Prevalence of human papillomavirus infection in esophageal and cervical cancers in the high incidence area for the two diseases from 2007 to 2009 in Linzhou of Henan Province, Northern China.

The etiological role of human papillomavirus (HPV) in cervical cancer has been well established. However, it is inconclusive whether HPV plays the same role in esophageal carcinogenesis. In this study, we detected HPV infection in 145 frozen esophageal tissues, including 30 normal epithelium (ENOR), 37 dysplasia (DYS) and 78 invasive squamous cell carcinoma (ESCC), and in 143 frozen cervical tissues composed of 30 normal epithelium (CNOR), 38 intraepithelial neoplasia (CIN) and 75 invasive squamous cell carcinoma (CSCC). The patients and symptom-free subjects enrolled in this study were from a high-incidence area for both ESCC and CSCC, Linzhou City, Northern China, from 2007 to 2009. The HPV infection analysis was conducted by using an HPV GenoArray Test Kit. We found that the high-risk HPV types accounted for more than 90 % of the HPV-positive lesions of esophagus and cervix tissues. The prevalence of high-risk HPV types increased significantly during the progression of both esophageal and cervical carcinogenesis (positive rate in esophageal tissues: 33 % ENOR, 70 % in DYS and 69 % in ESCC; positive rate in cervical tissues: 27 % in CNOR, 82 % in CIN and 88 % in CSCC; P < 0.001, respectively). Infection with the high-risk HPV types increased the risk for both DYS and ESCC by 4-fold (DYS vs. ENOR: OR = 4.73, 95 %CI = 1.68-13.32; ESCC vs. ENOR: OR = 4.50, 95 %CI = 1.83-11.05) and increased the risk for both CIN and CSCC by 12-fold and 20-fold (CIN vs. CNOR: OR = 12.18, 95 %CI = 3.85-38.55; CSCC vs. CNOR: OR = 20.17, 95 %CI = 6.93-58.65), respectively. The prevalence of high-risk types in ESCC patients was lower than that in CSCC patients (P = 0.005) and was significantly associated with the degree of ESCC tumor infiltration (P = 0.001). HPV 16 was the most prevalent subtype in both esophageal and cervical tissues. Single HPV infection increased significantly along with the progression of ESCC and maintained a high level in cervical tissues, regardless of whether they were CNOR or CSCC tissues. Our results showed that infection with HPV, especially the high-risk types, was positively associated with both esophageal and cervical cancers, suggesting that HPV also plays a role in the etiology of ESCC in the high-incidence area.

Selective expression of CYP2A13 in human pancreatic α-islet cells.

Exposure to cigarette smoke is an etiological factor of human pancreatic cancer and has been associated with an increased risk of pancreatic diseases, including pancreatitis and diabetes. The toxicants in cigarette smoke can reach pancreatic tissue, and most of the toxicants require cytochrome P450 (P450)-mediated metabolic activation to exert their toxicity. Among all the human P450 enzymes, CYP2A13 is the most efficient enzyme in the metabolic activation of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), a major tobacco-specific toxicant and a suspected human carcinogen. It also metabolically activates 4-aminobiphenyl, another toxicant in cigarette smoke. Immunohistochemical analysis in this study demonstrated that CYP2A13 was selectively expressed in the islets but not in the exocrine portion of adult human pancreas. Further study using dual immunofluorescence labeling technique showed that CYP2A13 protein was mainly expressed in the α-islet but not in ß-islet cells. The selective expression of CYP2A13 in human pancreatic α-islet cells suggests that these islet cells could be damaged by the toxicants existing in cigarette smoke through CYP2A13-mediated in situ metabolic activation. Our result provides a mechanistic insight for human pancreatic diseases that have been associated with cigarette smoke exposure.

Metabolism of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) by human CYP1B1 genetic variants.

Human cytochrome P450 1B1 (CYP1B1) plays a critical role in the metabolic activation of a variety of procarcinogens, including 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). The existence of human CYP1B1 missense genetic variants has been demonstrated, but their activities in metabolizing PhIP are unknown. In this study, we expressed 15 naturally occurring CYP1B1 variants (with either single or multiple amino acid substitutions) and determined their activity changes in metabolizing PhIP to its two major metabolites, 2-hydroxyamino-PhIP and 4'-hydroxy-PhIP. Although the PhIP-metabolizing activities of four variants (Ala(119)Ser, Pro(379)Leu, Ala(443)Gly, Arg(48)Gly/Leu(432)Val) were comparable with that of the expressed wild-type CYP1B1, five variants (Trp(57)Cys, Gly(61)Glu, Arg(48)Gly/Ala(119)Ser, Arg(48)Gly/Ala(119)Ser/Leu(432)Val, Arg(48)Gly/Ala(119)Ser/Leu(432)Val/Ala(443)Gly) exhibited more than 2-fold decrease in activity and a reduction in the catalytic efficiency (V(max)/K(m)) for both N- and 4-hydroxylation of PhIP. Six variants (Gly(365)Trp, Glu(387)Lys, Arg(390)His, Pro(437)Leu, Asn(453)Ser, Arg(469)Trp) showed little activity in PhIP metabolism, but the molecular mechanisms involved are apparently different. The microsomal CYP1B1 protein level was significantly decreased for the Trp(365), Lys(387), and His(390) variants and was not detectable for the Ser(453) variant. In contrast, there was no difference between the Trp(469) variant and the wild-type in the microsomal CYP1B1 protein level and P450 content but the Trp(469) variant totally lost its metabolic activity toward PhIP. The Leu(437) variant also had a substantial amount of CYP1B1 protein in the microsomes, but there was a lack of detectable P450 peak and activity. Our results should be useful in selecting appropriate CYP1B1 variants as cancer susceptibility biomarkers for human population studies related to PhIP exposure.

Arsenic Exposure and Cancer Risk Reduction with Local Ordinance Requiring Whole-House Dual-Tank Water Treatment Systems.

Arsenic, a known human carcinogen, occurs naturally in groundwater in New Jersey and many other states and countries. A number of municipalities in the Piedmont, Highlands, and Valley and Ridge Physiographic Provinces of New Jersey have a high proportion of wells that exceed the New Jersey maximum contaminant level (MCL) of 5 µg/L. Hopewell Township, located in Mercer County and the Piedmont Province, has a progressive local ordinance which requires the installation of dual-tank, point-of-entry treatment systems on affected wells.Thisprovided a unique study opportunity. Of the 55 homes with dual-tank POE treatment systems recruited into this study, 51 homes (93%) had arsenic levels under the MCL at the kitchen sink, regardless of years in service and/or maintenance schedule adherence. Based on the study participants' water consumption and arsenic concentrations, we estimate that Hopewell's arsenic water treatment ordinance, requiring POE dual-tank arsenic treatment, reduced the incidence of excess lifetime (70-year) bladder and lung cancers from 121 (1.7 cancer cases/year) to 16 (0.2 cancer cases/year) preventing 105 lifetime cancer cases (1.5 cases/year). Because the high risk of cancer from arsenic can be mitigated with effective arsenic water treatment systems, this ordinance should be considered a model for other municipalities.

The missense genetic polymorphisms of human CYP2A13: functional significance in carcinogen activation and identification of a null allelic variant.

Cytochrome P450 2A13 (CYP2A13), an enzyme predominantly expressed in human respiratory tissues, is highly efficient for the metabolic activation of two suspected human lung carcinogens 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and aflatoxin B1 (AFB1). Functional genetic polymorphisms of CYP2A13 may therefore be an important factor in human susceptibility to related lung cancers. Among the reported CYP2A13 polymorphisms with missense variations, only CYP2A13*2 variant (containing either a single or double variation of R25Q and R257C) was studied for its NNK-metabolizing activity. The present study demonstrated that there was no remarkable difference in AFB1- and NNK-induced toxicity between the Flp-In Chinese Hamster Ovary (CHO) cells stably expressing wild-type CYP2A13 and the cells expressing the individual polymorphic variants R25Q, D158E, R257C, R25Q/R257C, V323L, F453Y, and R494C. In contrast, cells transfected with R101Q variant complementary DNA (cDNA), same as the vector control cells, showed no significant death even at highest concentrations of AFB1 (10microM) and NNK (200microM). This result correlated with the lack of CYP2A13 protein in the R101Q-CHO cells, although the genomic integration of transfected R101Q cDNA and the expression of R101Q messenger RNA were clearly demonstrated in these stable transfectants. Consistent with the possibility that the variation might reduce the protein stability, R101Q variant protein expressed in insect cells showed a loss of P450 peak and coumarin 7-hydroxylase activity as well as an increased susceptibility to limited protein digestion. Thus, the R101Q polymorphic change results in a null allelic variant of CYP2A13. Our results should be useful in designing and interpreting molecular epidemiological studies related to CYP2A13 genetic polymorphisms.

Effect of genetic variation on human cytochrome p450 reductase-mediated paraquat cytotoxicity.

Paraquat (1,1'-dimethyl-4,4'-bipyridylium dichloride) is a widely used herbicide and is highly toxic to human and animals. The mechanisms of paraquat toxicity involve the generation of superoxide anion through the process of redox cycling. NADPH-cytochrome P450 oxidoreductase (POR) has been reported to be a major enzyme for one-electron reduction of paraquat that initiates the redox cycling. Recently, a total of six missense variants of human POR have been identified in patients with discorded steroidogenesis. However, the effect of these genetic variations on POR-mediated paraquat toxicity is not known. Using the Flp-In Chinese hamster ovary (CHO) cells stably expressing either mouse or human POR and the cells with POR knockdown by siRNA, we confirmed that POR is responsible for paraquat-induced cytotoxicity. We further used this validated system to compare paraquat-induced toxicity among the cells that stably expressed wild-type human POR and its natural variants. While there was no difference in paraquat-induced toxicity between the cells expressing wild-type human POR and the Cys569Tyr variant, the toxicity in cells expressing all the other variants (Tyr181Asp, Ala287Pro, Arg457His, Val492Glu, and Val608Phe) was significantly decreased. Our results provide further evidence on the important role of POR in paraquat-induced toxicity and suggest that individuals carrying the functional variant POR alleles may have an altered susceptibility to paraquat exposure.

[Study of Papaver somniferum cultivars identification by TD-RAPD technique].

OBJECTIVE: The feasibility of Papaver somniferum L. cultivars identification was explored by TD-RAPD technique. METHOD: Genomic DNA was extracted by improved CTAB method. One sample of species from Papaver somniferum L in xishuangbanna area. was studied by using TD-RAPD method. RESULT: We established an optimal method of extracting genomic DNA. Six primers were picked out from 10 primers. CONCLUSION: TD-RAPD could be applied to researches of molecular marker of Papaver somniferum L. TD-RAPD technique provide a method to constitute DNA database of Papaver somniferum L. and conclude the source of opium poppy.

Genetic polymorphisms of CYP2E1, GSTT1, GSTP1, GSTM1, ALDH2, and ODC and the risk of advanced precancerous gastric lesions in a Chinese population.

There have been few studies of the associations of genetic polymorphisms with precancerous gastric lesions. We conducted a cross-sectional study to compare the prevalences of several genetic polymorphisms in 302 subjects with mild chronic atrophic gastritis with prevalences in 606 subjects with deep intestinal metaplasia or dysplasia. This stratified random sample of 908 subjects was selected and analyzed for genetic polymorphisms from 2,628 individuals who had gastric biopsies with histopathology in 1989 in Linqu County, Shandong Province, China. In subjects with mild chronic atrophic gastritis, the frequencies of the variant (less common) alleles of CYP2E1 RsaI, CYP2E1 DraI, GSTP1, ALDH2, and ODC were, respectively, 0.156, 0.201, 0.189, 0.190, and 0.428. The frequencies of the null genotypes of GSTM1 and GSTT1 in the mild chronic atrophic gastritis group were 0.509 and 0.565, respectively. Comparing mild chronic atrophic gastritis with deep intestinal metaplasia or any degree of dysplasia, we found no statistically significant associations with any genotype from these loci for dominant, additive, or recessive inheritance models. There was no statistically significant evidence of multiplicative interactions between any pair of genotypes based on CYP2E1 RsaI, CYP2E1 DraI, GSTP1, GSTM1, or GSTT1; nor between Helicobacter pylori status and any of these five loci; nor between smoking status and GSTP1, GSTM1, or GSTT1; nor between alcohol consumption and ALDH2. Statistically significant interactions were noted between salt consumption and GSTP1 and between sour pancake consumption and CYP2E1 RsaI. There was, moreover, a statistically significant interaction (odds ratio, 1.78; 95% confidence interval, 1.03-3.08) between CYP2E1 DraI and smoking at least one cigarette per day. A positive but not statistically significant interaction was also seen between CYP2E1 RsaI and smoking status. These polymorphisms do not seem to govern progression from mild chronic atrophic gastritis to advanced precancerous gastric lesions, but the effects of smoking may be accentuated in individuals carrying variants of CYP2E1.

Changes of multiple biotransformation phase I and phase II enzyme activities in human fetal adrenals during fetal development.

AIM: Fetal adrenal, which synthesizes steroid hormones, is critical to fetal growth and development. Our recent research showed that some xenobiotics could interfere with steroidogenesis and induce intrauterine growth retardation in rats. The study on the characteristics of biotransformation enzymes in fetal adrenals still seems to be important with respect to possible significance in xenobiotic-induced fetal development toxicity. In this study, the activities of several important xenobiotic-related phase I and phase II enzymes in human fetal adrenals were examined and compared with those in fetal livers. METHODS: The activity and mRNA expression were determined by enzymatic analysis and RT-PCR. RESULTS: The levels of cytochrome (CYP)2A6, CYP2E1, and CYP3A7 isozymes in fetal adrenals were 82%, 92%, and 33% of those in fetal livers, respectively. There was a good positive correlation between adrenal CYP2A6 activity and gestational time. The values of alpha glutathione S-transferase (GST), pi-GST, and microGST in adrenals were 0.5, 4.4, and 8.3-fold of those in the livers, respectively, and the activity of adrenal pi-GST was negatively correlated with gestational time. The uridine diphosphoglucuronyl transferase activities, which were measured using p-hydroxy-biphenyl and 7-hydroxy-4-methylcoumarin as substrates, were 9% and 3%, respectively, of those in the fetal livers. CONCLUSION: Our investigation suggested that adrenal could be an important xenobiotic-metabolizing organ in fetal development and may play a potential role in xenobiotic-induced fetal development toxicity.

Multiplex genotyping of cytochrome p450 single-nucleotide polymorphisms by use of MALDI-TOF mass spectrometry.

BACKGROUND: Polymorphisms in cytochrome P450 (CYP450) genes contribute to interindividual differences in the metabolism of xenobiotic chemicals, including the vast majority of drugs, and may lead to toxicity and adverse drug reactions. Studies on these polymorphisms in research and diagnostic settings typically involve large-scale genotyping and hence require high-throughput assays. METHODS: We used the previously developed solid-phase capture-single-base extension (SPC-SBE) approach for concurrent analysis of 40 single-nucleotide polymorphisms (SNPs) of CYP2C9 and 50 SNPs of CYP2A13, both genes belonging to the CYP450 family. Desired SNP-containing regions for each gene were amplified in a single-step multiplex PCR. We designed a library of primers to anneal immediately upstream of the selected SNPs and extended it with biotinylated terminators using PCR products as templates. Biotinylated extension products were isolated by affinity purification and analyzed with MALDI-TOF mass spectrometry to determine SNP genotypes. RESULTS: We analyzed 11 samples for CYP2C9 and 14 samples for CYP2A13 with unambiguous detection of SNPs in all samples. Many samples showed a high occurrence of heterozygotes for both genes, with as many as 10 of 50 SNPs appearing as heterozygotes in 1 sample genotyped for CYP2A13. CONCLUSIONS: The SPC-SBE method provides an efficient means for genotyping SNPs from the CYP450 family. This approach is suitable for automation and can be extended to other genotyping applications.

Genetic variation of human cytochrome p450 reductase as a potential biomarker for mitomycin C-induced cytotoxicity.

The importance of genetic variation in clinical response to various drugs is now well recognized. Identification of genetic biomarkers that can predict efficacy and toxicity of chemotherapeutic drugs in cancer patients holds great promise in treatment improvement and cost reduction. Mitomycin C (MMC) is a common anticancer drug used for the treatment of numerous types of tumors. Metabolism-mediated activation, by either one-electron or two-electron reduction, plays a critical role in the chemotherapeutic action of MMC. NADPH-cytochrome P450 (oxido)reductase (POR) is a major enzyme responsible for MMC activation through the one-electron reductive pathway, which leads to the production of semiquinone anion radicals and subsequent DNA damage in the cells. Recently, a total of six naturally occurring human POR variants with single amino acid changes (Y181D, A287P, R457H, V492E, C569Y, and V608F) have been identified. Although the catalytic efficiency of these variants in reduction of cytochrome c was reported to be altered, their capability in activating MMC, a direct substrate of POR, has not been examined. In the present study, we demonstrated that except for the C569Y variant, MMC-induced toxicity assayed as cell viability and proliferative capability was significantly decreased in the Flp-In Chinese hamster ovary cells stably expressing all the other POR variants in comparison with the cells expressing wild-type human POR. Cells expressing the V608F and Y181D variants had a complete loss of the capability to activate MMC. Our finding suggests that these functional POR genetic variations may serve as a potential biomarker to predict the chemotherapeutic response to MMC.

Paraquat increases cyanide-insensitive respiration in murine lung epithelial cells by activating an NAD(P)H:paraquat oxidoreductase: identification of the enzyme as thioredoxin reductase.

Pulmonary fibrosis is one of the most severe consequences of exposure to paraquat, an herbicide that causes rapid alveolar inflammation and epithelial cell damage. Paraquat is known to induce toxicity in cells by stimulating oxygen utilization via redox cycling and the generation of reactive oxygen intermediates. However, the enzymatic activity mediating this reaction in lung cells is not completely understood. Using self-referencing microsensors, we measured the effects of paraquat on oxygen flux into murine lung epithelial cells. Paraquat (10-100 microm) was found to cause a 2-4-fold increase in cellular oxygen flux. The mitochondrial poisons cyanide, rotenone, and antimycin A prevented mitochondrial- but not paraquat-mediated oxygen flux into cells. In contrast, diphenyleneiodonium (10 microm), an NADPH oxidase inhibitor, blocked the effects of paraquat without altering mitochondrial respiration. NADPH oxidases, enzymes that are highly expressed in lung epithelial cells, utilize molecular oxygen to generate superoxide anion. We discovered that lung epithelial cells possess a distinct cytoplasmic diphenyleneiodonium-sensitive NAD(P)H:paraquat oxidoreductase. This enzyme utilizes oxygen, requires NADH or NADPH, and readily generates the reduced paraquat radical. Purification and sequence analysis identified this enzyme activity as thioredoxin reductase. Purified paraquat reductase from the cells contained thioredoxin reductase activity, and purified rat liver thioredoxin reductase or recombinant enzyme possessed paraquat reductase activity. Reactive oxygen intermediates and subsequent oxidative stress generated from this enzyme are likely to contribute to paraquat-induced lung toxicity.

Efficient activation of aflatoxin B1 by cytochrome P450 2A13, an enzyme predominantly expressed in human respiratory tract.

The worldwide human exposure to aflatoxin B1 (AFB1), particularly in developing countries, remains to be a serious public health concern. Although AFB1 is best known as a hepatocarcinogen, epidemiological studies have shown a positive association between human lung cancer occurrence and inhalation exposure to AFB1. Cytochrome P450 (CYP)-catalyzed metabolic activation is required for AFB1 to exert its carcinogenicity. Previous studies have identified CYP1A2 and CYP3A4 as the major enzymes for AFB1 activation in human liver. However, the key CYP enzymes in human lung that can efficiently activate AFB1 in situ are unknown. In the present study, we demonstrate that CYP2A13, an enzyme predominantly expressed in human respiratory tract, has a significant activity in metabolizing AFB1 to its carcinogenic/toxic AFB1-8,9-epoxide and AFM1-8,9-epoxide at both low (15 microM) and high (150 microM) substrate concentrations. Under the same conditions, there was no detectable AFB1 epoxide formation by CYP2A6, which was also reported to be involved in the metabolic activation of AFB1. Consistent with the activity data, there was an approximately 800-fold difference in LC50 values of AFB1 (48-hr treatment) between Chinese hamster ovary (CHO) cells expressing CYP2A13 and CYP2A6 (50 nM versus 39 microM). We further demonstrate that amino acid residues Ala117 and His372 in CYP2A13 protein are important for AFB1 epoxidation and its related cytotoxicity. Our results suggest that CYP2A13-catalyzed metabolic activation in situ may play a critical role in human lung carcinogenesis related to inhalation exposure to AFB1.

CYP2A13 in human respiratory tissues and lung cancers: an immunohistochemical study with a new peptide-specific antibody.

Human cytochrome P450 2A13 (CYP2A13) is highly efficient in the metabolic activation of a tobacco-specific carcinogen, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), and another potent carcinogen, aflatoxin B1 (AFB1). Although previous studies demonstrated that CYP2A13 mRNA is predominantly expressed in human respiratory tissues, expression of CYP2A13 protein in these tissues and the involved cell types have not been determined because of the lack of CYP2A13-specific antibodies. To explore the toxicological and physiological function of CYP2A13, it is important to understand the tissue/cellular distribution of CYP2A13 protein. In this study, we generated a peptide-specific antibody against human CYP2A13 and demonstrated by immunoblot analysis that this antibody does not cross-react with heterologously expressed human CYP2A6 and mouse CYP2A5 proteins, both sharing a high degree of amino acid sequence similarity with CYP2A13. Nor does the antibody cross-react with heterologously expressed human CYP3A4, CYP2S1, or any of the cytochrome P450 enzymes present in the human liver microsomes. Using this highly specific antibody for immunohistochemical staining, we detected a high level of CYP2A13 protein expression in the epithelial cells of human bronchus and trachea, but a rare distribution in the alveolar cells. There was little expression of CYP2A13 protein in different types of lung cancers. In consideration of the high efficiency of CYP2A13 in NNK metabolic activation, our result is consistent with the reported observations that most smoking-related human lung cancers are bronchogenic and supports that CYP2A13-catalyzed in situ activation may play a critical role in human lung carcinogenesis related to NNK and AFB1 exposure.

Human cytochrome p450 2s1: lack of activity in the metabolic activation of several cigarette smoke carcinogens and in the metabolism of nicotine.

Cytochrome P450 (P450) enzymes play a critical role in the metabolic activation of a wide variety of environmental carcinogens. Recently, a novel human P450 enzyme, CYP2S1, has been identified. It is inducible by dioxin and other classical aryl hydrocarbon receptor ligands. However, little is known regarding the substrates and the functional role of CYP2S1. Since CYP2S1 is predominantly expressed in human lung and trachea, it is reasonable to speculate that CYP2S1 may play an important role in metabolizing the environmental chemicals to which human respiratory tissues are exposed. In the present study, we examined the activity of human CYP2S1 in the metabolism of nicotine and in the activation of three potent carcinogens in cigarette smoke, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), benzo[a]pyrene (BaP), and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). The full-length CYP2S1 cDNA was amplified by nested polymerase chain reaction from a human lung cDNA library and was expressed in both Chinese hamster ovary (CHO) cells and Sf9 insect cells. In contrast to the positive controls, i.e., CHO cells expressing human CYP2A13 (for NNK activation) or human CYP1A1 (for BaP activation), there was no increase in NNK- or BaP-induced toxicity in the CHO cells expressing CYP2S1. The heterologously expressed CYP2S1 proteins showed no detectable activity in metabolizing nicotine and PhIP. These results clearly demonstrate that CYP2S1 does not catalyze the metabolism of nicotine and the metabolic activation of these lung carcinogens.

Metabolism of nicotine and cotinine by human cytochrome P450 2A13.

Nicotine, a major constituent of tobacco, plays a critical role in smoking addiction. In humans, nicotine is primarily metabolized to cotinine, which is further metabolized to trans-3'-hydroxycotinine. Recently, we have demonstrated that heterologously expressed human CYP2A13 is highly active in the metabolism of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), a nicotine-derived carcinogen. In the present study, CYP2A13-catalyzed NNK metabolism was found to be inhibited competitively by nicotine and N'-nitrosonornicotine (NNN), suggesting that both nicotine and NNN are also substrates of CYP2A13. We have further demonstrated that human CYP2A13 is indeed an efficient enzyme in catalyzing C-oxidation of nicotine to form cotinine, with the apparent K(m) and V(max) values of 20.2 microM and 8.7 pmol/min/pmol, respectively. CYP2A13 also catalyzes the 3'-hydroxylation of cotinine to form trans-3'-hydroxycotinine, with the apparent K(m) and V(max) values of 45.2 microM and 0.7 pmol/min/pmol, respectively. The importance of CYP2A13-catalyzed nicotine and cotinine metabolism in vivo remains to be determined.

Metabolic and biochemical issues in the molecular epidemiology of cancer.

In this chapter, we highlight the significance of some metabolic and biochemical issues in molecular epidemiology. We discuss biomarkers and biologically reactive intermediates and describe the significance of polymorphisms in xenobiotic metabolizing enzymes, with emphasis on the role of some of these enzymes in the generation of reactive metabolites and on the related issue of susceptibility biomarkers. In the final section, we have selected an example of a specific compound, benzene, for which a variety of different biomarkers have been studied.

Principles of safety, efficacy and stability in gene therapy--review.

Somatic gene therapy based on nonviral and viral vectors is an attractive approach for treatment of human diseases. It has developed from preliminary animal experiments to clinical trials. Vectors and target genes used in gene therapy are mainly focused on viral, nonviral vector and single target gene or monogene and each vector system has a series of advantages and limitations. However, the application of gene therapy to human disease is currently hampered by potential hazards of methods of gene delivery, the relatively low efficiency and intracellular stability of target gene. Safety, efficacy and stability are three problems that people often met and also three principles that people should be followed in gene therapy.

Lipopolysaccharide-mediated modulation of cytochromes P450 in Stat1 null mice.

Signal transducer and activator of transcription (Stat), a family of transcriptional factors, has been demonstrated to play a critical role in gene regulation in response to inflammatory cytokines, such as interferon and interleukin-6. Inflammatory cytokines and bacterial endotoxin are known to suppress, in most of cases, the constitutive or induced cytochromes P450 (P450) in animals and humans. However, it is not clear if the suppression of P450 by cytokines is through the Stat-signaling pathway. In the present study, we determined whether Stat1 is involved in lipopolysaccharide (LPS)-mediated modulation of P450 in mouse liver. In both Stat1(+/+) (wild type) and Stat1(-/-) (null) mice, a single dose of LPS treatment (1 mg/kg of body weight, i.p.) significantly reduced the expression of CYP3A11, 2C29, and 1A2 mRNA to 8 to 40% of the control levels as determined by real-time quantitative reverse transcription-polymerase chain reaction. The reduction was supported by Western blot analysis. In contrast, LPS significantly induced the level of CYP4A10 mRNA in both Stat1(+/+) (338% of control) and Stat1(-/-) mice (264% of control). Although suppression of mRNA levels of CYP2E1, and 2D9 was not observed in either LPS-treated Stat1 null or wild-type animals, LPS treatment resulted in a reduction of CYP2E1 protein content, which was more significant in Stat1(+/+) (23% of control) than in Stat1(-/-) mice (67% of control). Consistent with this result, the chlorzoxazone 6-hydroxylase and lauric acid 11-hydroxylase activities, as CYP2E1 representative activities, were reduced markedly by LPS in Stat1(+/+) but not in Stat1(-/-) mice. The ethoxyresorufin O-deethylase activity, as a representative CYP1A activity, was also reduced significantly only in LPS-treated Stat1(+/+) mice. These data clearly demonstrate that LPS-mediated modulation of CYP3A11, 2B10, 2C29, 1A2, and 4A10 in mouse liver is Stat1-independent. However, the significant difference between the LPS-treated Stat1(+/+) and Stat1(-/-) mice in the levels of CYP2E1 protein and activity as well as in the activity level of CYP1A suggests that Stat1 may be indirectly involved in the post-transcriptional modulation of these two mouse P450 enzymes.

Identification of critical amino acid residues of human CYP2A13 for the metabolic activation of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, a tobacco-specific carcinogen.

Among all the known human cytochrome P450 enzymes, CYP2A13 has the highest efficiency in catalyzing the metabolic activation (keto aldehyde and keto alcohol formation) of the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), a potent lung carcinogen in animals and a suspected human lung carcinogen. As part of the structure-activity relationship (SAR) study, the present work was done to identify the key amino acid residues in CYP2A13 that are responsible for this high catalytic efficiency by using a series of mutants (Ala117Val, His164Gly, Ser208Ile, His372Arg, and Pro465Ser). In these CYP2A13 mutants, the amino acid residues were substituted by the residues at the corresponding positions of CYP2A6, which shares 93.5% amino acid sequence identity with CYP2A13 but is significantly less active (<5%) than CYP2A13 in NNK alpha-hydroxylation. We demonstrated that, except for the His164Gly mutant, all the CYP2A13 mutant proteins showed a significant decrease in the catalytic efficiency (Vmax/Km) for NNK alpha-hydroxylation. The His372 to Arg substitution resulted in a 20-fold increase in the Km value and a 7-fold decrease in the Vmax value for keto aldehyde formation as well as a total loss of detectable keto alcohol formation. The Ala117 to Val substitution, however, only caused a selective decrease in the Vmax value for keto aldehyde formation. The role of these amino acid residues in CYP2A13-catalyzed reactions is clearly substrate-dependent, since the same Ala117Val and His372Arg mutants showed a 9-fold increase in the catalytic efficiency for coumarin 7-hydroxylation. Together with the computational substrate docking, our study provides new SAR in formation of human CYP2A13.