The worldwide human exposure to aflatoxin B 1 (AFB 1 ), particularly in developing countries, remains to be a serious public health concern. Although AFB 1 is best known as a hepatocarcinogen, epidemiological studies have shown a positive association between human lung cancer occurrence and inhalation exposure to AFB 1 . Cytochrome P450 (CYP)-catalyzed metabolic activation is required for AFB 1 to exert its carcinogenicity. Previous studies have identified CYP1A2 and CYP3A4 as the major enzymes for AFB 1 activation in human liver. However, the key CYP enzymes in human lung that can efficiently activate AFB 1 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 AFB 1 to its carcinogenic/toxic AFB 1 -8,9-epoxide and AFM 1 -8,9-epoxide at both low (15 lM) and high (150 lM) substrate concentrations. Under the same conditions, there was no detectable AFB 1 epoxide formation by CYP2A6, which was also reported to be involved in the metabolic activation of AFB 1 . Consistent with the activity data, there was an 800-fold difference in LC 50 values of AFB 1 (48-hr treatment) between Chinese hamster ovary (CHO) cells expressing CYP2A13 and CYP2A6 (50 nM versus 39 lM). We further demonstrate that amino acid residues Ala 117 and His 372 in CYP2A13 protein are important for AFB 1 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 AFB 1. ' 2005 Wiley-Liss, Inc.Key words: cytochrome P450 2A13; aflatoxin B 1 ; metabolic activation; cytotoxicity Aflatoxin B 1 (AFB 1 ), a mycotoxin produced mainly by Aspergillus flavus and Aspergillus parasitcus, is a potent carcinogen in humans and animals.1,2 Mutation-induced inactivation of p53 tumor suppressor gene and/or activation of K-ras oncogene has been proposed as the major molecular mechanism in AFB 1 -induced cancers.3-6 The worldwide human exposure to AFB 1 , particularly in developing countries, remains to be a serious public health problem. Since AFB 1 is a major etiological agent of human liver cancer, extensive studies have been focused on dietary exposure to AFB 1 and AFB 1 -induced liver cancer. 7,8 However, there are epidemiological evidences to suggest that human respiratory tract is also a target for AFB 1 carcinogenicity. Occupational exposure to inhaled AFB 1 , particularly in the form of contaminated grain dusts, is associated with increased respiratory cancers. 2,8-12The reported highest amount of AFB 1 in respirable grain dusts was 52 ppm. 13 Human lung is also at risk of cancer for dietary AFB 1 exposure.14 AFB 1 also induces lung tumors in laboratory animals. 15-17Metabolic activation is required for AFB 1 to exert its carcinogenicity and toxicity (Fig. 1). The major carcinogenic and mutagenic metabolites of AFB 1 are AFB 1 -8,9-epoxide and AFM 1 -8,9-epoxide, although the latter is relatively less...
ABSTRACT: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 Nnitrosonornicotine (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 M 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 M and 0.7 pmol/min/pmol, respectively. The importance of CYP2A13-catalyzed nicotine and cotinine metabolism in vivo remains to be determined.Nicotine is a major constituent of tobacco, playing a critical role in establishing and maintaining tobacco dependence (Henningfield et al., 1985). The major pathway of nicotine metabolism in humans is the C-oxidation to form cotinine, which is a two-step process. The first step is catalyzed by the cytochrome P450 (P450) system to produce the intermediate nicotine-⌬ Ϫ1Ј(5Ј) -iminium ion, which is further oxidized to cotinine by cytosolic aldehyde oxidase (Gorrod and Hibberd, 1982;Peterson et al., 1987;Williams et al., 1990). Other major metabolites of nicotine identified in human urine so far include trans-3Ј-hydroxycotinine and nicotine NЈ-oxide (Jacob et al., 1988). The conversion of cotinine to 3Ј-hydroxycotinine is highly stereospecific in vivo, with the trans isomer as a predominating product (Jacob et al., 1990). trans-3Ј-Hydroxycotinine is excreted in the urine to a much greater extent than cotinine itself. Cotinine may also be metabolized to 5Ј-hydroxycotinine, norcotinine, and cotinine N-oxide (Murphy et al., 1999).In humans, liver is the primary site of nicotine metabolism. Nicotine is also metabolized to some extent in the lung and kidney (Benowitz et al., 1987). Previous studies demonstrated that human CYP2A6 is a key enzyme for the metabolism of nicotine to cotinine, and cotinine to trans-3Ј-hydroxycotinine (Flammang et al., 1992;McCracken et al., 1992; Nakajima et al., 1996a,b;Shimada et al., 1996;Messina et al., 1997). CYP2A6 is the best characterized member in the CYP2A subfamily (Pelkonen and Raunio, 1995; FernandezSalguero and Gonzalez, 1995;Pelkonen et al., 2000). The other two members are CYP2A7 and CYP2A13, which share a high degree of sequence identity with CYP2A6 (Fernandez-Salguero and Gonzalez, 1995). Although the transcripts of these three genes were all found in the liver, CYP2A6 was more abundant than CYP2A7 and CYP2A13 (Koskela et al., 1999). The highest level of CYP2A13 mRNA was found in huma...
ABSTRACT: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.
Structure-based rational design led to the synthesis of a novel series of potent PI3K inhibitors. The optimized pyrrolopyridine analogue 63 was a potent and selective PI3Kβ/δ dual inhibitor that displayed suitable physicochemical properties and pharmacokinetic profile for animal studies. Analogue 63 was found to be efficacious in animal models of inflammation including a keyhole limpet hemocyanin (KLH) study and a collagen-induced arthritis (CIA) disease model of rheumatoid arthritis. These studies highlight the potential therapeutic value of inhibiting both the PI3Kβ and δ isoforms in the treatment of a number of inflammatory diseases.
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