Biomedical subjects

F F Kadlubar

Publications and source records attributed to F F Kadlubar.

At least 37 records · Page 2Linked to original sources

PubMed2000

Human CYP1B1 Leu432Val gene polymorphism: ethnic distribution in African-Americans, Caucasians and Chinese; oestradiol hydroxylase activity; and distribution in prostate cancer cases and controls.

Cytochrome P4501B1 (CYP1B1) is involved in the activation of many carcinogens and in the metabolism of steroid hormones, including 17beta-oestradiol (E2) and testosterone. We report a significant difference in the allele frequencies of two point mutations in the coding region of the CYP1B1 gene among Caucasian (n = 189), African-American (n = 52) and Chinese (Linxian) (n = 109) populations. A (C to G) transversion at position 1666 in exon 3, which results in an amino acid substitution of Leu432 to Val, was present in African-Americans with an allele frequency for Va1432 of 0.75, in Caucasians of 0.43, and in Chinese of 0.17. A (C to T) transition at position 1719 in exon 3, with no amino acid change (Asp449), appeared to be closely linked with the Val432 variant. Results using human lung microsomal preparations from individuals with the CYP1B1Val/Val and CYP1B1Leu/Leu genotypes indicate that Val432 variant may be a high activity allele and thus may contribute to the interindividual differences in CYP1B1 activity. Because CYP1B1 is involved in hormone and carcinogen metabolism, and given the disparate rates of prostate cancer among ethnic groups, we also evaluated the association of the CYP1B1 Leu432Val polymorphism with prostate cancer risk in a pilot case-control study. Among Caucasians, 34% of men with cancer (n = 50) were homozygous for the Val432 polymorphism, while only 12% of matched control subjects (n = 50) had this genotype. These preliminary data indicate that genetic polymorphisms in CYP1B1 might play an important role in human prostate carcinogenesis.

Adult↗

PubMed2000

Relationship of phenol sulfotransferase activity (SULT1A1) genotype to sulfotransferase phenotype in platelet cytosol.

Sulfation catalysed by human cytosolic sulfotransferases is generally considered to be a detoxification mechanism. Recently, it has been demonstrated that sulfation of heterocyclic aromatic amines by human phenol sulfotransferase (SULT1A1) can result in a DNA binding species. Therefore, sulfation capacity has the potential to influence chemical carcinogenesis in humans. To date, one genetic polymorphism (Arg213His) has been identified that is associated with reduced platelet sulfotransferase activity. In this study, data on age, race, gender, SULT1A1 genotype and platelet SULT1A1 activity were available for 279 individuals. A simple colorimetric phenotyping assay, in conjunction with genotyping, was employed to demonstrate a significant correlation (r = 0.23, P < 0.01) of SULT1A1 genotype and platelet sulfotransferase activity towards 2-naphthol, a marker substrate for this enzyme. There was also a difference in mean sulfotransferase activity based on gender (1.28 nmol/min/mg, females; 0.94 nmol/min/mg, males, P = 0.001). DNA binding studies using recombinant SULT1A1*1 and SULT1A1*2 revealed that SULT1A1*1 catalysed N-hydroxy-aminobiphenyl (N-OH-ABP) DNA adduct formation with substantially greater efficiency (5.4 versus 0.4 pmol bound/mg DNA/20 min) than the SULT1A1*2 variant. A similar pattern was observed with 2-hydroxyamino-1-methyl-6-phenylimidazo[4,5b]pyridine (N-OH-PhIP) (4.6 versus 1.8 pmol bound/mg DNA/20 min).

Age Factors↗

PubMed2000

Cancer therapy and polymorphisms of cytochromes P450.

Cytochrome P450 (CYP) enzymes are important in the metabolism of some endogenous compounds, environmental and dietary xenobiotics and many drugs. Many of these enzymes have genetic polymorphisms that produce significant changes in metabolic activity, however the function of other polymorphisms is unknown. Genetic polymorphisms have important influences on variability in human pharmacokinetics, including intra-individual differences in drug toxicity, drug interactions and response to chemotherapy. Other factors that influence drug metabolism include differences in enzyme expression due to differences in age, gender, smoking status, exposure to dietary or environmental xenobiotics or co-administration of other drugs. In addition, some xenobiotics and drugs can directly inhibit or induce the activity of CYPs. All of these factors can produce differences in metabolic capacities among individuals which can produce toxicity in some patients and sub-effective dosing in others. Maximum clinical benefit will require a more complete understanding of the influence of these polymorphisms on allele function and their interaction with inducers and inhibitors of enzyme expression or activity. This effort will permit the pharmacogenetic screening of patients before the administration of drugs and result in the identification of individuals who are prone to adverse reactions or poor response, resulting in more effective individualized therapy.

Antineoplastic Agents↗

PubMed2000

Characterization of an ATP-dependent pathway of activation for the heterocyclic amine carcinogen N-hydroxy-2-amino-3-methylimidazo[4, 5-f]quinoline.

2-amino-3-methylimidazo[4,5-f]quinoline (IQ) is one of several mutagenic and carcinogenic heterocyclic amines formed during the cooking process of protein-rich foods. These compounds are highly mutagenic and have been shown to produce tumours in various tissues in rodents and non-human primates. Metabolic activation of IQ is a two-step process involving N-hydroxylation by CYP1A2 followed by esterification to a more reactive species capable of forming adducts with DNA. To date, acetylation and sulphation have been proposed as important pathways in the formation of N-hydroxy esters. In this study we have demonstrated the presence of an ATP-dependent activation pathway for N-hydroxy-IQ (N-OH-IQ) leading to DNA adduct formation measured by covalent binding of [(3)H]N-OH-IQ to DNA. ATP-dependent DNA binding of N-OH-IQ was greatest in the cytosolic fraction of rat liver, although significant activity was also seen in colon, pancreas and lung. ATP was able to activate N-OH-IQ almost 10 times faster than N-hydroxy-2-amino-1-methyl-6-phenylimidazo[4, 5-b]pyridine (7.7 +/- 0.3 and 0.9 +/- 0.1 pmol/mg protein/min, respectively). Using reported intracellular concentrations of cofactor, the ability of ATP to support DNA binding was similar to that seen with 3'-phosphoadenosine 5'-phosphosulphate and approximately 50% of that seen with acetyl coenzyme A (AcCoA). In addition to DNA binding, HPLC analysis of the reaction mixtures using ATP as co-factor showed the presence of two stable, polar metabolites. With AcCoA, only one metabolite was seen. The kinase inhibitors genistein, tyrphostin A25 and rottlerin significantly inhibited both DNA binding and metabolite formation with ATP. However, inhibition was unlikely to be due to effects on enzyme activity since the broad spectrum kinase inhibitor staurosporine had no effect and the inactive analogue of genistein, daidzein, was as potent as genistein. The effects of genistein and daidzein, which are naturally occurring isoflavones from soy and other food products, on DNA adduct formation may potentially be useful in the prevention of heterocyclic amine-induced carcinogenesis.

Acetylation↗

PubMed2000

Impact of genetic polymorphisms in cytochrome P450 2E1 and glutathione S-transferases M1, T1, and P1 on susceptibility to esophageal cancer among high-risk individuals in China.

Esophageal cancer, which is prevalent in China, is believed to be induced by environmental carcinogens such as nitrosamines and other agents. The disproportionate geographical distribution of this cancer among individuals suggests a role for gene-environment interactions in developing the disease. We have shown in our preliminary study that a genetic polymorphism in cytochrome P450 2E1 (CYP2E1) that is known to activate nitrosamines may be a susceptibility factor involved in the early events leading to the development of esophageal cancer (Lin et al., Cancer Epidemiol. Biomark. Prev., 7: 1013-1018, 1998). This relatively larger study was conducted to compare the results with our previous findings. One hundred and fifty cases with esophageal cancer, 146 cases with esophageal dysplasia, and 150 normal controls were residents of Linxian, China, a high-risk area. Genomic DNA samples were assayed for restriction fragment length polymorphisms in the CYP2E1 and GSTP1 loci by PCR amplification followed by digestion with RsaI and Alw26I, respectively. Deletion of the GSTM1 and GSTT1 genes was detected by multiplex PCR. The distribution of CYP2E1 c1/c1 allele frequency was found to be significantly different between controls (44.0%) and cases with cancer (71.3%) or cases with dysplasia (70.6%; P < 0.0001). Individuals having the c1/c1 genotype were at a 3.1-fold [95% confidence interval (CI), 2.4-3.9] increased risk of developing dysplasia and a 3.2-fold (95% CI, 2.5-4.1) increased risk of developing squamous cell carcinoma of the esophagus. Although polymorphisms in the GSTT1 and GSTP1 were not significantly different between cases with cancer or cases with dysplasia and controls, the frequency of the GSTM1 non-null (+/+ and +/0) genotypes appeared to be overrepresented in cases with cancer compared with controls (odds ratio, 2.3; 95% CI, 1.8-3.0). Furthermore, a joint effect of the CYP2E1 c1/c1 genotype and GSTM1 non-null genotype on the cancer risk was observed, showing an odds ratio of 8.5 (95% CI, 3.7-19.9). These results demonstrate that CYP2E1 and perhaps GSTM1 are genetic determinants in the development of squamous cell carcinoma of the esophagus.

Carcinoma, Squamous Cell↗

PubMed2000

In vitro bioactivation of N-hydroxy-2-amino-alpha-carboline.

2-Amino-alpha-carboline (A alpha C) is a mutagenic and carcinogenic heterocyclic amine present in foods cooked at high temperature and in cigarette smoke. The mutagenic activity of A alpha C is dependent upon metabolic activation to N-hydroxy-A alpha C (N-OH-A alpha C); however, the metabolism of N-OH-A alpha C has not been studied. We have synthesized 2-nitro-alpha-carboline and N-OH-A alpha C and have examined in vitro bioactivation of N-OH-A alpha C by human and rodent liver cytosolic sulfotransferase(s) and acetyltransferase(s) and by recombinant human N-acetyltransferases, NAT1 and NAT2. The sulfotransferase-dependent bioactivation of N-OH-A alpha C by human liver cytosol exhibited large inter-individual variation (0.5-75, n = 14) and was significantly higher than bioactivation of N-hydroxy-2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (N-OH-PhIP). Correlation and inhibition studies suggested that the isoform of sulfotransferase primarily responsible for bioactivation of N-OH-A alpha C in human liver cytosol is SULT1A1. O-Acetyltransferase-dependent bioactivation of N-OH-A alpha C by human liver cytosol also exhibited large inter-individual variation (16-192, n = 18). In contrast to other N-hydroxy heterocyclic amines, which are primarily substrates only for NAT2, both NAT1 and NAT2 catalyzed bioactivation of N-OH-A alpha C. The rate of bioactivation of N-OH-A alpha C by both NAT1 and NAT2 was significantly higher than that for N-OH-PhIP. In rat and mouse liver cytosols, the level of sulfotransferase-dependent bioactivation of N-OH-A alpha C was similar to the level in the high sulfotransferase activity human liver cytosol. The level of O-acetyltransferase-dependent bioactivation of N-OH-A alpha C in rat liver cytosol was also comparable with that in the high acetyltransferase activity human liver cytosol. However, the level of O-acetyltransferase-dependent bioactivation of N-OH-A alpha C in mouse liver cytosol was comparable with that in the low acetyltransferase activity human liver cytosol. In contrast to N-OH-PhIP, bioactivation of N-OH-A alpha C was not inhibited by glutathione S-transferase activity; however, DNA binding of N-acetoxy-A alpha C was inhibited 20% in the presence of GSH. These results suggest that bioactivation of N-OH-A alpha C may be a significant source of DNA damage in human tissues after dietary exposure to AalphaC and that the relative contribution of each pathway to bioactivation or detoxification of N-OH-A alpha C differs significantly from other N-hydroxy heterocyclic or aromatic amines.

Acetylation↗

PubMed2000

A simple colorimetric assay for phenotyping the major human thermostable phenol sulfotransferase (SULT1A1) using platelet cytosols.

A thermostable phenol sulfotransferase, SULT1A1, has been implicated in numerous detoxification and bioactivation pathways; however, little is known regarding its endogenous function or its putative role in mediating risk for human environmental disease. A simple endpoint colorimetric assay is described that can be used for rapid phenotyping of SULT1A1 activity in human populations. The assay utilizes a microtiter-plate format and relatively small amounts of platelet cytosol-derived enzyme. The enzyme catalyzes the synthesis of 2-naphthylsulfate from 2-naphthol and 5'-phosphoadenosine 3'-phosphosulfate (PAPS), whereas addition of p-nitrophenyl sulfate to the assay contributes to an effective PAPS-regenerating system. In contrast to other sulfotransferase assay methods, 3'-phosphoadenosine 5'-phosphate (PAP) does not accumulate during the incubation to interfere with enzyme activity, but instead serves as a cofactor to cause the removal of sulfate from p-nitrophenyl sulfate to regenerate PAPS. This reaction concomitantly results in generation of p-nitrophenol that can be quantified colorimetrically at 405 nm (epsilon = 18,200 M(-1)) to give an indirect measure of sulfotransferase activity. Using platelet enzyme preparations from adult human subjects, sulfation rates of two prototypical thermostable phenol sulfotransferase substrates (2-naphthol and p-nitrophenol) and one thermolabile phenol sulfotransferase substrate (dopamine) were determined using standard radiochemical protocols. These data were then compared with results from the colorimetric assay using 2-naphthol as substrate. There was a good correlation between the phenotyping assay and radiochemical assays for both 2-naphthol sulfotransferase and p-nitrophenol sulfotransferase activity (r = 0.85 and 0.69, respectively). However, SULT1A1 activity was approximately 10 to 20 times higher with the colorimetric determination. As anticipated, there was no correlation between SULT1A1 activity and dopamine sulfotransferase activity (r = 0.07) in these human platelet preparations. This inexpensive and rapid method for phenotyping SULT1A1 activity may help investigators assess a role for this enzyme in disease susceptibility.

Adult↗

PubMed2000

Pharmacogenetics of the arylamine N-acetyltransferases: a symposium in honor of Wendell W. Weber.

This article is a report on a symposium sponsored by the American Society for Pharmacology and Experimental Therapeutics presented at the joint meeting of the American Society for Biochemistry and Molecular Biology and the American Society for Pharmacology and Experimental Therapeutics, June 4-8, Boston, Massachusetts. The presentations focused on the pharmacogenetics of the NAT1 and NAT2 arylamine N-acetyltransferases, including developmental regulation, structure-function relationships, and their possible role in susceptibility to breast, colon, and pancreatic cancers. The symposium honored Wendell W. Weber for over 35 years of leadership and scientific advancement in pharmacogenetics and was highlighted by his overview of the historical development of the field.

Animals↗

PubMed1999

Detoxification of carcinogenic aromatic and heterocyclic amines by enzymatic reduction of the N-hydroxy derivative.

The metabolic activation pathways associated with carcinogenic aromatic and heterocyclic amines have long been known to involve N-oxidation, catalyzed primarily by cytochrome P4501A2, and subsequent O-esterification, often catalyzed by acetyltransferases (NATs) and sulfotransferases (SULTs). We have found a new enzymatic mechanism of carcinogen detoxification: a microsomal NADH-dependent reductase that rapidly converts the N-hydroxy arylamine back to the parent amine. The following N-OH-arylamines and N-OH-heterocyclic amines were rapidly reduced by both human and rat liver microsomes: NOH-4-aminoazobenzene, N-OH-4-aminobiphenyl (N-OH-ABP), N-OH-aniline, N-OH-2-naphthylamine, N-OH-2-aminofluorene, N-OH-4,4'-methylenebis(2-chloroaniline) (N-OH-MOCA), N-OH-1-naphthyamine, N-OH-2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (N-OH-PhIP), N-OH-2-amino-alpha-carboline (N-OH-AalphaC), N-OH-2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (N-OH-MeIQx), and N-OH-2-amino-3-methylimidazo[4,5-f]quinoline (N-OH-IQ). In addition, primary rat hepatocytes and human HepG2 cells efficiently reduced N-OH-PhIP to PhIP. This previously unrecognized detoxification pathway may limit the bioavailability of carcinogenic N-OH heterocyclic and aromatic amines for further activation, DNA adduct formation, and carcinogenesis.

Animals↗

PubMed1999

The effects of phytoestrogens on human pancreatic tumor cells in vitro.

Diet has been implicated as a possible link to the etiology, promotion and/or progression of many diseases, including cancer. Recently, interest has been focused on the cancer-protective role of several of the hormone-like diphenolic phytoestrogens, lignans, and isoflavonoids. This study examined the chemoprotective effects of genistein, biochanin A, equol, and coumestrol on human pancreatic adenocarcinoma cells in vitro. Two human adenocarcinoma cell lines, HPAF-11 from a male and Su 86.86 from a female, were used. HPAF-11 cells were exposed for 24 h to these agents at concentrations of 1 and 10 microM. Su 86.86 cells were exposed for 24 h at a concentration of 1 microM. Coumestrol and equol at higher concentrations were toxic to the Su 86.86 cells. These agents displayed marked differences between cell lines in inhibition of growth. Equol and coumestrol inhibited the growth of the female pancreatic tumor cells by 95%; however, these agents stimulated the growth of pancreatic tumor cells from the male. Genistein also stimulated growth in the male pancreatic tumor cells, but had little effect on pancreatic tumor cells from the female. Biochanin A inhibited growth of both male and female tumor cells, but to a lesser extent than other agents. This study also indicated a difference in K-ras expression in pancreatic tumors cells treated with these agents. Equol and coumestrol decreased K-ras expression in the female tumor cell line. Genistein increased expression of K-ras in both male and female pancreatic tumor cells. Genistein also increased expressions of the multidrug resistant (mdr-1) gene in the male tumor-cell line, while coumestrol and biochanin A decreased expression. Equol had no effect on mdr-1 expression. Whether the chemoprotective potential of equol and coumestrol against pancreatic cancer is greater in females than males is being further studied.

Analysis of Variance↗

PubMed1999

Comparison of DNA adduct levels associated with exogenous and endogenous exposures in human pancreas in relation to metabolic genotype.

Recently, we examined normal human pancreas tissue for DNA adducts derived from either exogenous chemical exposure and/or endogenous agents. In an effort to explain the different types and levels of DNA adducts formed in the context of individual susceptibility to cancer, we have focused on gene-environment interactions. Here, we report on the levels of hydrophobic aromatic amines (AAs), specifically those derived from 4-aminobiphenyl (ABP), and DNA adducts associated with oxidative stress in human pancreas. Although these adducts have been reported in several human tissues by different laboratories, a comparison of the levels of these adducts in the same tissue samples has not been performed. Using the same DNA, the genotypes were determined for N-acetyltransferase 1 (NAT1), the glutathione S-transferase (GST) M1, GSTP1, GSTT1, and NAD(P)H quinone reductase-1 (NQO1) as possible modulators of adduct levels because their gene products are involved in the detoxification of AAs, lipid peroxidation products and in redox cycling. These results indicate that ABP-DNA adducts, malondialdehyde-DNA adducts, and 8-oxo-2'-deoxyguanosine (8-oxo-dG) adducts are present at similar levels. Of the metabolic genotypes examined, the presence of ABP-DNA adducts was strongly associated with the putative slow NAT1*4/*4 genotype, suggesting a role for this pathway in ABP detoxification.

Aminobiphenyl Compounds↗

PubMed1999

An FDA review of sulfamethazine toxicity.

Recently, changes have been proposed in the criteria historically used in the evaluation of the applicability to humans of some of the results obtained from the rodent carcinogenicity bioassay data. These questions center on the suitability of the rodent model for agents that exert their toxic effects via specific enzyme interactions and endocrine mechanisms which appear to be inoperative within humans. Within the U.S. Food and Drug Administration (FDA), this issue has been brought to the forefront of concern with the recent application for a New Animal Drug Application for sulfamethazine (SMZ). A panel of FDA experts from the National Center for Toxicological Research (NCTR), the Center for Veterinary Medicine (CVM), and the Center for Food Safety and Applied Nutrition has reviewed the sum of the scientific evidence available on the toxicology of SMZ. They noted that, in previous feeding studies at NCTR, high doses of SMZ were associated with significant incidences of thyroid tumors in mice and rats. The panel also notes that the tumorigenic activity of SMZ in rodents was due to its goitrogenic activity, resulting in constant stimulation of the thyroid by TSH. Humans, on the other hand, were found to be insensitive to the SMZ-like inhibition of thyroid function. Further, apart from X-irradiation and radioactive iodine, there are no other physical or chemical agents known to cause thyroid tumors in humans. Thus, the expert panel concludes that the best scientific information available indicates that elevated levels of TSH and the consequent thyroid tumors would not be produced under approved use conditions of SMZ. This conclusion is in agreement with recommendations made by three other panels, viz. the World Health Organization, the U.S. Environmental Protection Agency, and CVM, which also evaluated the public health risk of SMZ.

Animals↗

PubMed1999

Formation and reactions of N(7)-aminoguanosine and derivatives.

Arylamines are mutagens and carcinogens and are thought to initiate tumors by forming adducts with DNA. The major adducts are C(8)-guanyl, and we have previously suggested a role for guanyl-N(7) intermediates in the formation process. N(7)-Aminoguanosine (Guo) was synthesized and characterized, with the position of the NH(2) at N7 established by two-dimensional rotating frame Overhauser enhancement NMR spectroscopy. In DMF, N(7)-NH(2)Guo formed C(8)-NH(2)Guo and the cyclic product C(8):5'-O-cycloGuo. In aqueous media, these products were formed along with 8-oxo-7,8-dihydroGuo, N(7)-NH(2)guanine, and a product characterized as a purine 8, 9-ring-opened derivative (N-aminoformamidopyrimidine). The rate of aqueous decomposition of N(7)-NH(2)Guo increased with pH, with a t(1/2) of 10 h at pH 7 and a t(1/2) of 2 h at pH 9. The rate of migration of NH(2) from N7 to C8 is fast enough to explain the formation of C(8)-NH(2)Guo from the reaction of 2, 4-dinitrophenoxyamine with Guo but not the formation of C(8)-(arylamino)Guo in the reaction of Guo with aryl hydroxylamine esters; however, the fluorenyl moiety may facilitate the proposed rearrangement by stabilizing an incipient negative charge in the transfer. In the reaction of Guo with N-hydroxy-2-aminofluorene and acetylsalicylic acid, a peak with the mass spectrum expected for N(7)-(2-aminofluorenyl)Guo was detected early in the reaction and was distinguished from C(8)-(2-aminofluorenyl)Guo. NMR experiments with [8-(13)C]Guo also provided some additional support for transient formation of N(7)-(2-aminofluorenyl)Guo. We conclude that a guanyl-N(7) intermediate is reasonable in the reaction of activated arylamines with nucleic acids, although an exact rate of transfer of an N(7)-arylamine group to the C8 position has not yet been quantified. The results provide an explanation for the numerous products associated with modification of DNA by activated arylamines. However, the contribution of "direct" reaction at the guanine C8 atom cannot be excluded.

Chromatography, High Pressure Liquid↗

PubMed1999

Increased expression of hepatic DNA methyltransferase in smokers.

The DNA methyltransferase enzyme (DNA MTase) catalyzes DNA methylation at cytosines in CpG dinucleotides. 5-Methylcytosine modification of DNA is important in gene regulation, DNA replication, chromatin organization and disease. Increased levels of DNA MTase have been associated with the initiation and promotion of cancer. This study was conducted to assess whether cigarette smoking and other factors, such as age and gender, influence DNA MTase expression in nontumorous tissue. DNA MTase was significantly (p<0.05) higher in samples from cigarette smokers; the mean level of DNA MTase mRNA was almost 2-fold higher in these samples than in those from nonsmokers. Levels of DNA MTase mRNA were higher in samples from females than in those from males, but the difference was not statistically significant. Age was not associated with DNA MTase levels. Increased levels of DNA MTase in individuals who smoke may indicate a greater susceptibility to the risk of cancer since increased levels of this enzyme are found in cancer cell lines and human tumors. The results of this study suggest that further investigations of increased expression of this enzyme as a predisposing factor for cancer susceptibility are needed.

Adolescent↗

PubMed1999

Effects of chemoprotective agents on the metabolic activation of the carcinogenic arylamines PhIP and 4-aminobiphenyl in human and rat liver microsomes.

Carcinogenic aromatic amines, including the heterocyclic amines, may pose a significant health risk to humans. To determine the potential for chemoprotective intervention against the carcinogenicity of these arylamines and to better understand their mechanism of action, a range of agents, most of them natural dietary constituents, was examined in vitro for their ability to modulate the N-hydroxylation of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and 4-aminobiphenyl (ABP), an initial step in their bioactivation. Experiments were conducted with rat and human liver microsomes. The agents (diallyl sulfide, indole-3-carbinol, alpha-angelicalactone, cafestol/kahweol palmitates, cafestol, kahweol, benzylisothiocyanate, genistin, formononetin, daidzin, equol, biochanin A, Oltipraz, tannic acid, quercetin, ethoxyquin, green tea, and black tea) comprised a variety of chemical classes that included sulfur-containing compounds, antioxidants, flavonoids, phytoestrogens, diterpenes, and polyphenols. Several of these agents, quercetin, ethoxyquin, and black tea, were found to strongly inhibit PhIP N-hydroxylation in rat liver microsomes, resulting in a nearly 85-90% decrease in activity at 100 microM or 0.2%. Tannic acid and green tea, in addition to these agents, were also strong inhibitors of ABP N-hydroxylation. In human liver microsomes, each of these agents was strongly inhibitory (approx 85-95% at 100 microM or 0.02%) of PhIP and ABP N-hydroxylation. Theaflavins and polyphenols were judged to be the primary inhibiting components in the teas, the theaflavins showing the most potent effect. These results demonstrate that chemoprotective agents can inhibit the bioactivation of carcinogenic arylamines, and this is likely to be one of the mechanisms of protection.

Aminobiphenyl Compounds↗

PubMed1999

Glucuronidation of 2-hydroxyamino-1-methyl-6-phenylimidazo[4, 5-b]pyridine by human microsomal UDP-glucuronosyltransferases: identification of specific UGT1A family isoforms involved.

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine is a heterocyclic aromatic amine found in cooked meats and dietary exposure to PhIP has been implicated in the etiology of colon cancer in humans. PhIP, along with other heterocyclic aromatic amines, requires metabolic activation to exhibit genotoxic effects. PhIP is initially oxidized by the activity of cytochrome P4501A2 to produce 2-hydroxyamino-1-methyl-6-phenylimidazo[4,5-b]pyridine (N-OH-PhIP), a reaction occurring primarily in the liver. Whereas subsequent biotransformation of N-OH-PhIP via acetylation or sulfation can produce reactive electrophiles that readily bind to DNA, N-glucuronidation, catalyzed by UDP-glucuronosyltransferases (UGTs), functions as a detoxification mechanism. Although hepatic glucuronidation of N-OH-PhIP has been well characterized, the extrahepatic metabolism of this compound is poorly understood. Studies in our laboratory now indicate that the intestinal tract, and particularly the colon, is a significant site of glucuronidation of N-OH-PhIP. When assays were performed with microsomes prepared from the mucosa of the intestinal tract, it was determined that glucuronidation of N-OH-PhIP occurs throughout the intestinal tract, with activity approximately three times higher in the colon as that found in the upper intestine. Glucuronidation rates from colon microsomes showed considerable interindividual variability and incubation with N-OH-PhIP yielded two glucuronides. HPLC analysis showed that the predominant product formed is the N-OH-PhIP-N2-glucuronide, while the N3-glucuronide accounts for <10% of the total glucuronidation product. These rates approach the rates found in human liver microsomes, demonstrating the significance of extrahepatic metabolism of this food-borne carcinogen. Subsequent assays with human recombinant UGTs demonstrated that at least four human UGT isoforms, all from the UGT1A subfamily, are capable of catalyzing the biotransformation of N-OH-PhIP. Members of the UGT2B family available for this study did not conjugate N-OH-PhIP, although immunoinhibition studies in human liver microsomes strongly suggest the involvement of a UGT2B isoform(s) in this organ.

Animals↗

PubMed1999

Analyses of bronchial bulky DNA adduct levels and CYP2C9, GSTP1 and NQO1 genotypes in a Hungarian study population with pulmonary diseases.

Carcinogen-DNA adducts may represent an intermediate end-point in the carcinogenic cascade and may reflect exposure to chemical carcinogens, as well as susceptibility and, ultimately, cancer risk. Interindividual variability in activity of enzymes involved in the metabolism of polycyclic aromatic hydrocarbons to mutagenic diol epoxides may predict adduct levels and, indirectly, lung cancer risk. Using 32P-postlabeling methods, the levels of bulky DNA adducts were determined in macroscopically normal bronchial tissues obtained from resected lobes of 143 Hungarian patients with lung malignancy and other pulmonary conditions. DNA from normal tissue was also evaluated for polymorphisms in cytochrome P450 2C9 (CYP2C9) at two sites, codons 144 (Arg/Cys) and 359 (Ile/Leu), for glutathione S-transferase P1 (GSTP1) at codon 105 and for NAD(P)H:quinone oxidoreductase (NQO1) at codon 187 (Pro/Ser). Using the Mann-Whitney U-test and analysis of variance, levels of adducts were evaluated in relation to variant genotypes, separately for smokers and non-smokers. As previously reported, bulky DNA adduct levels in smokers (n = 104) were estimated to be 54% higher than in non-smokers (n = 39) (8.6 +/- 4.2 versus 5.6 +/- 3.3 per 10(8) nucleotides, respectively, P < 0.01). Adduct levels were 16-29% higher in individuals with the homozygous Ile359/Ile359 CYP2C9 allele than in those heterozygous for the variant allele (Ile359/Leu359) [8.8 +/- 4.3 (n = 84) versus 7.6 +/- 3.5 (n = 20) for smokers and 5.8 +/- 3.5 (n = 32) versus 4.5 +/- 1.3 (n = 7) for non-smokers], although differences were not statistically significant. There were no clear differences in adduct levels in relation to genotypes of NQO1 or GSTP1. Although numbers of patients in this study are large in relation to many studies of carcinogen-DNA adducts, it is still possible that significant differences were not noted for polymorphisms in xenobiotic metabolizing enzymes due to relatively small numbers in stratified data.

Aryl Hydrocarbon Hydroxylases↗

PubMed1999

CYP1A2 is not the primary enzyme responsible for 4-aminobiphenyl-induced hepatocarcinogenesis in mice.

4-Aminobiphenyl (4-ABP), a potent carcinogen in rodents (liver cancer) and human (bladder cancer), is found as an environmental contaminant and in tobacco smoke. Hemoglobin adducts and lung DNA adducts of 4-ABP are found in tobacco smokers. In vitro metabolism studies with human and rat liver microsomes have shown that CYP1A2 is primarily responsible for catalyzing N-hydroxylation, the initial step in the metabolic activation of 4-ABP. To determine whether this P450 is a rate limiting pathway for hepatocarcinogenesis, CYP1A2-null mice were analyzed at 16 months of age and were compared with wild-type mice in their response to 4-ABP using the neonatal mouse bioassay and two different doses of the carcinogen. Overall differences in incidences of hepatocellular adenoma, carcinoma and preneoplastic foci were not significant between either genotypes or 4-ABP doses used, whereas small, but significant, differences were found for specific types of foci. These results suggest that while CYP1A2 levels may not be rate limiting for 4-ABP metabolism to produce tumors and foci, it may modulate the induction process of some types of liver foci in either a positive or negative manner. In vitro studies using CYP1A2-null and wild-type mouse liver microsomes revealed that CYP1A2 is not the sole P450 required for 4-ABP N-hydroxylation and that another, yet to be identified, P450 is likely to be involved.

Adenoma↗