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ABSTRACT: 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is the most abundant heterocyclic aromatic amine found in cooked meat. It is metabolically activated by the human cytochrome P450 enzymes to form the carcinogenic metabolite N2-OH-PhIP. PhIP has been found to induce tumors in rats and is a suspected human carcinogen. In the present work, we have developed and validated a liquid chromatography-electrospray ionization/ion trap mass spectrometry (LC-ESI/ITMS) method for the determination of PhIP and N2-OH-PhIP. PhIP was incubated with microsomes prepared from the human liver; the PhIP and N2-OH-PhIP formed were isolated from the biomatrices by solid-phase extraction using C18 cartridges, with recoveries greater than 86%. Subsequently, the products were separated on a microbore reversed-phase C18 liquid chromatograph coupled to an ESI-ITMS. The ESI interface and the ITMS were tuned for various parameters, and data acquisition was performed in selective ion monitoring mode. The detection limit of PhIP and N2-OH-PhIP was 1 and 10 pg, respectively. The method is highly sensitive and selective, has simple sample preparation protocols, and should be applicable to the study of the metabolic activation of PhIP in various human tissues.
Analytical Biochemistry 12/2001; 298(2):306-13. · 3.00 Impact Factor
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ABSTRACT: To reduce the production of carbon monoxide and other pollutants in motor vehicle exhaust, methyl tert-butyl ether (MTBE*), ethyl tert-butyl ether (ETBE), and tert-amyl methyl ether (TAME) are added to gasoline as oxygenates for more complete combustion. Among them, MTBE is the most widely used. The possible adverse effect of MTBE in humans is a public concern, but the human enzymes responsible for metabolism of these gasoline ethers and the causes or factors for increased sensitivity to MTBE in certain individuals are totally unknown. This information is important to understanding the health effects of MTBE in humans and to assessing the human relevance of pharmacokinetics and toxicity data obtained from animals. In the present study, we demonstrated that human liver is active in metabolizing MTBE to tert-butyl alcohol (TBA), a major circulating metabolite and an exposure marker of MTBE. The activity is localized in the microsomal fraction but not in the cytosol. Formation of TBA in human liver microsomes is NADPH-dependent and is significantly inhibited by carbon monoxide, which inhibits cytochrome P450 (CYP) enzymes. These results provide strong evidence that CYP enzymes play a critical role in the metabolism of MTBE in human livers. Human liver is also active in the oxidative metabolism of 2 other gasoline ethers, ETBE and TAME. We observed a large interindividual variation in metabolizing these gasoline ethers in 15 microsomal samples prepared from normal human livers. The activity level (pmol metabolite/min/mg) ranged from 204 to 2,890 for MTBE; 179 to 3,134 for ETBE; and 271 to 8,532 for TAME. The microsomal activities in metabolizing MTBE, ETBE, and TAME correlated highly with each other (r = 0.91 to 0.96), suggesting that these ethers are metabolized by the same enzyme(s). Correlation analysis of the ether-metabolizing activities with individual CYP enzyme activities in the human liver microsomes showed that the highest degree of correlation was with CYP isoform 2A6 (CYP2A6)+ (r = 0.94 for MTBE, 0.95 for ETBE, and 0.90 for TAME), which is constitutively expressed in human livers and known to be polymorphic. CYP2A6 displayed the highest turnover number in metabolizing gasoline ethers among a battery of human CYP enzymes expressed in human B-lymphoblastoid cells. CYP2A6 coexpressed with human CYP reductase by a baculovirus expression system was also more active than CYP isoform 2E1 (CYP2E1) in the metabolism of MTBE, ETBE, and TAME. Kinetic studies on MTBE metabolism with human liver microsomes (n = 3) exhibited an apparent Michaelis constant (Km) of 28 to 89 microM and a maximum rate of metabolism (Vmax) of 215 to 783 pmol/min/mg. Metabolism of MTBE, ETBE, and TAME by human liver microsomes was inhibited by coumarin, a known substrate of human CYP2A6, in a concentration-dependent manner. Monoclonal antibody against human CYP2A6 caused a significant inhibition (75% to 95%) of the metabolism of MTBE, ETBE, and TAME in human liver microsomes. Taken together, these results clearly indicate that, in human liver, CYP2A6 is a major enzyme responsible for metabolism of MTBE, ETBE, and TAME. Although CYP2E1 metabolizes diethyl ether and was previously suggested to be involved
Research report (Health Effects Institute) 06/2001;
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ABSTRACT: Diallyl sulfide (DAS) is a flavor compound derived from garlic and is sequentially converted to diallyl sulfoxide (DASO) and diallyl sulfone (DASO(2)) by cytochrome P(450) 2E1 (CYP2E1). These compounds have been shown to reduce the incidence of a multitude of chemically induced tumors in animal models. The impediment of phase I activation of these carcinogens is hypothesized to be accountable for the reduction in tumor incidence. Indeed, DAS, DASO and DASO(2) are competitive inhibitors of CYP2E1. DASO(2), in addition, is a suicide inhibitor of CYP2E1. These compounds have been shown to reduce carbon tetrachloride-, N-nitrosodimethylamine- and acetaminophen-induced toxicity in rodents. All three chemicals are substrates for CYP2E1. The protective effect was observed when the organosulfur compounds were given before, during or soon after chemical treatment. DAS and DASO(2) inhibited the bioactivation of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and related lung tumorigenesis in A/J mice. Because CYP2E1 does not play a key role in NNK activation, the inhibition of other CYP enzymes active in NNK metabolism is likely. DAS also has been shown to induce other CYP and phase II enzymes as well as decrease hepatic catalase activity. All of these effects are observed at concentrations much higher than what is normally ingested by humans. The biological activities of garlic and its related compounds at lower concentrations that mimic human consumption remain to be studied further.
Journal of Nutrition 04/2001; 131(3s):1041S-5S. · 3.92 Impact Factor
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ABSTRACT: The human CYP2A subfamily comprises three genes, CYP2A6, CYP2A7, and CYP2A13. CYP2A6 is active toward many carcinogens and is the major coumarin 7-hydroxylase and nicotine C-oxidase in the liver, whereas CYP2A7 is not functional. The function of CYP2A13 has not been characterized. In this study, a CYP2A13 cDNA was prepared by RNA-PCR from human nasal mucosa and was translated using a baculovirus expression system. In a reconstituted system, the expressed CYP2A13 was more active than CYP2A6 in the metabolic activation of hexamethylphosphoramide, N,N-dimethylaniline, 2'-methoxyacetophenone, and N-nitrosomethylphenylamine but was much less active than CYP2A6 in coumarin 7-hydroxylation. Of particular interest, CYP2A13 was highly active in the metabolic activation of a major tobacco-specific carcinogen, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, with a catalytic efficiency much greater than that of other human cytochrome P450 isoforms examined previously. The tissue distribution of CYP2A13 was determined with isoform-specific RNA-PCR. CYP2A13 mRNA was detected in liver and a number of extrahepatic tissues, including nasal mucosa, lung, trachea, brain, mammary gland, prostate, testis, and uterus, but not in heart, kidney, bone marrow, colon, small intestine, spleen, stomach, thymus, or skeletal muscle. Quantitative PCR analysis further revealed that CYP2A13 mRNA is expressed at the highest level in the nasal mucosa, followed by the lung and the trachea. Together, these findings suggest that CYP2A13 plays important roles in xenobiotic toxicity and tobacco-related tumorigenesis in the human respiratory tract.
Cancer Research 10/2000; 60(18):5074-9. · 7.86 Impact Factor
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ABSTRACT: To reduce the production of carbon monoxide and other pollutants in motor vehicle exhaust, methyl tert-butyl ether (MTBE), ethyl tert-butyl ether (ETBE), and tert-amyl methyl ether (TAME) are added to gasoline as oxygenates for more complete combustion. Previously, we demonstrated that human liver is active in metabolizing MTBE to tert-butyl alcohol (TBA) and that cytochrome P450 (CYP) enzymes play a critical role in the metabolism of MTBE. The present study demonstrates that human liver is also active in the oxidative metabolism of ETBE and TAME. A large interindividual variation in metabolizing these gasoline ethers was observed in 15 human liver microsomal samples. The microsomal activities in metabolizing MTBE, ETBE, and TAME were highly correlated among each other (r, 0.91-0. 96), suggesting that these ethers are metabolized by the same enzyme(s). Correlation analysis of the ether-metabolizing activities with individual CYP enzyme activities in the liver microsomes showed that the highest degree of correlation was with human CYP2A6 (r, 0. 90-0.95), which is constitutively expressed in human livers and known to be polymorphic. CYP2A6 displayed the highest turnover number in metabolizing gasoline ethers among a battery of human CYP enzymes expressed in human B-lymphoblastoid cells. Kinetic studies on MTBE metabolism with three human liver microsomes exhibited apparent Km values that ranged from 28 to 89 microM and the V(max) values from 215 to 783 pmol/min/mg, with similar catalytic efficiency values (7.7 to 8.8 microl/min/mg protein). Metabolism of MTBE, ETBE, and TAME by human liver microsomes was inhibited by coumarin, a known substrate of human CYP2A6, in a concentration-dependent manner. Monoclonal antibody against human CYP2A6 caused a significant inhibition (75% to 95%) of the metabolism of MTBE, ETBE, and TAME in human liver microsomes. Taken together, these results clearly indicate that in human liver, CYP2A6 is the major enzyme responsible for the metabolism of MTBE, ETBE, and TAME.
Toxicology and Applied Pharmacology 11/1999; 160(1):43-8. · 4.45 Impact Factor
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ABSTRACT: Previous experiments in vitro have suggested that cytochrome P450 2E1 (CYP2E1) is involved in acetone catabolism by converting acetone to acetol and then to methylglyoxal, both intermediates in the gluconeogenic pathway. In the present study, CYP2E1-null mice were used to demonstrate the role of CYP2E1 in acetone catabolism in vivo. The blood acetone level in male CYP2E1-null mice was 3.3 +/- 0.9 microg/mL, which was similar to levels of their sex- and age-matched parental lineage strains C57BL/6N (2.3 +/- 0.2 microg/mL) and 129/Sv (3.5 +/- 0.3 microg/mL) mice (both are CYP2E1 wild-type). After fasting for 48 hr, the blood acetone levels in the CYP2E1 wild-type mice were increased by 2.5- to 4.4-fold, but that in the CYP2E1-null mice increased 28-fold. These results clearly demonstrate that CYP2E1 plays a vital role in the catabolism of acetone under fasting conditions.
Biochemical Pharmacology 09/1999; 58(3):461-3. · 4.70 Impact Factor
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ABSTRACT: To reduce the production of pollutants in motor vehicle exhaust, methyl tert-butyl ether (MTBE) and other ethers such as ethyl tert-butyl ether (ETBE) and tert-amyl methyl ether (TAME) are added to gasoline as oxygenates for more complete combustion. Metabolism of these gasoline ethers is catalyzed by cytochrome P450 (P450) enzymes. P450 2E1, which metabolizes diethyl ether, was suggested to be an enzyme involved. The present study used 2E1 knock-out mice (2E1-/-) to assess the contribution of 2E1 to the metabolism of MTBE, ETBE and TAME. Liver microsomes prepared from the 2E1 knock-out mice lacked 2E1 activity (assayed as N-nitrosodimethylamine demethylation), but were still active in metabolizing all three gasoline ethers. The levels of ether-metabolizing activity (nmol/min per mg) in the liver microsomes from 7 week old female 2E1 knock-out mice were 0.54+/-0.17 for MTBE, 0.51+/-0.24 for ETBE and 1.14+/-0.25 for TAME at a 1 mM substrate concentration. These activity levels were not significantly different from those of the sex- and age-matched C57BL/6N and 129/Sv mice, which are the parental lineage strains of the 2E1 knock-out mice and are both 2E1+/+. Our results clearly demonstrate that 2E1 plays a negligible role in the metabolism of MTBE, ETBE and TAME in mouse livers.
Toxicology Letters 04/1999; 105(1):83-8. · 3.23 Impact Factor
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ABSTRACT: O6-Alkylguanine-DNA alkyltransferase (AGT, EC 2.1.1.63) is a principle DNA repair protein in repairing O6-alkylguanine in DNA, a major premutagenic lesion produced by environmental and therapeutic alkylating agents. AGT plays a critical role in protecting cells against mutation and cytotoxicity induced by these alkylating agents. The existence of a large interindividual variation in human AGT activity level has been observed and we hypothesize that genetic polymorphism of AGT could be an important determinant for this variation. The present study reports the identification of a novel missense polymorphism in the human AGT gene. The polymorphic alteration occurs at codon 143 in exon 5, converting isoleucine (ATC) to valine (GTC). Because Ile143 is adjacent to the alkyl acceptor Cys145 of the AGT active site and is conserved among mammalian AGTs, amino acid substitution at this position may affect the function of AGT. The codon 143 polymorphism appears to be linked to another new polymorphic alteration at codon 178, which converts lysine (AAG) to arginine (AGG). Because it has been reported that human AGT can be truncated at position 176 without loss of activity, the codon 178 polymorphism may not affect AGT activity. The codon 143/178 polymorphism was found in two of 90 (2%) esophageal cancer patients residing in a high incidence area of China, but was not detected in 60 normal individuals residing in the same area. Six of 28 (210%) non-cancer Caucasian individuals, however, were found to carry this polymorphic allele, suggesting a significant ethnic difference in distribution of this codon 143/178 polymorphism between Chinese and Caucasian individuals. In addition, we confirmed the existence of a codon 84 genetic polymorphism previously identified in a Japanese population, which converts leucine (CTT) to phenylalanine (TTT). The distribution of codon 84 polymorphism was 16%, 20% and 36%, respectively, in the Chinese esophageal cancer patients, Chinese and Caucasian non-cancer individuals. Coexistence of codons 84 and 143/178 polymorphic alterations was found in one Caucasian individual. In all the Chinese (n = 150) and Caucasian (n = 28) samples examined, we were unable to detect a previously reported codon 160 polymorphism (Gly to Arg) which occurred in 10-25% of the Japanese individuals and was shown to affect the reaction of AGT with the drug O6-benzylguanine. The functional significance of the codon 143/178 genetic polymorphism of human AGT and its role in determining an individual's susceptibility to environmental alkylating carcinogens and response to alkylating chemotherapeutic drugs both remain to be studied.
Pharmacogenetics 03/1999; 9(1):81-7.
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ABSTRACT: Diallyl sulfide (DAS) is a flavor compound derived from garlic and is active in the inhibition of chemically induced cytotoxicity and carcinogenicity in animal models. This study was conducted to examine the effects of the treatment of DAS and garlic homogenates on the activities of catalase, glutathione peroxidase, and superoxide dismutase. Male Sprague-Dawley rats were treated with DAS i.g. at daily doses of 50 or 200 mg/kg for 8 days, causing the hepatic catalase activity to decrease by 55 and 95%, respectively. Such a decrease in hepatic catalase activity was also observed when the DAS treatment was extended to 29 days. Western blot analysis showed that the DAS treatments resulted in corresponding decreases in the liver catalase protein level. No significant change in the catalase activity in the kidney, lung, and brain was observed with the treatments, but a slight decrease in heart catalase activity was observed. These treatments did not cause significant changes in superoxide dismutase and glutathione peroxidase activities in these tissues. Treatment with DAS at a daily dose of 200 mg/kg for 1-7 days resulted in a gradual decrease in the liver catalase activity to 5% of the control level, but it did not decrease the erythrocyte catalase activity. Treatment of rats with fresh garlic homogenates (2 or 4 g/kg, i.g., daily for 7 days) caused a 35% decrease in liver catalase activity. A/J mice treated with DAS and garlic homogenates also showed a decrease in the liver catalase activity. Diallyl sulfone (DASO2), a DAS metabolite, however, did not effectively decrease catalase activity in mice. The catalase activity was not inhibited by either DAS or DASO2 in vitro. The present results demonstrate that treatment with DAS and garlic homogenates decrease the hepatic catalase level in rats and mice.
Journal of Biochemical and Molecular Toxicology 02/1999; 13(3-4):127-34. · 1.38 Impact Factor
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ABSTRACT: Methyl tert-butyl ether (MTBE) is a widely used gasoline oxygenate. Two other ethers, ethyl tert-butyl ether (ETBE) and tert-amyl methyl ether (TAME), are also used in reformulated gasoline. Inhalation is a major route for human exposure to MTBE and other gasoline ethers. The possible adverse effects of MTBE in humans are a public concern and some of the reported symptoms attributed to MTBE exposure appear to be related to olfactory sensation. In the present study, we have demonstrated that the olfactory mucosa of the male Sprague-Dawley rat possesses the highest microsomal activities, among the tissues examined, in metabolizing MTBE, ETBE, and TAME. The metabolic activity of the olfactory mucosa was 46-fold higher than that of the liver in metabolizing MTBE, and 37- and 25-fold higher, respectively, in metabolizing ETBE and TAME. No detectable activities were found in the microsomes prepared from the lungs, kidneys, and olfactory bulbs of the brain. The observations that the metabolic activity was localized exclusively in the microsomal fraction, depended on the presence of NADPH, and was inhibitable by carbon monoxide are consistent with our recent report on MTBE metabolism in human and mouse livers (Hong et al., 1997) and further confirm that cytochrome P450 enzymes play a critical role in the metabolism of MTBE, ETBE, and TAME. The apparent K(m) and Vmax values for the metabolism of MTBE, ETBE, and TAME in rat olfactory microsomes were very similar, ranging from 87 to 125 microM and 9.8 to 11.7 nmol/min/mg protein, respectively. Addition of TAME (0.1 to 0.5 mM) into the incubation mixture caused a concentration-dependent inhibition of the metabolism of MTBE and ETBE. Coumarin (50 microM) inhibited the metabolism of these ethers by approximately 87%. Further comparative studies with human nasal tissues on the metabolism of these ethers are needed in order to assess the human relevance of our present findings.
Fundamental and Applied Toxicology 01/1998; 40(2):205-10.
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ABSTRACT: Cytochrome P450 (CYP) enzymes are responsible for the metabolism of numerous xenobiotics and endogenous compounds, including the metabolic activation of most environmental toxic chemicals and carcinogens. Both metabolic and genetic polymorphisms have been identified for human CYP enzymes. The association of CYP genetic polymorphism and human cancer risk, and susceptibility to environmental hazards, have received increasing attention. This article briefly reviews the approaches and methods currently used in CYP genetic polymorphism studies. In addition, the current status and perspectives of using CYP genetic polymorphism as a biomarker of individual susceptibility to cancer and environmental toxicity are discussed.
Environmental Health Perspectives 07/1997; 105 Suppl 4:759-62. · 7.04 Impact Factor
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ABSTRACT: Methyl tert-butyl ether (MTBE) is widely used as a gasoline oxygenate for more complete combustion in order to reduce the air pollution caused by motor vehicle exhaust. The possible adverse effects of MTBE on human health is a major public concern. However, information on the metabolism of MTBE in human tissues is lacking. The present study demonstrates that human liver is active in metabolizing MTBE to tert-butyl alcohol (TBA), a major circulating metabolite and a marker for exposure to MTBE. The activity is localized in the microsomal fraction (125 +/- 11 pmol TBA/ min per mg protein, n = 8) but not in the cytosol. This activity level in human liver microsomes is approximately one-half of the value in rat and mouse liver microsomes. Formation of TBA in human liver microsomes is NADPH-dependent, and is significantly inhibited by carbon monoxide (CO), an inhibitor of cytochrome P450 (CYP) enzymes, suggesting that CYP enzymes play a critical role in the metabolism of MTBE in human livers. Both CYP2A6 and 2E1 are known to be constitutively expressed in human livers. To examine their involvement in MTBE metabolism, human CYP2A6 and 2E1 cDNAs were individually co-expressed with human cytochrome P450 reductase by a baculovirus expression system and the expressed enzymes were used for MTBE metabolism. The turnover number for CYP2A6 and 2E1 was 6.1 and 0.7 nmol TBA/min per nmol P450, respectively. The heterologously expressed human CYP2A6 was also more active than 2E1 in the metabolism of two other gasoline ethers, ethyl tert-butyl ether (ETBE) and tert-amyl methyl ether (TAME). Although the contributions of other human CYP forms to MTBE metabolism remain to be determined, these results strongly suggest that CYP enzymes play an important role in the metabolism of MTBE in human livers.
Archive für Toxikologie 02/1997; 71(4):266-9. · 4.67 Impact Factor
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ABSTRACT: Multifocal occurrence of precancerous lesions of the esophagus has been observed among individuals in a high incidence area for esophageal carcinoma in Henan Province, China. Results from recent studies suggest that p53 protein accumulation and mutation occur early in the pathogenesis of esophageal carcinoma. Discordant p53 gene mutations have been observed in invasive carcinoma and preinvasive lesions from a patient with esophageal carcinoma. The p53 alterations in the multifocal precancerous lesions from symptom-free subjects, however, have not been investigated.
Two biopsy samples, one each from the middle-third and the lower-third of the esophagus, from each subject, were taken from 55 symptom-free subjects in a high incidence area for esophageal cancer in Huixian, Henan Province, China. p53 protein accumulation and p53 gene mutation were analyzed in the multifocal esophageal precancerous lesions from these subjects.
Histopathologic examination showed that among the 110 biopsies, 20 had dysplasia, 72 had basal cell hyperplasia, and 18 had normal epithelia. Concurrent lesions at the middle- and lower-third biopsy occurred in 2 subjects with dysplasia (2 of 55 subjects, 4%) and 26 subjects with basal cell hyperplasia (26 of 55 subjects, 47%). Analysis by immunohistochemistry showed high concurrent rates of p53 protein accumulation (51 of 55 subjects, 93%). p53 sequence analysis of 32 samples from 16 subjects identified missense mutations in 5. In one subject, there were three different mutations in the middle-third biopsy (codon 161, GCC-->GAC) and the lower-third biopsy (codon 159, GCC-->CCC). A single mutation was detected in the other four subjects in either the middle- or lower-third biopsy.
The present findings indicate that p53 protein accumulation and mutations occur in the early stages of human esophageal carcinogenesis. Independent somatic mutations of the p53 tumor suppressor gene and protein accumulation in different regions of the esophageal "field" might be key molecular events in multifocal esophageal carcinogenesis.
Cancer 04/1996; 77(7):1244-9. · 4.77 Impact Factor
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Annals of the New York Academy of Sciences 10/1995; 768:82-90. · 3.15 Impact Factor
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ABSTRACT: Previous studies have demonstrated that cytochrome P450 2E1 (P450 2E1) catalyzes the oxidation of acetone in vitro. The present study was designed to determine the importance of P450 2E1 in the catabolism of acetone in rats using diallyl sulfide (DAS) as an inhibitor of this enzyme. After a single intragastric dose of DAS, blood samples were collected from rats at different time points, and blood acetone concentrations were measured by gas chromatography. In a low DAS dose (50 mg/kg body weight) group, the maximum acetone level of 6-fold higher than the normal level was reached at 6 hr; the acetone level returned to normal at 48 hr. In a high dose (200 mg/kg) group, the maximum acetone level of 9-fold higher than the normal level was reached at 12 hr; the acetone level returned to normal at 60 hr. The turnover time and fractional turnover rate of elevated acetone were 15.8 +/- 0.5 hr and 0.054 +/- 0.001 hr-1, respectively, for the low dose, and 19.2 +/- 0.6 hr and 0.046 +/- 0.005 hr-1, respectively, for the high dose. In a chronic experiment, DAS (50 and 200 mg/kg, i.g.) was given to rats daily for 29 days, and elevated blood acetone levels were observed during the entire experimental period: 2.0 to 2.8 micrograms/mL for the low dose and 3.4 to 3.9 micrograms/mL for the high dose at 24 hr after the 1st, 7th, 14th and 28th doses versus 0.8 to 0.9 micrograms/mL for the control. The increase of blood acetone level was closely related to the decreases of N-nitrosodimethylamine (NDMA) demethylase activity and P450 2E1 content in liver microsomes. Consistent with the lack of cumulative effect from the multiple doses of DAS on acetone level, rather stable levels of the DAS metabolites, diallyl sulfoxide (45.0 micrograms/mL, range: 33.8 to 58.6 micrograms/mL) and diallyl sulfone (11.7 micrograms/mL, range: 6.9 to 15.6 micrograms/mL), were observed at 24 hr after the 1st, 7th, 21st and 28th doses with DAS (200 mg/kg) in the chronic experiment. It is likely that the inactivation and inhibition of P450 2E1 by DAS and its metabolites block the oxidation of acetone and cause its elevation in blood. The results strongly suggest an important role of P450 2E1 in acetone catabolism under physiological conditions.
Biochemical Pharmacology 01/1995; 48(12):2199-205. · 4.70 Impact Factor
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ABSTRACT: The objective of this study was to quantify the changes in p53 and cyclin D1 protein levels in different stages of human esophageal and gastric cardia carcinogenesis in a high-risk population in Henan, China. Immunoreactivity of p53, cyclin D1 and proliferating-cell nuclear antigen (PCNA) was observed in the cell nuclei of esophageal and gastric cardia biopsies. The number of p53-immunostaining-positive cells was low in normal epithelia, slightly increased in basal-cell hyperplasia (BCH), markedly increased in dysplasia (DYS) (10-fold), and further increased in squamous-cell carcinoma (SCC) (40-fold). This pattern of change was similar to that of cell proliferation as indicated by PCNA immunostaining. On the other hand, the number of cyclin D1-immunostaining-positive cells did not increase from BCH to DYS, although a slight increase from DYS to SCC was noted. In the gastric cardia, again, the pattern of change of p53-positive cells in different stages of lesions paralleled the pattern of cell proliferation. The number of p53-positive cells was very low, much lower than that of PCNA-positive cells, in normal, chronic superficial gastritis (CSG) and chronic atrophic gastritis (CAG); therefore, the increase of p53-positive cells from CAG to DYS was more dramatic (100-fold). From DYS to adenocarcinoma (AC), the p53-positive and the PCNA-positive cells increased 4-fold. On the other hand, the number of cyclin D1-positive cells did not increase in pre-cancerous lesions, but increased slightly in AC. This study demonstrates that p53 protein accumulation increased with the progression of pre-cancerous lesions, especially in the genesis of dysplasia, both in the esophagus and in the gastric cardia. Our approach of quantitative immunohistochemistry sheds light on the mechanisms of genesis of esophageal and gastric-cardia cancers, which frequently occur together in many high-incidence areas.
International Journal of Cancer 12/1994; 59(4):514-9. · 5.44 Impact Factor
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ABSTRACT: Previous studies in our laboratory showed that decaffeinated green tea and black tea extracts inhibited 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-induced tumorigenicity in A/J female mice. In order to understand the mechanism of the inhibitory action, we examined the effects of decaffeinated green tea, black tea, and tea components on the metabolic activation of NNK in vitro and in vivo in this animal model. When added to incubation mixtures containing mouse lung microsomes, decaffeinated green tea and black tea extracts and their fractions, at concentrations up to 0.4 mg/ml, inhibited NNK oxidation and NNK-induced DNA methylation. Among the tea components examined, (-)-epigallocatechin-3-gallate was the most potent inhibitor with 50% inhibitory concentrations of about 0.12 mM for both NNK oxidation and DNA methylation. At these concentrations, (-)-epigallocatechin-3-gallate inhibited the catalytic activities of several P450 enzymes and was more potent against P450 1A and 2B1 than 2E1. When decaffeinated green or black tea extracts were given to female A/J mice as the sole source of drinking fluid before an i.p. injection of NNK (100 mg/kg body weight), a statistically significant inhibition of lung DNA methylation, however, was not observed, although a significant reduction in lung tumor multiplicity was observed. The results suggest that, although inhibition of the metabolic activation of NNK and the subsequent DNA alkylation by tea extracts can be demonstrated in vitro, this mechanism may not be important for the inhibitory action of tea against lung tumorigenesis.
Cancer Research 10/1994; 54(17):4641-7. · 7.86 Impact Factor
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ABSTRACT: To understand whether p53 gene mutation is an early or late event in esophageal carcinogenesis, biopsy samples of esophageal epithelium from symptom-free subjects in a high incidence area, Huixian county of Henan Province, China, were analyzed. Mutations in exons 5, 6, 7, and 8 of p53 were analyzed by single-strand conformation polymorphism analysis and DNA sequencing. Among the 37 biopsy samples showing accumulation of p53 protein in immunohistochemical staining, missense mutations of p53 gene were detected in 1 of 3 samples with normal epithelia, 3 of 23 samples with basal cell hyperplasia, and 4 of 11 samples with dysplasia. All mutations occurred at exon 5 with 3 at codon 175, 2 at codon 176, and 1 each at codons 159, 135, and codon 132. Of the 8 mutations, there were 3 G to A transitions and 3 G to T transversions. To understand the mutation spectrum and possible causative factors of esophageal cancer in this area, surgically resected human primary esophageal carcinomas from Linxian county were analyzed for p53 gene mutations in exons 5, 6, 7, and 8. Mutations were detected in 16 of 29 samples (55%). Twelve samples contained different missense point mutations, with 75% transitions (7 G to A and 2 A to G) and 25% transversions (2 G to T and 1 G to C). Most of the mutations were located at either exon 5 or exon 7. A deletion and an insertion of nucleotides leading to frame-shift mutations were detected in each of two other samples. The results demonstrate that p53 protein accumulation and gene mutation may occur at very early stages of esophageal carcinogenesis. In carcinomas, there was a higher frequency of p53 gene mutations, which accounts for most of the cases with p53 protein accumulation.
Cancer Research 09/1994; 54(16):4342-6. · 7.86 Impact Factor
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Cancer Research 05/1994; 54(7 Suppl):1982s-1986s. · 7.86 Impact Factor
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Advances in experimental medicine and biology 02/1994; 354:113-22. · 1.09 Impact Factor
