RESUMO
[Figure: see text].
Assuntos
2-Metoxiestradiol/farmacologia , Angiotensina II/farmacologia , Fosfolipases A2 do Grupo IV/antagonistas & inibidores , Hipertensão/tratamento farmacológico , Ácido 5,8,11,14-Eicosatetrainoico/farmacologia , Animais , Colágeno/metabolismo , Citocromo P-450 CYP1A1/fisiologia , Feminino , Fosfolipases A2 do Grupo IV/genética , Hipertensão/induzido quimicamente , Camundongos , Espécies Reativas de Oxigênio/metabolismo , Receptores de Prostaglandina E Subtipo EP3/antagonistas & inibidores , Receptores de Prostaglandina E Subtipo EP3/fisiologiaRESUMO
SCOPE: This study investigates the mechanism of action and functional effects of coffee extracts in colonic cells, on intestinal stem cell growth, and inhibition of dextran sodium sulfate (DSS)-induced intestinal barrier damage in mice. METHODS AND RESULTS: Aqueous coffee extracts induced Ah receptor (AhR) -responsive CYP1A1, CYP1B1, and UGT1A1 gene expression in colon-derived Caco2 and YAMC cells. Tissue-specific AhR knockout (AhRf/f x Lgr5-GFP-CreERT2 x Villin-Cre), wild-type (Lgr5-CreERT2 x Villin-Cre) mice are sources of stem cell enriched organoids and both coffee extracts and norharman, an AhR-active component of these extracts inhibited stem cell growth. Coffee extracts also inhibit DSS-induced damage to intestinal barrier function and DSS-induced mucosal inflammatory genes such as IL-6 and TGF-ß1 in wild-type (AhR+/+ ) but not AhR-/- mice. In contrast, coffee does not exhibit protective effects in intestinal-specific AhR knockout mice. Coffee extracts also enhanced overall formation of AhR-active microbial metabolites. CONCLUSIONS: In colon-derived cells and in the mouse intestine, coffee induced several AhR-dependent responses including gene expression, inhibition of intestinal stem cell-enriched organoid growth, and inhibition of DSS-induced intestinal barrier damage. We conclude that the anti-inflammatory effects of coffee in the intestine are due, in part, to activation of AhR signaling.
Assuntos
Café , Colo/efeitos dos fármacos , Extratos Vegetais/farmacologia , Receptores de Hidrocarboneto Arílico/fisiologia , Animais , Células CACO-2 , Colo/metabolismo , Citocromo P-450 CYP1A1/fisiologia , Citocromo P-450 CYP1B1/fisiologia , Sulfato de Dextrana/toxicidade , Feminino , Humanos , Masculino , CamundongosRESUMO
The hydroxylase cytochrome P450 1A1 (CYP1A1) is regulated by the inflammation-limiting aryl hydrocarbon receptor (AhR), but CYP1A1 immune functions remain unclear. We observed CYP1A1 overexpression in peritoneal macrophages (PMs) isolated from mice following LPS or heat-killed Escherichia. coli (E. coli) challenge. CYP1A1 overexpression augmented TNF-α and IL-6 production in RAW264.7 cells (RAW) by enhancing JNK/AP-1 signalling. CYP1A1 overexpression also promoted 12S-hydroxy-5Z,8Z,10E,14Z-eicosatetraenoic acid (12(S)-HETE) production in activated RAW, while a 12(S)-HETE antibody attenuated and 12(S)-HETE alone induced inflammatory responses. Macrophages harbouring hydroxylase-deficient CYP1A1 demonstrated reduced 12(S)-HETE generation and LPS-induced TNF-α/IL-6 secretion. CYP1A1 overexpression also impaired phagocytosis of bacteria via decreasing the expression of scavenger receptor A (SR-A) in PMs. Mice injected with CYP1A1-overexpressing PMs were more susceptible to CLP- or E. coli-induced mortality and bacteria invading, while Rhapontigenin, a selective CYP1A1 inhibitor, improved survival and bacteria clearance of mice in sepsis. CYP1A1 and 12(S)-HETE were also elevated in monocytes and plasma of septic patients and positively correlated with SOFA scores. Macrophage CYP1A1 disruption could be a promising strategy for treating sepsis. Video abstract.
Assuntos
Citocromo P-450 CYP1A1/fisiologia , MAP Quinase Quinase 4/metabolismo , Macrófagos Peritoneais , Fagocitose , Sepse/metabolismo , Ácido 12-Hidroxi-5,8,10,14-Eicosatetraenoico/metabolismo , Adulto , Idoso , Animais , Escherichia coli , Humanos , Inflamação , Macrófagos Peritoneais/metabolismo , Macrófagos Peritoneais/microbiologia , Macrófagos Peritoneais/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Células RAW 264.7 , Adulto JovemRESUMO
The polarization of macrophages is critical to inflammation and tissue repair, with unbalanced macrophage polarization associated with critical dysfunctions of the immune system. Cytochrome P450 1A1 (CYP1A1) is a hydroxylase mainly controlled by the inflammation-limiting aryl hydrocarbon receptor (AhR), which plays a critical role in mycoplasma infection, oxidative stress injury, and cancer. Arginase-1 (Arg-1) is a surrogate for polarized alternative macrophages and is important to the production of nitric oxide (NO) by the modulation of arginine. In the present study, we found CYP1A1 to be upregulated in IL-4-stimulated mouse peritoneal macrophages (PMs) and human peripheral blood monocytes. Using CYP1A1-overexpressing RAW264.7 cells (CYP1A1/RAW) we found that CYP1A1 augmented Arg-1 expression by strengthening the activation of the JAK1/STAT6 signaling pathway in macrophages treated with IL-4. 15(S)-HETE, a metabolite of CYP1A1 hydroxylase, was elevated in IL-4-induced CYP1A1/RAW cells. Further, in macrophages, the loss-of-CYP1A1-hydroxylase activity was associated with reduced IL-4-induced Arg-1 expression due to impaired 15(S)-HETE generation. Of importance, CYP1A1 overexpressing macrophages reduced the inflammation associated with LPS-induced peritonitis. Taken together, these findings identified a novel signaling axis, CYP1A1-15(S)-HETE-JAK1-STAT6, that may be a promising target for the proper maintenance of macrophage polarization and may also be a means by which to treat immune-related disease due to macrophage dysfunction.
Assuntos
Arginase/biossíntese , Citocromo P-450 CYP1A1/fisiologia , Janus Quinase 1/antagonistas & inibidores , Macrófagos Peritoneais/efeitos dos fármacos , Peritonite/prevenção & controle , Fator de Transcrição STAT6/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Ácido 12-Hidroxi-5,8,10,14-Eicosatetraenoico/farmacologia , Transferência Adotiva , Animais , Araquidonato 15-Lipoxigenase/fisiologia , Arginase/genética , Citocromo P-450 CYP1A1/biossíntese , Citocromo P-450 CYP1A1/genética , Endotoxinas/toxicidade , Humanos , Ácidos Hidroxieicosatetraenoicos/biossíntese , Ácidos Hidroxieicosatetraenoicos/genética , Ácidos Hidroxieicosatetraenoicos/farmacologia , Interleucina-4/farmacologia , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/metabolismo , Leucócitos Mononucleares/transplante , Macrófagos Peritoneais/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Peritonite/induzido quimicamente , Células RAW 264.7 , Interferência de RNA , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Receptores Citoplasmáticos e Nucleares/biossíntese , Receptores Citoplasmáticos e Nucleares/genética , Células THP-1 , Regulação para Cima/efeitos dos fármacosRESUMO
As the Coronavirus situation (COVID-19) continues to evolve, many questions concerning the factors relating to the diffusion and severity of the disease remain unanswered.Whilst opinions regarding the weight of evidence for these risk factors, and the studies published so far are often inconclusive or offer contrasting results, the role of comorbidities in the risk of serious adverse outcomes in patients affected with COVID-19 appears to be evident since the outset. Hypertension, diabetes, and obesity are under discussion as important factors affecting the severity of disease. Air pollution has been considered to play a role in the diffusion of the virus, in the propagation of the contagion, in the severity of symptoms, and in the poor prognosis. Accumulating evidence supports the hypothesis that environmental particulate matter (PM) can trigger inflammatory responses at molecular, cellular, and organ levels, sustaining respiratory, cardiovascular, and dysmetabolic diseases.To better understand the intricate relationships among pre-existing conditions, PM, and viral infection, we examined the response at the molecular level of T47D human breast adenocarcinoma cells exposed to different fractions of PM. T47D cells express several receptors, including the aryl hydrocarbon receptor (AhR), and ACE2, the main - but not the only - receptor for SARS-CoV-2 entry.PM samples were collected in an urban background site located in the Northern area of the City of Bologna (Emilia-Romagna Region, Northern Italy) during winter 2013. T47D cells were exposed to organic or aqueous (inorganic) extracts at the final concentration of 8 m3 for a 4-hour duration. Both the concentration and the exposure time were chosen to resemble an average outdoor exposure. RNA was extracted from cells, purified and hybridised on 66k microarray slides from Agilent.The lists of differentially expressed genes in PM organic extracts were evaluated by using Metacore, and an enrichment analysis was performed to identify pathways maps, process networks, and disease by biomarkers altered after T47D treatment.The analysis of the modulated genes gave evidence for the involvement of PM in dysmetabolic diseases, including diabetes and obesity, and hypertension through the activation of the aryl hydrocarbon receptor (AhR) canonical pathway.On the basis of current knowledge, existing data, and exploratory experimental evidence, we tease out the likely molecular interplay that can ultimately tip the disease outcome into severity. Looking beyond ACE2, several additional key markers are identified. Disruption of these targets worsens pre-existing conditions and/or exacerbates the adverse effects induced by SARS-CoV-2 infection. Whilst appropriately designed, epidemiological studies are very much needed to investigate these associations based on our hypothesis of investigation, by reviewing recent experimental and epidemiological evidence, here we speculate and provide new insights on the possible role of environmental pollution in the exacerbation of effects by SARS-CoV-2 and other respiratory viruses. This work is intended to assist in the development of appropriate investigative approaches to protect public health.
Assuntos
Poluição do Ar/efeitos adversos , COVID-19/epidemiologia , Material Particulado/efeitos adversos , SARS-CoV-2/fisiologia , Enzima de Conversão de Angiotensina 2/fisiologia , COVID-19/etiologia , Linhagem Celular Tumoral , Comorbidade , Coronaviridae/fisiologia , Citocromo P-450 CYP1A1/fisiologia , Diabetes Mellitus/epidemiologia , Diabetes Mellitus/genética , Diabetes Mellitus/metabolismo , Suscetibilidade a Doenças , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Hipertensão/epidemiologia , Hipertensão/genética , Hipertensão/metabolismo , Inflamação/epidemiologia , Inflamação/genética , Inflamação/metabolismo , Itália , Obesidade/epidemiologia , Obesidade/genética , Obesidade/metabolismo , Material Particulado/farmacologia , Receptores de Hidrocarboneto Arílico/fisiologia , Receptores Virais/fisiologia , Risco , SARS-CoV-2/ultraestrutura , Transdução de SinaisRESUMO
BACKGROUND: A cytochrome P450 active site is buried within the protein molecule and several channels connect the catalytic cavity to the protein surface. Their role in P450 catalysis is still matter of debate. The aim of this study was to understand the possible relations existing between channels and substrate specificity. METHODS: Time course studies were carried out with a collection of polycyclic substrates of increasing sizes assayed with a library of wild-type and chimeric CYP1A enzymes. This resulted in a matrix of activities sufficiently large to allow statistical analysis. Multivariate statistical tools were used to decipher the correlation between observed activity shifts and sequence segment swaps. RESULTS: The global kinetic behavior of CYP1A enzymes toward polycyclic substrates is significantly different depending on the size of the substrate. Mutations which are close or lining the P450 channels significantly affect this discrimination, whereas mutations distant from the P450 channels do not. CONCLUSIONS: Size discrimination is taking place for polycyclic substrates at the entrance of the different P450 access channels. It is thus hypothesized that channels differentiate small from large substrates in CYP1A enzymes, implying that residues located at the surface of the protein may be implied in this differential recognition. GENERAL SIGNIFICANCE: Catalysis thus occurs after a two-step recognition process, one at the surface of the protein and the second within the catalytic cavity in enzymes with a buried active site.
Assuntos
Citocromo P-450 CYP1A1/química , Citocromo P-450 CYP1A2/química , Sítios de Ligação , Catálise , Citocromo P-450 CYP1A1/fisiologia , Citocromo P-450 CYP1A2/fisiologia , Simulação de Dinâmica Molecular , Especificidade por SubstratoRESUMO
The mechanisms that mediate the cardiovascular protective effects of omega 3 (n-3) polyunsaturated fatty acids (PUFAs) have not been fully elucidated. Cytochrome P450 1A1 efficiently metabolizes n-3 PUFAs to potent vasodilators. Thus, we hypothesized that dietary n-3 PUFAs increase nitric oxide (NO)-dependent blood pressure regulation and vasodilation in a CYP1A1-dependent manner. CYP1A1 wild-type (WT) and knockout (KO) mice were fed an n-3 or n-6 PUFA-enriched diet for 8 weeks and were analyzed for tissue fatty acids and metabolites, NO-dependent blood pressure regulation, NO-dependent vasodilation of acetylcholine (ACh) in mesenteric resistance arterioles, and endothelial NO synthase (eNOS) and phospho-Ser1177-eNOS expression in the aorta. All mice fed the n-3 PUFA diet showed significantly higher levels of n-3 PUFAs and their metabolites, and significantly lower levels of n-6 PUFAs and their metabolites. In addition, KO mice on the n-3 PUFA diet accumulated significantly higher levels of n-3 PUFAs in the aorta and kidney without a parallel increase in the levels of their metabolites. Moreover, KO mice exhibited significantly less NO-dependent regulation of blood pressure on the n-3 PUFA diet and significantly less NO-dependent, ACh-mediated vasodilation in mesenteric arterioles on both diets. Finally, the n-3 PUFA diet significantly increased aortic phospho-Ser1177-eNOS/eNOS ratio in the WT compared with KO mice. These data demonstrate that CYP1A1 contributes to eNOS activation, NO bioavailability, and NO-dependent blood pressure regulation mediated by dietary n-3 PUFAs.
Assuntos
Pressão Sanguínea/efeitos dos fármacos , Pressão Sanguínea/fisiologia , Citocromo P-450 CYP1A1/fisiologia , Ácidos Graxos Ômega-3/administração & dosagem , Vasodilatação/efeitos dos fármacos , Vasodilatação/fisiologia , Animais , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Óxido Nítrico/metabolismoRESUMO
The abundance of dioxins and dioxin-like pollutants has massively increased in the environment due to human activity. These chemicals are particularly persistent and accumulate in the food chain, which raises major concerns regarding long-term exposure to human health. Most dioxin-like pollutants activate the aryl hydrocarbon receptor (AhR) transcription factor, which regulates xenobiotic metabolism enzymes that belong to the cytochrome P450 1A family (that includes CYP1A1 and CYP1B1). Importantly, a crosstalk exists between estrogen receptor α (ERα) and AhR. More specifically, ERα represses the expression of the CYP1A1 gene, which encodes an enzyme that converts 17ß-estradiol into 2-hydroxyestradiol. However, (ERα) does not repress the CYP1B1 gene, which encodes an enzyme that converts 17ß-estradiol into 4-hydroxyestradiol, one of the most genotoxic estrogen metabolites. In this review, we discuss how chronic exposure to xenobiotic chemicals, such as pesticides, might affect the expression of genes regulated by the AhR-ERα crosstalk. Here, we focus on recent advances in the understanding of molecular mechanisms that mediate this crosstalk repression, and particularly on how ERα represses the AhR target gene CYP1A1, and could subsequently promote breast cancer. Finally, we propose that genes implicated in this crosstalk could constitute important biomarkers to assess long-term effects of pesticides on human health.
Assuntos
Biomarcadores , Carcinógenos Ambientais/toxicidade , Transformação Celular Neoplásica/efeitos dos fármacos , Praguicidas/toxicidade , Neoplasias da Mama/etiologia , Cocarcinogênese , Citocromo P-450 CYP1A1/biossíntese , Citocromo P-450 CYP1A1/genética , Citocromo P-450 CYP1A1/fisiologia , Dieta , Receptor alfa de Estrogênio/efeitos dos fármacos , Receptor alfa de Estrogênio/fisiologia , Estrogênios , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Genes Neoplásicos/efeitos dos fármacos , Humanos , Ligantes , Masculino , Neoplasias Hormônio-Dependentes/etiologia , Receptor Cross-Talk/efeitos dos fármacos , Receptores de Hidrocarboneto Arílico/efeitos dos fármacos , Receptores de Hidrocarboneto Arílico/fisiologia , Transdução de Sinais/efeitos dos fármacos , Xenobióticos/toxicidadeRESUMO
The primary objective of this study was to determine specific cytochrome P450 isozyme(s) involved in the metabolism of aldrin to its toxic metabolite dieldrin in flathead mullet (Mugil cephalus) liver microsomes. To identify the cytochrome P450 isozyme responsible for the aldrin metabolism in mullet liver, the effects of mammalian-specific cytochrome P450 inhibitors and substrates were determined in the epoxidation reaction of aldrin. CYP3A-related inhibitors, ketoconazole, SKF-525A, and cimetidine, inhibited the metabolism of aldrin. The contribution of CYP1A to the aldrin metabolism was shown by the inhibition of 7-ethoxyresorufin-O-deethylase activity in the presence of aldrin. The results indicate that CY1A and CYP3A are the cytochrome P450s involved in aldrin epoxidase activity in mullet. In addition, the suitability of aldrin epoxidase activity for monitoring of environmental pollution was also assessed in the fish samples caught from four different locations of the West Black Sea coast of Turkey.
Assuntos
Aldrina/metabolismo , Citocromo P-450 CYP1A1/fisiologia , Citocromo P-450 CYP3A/fisiologia , Proteínas de Peixes/fisiologia , Inseticidas/metabolismo , Smegmamorpha/metabolismo , Poluentes Químicos da Água/metabolismo , Animais , Benzoflavonas/farmacologia , Citocromo P-450 CYP1A1/antagonistas & inibidores , Inibidores do Citocromo P-450 CYP3A/farmacologia , Compostos de Epóxi/metabolismo , Inativação Metabólica , Microssomos Hepáticos/enzimologia , RiosRESUMO
Given the fact that increased dietary intake of polycyclic aromatic hydrocarbons (PAHs; a family of environmental toxicants) leads to the formation and development of colon tumors, the ability of the gastrointestinal tract to process these compounds is important from the viewpoint of toxicity/carcinogenesis. Benzo(a)pyrene (BaP), a prototypical PAH compound is released into the environment from automobile exhausts, cigarette smoke, and industrial emissions. Additionally, considerable intake of BaP is expected in people who consume barbecued foods and a diet rich in saturated fat. In exposed animals, BaP becomes activated to potent metabolites that interfere with target organ function and as a consequence cause toxicity and cancer. Therefore, knowledge of BaP metabolism in the digestive system will be of importance in the management of cancers of the digestive tract. The objective of our study was to study the metabolism of BaP by subcellular fractions (nuclear, cytosolic, mitochondrial, and microsomal) of the gastrointestinal tract and liver. Subcellular fractions were isolated by differential centrifugation from the stomach, jejunum, colon, and liver tissues of Apc(Min) mice that received a subchronic dose of 25 µg/kg BaP. The fractions were incubated with 1 and 3 µM BaP. Subsequent to incubation, samples were extracted with ethyl acetate and analyzed for BaP metabolites by reverse-phase HPLC equipped with fluorescence detection. Among the different fractions tested, microsomal BaP metabolism was higher than the rest of the fractions in all the samples analyzed. Additionally, a BaP exposure concentration-dependent effect on metabolite levels generated by the subcellular fractions was recorded. The BaP metabolites identified were the following: BaP-9,10-diol; BaP-4,5-diol; BaP-7,8-diol; 9(OH) BaP; 3(OH) BaP; BaP-3,6-dione; and BaP-6,12-dione. While the diol group of metabolites was frequently detected, among diones, the 3,6 and 6,12-dione metabolites were infrequently detected. Among the diol metabolites, the preponderance of BaP-7,8-dihydrodiol is interesting, since this metabolite is a precursor to the DNA-reactive BaP-7,8-dihydrodiol epoxide (BPDE) that has been linked to BaP-induced cancer.
Assuntos
Benzo(a)pireno/metabolismo , Neoplasias do Colo/metabolismo , Trato Gastrointestinal/metabolismo , Fígado/metabolismo , Animais , Citocromo P-450 CYP1A1/fisiologia , Trato Gastrointestinal/ultraestrutura , Fígado/ultraestrutura , Masculino , CamundongosRESUMO
Omeprazole (OME) induces the expression of genes encoding drug-metabolizing enzymes, such as CYP1A1, via activation of the aryl hydrocarbon receptor (AhR) both in vivo and in vitro. However, the precise mechanism of OME-mediated AhR activation is still under investigation. While elucidating species-specific susceptibility to dioxin, we found that OME-mediated AhR activation was mammalian species specific. Moreover, we previously reported that OME has inhibitory activity toward CYP1A1 enzymes. From these observations, we speculated that OME-mediated AhR target gene transcription is due to AhR activation by increasing amounts of putative AhR ligands in serum by inhibition of CYP1A1 activity. We compared the amino acid sequences of OME-sensitive rabbit AhR and nonsensitive mouse AhR to identify the residues responsible for the species-specific response. Chimeric AhRs were constructed by exchanging domains between mouse and rabbit AhRs to define the region required for the response to OME. OME-mediated transactivation was observed only with the chimeric AhR that included the ligand-binding domain (LBD) of the rabbit AhR. Site-directed mutagenesis revealed three amino acids (M328, T353, and F367) in the rabbit AhR that were responsible for OME-mediated transactivation. Replacing these residues with those of the mouse AhR abolished the response of the rabbit AhR. In contrast, substitutions of these amino acids with those of the rabbit AhR altered nonsensitive mouse AhR to become sensitive to OME. These results suggest that OME-mediated AhR activation requires a specific structure within LBD that is probably essential for binding with enigmatic endogenous ligands.
Assuntos
Omeprazol/farmacologia , Inibidores da Bomba de Prótons/farmacologia , Receptores de Hidrocarboneto Arílico/química , Sequência de Aminoácidos , Animais , Sítios de Ligação , Células Cultivadas , Citocromo P-450 CYP1A1/antagonistas & inibidores , Citocromo P-450 CYP1A1/genética , Citocromo P-450 CYP1A1/fisiologia , Cobaias , Humanos , Ligantes , Camundongos , Dados de Sequência Molecular , Dibenzodioxinas Policloradas/farmacologia , Receptores de Hidrocarboneto Arílico/metabolismo , Especificidade da EspécieRESUMO
Eupatorin-5-methyl ether (E5M) is a flavone containing 4 methoxy groups that is present in plants with medicinal activity, whereas luteolin (L) is a polyhydroxylated flavone commonly encountered in dietary products. In the present study we investigated the interaction of the two flavonoids with cytochrome P450 CYP1 enzymes in breast cancer MCF7 cells. Both compounds induced a dose dependent increase in CYP1A1 and CYP1B1 mRNA levels, as well as in EROD activity, a marker of CYP1 enzyme activity. Induction of cytochrome P450 CYP1 expression by E5M was accompanied by translocation of the ligand-activated transcription factor AhR to the nucleus, as demonstrated by confocal immunofluoresence. More importantly, although E5M was less active than L in inhibiting proliferation of MCF7 cells, when the cells were pretreated with the CYP1 inducer Benzo[a]pyrene (BaP) the potency of E5M was augmented. HPLC and LC-MS analysis revealed that E5M was metabolized to a major conversion product assigned E5M1 resulting from one step demethylation reaction in MCF7 cells whereas L metabolism by recombinant CYP1A1 did not reveal any metabolites. E5M1 production in BaP-induced MCF7 cells was attenuated in the presence of the CYP1A1 inhibitor α-napthoflavone. E5M further induced a dose dependent increase in the cell signaling proteins p21, JNK and p-JNK in MCF7 cells. This effect was enhanced in BaP pretreated cells and was associated with G1 arrest and a small percentage of apoptosis (3.5%). E5M antiproliferative effect in BaP pretreated cells was attenuated in the presence of the CYP1A1 inhibitor α-napthoflavone, as demonstrated by Western blotting and FACS analysis. Taken together the results demonstrate that BaP sensitizes MCF7 cells to E5M antiproliferative activity via enhanced induction of p21, JNK and p-JNK that in turn results by cytochrome P450 CYP1-mediated conversion to the metabolite E5M1.
Assuntos
Benzo(a)pireno/toxicidade , Citocromo P-450 CYP1A1/fisiologia , Citocromo P-450 CYP1B1/fisiologia , Flavonas/farmacologia , Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos dos fármacos , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Relação Dose-Resposta a Droga , Ativação Enzimática , Feminino , Humanos , MAP Quinase Quinase 4/metabolismo , Células MCF-7RESUMO
Benzo[a]pyrene (BaP) is a prototypical polycyclic aromatic hydrocarbon (PAH) found in combustion processes. Cytochrome P450 1A1 and 1B1 enzymes (CYP1A1, CYP1B1) and other enzymes can activate PAHs to reactive oxygenated intermediates involved in mutagenesis and tumor initiation; also, CYP1 enzymes can detoxify PAHs. Cyp1(+/+) wild-type (WT) and Cyp1b1(-/-) knockout mice receiving oral BaP (12.5 mg/kg/day) remain healthy for >12 months. In contrast, we found that global knockout of the Cyp1a1 gene (1a1KO) results in proximal small intestine (PSI) adenocarcinoma within 8-12 weeks on this BaP regimen; striking compensatory increases in PSI CYP1B1 likely participate in initiation of adenocarcinoma in 1a1KO mice. Cyp1a1/1b1(-/-) double-knockout (DKO) mice on this BaP regimen show no PSI adenocarcinoma, but instead preputial gland duct (PGD) squamous cell carcinoma (SCC) occurs by 12 weeks. Herein, we compare microarray expression of PGD genes in WT, 1a1KO and DKO mice at 0, 4, 8, 12 and 16 weeks of oral BaP; about four dozen genes up- or down-regulated during most critical time-points were further verified by qRT-PCR. In DKO mice, CYP3A59 was unequivocally identified as the BaP-inducible and BaP-metabolizing best candidate responsible for initiation of BaP-induced SCC. Striking increases or decreases were found in 26 cancer-related genes plus eight Serpin genes in DKO, but not in 1a1KO or WT, mice on this BaP regimen; of the 26, 8 were RAS-related oncogenes. The mechanism by which cancer-related genes are responsible for SCC tumor progression in the PGD remains to be elucidated.
Assuntos
Adenocarcinoma/genética , Hidrocarboneto de Aril Hidroxilases/fisiologia , Biomarcadores Tumorais/genética , Carcinoma de Células Escamosas/genética , Citocromo P-450 CYP1A1/fisiologia , Perfilação da Expressão Gênica , Glândulas Odoríferas/patologia , Adenocarcinoma/tratamento farmacológico , Adenocarcinoma/patologia , Animais , Carcinoma de Células Escamosas/tratamento farmacológico , Carcinoma de Células Escamosas/patologia , Citocromo P-450 CYP1B1 , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Análise de Sequência com Séries de Oligonucleotídeos , Glândulas Odoríferas/efeitos dos fármacos , Glândulas Odoríferas/metabolismoRESUMO
Microsomal cytochrome P450 (P450) enzymes, which are important in the metabolism of carcinogens, are expressed in both epithelial and stromal cells in the mammary gland. The aim of this study was to investigate the roles of mammary epithelial P450 enzymes in the bioactivation and disposition of 7,12-dimethylbenz(a)anthracene (DMBA), a breast carcinogen, in the mammary gland. A new mouse model (named MEpi-Cpr-null) was produced, wherein P450 activities in the mammary epithelial cells are suppressed through tissue-specific deletion of the gene for P450 reductase (Cpr), an enzyme required for the activities of all microsomal P450 enzymes. Comparisons between wild-type and MEpi-Cpr-null mice showed that the tissue-specific deletion of Cpr in the mammary epithelial cells was accompanied by significant increases in the levels of DMBA and DMBA-DNA adduct in the mammary gland following a single intraperitoneal injection of DMBA at 50mg/kg. Immunohistochemical and immunoblot analysis further revealed greater induction of CYP1B1 expression by the DMBA treatment in the mammary stroma of the MEpi-Cpr-null mice than in that of the WT mice. These findings not only demonstrate that the epithelial P450 enzymes play important roles in the clearance of DMBA, but also suggest that P450 enzymes in both mammary epithelial and stromal cells contribute to carcinogen-mediated DNA damage.
Assuntos
9,10-Dimetil-1,2-benzantraceno/farmacocinética , Hidrocarboneto de Aril Hidroxilases/fisiologia , Carcinógenos/farmacocinética , Citocromo P-450 CYP1A1/fisiologia , Glândulas Mamárias Animais/metabolismo , Animais , Biotransformação , Citocromo P-450 CYP1B1 , Adutos de DNA/análise , Epitélio/metabolismo , Feminino , Taxa de Depuração Metabólica , CamundongosRESUMO
Food-drug interaction is an emerging phenomenon, comprising pharmacokinetic or toxicokinetic interactions between food constituents and drugs. The mechanisms include inhibition of enzymes and transporters, and induction of drug metabolizing enzymes. A prominent regulator of drug-metabolizing enzymes is an aryl hydrocarbon receptor (AhR) that transcriptionally regulates CYP1 enzymes, phase II enzymes and many other genes. In the current paper, we have examined the effects of 28 different flavored mineral waters on AhR-CYP1A1 signaling pathway in primary cultures of human hepatocytes and in human cancer cell lines HepG2 (hepatic) and LS174T (intestinal). The techniques of Western blot, RT-PCR and gene reporter assays were employed to determine the expression of CYP1A1 mRNA, protein and activation of AhR, respectively. We have identified four flavored mineral waters which activated AhR and/or induced CYP1A1. These data imply a potential of some flavored mineral waters to cause food-drug interactions. In addition, activation of AhR-CYP1A1 signaling may result in chemically-induced carcinogenesis and alteration of intermediary metabolism.
Assuntos
Citocromo P-450 CYP1A1/fisiologia , Hepatócitos/efeitos dos fármacos , Águas Minerais , Receptores de Hidrocarboneto Arílico/fisiologia , Western Blotting , Linhagem Celular Tumoral , Citocromo P-450 CYP1A2/fisiologia , Interações Alimento-Droga , Genes Reporter/efeitos dos fármacos , Humanos , Neoplasias Intestinais/patologia , Neoplasias Hepáticas/patologia , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase em Tempo Real , Transdução de Sinais/efeitos dos fármacos , Transcrição Gênica/efeitos dos fármacosRESUMO
JI-101, chemically 1-[1-(2-amino-pyridin-4-ylmethyl)-1H-indol-4-yl]-3-(5-bromo-2-methoxy-phenyl)-urea hydrochloride, is a novel orally active kinase inhibitor, which has shown potent in vitro and in vivo anticancer activity against a variety of cancer cell lines and xenografts. It is currently entering Phase II clinical development for the treatment of solid tumors. The aim of the study is to assess the metabolic stability of JI-101 in various pre-clinical and human liver microsomes, to identify the major CYPs (cytochrome ß450) involved in the metabolism of JI-101 and identification of putative metabolites. We have also studied the pharmacokinetics, tissue distribution and excretion of JI-101 in Sprague Dawley rats. JI-101 was found to be stable in various liver microsomes tested. JI-101 is highly permeable and not a substrate for P-gp (permeability glycoprotein). JI-101 excreted through bile along with its mono- and di-hydroxy metabolites. Following oral administration, JI-101 was rapidly absorbed, reaching Cmax within 2 h. The t½ of JI-101 with intravenous and oral route was found to be 1.75 ± 0.79 and 2.66 ± 0.13 h, respectively. The Cl and Vd by intravenous route for JI-101 were found to be 13.0 ± 2.62 mL/min/kg and 2.11 ± 1.42 L/kg, respectively. The tissue distribution of JI-101 was extensive with rapid and preferred uptake into lung tissue. Overall, the oral bioavailability of JI-101 is 55% and the primary route of elimination for JI-101 is feces.
Assuntos
Indóis/farmacocinética , Compostos de Fenilureia/farmacocinética , Inibidores de Proteínas Quinases/farmacocinética , Receptor EphB4/antagonistas & inibidores , Animais , Bile/metabolismo , Células CACO-2 , Cromatografia Líquida , Citocromo P-450 CYP1A1/fisiologia , Humanos , Masculino , Fenótipo , Ratos , Ratos Sprague-Dawley , Espectrometria de Massas em Tandem , Distribuição TecidualRESUMO
Carcinogenic and toxic molecules produce DNA adducts that contribute to the development of atherosclerosis. Genetic polymorphisms of xenobiotic-detoxified enzymes, which control the level of DNA adducts, may affect both enzymatic activity and individual susceptibility to coronary artery disease (CAD). In this study we investigated the effects of genetic polymorphisms of the CYP1A1*2C, GSTT1, and GSTM1 enzymes on CAD risk in a Turkish population. Genotypes were determined for 132 CAD patients and 151 healthy controls by the polymerase chain reaction/restriction fragment length polymorphism method. There were no significant differences between patients and controls in terms of CYP1A1, GSTT1, and GSTM1 genotypes. Analysis of the possible interactions between the genotypes, after adjustment for the risk factors, demonstrated that individuals carrying CYP1A1 variant GSTT1 null genotypes had an 8.907-fold increased CAD risk compared to their wild status (p<0.05). We suggest that genetic polymorphisms of xenobiotic-metabolizing enzymes could play an important role in CAD. Therefore, CYP1A1 and GSTM1 polymorphisms should be considered as important parameters for the prediction of CAD.
Assuntos
Doença da Artéria Coronariana/genética , Citocromo P-450 CYP1A1/genética , Glutationa Transferase/genética , Idoso , Estudos de Casos e Controles , Citocromo P-450 CYP1A1/fisiologia , Feminino , Frequência do Gene , Predisposição Genética para Doença , Variação Genética/fisiologia , Genótipo , Glutationa Transferase/fisiologia , Humanos , Masculino , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único , RiscoRESUMO
INTRODUCTION: Cyp1a1-Ren2 transgenic rats [strain name: TGR(Cyp1a1Ren2)], administered indole-3-carbinol (I3C) develop angiotensin (ANG) II-dependent hypertension due to hepatic expression of the Ren2 renin gene. Although AT1 receptor blockade prevents the development of hypertension and normalizes the elevated arterial blood pressure of Cyp1-Ren2 rats, little information is available regarding the blood pressure and renal functional responses to direct inhibition of renin in this high circulating renin model of ANG II-dependent hypertension. This study was performed to determine the effects of acute direct renin inhibition with aliskiren on blood pressure and renal hemodynamics in Cyp1a1-Ren2 rats with ANG II-dependent malignant hypertension. METHODS: Mean arterial pressure (MAP) and renal hemodynamics were measured in pentobarbital-anesthetized male Cyp1a1-Ren2 rats during control conditions and after administration of the renin inhibitor, aliskiren (10 mg/kg, intravenous). RESULTS: Rats induced with I3C had higher MAP (194 ± 7 versus 141 ± 2 mm Hg, P < 0.001), lower renal plasma flow (RPF; 2.47 ± 0.23 versus 4.17 ± 0.35 mL/min/g, P < 0.001) and lower glomerular filtration rate (GFR; 1.01 ± 0.07 versus 1.34 ± 0.06 mL/min/g, P = 0.01) than noninduced Cyp1a1-Ren2 rats (n = 5). Aliskiren administration decreased MAP (194 ± 7 to 136 ± 2 mm Hg, P < 0.001) and increased RPF (2.47 ± 0.23 versus 4.31 ± 0.20 mL/min/g, P < 0.001) in hypertensive but not in normotensive rats, without altering GFR. CONCLUSIONS: Acute renin inhibition with aliskiren normalizes MAP and RPF in Cyp1a1-Ren2 rats with malignant hypertension. The normalization of MAP and RPF after acute renin inhibition indicates that renin generated by expression of the Ren2 gene is responsible for the maintenance of malignant hypertension and the associated reduction in renal hemodynamic function in Cyp1a1-Ren2 rats.
Assuntos
Amidas/farmacologia , Angiotensina II/fisiologia , Pressão Sanguínea/efeitos dos fármacos , Citocromo P-450 CYP1A1/genética , Fumaratos/farmacologia , Hipertensão Maligna/fisiopatologia , Renina/antagonistas & inibidores , Animais , Pressão Sanguínea/fisiologia , Citocromo P-450 CYP1A1/fisiologia , Modelos Animais de Doenças , Taxa de Filtração Glomerular/efeitos dos fármacos , Taxa de Filtração Glomerular/fisiologia , Rim/irrigação sanguínea , Rim/efeitos dos fármacos , Rim/fisiopatologia , Masculino , Ratos , Ratos Transgênicos , Fluxo Sanguíneo Regional/efeitos dos fármacos , Fluxo Sanguíneo Regional/fisiologia , Renina/genética , Renina/fisiologia , Resistência Vascular/efeitos dos fármacos , Resistência Vascular/fisiologiaRESUMO
2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), the most abundant heterocyclic amine produced during the cooking of meats and fish, is suspected to be a human carcinogen. Metabolic activation of PhIP is primarily mediated by the enzyme cytochrome P450 (CYP) 1A2. Metabolism of PhIP by CYP1A2 differs considerably between humans and rodents, with more N(2)-hydroxylation (activation) and less 4'-hydroxylation (detoxication) in humans. Transgenic CYP1A-humanized mice (hCYP1A-mice), which have the human CYP1A1 and CYP1A2 genes but lack the murine orthologs Cyp1a1 and Cyp1a2, provide an excellent opportunity to develop a relevant model to study dietary-induced colon carcinogenesis. The treatment with 200 mg/kg PhIP by oral gavage, followed by 1.5% dextran sodium sulfate (DSS) in the drinking water for 7 days, was found to be an effective combination to induce colon carcinogenesis in hCYP1A-mice. Tumor multiplicity at week 6 was calculated to be 3.75 ± 0.70 and for week 10 was 3.90 ± 0.61 with 80-95% of the tumors being adenocarcinomas. No tumors were found in the similarly treated wild-type mice. Western blots revealed overexpression of ß-catenin, c-Myc, cyclin D1, inducible nitric oxide synthase and cyclooxygenase-2 in colon tumor samples. Strong nuclear localization of ß-catenin was observed in tumors. These results illustrate that PhIP and DSS combination produces rapid colon carcinogenesis in hCYP1A-mice and this is an effective model to mimic human colon carcinogenesis.
Assuntos
Carcinógenos/toxicidade , Neoplasias do Colo/induzido quimicamente , Citocromo P-450 CYP1A1/fisiologia , Citocromo P-450 CYP1A2/fisiologia , Sulfato de Dextrana/toxicidade , Imidazóis/toxicidade , Animais , Biotransformação , Neoplasias do Colo/mortalidade , Feminino , Humanos , Imidazóis/farmacocinética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Taxa de Sobrevida , beta Catenina/genéticaRESUMO
The environmental pollutant 2,3,7,8-tetracholorodibenzo-p-dioxin (TCDD) is known to cause a wide variety of toxic effects, including hepatotoxicity, by way of the aryl hydrocarbon receptor (AHR). Although inducible expression of cytochrome P450 (CYP) 1A1 and CYP1A2 is associated with liver injury caused by high-dose TCDD, the specific role of the AHR-CYP1 cascade in hepatotoxicity remains unclear. We investigated the effects of AHR activation under conditions of cholestasis. We administered oral TCDD to mice at a dose that can effectively induce Cyp1 gene expression without overt liver toxicity and then ligated their bile ducts. TCDD pretreatment enhanced bile duct ligation (BDL)-induced increases in liver and plasma bile acids, bilirubin, and aminotransferases. Histology of TCDD-pretreated BDL mice revealed massive hepatic necrosis without any increase in number of apoptotic cells. Whereas induction of AHR-target genes by TCDD was observed similarly in sham-operated as well as in BDL mice, TCDD pretreatment of BDL mice altered the expression of hepatic genes involved in bile acid synthesis and transport. Increased plasma proinflammatory cytokines, tumor necrosis factor and interleukin-1ß, in BDL mice were further elevated by TCDD pretreatment. Liver injury by TCDD plus BDL, such as increased plasma bile acids, bilirubin and aminotransferases, liver necrosis, and increased tumor necrosis factor production, was exaggerated in Cyp1a1/1a2(-/-) double knockout mice. These findings indicate that TCDD aggravates cholestatic liver damage and that the presence of CYP1A1 and CYP1A2 plays a protective role in liver damage caused by TCDD and BDL.