organism, tissue, and cellular levels of integration both during natural aging and carcinogenesis. Some results of study on effects of geroprotectors on spontaneous carcinogenesis are summarized. The geroprotectors, which were shown to delay the realization of the genetic programme of aging and age pathology development, prolong lifespan and inhibit carcinogenesis more effectively than those influencing the initiatial stage of spontaneous carcinogenesis. Finally, the integral scheme of events taking place in cell, tissue, and organism during carcinogenesis is presented.