RESUMO
This study was designed to assess the ameliorative effects of eugenol and to propose the possible mechanisms of action of eugenol in diethylnitrosamine (DENA)/acetylaminofluorene (AAF)-caused lung cancer in Wistar rats. To induce lung cancer, DENA at a dose of 150 mg/kg body weight (b.wt) for 2 weeks were intraperitoneally injected once each week and AAF was administered orally at a dose of 20 mg/kg b.wt. four times each week for the next 3 weeks. DENA/AAF-administered rats were orally supplemented with eugenol at a dose of 20 mg/kg b.wt administered once a day until 17 weeks starting from the 1st week of DENA administration. Lung histological lesions, including sheets of tumor cells, micropapillary adenocarcinoma, and apoptotic cells, resulting from the DENA/AAF dosage, were ameliorated by eugenol treatment. However, a significant drop in the levels of LPO in the lungs and a remarkable rise in GSH content and GPx and SOD activities were observed in DENA/AAF-administered rats treated with eugenol compared with those in DENA/AAF-administered controls. Moreover, in DENA/AAF-administered rats, eugenol supplementation significantly reduced TNF-α and IL-1ß levels and mRNA expression levels of NF-κB, NF-κB p65, and MCP-1 but significantly elevated the level of Nrf2. Furthermore, the DENA/AAF-administered rats treated with eugenol exhibited a significant downregulation of Bcl-2 expression levels in addition to a significant upregulation in P53 and Bax expression levels. Otherwise, the administration of DENA/AAF elevated the protein expression level of Ki-67, and this elevation was reversed by eugenol treatment. In conclusion, eugenol has effective antioxidant, anti-inflammatory, proapoptotic, and antiproliferative properties against lung cancer.
Assuntos
Anticarcinógenos , Neoplasias Hepáticas Experimentais , Neoplasias Pulmonares , Ratos , Animais , Ratos Wistar , Anticarcinógenos/farmacologia , Anticarcinógenos/uso terapêutico , 2-Acetilaminofluoreno/efeitos adversos , 2-Acetilaminofluoreno/metabolismo , Dietilnitrosamina/toxicidade , Dietilnitrosamina/metabolismo , Eugenol/efeitos adversos , NF-kappa B/genética , NF-kappa B/metabolismo , Apoptose , Neoplasias Pulmonares/induzido quimicamente , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/metabolismo , Fígado/patologia , Neoplasias Hepáticas Experimentais/induzido quimicamente , Neoplasias Hepáticas Experimentais/metabolismo , Neoplasias Hepáticas Experimentais/patologiaRESUMO
This study was designed to assess the preventive effects and to suggest the probable mechanisms of action of quercetin and naringein in diethylnitrosamine (DEN)/2-acetylaminofluorene (2AAF)-induced hepatocarcinogenesis in Wistar male rats. The chemical-induction of hepatocarcinogenesis was performed by injection of DEN intraperitoneally at 150 mg/kg body weight (b.w.) twice/week for two weeks, followed by oral administration of 2AAF at 20 mg/kg body weight (b.w.) 4 times/week for 3 weeks. The DEN/2AAF-administered rats were co-treated with quercetin and naringenin at dose level of 10 mg/kg b. w. by oral gavage for 20 weeks. The treatment of DEN/2AAF-administered rats with quercetin and naringenin significantly prevented the elevations in serum levels of liver function indicators (ALT, AST, ALP, γ-GT, total bilirubin and albumin) and liver tumor biomarkers including AFP, CEA and CA19.9. The cancerous histological lesions and inflammatory cells infiltration in liver of DEN/2AAF-administered rats were remarkably suppressed by treatments with quercetin and naringenin. The hepatic oxidative stress markers including NO level and lipid peroxidation significantly decreased while the SOD, GPx and CAT activities and GSH content significantly increased in DEN/2AAF-administered rats treated with quercetin and naringenin when compared to DEN/2AFF-administered control rats. Furthermore, the lowered mRNA expression of liver IL-4, P53 and Bcl-2 in of DEN/2AAF-administered rats were significantly counteracted by treatment with quercetin and naringenin. Taken together, our results demonstrate that quercetin and naringenin may abate hepatocarcinogenesis via enhancement of anti-inflammatory, anti-oxidant and apoptotic actions.
Assuntos
2-Acetilaminofluoreno , Dietilnitrosamina , 2-Acetilaminofluoreno/metabolismo , 2-Acetilaminofluoreno/toxicidade , Animais , Apoptose , Dietilnitrosamina/toxicidade , Flavanonas , Inflamação , Fígado/metabolismo , Masculino , Estresse Oxidativo , Quercetina/farmacologia , Ratos , Ratos WistarRESUMO
N-acetyl-2-aminofluorene (AAF) is a procarcinogen used widely in physiological investigations of chemical hepatocarcinogenesis. Its metabolic pathways have been described extensively, yet little is known about its biochemical processing, growth cycle expression, and pharmacological properties inside living hepatocytes-the principal cellular targets of this hepatocarcinogen. In this report, primary monolayer adult rat hepatocyte cultures and high specific-activity [ring G-3 H]-N-acetyl-2-aminofluorene were used to extend previous observations of metabolic activation of AAF by highly differentiated, proliferation-competent hepatocytes in long-term cultures. AAF metabolism proceeded by zero-order kinetics. Hepatocytes processed significant amounts of procarcinogen (≈12 µg AAF/106 cells/day). Five ring-hydroxylated and one deacetylated species of AAF were secreted into the culture media. Extracellular metabolite levels varied during the growth cycle (days 0-13), but their rank quantitative order was time invariant: 5-OH-AAF > 7-OH-AAF > 3-OH-AAF > N-OH-AAF > aminofluorene (AF) > 1-OH-AAF. Lineweaver-Burk analyses revealed two principal classes of metabolism: System I (high-affinity and low-velocity), Km[APPARENT] = 1.64 × 10-7 M and VMAX[APPARENT] = 0.1 nmol/106 cells/day and System II (low-affinity and high-velocity), Km[APPARENT] = 3.25 × 10-5 M and VMAX[APPARENT] = 1000 nmol/106 cells/day. A third system of metabolism of AAF to AF, with Km[APPARENT] and VMAX[APPARENT] constants of 9.6 × 10-5 M and 4.7 nmol/106 cells/day, was also observed. Evidence provided in this report and its companion paper suggests selective roles and intracellular locations for System I- and System II-mediated AAF metabolite formation during hepatocarcinogenesis, although some of the molecules and mechanisms responsible for multi-system processing remain to be fully defined.
Assuntos
2-Acetilaminofluoreno/metabolismo , Carcinógenos/metabolismo , Hepatócitos/metabolismo , Fígado/metabolismo , Animais , Autorradiografia , Células Cultivadas , Meios de Cultura/metabolismo , Cinética , Masculino , Cultura Primária de Células , Ratos Endogâmicos F344RESUMO
Long-term cultures of primary adult rat hepatocytes were used to study the effects of N-acetyl-2-aminofluorene (AAF) on hepatocyte proliferation during the growth cycle; on the initiation of hepatocyte DNA synthesis in quiescent cultures; and, on hepatocyte DNA replication following the initiation of DNA synthesis. Scatchard analyses were used to identify the pharmacologic properties of radiolabeled AAF metabolite binding to hepatocyte macromolecules. Two classes of growth cycle-dependent AAF metabolite binding sites-a high-affinity low-capacity site (designated Site I) and a low-affinity high-capacity site (designated Site II)-associated with two spatially distinct classes of macromolecular targets, were revealed. Based upon radiolabeled AAF metabolite binding to purified hepatocyte genomic DNA or to DNA, RNA, proteins, and lipids from isolated nuclei, Site IDAY 4 targets (KD[APPARENT] ≈ 2-4×10-6 M and BMAX[APPARENT] ≈ 6 pmol/106 cells/24 h) were consistent with genomic DNA; and with AAF metabolized by a nuclear cytochrome P450. Based upon radiolabeled AAF binding to total cellular lysates, Site IIDAY 4 targets (KD[APPARENT] ≈ 1.5×10-3 M and BMAX[APPARENT] ≈ 350 pmol/106 cells/24 h) were consistent with cytoplasmic proteins; and with AAF metabolized by cytoplasmic cytochrome P450s. DNA synthesis was not inhibited by concentrations of AAF that saturated DNA binding in the neighborhood of the Site I KD. Instead, hepatocyte DNA synthesis inhibition required higher concentrations of AAF approaching the Site II KD. These observations raise the possibility that carcinogenic DNA adducts derived from AAF metabolites form below concentrations of AAF that inhibit replicative and repair DNA synthesis.
Assuntos
2-Acetilaminofluoreno/metabolismo , Carcinógenos/metabolismo , Proliferação de Células/efeitos dos fármacos , Replicação do DNA/efeitos dos fármacos , DNA/biossíntese , Hepatócitos/metabolismo , Animais , Autorradiografia , Sítios de Ligação , Células Cultivadas , Hepatócitos/patologia , Cultura Primária de Células , Ratos Endogâmicos F344 , Ratos Sprague-DawleyRESUMO
Nucleotide excision repair (NER) is responsible for the removal of a large variety of structurally diverse DNA lesions. Mutations of the involved proteins cause the xeroderma pigmentosum (XP) cancer predisposition syndrome. Although the general mechanism of the NER process is well studied, the function of the XPA protein, which is of central importance for successful NER, has remained enigmatic. It is known, that XPA binds kinked DNA structures and that it interacts also with DNA duplexes containing certain lesions, but the mechanism of interactions is unknown. Here we present two crystal structures of the DNA binding domain (DBD) of the yeast XPA homolog Rad14 bound to DNA with either a cisplatin lesion (1,2-GG) or an acetylaminofluorene adduct (AAF-dG). In the structures, we see that two Rad14 molecules bind to the duplex, which induces DNA melting of the duplex remote from the lesion. Each monomer interrogates the duplex with a ß-hairpin, which creates a 13mer duplex recognition motif additionally characterized by a sharp 70° DNA kink at the position of the lesion. Although the 1,2-GG lesion stabilizes the kink due to the covalent fixation of the crosslinked dG bases at a 90° angle, the AAF-dG fully intercalates into the duplex to stabilize the kinked structure.
Assuntos
Dano ao DNA , Enzimas Reparadoras do DNA/química , Reparo do DNA , Proteínas de Saccharomyces cerevisiae/química , 2-Acetilaminofluoreno/química , 2-Acetilaminofluoreno/metabolismo , Sequência de Aminoácidos , Cisplatino/química , Cisplatino/metabolismo , Cristalografia por Raios X , Enzimas Reparadoras do DNA/genética , Enzimas Reparadoras do DNA/metabolismo , DNA Fúngico/química , DNA Fúngico/genética , DNA Fúngico/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Desnaturação de Ácido Nucleico , Ligação Proteica , Estrutura Terciária de Proteína , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Homologia de Sequência de Aminoácidos , Termodinâmica , Temperatura de TransiçãoRESUMO
DNA lesion bypass polymerases process different lesions with varying fidelities, but the structural, dynamic, and mechanistic origins of this phenomenon remain poorly understood. Human DNA polymerase κ (Polκ), a member of the Y family of lesion bypass polymerases, is specialized to bypass bulky DNA minor groove lesions in a predominantly error-free manner, by housing them in its unique gap. We have investigated the role of the unique Polκ gap and N-clasp structural features in the fidelity of minor groove lesion processing with extensive molecular modeling and molecular dynamics simulations to pinpoint their functioning in lesion bypass. Here we consider the N(2)-dG covalent adduct derived from the carcinogenic aromatic amine, 2-acetylaminofluorene (dG-N(2)-AAF), that is produced via the combustion of kerosene and diesel fuel. Our simulations reveal how the spacious gap directionally accommodates the lesion aromatic ring system as it transits through the stages of incorporation of the predominant correct partner dCTP opposite the damaged guanine, with preservation of local active site organization for nucleotidyl transfer. Furthermore, flexibility in Polκ's N-clasp facilitates the significant misincorporation of dTTP opposite dG-N(2)-AAF via wobble pairing. Notably, we show that N-clasp flexibility depends on lesion topology, being markedly reduced in the case of the benzo[a]pyrene-derived major adduct to N(2)-dG, whose bypass by Polκ is nearly error-free. Thus, our studies reveal how Polκ's unique structural and dynamic properties can regulate its bypass fidelity of polycyclic aromatic lesions and how the fidelity is impacted by lesion structures.
Assuntos
DNA Polimerase Dirigida por DNA/química , DNA Polimerase Dirigida por DNA/metabolismo , 2-Acetilaminofluoreno/análogos & derivados , 2-Acetilaminofluoreno/química , 2-Acetilaminofluoreno/metabolismo , Pareamento Incorreto de Bases , Domínio Catalítico , Adutos de DNA/química , Adutos de DNA/metabolismo , Desoxiguanosina/análogos & derivados , Desoxiguanosina/química , Desoxiguanosina/metabolismo , Humanos , Modelos Moleculares , Simulação de Dinâmica Molecular , Conformação Proteica , Especificidade por SubstratoRESUMO
2-Acetylaminofluorene (2-AAF) is a carcinogenic and mutagenic derivative of fluorene. It is used as a biochemical tool in the study of carcinogenesis. Studies have shown that it induces tumors in a number of species in the liver, bladder, and kidney. It is thought that 2-AAF-DNA adduct formation leads to mutation, and eventually tumor formation. The aim of this study was to examine the interactions of AAF with calf-thymus DNA in aqueous solution at physiological conditions, using constant DNA concentration (12.5 mM) and various AAF/polynucleotide (phosphate) ratios of 1/120, 1/80, 1/40, 1/20, 1/10, 1/5, 1/2, and 1/1. Fourier transform infrared and UV-visible spectroscopic methods and molecular modeling were used to determine the ligand binding mode, the binding constant, and the stability of AAF-DNA complexes in aqueous solution. Spectroscopic evidence showed both intercalation and external binding of AAF to DNA with an overall binding constant of K(AAF-DNA) = 2.33 × 10(7) M(-1). 2-AAF induced a partial B to A-DNA transition and DNA aggregation was observed at high AAF content.
Assuntos
2-Acetilaminofluoreno/metabolismo , Carcinógenos/metabolismo , DNA/metabolismo , 2-Acetilaminofluoreno/farmacologia , Animais , Sequência de Bases , Carcinógenos/farmacologia , Bovinos , DNA/química , DNA/genética , Modelos Moleculares , Conformação de Ácido Nucleico/efeitos dos fármacos , Espectrofotometria Ultravioleta , Espectroscopia de Infravermelho com Transformada de FourierRESUMO
Earlier studies conducted by our laboratory have shown that suppression of transforming growth factor-beta (TGFbeta)-mediated upregulation of connective tissue growth factor (CTGF) by iloprost resulted in a greatly diminished oval cell response to 2-acetylaminofluorene/partial hepatectomy (2AAF/PH) in rats. We hypothesized that this effect is due to decreased activation of hepatic stellate cells. To test this hypothesis, we maintained rats on a diet supplemented with 2% L-cysteine as a means of inhibiting stellate cell activation during the oval cell response to 2AAF/PH. In vitro experiments show that L-cysteine did, indeed, prevent the activation of stellate cells while exerting no direct effect on oval cells. Desmin immunostaining of liver sections from 2AAF/PH animals indicated that maintenance on the L-cysteine diet resulted in an 11.1-fold decrease in the number of activated stellate cells within the periportal zones. The total number of cells proliferating in the periportal zones of livers from animals treated with L-cysteine was drastically reduced. Further analyses showed a greater than fourfold decrease in the magnitude of the oval cell response in animals maintained on the L-cysteine diet as determined by immunostaining for both OV6 and alpha-fetoprotein (AFP). Global liver expression of AFP as measured by real-time PCR was shown to be decreased 4.7-fold in the L-cysteine-treated animals. These data indicate that the activation of hepatic stellate cells is required for an appropriate oval cell response to 2AAF/PH.
Assuntos
Células Estreladas do Fígado/fisiologia , Regeneração Hepática/fisiologia , Células-Tronco/metabolismo , 2-Acetilaminofluoreno/metabolismo , 2-Acetilaminofluoreno/farmacologia , Animais , Fator de Crescimento do Tecido Conjuntivo , Cisteína/metabolismo , Cisteína/farmacologia , Hepatectomia , Fígado/citologia , Fígado/efeitos dos fármacos , Fígado/metabolismo , Hepatopatias/metabolismo , Regeneração Hepática/efeitos dos fármacos , Masculino , Ratos , Ratos Endogâmicos F344 , Fator de Crescimento Transformador beta/metabolismo , Fator de Crescimento Transformador beta/farmacologia , alfa-Fetoproteínas/metabolismo , alfa-Fetoproteínas/farmacologiaRESUMO
In the investigation of oligonucleotides, DNA and their adducts by LC-MS, a myriad of data are generated that make manual data processing quite difficult. This paper describes a 'reversed pseudo-combinatorial' approach for fragment identification and the software implementation of this approach. Combinatorial isomer libraries are generated in silico to represent the digestion products of oligonucleotides, DNA or DNA adducts of various sizes. The software automatically calculates ion masses of each isomeric segment of the library, searches for them in complicated LC-MS data, lists their intensities and plots extracted ion chromatograms (EIC). This customized new data analysis tool has enabled a study of the enzymatic behavior of a nuclease system in the digestion of normal and adducted DNA, and in the recognition of oligomers containing a carcinogen bound to a nucleobase. The software program potentially can be further expanded to postulate unknown DNA sequences and recognize the adduction sites.
Assuntos
DNA/análise , Espectrometria de Massas/métodos , Oligonucleotídeos/análise , Software , 2-Acetilaminofluoreno/metabolismo , Acetoxiacetilaminofluoreno/metabolismo , Fosfatase Alcalina/metabolismo , Sequência de Bases , Cromatografia Líquida de Alta Pressão/métodos , DNA/metabolismo , Endodesoxirribonucleases/metabolismo , Endorribonucleases/metabolismo , Dados de Sequência Molecular , Oligonucleotídeos/metabolismoRESUMO
AIM: To elucidate the anticancer mechanism of Huqi San by assessing the expression of G-6-Pase, SDH, ATPase and AFP in N-nitrosodiethylamine-mediated hepatocarcinogenesis in rats. METHODS: A Solt-Farber two-step test model of hepatocarcino genesis was established by diethylnitrosamine (DEN) and 2-acetylaminofluorene (AAF) in rats to investigate the modifying effects of expression of 6-glucosephosphatase (G-6-Pase), succinodehydrogenase (SDH), adenosine triphosphatase (ATPase) in N-nitrosodiethylamine-mediated hepatocarcinogenesis. Hu Qisan compounded by eight medicinal herbs was prepared in glycoprival granules with wich 0.38 g crude herbs/mL solution was prepared for administration. gamma-Glutamy-transpeptidase (gamma-GT), G-6-Pase, SDH and ATPase were immunohistochemically determined. The activity of alpha-fetoglobulin (AFP) in the livers was measured with Immunofluorescence. RESULTS: Huqi San treated rats showed significant decrease in areas of gamma-GT positive foci (P< 0.001). On the other hand, the expression of G-6-Pase, SDH and ATPase has obviously altered in Huqi San treated group. The activity of AFP also significantly decreased after the treatment with Huqi San (8 g/kg body weight or 4 g/kg body weight) or total alkali of mistletoe (0.12 g/kg body weight). CONCLUSIONS: Huqi San can obviously increase these activities of G-6-Pase, SDH and ATPase, and at the same time significantly decrease the expression of gamma-GT and AFP. Therefore, it can obstruct or inhibit the rat's liver preneolastic lesion induced by DEN.
Assuntos
Dietilnitrosamina/farmacologia , Medicamentos de Ervas Chinesas/farmacologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Neoplasias Hepáticas Experimentais/enzimologia , 2-Acetilaminofluoreno/metabolismo , Adenosina Trifosfatases , Animais , Medicamentos de Ervas Chinesas/uso terapêutico , Metabolismo Energético/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Glucose-6-Fosfatase/metabolismo , Fígado/efeitos dos fármacos , Fígado/enzimologia , Fígado/metabolismo , Neoplasias Hepáticas Experimentais/induzido quimicamente , Neoplasias Hepáticas Experimentais/tratamento farmacológico , Masculino , Ratos , Ratos Wistar , Succinato Desidrogenase/metabolismo , gama-GlutamiltransferaseRESUMO
The report from the 2002 International Workshop on Genotoxicity Tests (IWGT) Strategy Expert Group emphasized metabolic considerations as an important area to address in developing a common strategy for genotoxicity testing. A working group convened at the 2005 4th IWGT to discuss this area further and propose practical strategy recommendations. To propose a strategy, the working group reviewed: (1) the current status and deficiencies, including examples of carcinogens "missed" in genotoxicity testing, established shortcomings of the standard in vitro induced S9 activation system and drug metabolite case examples; (2) the current status of possible remedies, including alternative S9 sources, other external metabolism systems or genetically engineered test systems; (3) any existing positions or guidance. The working group established consensus principles to guide strategy development. Thus, a human metabolite of interest should be represented in genotoxicity and carcinogenicity testing, including evaluation of alternative genotoxicity in vitro metabolic activation or test systems, and the selection of a carcinogenicity test species showing appropriate biotransformation. Appropriate action triggers need to be defined based on the extent of human exposure, considering any structural knowledge of the metabolite, and when genotoxicity is observed upon in vitro testing in the presence of metabolic activation. These triggers also need to be considered in defining the timing of human pharmaceutical ADME assessments. The working group proposed two strategies to consider; a more proactive approach, which emphasizes early metabolism predictions to drive appropriate hazard assessment; and a retroactive approach to manage safety risks of a unique or "major" metabolite once identified and quantitated from human clinical ADME studies. In both strategies, the assessment of the genotoxic potential of a metabolite could include the use of an alternative or optimized in vitro metabolic activation system, or direct testing of an isolated or synthesized metabolite. The working group also identified specific areas where more data or experiences need to be gained to reach consensus. These included defining a discrete exposure action trigger for safety assessment and when direct testing of a metabolite of interest is warranted versus the use of an alternative in vitro activation system, a universal recommendation for the timing of human ADME studies for drug candidates and the positioning of metabolite structural knowledge (through in silico systems, literature, expert analysis) in supporting metabolite safety qualification. Lastly, the working group outlined future considerations for refining the initially proposed strategies. These included the need for further evaluation of the current in vitro genotoxicity testing protocols that can potentially perturb or reduce the level of metabolic activity (potential alterations in metabolism associated with both the use of some solvents to solubilize test chemicals and testing to the guidance limit dose), and proposing broader evaluations of alternative metabolic activation sources or engineered test systems to further challenge the suitability of (or replace) the current induced liver S9 activation source.
Assuntos
Redes e Vias Metabólicas , Testes de Mutagenicidade/métodos , 2-Acetilaminofluoreno/metabolismo , 2-Acetilaminofluoreno/toxicidade , Animais , Carcinógenos/toxicidade , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Indústria Farmacêutica , Enzimas/química , Guias como Assunto , Humanos , Fígado/metabolismo , Testes de Mutagenicidade/normas , Testes de Mutagenicidade/tendências , Extratos Vegetais/metabolismo , Proteínas Recombinantes/efeitos dos fármacos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Solventes/química , Estados Unidos , United States Food and Drug AdministrationRESUMO
Despite extensive studies, the hematopoietic versus hepatic origin of liver progenitor oval cells remains controversial. The aim of this study was to determine the origin of such cells after liver injury and to establish an oval cell line. Rat liver injury was induced by subcutaneous insertion of 2-AAF pellets for 7 days with subsequent injection of CCl(4). Livers were removed 9 to 13 days post-CCl(4) treatment. Immunohistochemistry was performed using anti-c-kit, OV6, Thy1, CK19, AFP, vWF and Rab3b. Isolated non-parenchymal cells were grown on mouse embryonic fibroblast, and their gene expression profile was characterized by RT-PCR. We identified a subpopulation of OV6/CK19/Rab3b-expressing cells that was activated in the periportal region of traumatized livers. We also characterized a second subpopulation that expressed the HSCs marker c-kit but not Thy1. Although we successfully isolated both cell types, OV6/CK19/Rab3b(+) cells fail to propagate while c-kit(+)-HSCs appeared to proliferate for up to 7 weeks. Cells formed clusters which expressed c-kit, Thy1 and albumin. Our results indicate that a bona fide oval progenitor cell population resides within the liver and is distinct from c-kit(+)-HSCs. Oval cells require the hepatic niche to proliferate, while cells mobilized from the circulation proliferate and transdifferentiate into hepatocytes without evidence of cell fusion.
Assuntos
Células-Tronco Hematopoéticas/citologia , Hepatócitos/citologia , Regeneração Hepática , Fígado/citologia , Células-Tronco/citologia , 2-Acetilaminofluoreno/metabolismo , 2-Acetilaminofluoreno/farmacologia , Animais , Antígenos de Diferenciação/metabolismo , Medula Óssea/metabolismo , Tetracloreto de Carbono/farmacologia , Células Cultivadas , Feminino , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/metabolismo , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Imuno-Histoquímica , Fígado/efeitos dos fármacos , Fígado/crescimento & desenvolvimento , Masculino , Camundongos , Ratos , Ratos Sprague-Dawley , Células-Tronco/metabolismo , Antígenos Thy-1/imunologia , Antígenos Thy-1/metabolismoRESUMO
The activation of ketoprofen, which inhibits the outgrowth of azoxymethane-induced aberrant crypt foci in the rat colon, on the inhibition of arylamine N-acetyltransferase (NAT) activity (N-acetylation of substrates), gene expression (mRNA NAT) and 2-aminofluorene (AF)-DNA adduct formation was studied in a human colon tumor (adenocarcinoma) cell line (colo 205). Cellular cytosols (9000 xg supernatant) and intact colon tumor cells were used. The NAT activity in colo 205 cells was inhibited by ketoprofen in a dose- and time -dependent manner in both examined systems. The data also indicated that ketoprofen decreased the apparent value of V(max) of NAT enzymes, being a competitive inhibitor of NAT enzymes. The AF-DNA adduct formation in colo 205 cells was also decreased by ketoprofen. Based on the results from PCR, it was shown that ketoprofen affected mRNA NAT expression in human colon colo 205 cells. The cells were stained with anti-NAT antibody, then analyzed by flow cytometry. The results showed that ketoprofen decreased the percentage of cells stained by anti-NAT. This report is the first to demonstrate that ketoprofen inhibits human colon tumor cell NAT activity, gene expression and DNA adduct formation.
Assuntos
Arilamina N-Acetiltransferase/antagonistas & inibidores , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/enzimologia , Cetoprofeno/farmacologia , 2-Acetilaminofluoreno/análogos & derivados , 2-Acetilaminofluoreno/metabolismo , Ácido 4-Aminobenzoico/metabolismo , Acetilação/efeitos dos fármacos , Arilamina N-Acetiltransferase/metabolismo , Linhagem Celular Tumoral , Neoplasias do Colo/genética , Neoplasias do Colo/metabolismo , Citosol/enzimologia , Adutos de DNA/biossíntese , Relação Dose-Resposta a Droga , Fluorenos , Expressão Gênica/efeitos dos fármacos , Humanos , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , para-AminobenzoatosRESUMO
Arylamine carcinogens and drugs are N-acetylated by cytosolic N-acetyltransferase (NAT), which uses acetyl-coenzyme A as a cofactor. NAT plays an initial role in the metabolism of these arylamine compounds. 2-Aminofluorene is one of the arylamine carcinogens which have been demonstrated to undergo N-acetylation in laboratory animals and humans. Our previous study showed that human cancer cell lines (colon cancer, colo 205; liver cancer, Hep G2; bladder cancer, T24; leukemia, HL-60; prostate cancer, LNCaP; osteogenic sarcoma, U-2 OS; malignant melanoma, A375.S2) displayed NAT activity, which was affected by aloe-emodin in human leukemia cells. The purpose of this study was to determine whether aloe-emodin could affect the enzyme activity and gene expression of NAT at the mRNA and protein levels in malignant human melanoma A375.S2 cells. The results showed that aloe-emodin inhibited NAT1 activity (decreased N-acetylation of 2-aminofluorene) in intact cells in a dose-dependent manner. The effect of aloe-emodin on NAT1 at the protein level was determined by Western blotting and the mRNA levels were examined by polymerase chain reaction (PCR) and cDNA microarray. These results clearly indicate that aloe-emodin inhibits the mRNA expression and enzyme activity of NAT1 in A375.S2 cells.
Assuntos
Arilamina N-Acetiltransferase/antagonistas & inibidores , Emodina/farmacologia , Inibidores Enzimáticos/farmacologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Isoenzimas/antagonistas & inibidores , Melanoma/enzimologia , 2-Acetilaminofluoreno/metabolismo , Antraquinonas , Arilamina N-Acetiltransferase/biossíntese , Arilamina N-Acetiltransferase/genética , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Fluorenos/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Isoenzimas/biossíntese , Isoenzimas/genética , Melanoma/genética , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
BACKGROUND: It has been shown that cytochrome P450 enzymes (CYPs) and acetyltransferase can be used as biomarkers of carcinogen-DNA adduct levels and human cancer susceptibility. The gastrointestinal tract is the portal of entry of foreign compounds and presents xenobiotic metabolizing N-acetyltransferase (NAT) and CYPs activities. 5-Methoxypsoralen (5-MOP) has been used in combination with UV radiation in skin photochemotherapy for decades. A number of studies have demonstrated that 5-MOP is inhibitory towards mouse and human CYP isoforms, but investigations on the direct effects on NAT activity in laboratory animals and human cancer cells are limited. The main objective of this study was to document the effects of 5-MOP on the modulation of NAT activities in the stomach and colon of rats and human stomach and colon tumor cell lines. MATERIALS AND METHODS: N-Acetylation of 2-aminofluorene (AF) to 2-acetylaminofluorene (AAF) by NAT in the stomach and colon of Sprague-Dawley (SD) rats and in human stomach (SC-M1) and colon (COLO 205) tumor cell lines was investigated. RESULTS: The data show that the metabolic activity of NAT in the rat colon was higher than that in the rat stomach, and the further metabolism of AAF was slower in the stomach than in the colon. 5-MOP increased the activity of NATand also increased the further metabolism of AAF at 24 h in the rat stomach. In the rat colon, no statistically significant changes caused by 5-MOP were observed in NAT activity, but 5-MOP increased the further metabolism of AAF at 24 to 72 h. 5-MOP decreased the activity of NAT only at 72-h incubation in SC-M1 cells. In COLO 205 cells, however, 5-MOP decreased the activity of NAT between 24 h and 72 h. The optimal concentrations of 5-MOP to induce decreased NAT activity in SC-M1 cells were 0.05 mM to 25 mM. In COLO 205 cells, the data indicate that the higher the concentrations of 5-MOP, the higher the acetylation of AF; a promotion effect of NAT activity occured at a higher dose (50 mM) of 5-MOP and an inhibition effect occured at lower doses (0.05-0.5 mM) of 5-MOP, while concentrations of 5-25 mM of 5-MOP showed no significant difference compared with the control regimen. CONCLUSION: The metabolic activity of NAT in the rat colon was higher than that in the rat stomach, and the results also showed a high degree of correspondence with SC-M1 cells and COLO 205 cells. 5-MOP more efficiently inhibited NAT activity in human stomach and colon tumor cell lines than in the stomach and colon of rats.
Assuntos
Arilamina N-Acetiltransferase/metabolismo , Colo/enzimologia , Neoplasias do Colo/enzimologia , Metoxaleno/análogos & derivados , Neoplasias Gástricas/enzimologia , Estômago/enzimologia , 2-Acetilaminofluoreno/metabolismo , 5-Metoxipsoraleno , Acetilação , Adenocarcinoma/patologia , Animais , Arilamina N-Acetiltransferase/análise , Linhagem Celular Tumoral , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/patologia , Citosol/química , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/administração & dosagem , Inibidores Enzimáticos/farmacologia , Fluorenos/metabolismo , Humanos , Cinética , Masculino , Metoxaleno/administração & dosagem , Metoxaleno/farmacologia , Ratos , Ratos Sprague-Dawley , Neoplasias Gástricas/tratamento farmacológico , Neoplasias Gástricas/patologiaRESUMO
It is well documented that arylamine carcinogens are N-acetylated by cytosolic N-acetyltransferase (NAT) enzyme. NAT plays an important role in the metabolizing of those arylamine compounds. 2-Aminofluorene (AF) is an arylamine carcinogen which has been demonstrated to induce carcinogenesis in laboratory animals. Our previous study has shown that a human promyelocytic leukemia cell line, HL-60, displays NAT activity. The purpose of the present study was to determine whether or not wogonin could affect the N-acetylation of AF in HL-60. N-acetylated and non-N-acetylated AF were determined by using high performance liquid chromatography. Wogonin displayed a dose-dependent inhibition of NAT activity in cytosols and intact cells. Wogonin also decreased AF-DNA adduct formation in these cells. The effects of wogonin on the NAT enzymes levels were also examined by Western blotting and flow cytometry and the changes of NAT gene expression were examined by polymerase chain reaction (PCR) and cDNA microarray. The results demonstrated that wogonin inhibited NAT1 mRNA gene expression and the level of NAT enzyme in HL-60 cells. This is the first demonstration that wogonin affects human leukemia cells' NAT activity in vitro.
Assuntos
2-Acetilaminofluoreno/análogos & derivados , Acetiltransferases/antagonistas & inibidores , Flavanonas/farmacologia , 2-Acetilaminofluoreno/metabolismo , Acetilação/efeitos dos fármacos , Acetiltransferases/genética , Acetiltransferases/metabolismo , Citosol/efeitos dos fármacos , Citosol/metabolismo , Adutos de DNA/biossíntese , Relação Dose-Resposta a Droga , Regulação para Baixo/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Fluorenos/metabolismo , Expressão Gênica/efeitos dos fármacos , Células HL-60 , Humanos , RNA Mensageiro/biossíntese , RNA Mensageiro/genéticaRESUMO
The present study was undertaken to determine the effect of acetylsalicylic acid acid on the in vitro N-acetyltransferase (NAT) enzyme activity and in vivo acetylation of 2-aminofluorene in laboratory rats. In the in vitro experiments, cytosols of blood, bladder, colon and liver cells, with or without acetylsalicylic acid co-treatment, showed different percentages of 2-aminofluorene acetylation. The data indicated that there was decreased NAT activity associated with increased acetylsalicylic acid in the cytosol reaction. In the in vitro experiments, values of apparent Km and Vmax decreased by 4% and 21%, respectively, for acetylation of AF in blood NAT, 28% and 31% for acetylation of AF in bladder NAT, 12% and 25% for acetylation of AF in colon NAT, and 50% and 35% for acetylation of AF in liver NAT. In the in vivo experiments, pretreatment with acetylsalicylic acid (50 mg/kg) 48 h prior to the administration of 2-aminofluorene (50 mg/kg) resulted in 24% and 28% decreases in the fecal and urinary recovery of N-acetyl-2-aminofluorene and a 26% decrease in the metabolic clearance of 2-aminofluorene to N-acetyl-2-aminofluorene. This is the first demonstration of acetylsalicylic acid (Aspirin) inhibition of arylamine N-acetyltransferase activity showing decreases in the N-acetylation of carcinogens in vivo.
Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Arilamina N-Acetiltransferase/metabolismo , Aspirina/farmacologia , Carcinógenos/farmacocinética , Fluorenos/farmacocinética , 2-Acetilaminofluoreno/metabolismo , Acetilação , Animais , Arilamina N-Acetiltransferase/sangue , Colo/enzimologia , Fígado/enzimologia , Masculino , Ratos , Ratos Sprague-Dawley , Bexiga Urinária/enzimologiaRESUMO
Avian embryos are a potential alternative model for chemical toxicity and carcinogenicity research. Because the toxic and carcinogenic effects of some chemicals depend on bioactivation, activities of biotransformation enzymes and formation of DNA adducts in embryonic turkey liver were examined. Biochemical analyses of 22-day in ovo turkey liver post-mitochondrial fractions revealed activities of the biotransformation enzymes 7-ethoxycoumarin de-ethylase (ECOD), 7-ethoxyresorufin de-ethylase (EROD), aldrin epoxidase (ALD), epoxide hydrolase (EH), glutathione S-transferase (GST), and UDP-glucuronyltransferase (GLUT). Following the administration of phenobarbital (24 mg/egg) on day 21, enzyme activities of ECOD, EROD, ALD, EH and GLUT, but not of GST, were increased by two-fold or higher levels by day 22. In contrast, acute administration of 3-methylcholanthrene (5 mg/egg) induced only ECOD and EROD activities. Bioactivation of structurally diverse pro-carcinogens was also examined using (32)P-postlabeling for DNA adducts. In ovo exposure of turkey embryos on day 20 of gestation to 2-acetylaminofluorene (AAF), 4,4'-methylenebis(2-chloroaniline) (MOCA), benzo[a]pyrene (BaP), and 2-amino-3,8-dimethylimidazo[4,5- f]quinoxaline (MeIQx) resulted in the formation of DNA adducts in livers collected by day 21. Some of the DNA adducts had (32)P-postlabeling chromatographic migration patterns similar to DNA adducts found in livers from Fischer F344 rats exposed to the same pro-carcinogens. We conclude that 21-day embryonic turkey liver is capable of chemical biotransformation and activation of genotoxic carcinogens to form DNA adducts. Thus, turkey embryos could be utilized to investigate potential chemical toxicity and carcinogenicity.
Assuntos
Alternativas aos Testes com Animais , Carcinógenos/metabolismo , Adutos de DNA/metabolismo , Embrião não Mamífero/enzimologia , Enzimas/metabolismo , Fígado/enzimologia , 2-Acetilaminofluoreno/metabolismo , 2-Acetilaminofluoreno/toxicidade , Animais , Benzo(a)pireno/metabolismo , Benzo(a)pireno/toxicidade , Biotransformação , Carcinógenos/toxicidade , Adutos de DNA/análise , Dano ao DNA , Relação Dose-Resposta a Droga , Embrião não Mamífero/efeitos dos fármacos , Feminino , Fígado/efeitos dos fármacos , Fígado/embriologia , Metilcolantreno/metabolismo , Metilcolantreno/toxicidade , Metilenobis (cloroanilina)/metabolismo , Metilenobis (cloroanilina)/toxicidade , Fenobarbital , Radioisótopos de Fósforo , Quinoxalinas/metabolismo , Quinoxalinas/toxicidade , Ratos , Ratos Endogâmicos F344 , Ratos Wistar , TurquiaRESUMO
Arylamine N-acetyltransferases (NATs) catalyze a variety of biotransformation reactions, including N-acetylation of arylamines and O-acetylation of arylhydroxylamines. Chemical modification of hamster recombinant NAT2 with 2-(bromoacetylamino)fluorene (Br-AAF) and bromoacetanilide revealed that Br-AAF is an affinity label for the enzyme whereas bromoacetanilide inactivates NAT2 through a bimolecular alkylation process. Electrospray ionization quadrupole time-of-flight mass spectrometry analysis of Br-AAF-treated NAT2 showed that a single molecule of 2-acetylaminofluorene had been adducted. Peptide sequencing with tandem mass spectrometry identified the catalytically essential Cys68 as the alkylated amino acid. Br-AAF exhibits similar affinity for hamster NAT1 and NAT2, but is a more effective inactivator of NAT1 because, subsequent to the formation of a reversible enzyme-Br-AAF complex, the rate of alkylation of NAT1 is greater than the rate of alkylation of NAT2. Bromoacetanilide alkylates Cys68 and, to a lesser extent, Cys237 of NAT2; it does not exhibit significant selectivity for either NAT1 or NAT2.
Assuntos
2-Acetilaminofluoreno/análogos & derivados , Arilamina N-Acetiltransferase/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Isoenzimas/antagonistas & inibidores , 2-Acetilaminofluoreno/química , 2-Acetilaminofluoreno/metabolismo , 2-Acetilaminofluoreno/farmacologia , Acetanilidas/farmacologia , Acetilação , Animais , Arilamina N-Acetiltransferase/química , Arilamina N-Acetiltransferase/metabolismo , Cricetinae , Cistina/química , Isoenzimas/química , Isoenzimas/metabolismo , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Especificidade por SubstratoRESUMO
Human epidemiological studies suggest an association between N-acetyltransferase (NAT) activity and the incidence of bladder and colorectal cancers. In this study, paclitaxel was selected to examine the inhibition of arylamine NAT activity, gene expression and 2-aminofluorene-DNA adduct formation in a human osteogenic sarcoma cell line (U-2 OS). The activity of NAT was determined by high performance liquid chromatography (HPLC) assay for the amounts of acetylated 2-aminofluorene (AF) and p-aminobenzoic acid (PABA) and nonacetylated AF and PABA. Human osteogenic sarcoma cell cytosols and intact cells were used to examine the NAT activity, gene expression and AF-DNA adduct formation. The results demonstrated that NAT activity percent of NAT in examined cells, gene expression (NAT1 mRNA) and AF-DNA adduct formation in human osteogenic sarcoma cells were inhibited and decreased by paclitaxel in a dose-dependent manner. The results also demonstrated that paclitaxel decreased the apparent values of Km and Vmax from intact human osteogenic sarcoma cells (U-2 OS). Thus, paclitaxel is an uncompetitive inhibitor of the NAT enzyme.