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Neovascularization contributes to various posterior eye segment diseases such as age-related macular degeneration and diabetic retinopathy. RNA nanoparticles were demonstrated previously to enter the corneal and retinal cells after subconjunctival injection for ocular delivery. In the present study, antiangiogenic aptamers (anti-vascular endothelial growth factor (VEGF) and anti-angiopoietin-2 (Ang2) aptamers) were conjugated to RNA nanoparticles. The objectives were to investigate the clearance and distribution of these angiogenesis-inhibiting RNA nanoparticles after subconjunctival injection in vivo and their antiangiogenic effects for inhibiting ocular neovascularization in vitro. The results in the whole-body fluorescence imaging study showed that the clearance of RNA nanoparticles was size-dependent with no significant differences between RNA nanoparticles with and without the aptamers except for pRNA-3WJ. The distribution study of RNA nanoparticles by confocal microscopy of the dissected eye tissues in vivo indicated cell internalization of the larger RNA nanoparticles in the retina and retinal pigment epithelium after subconjunctival injection, and the larger nanoparticles with aptamers showed higher levels of cell internalization than those without. In the cell proliferation assay in vitro, RNA nanoparticles with multiple aptamers had higher antiangiogenic effects. With both longer retention time and high antiangiogenic effect, SQR-VEGF-Ang2 could be a promising RNA nanoparticle for posterior eye delivery.
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Inhibidores de la Angiogénesis , Nanopartículas , ARN , Factor A de Crecimiento Endotelial Vascular , Animales , Nanopartículas/química , Inhibidores de la Angiogénesis/administración & dosificación , Inhibidores de la Angiogénesis/farmacología , Inhibidores de la Angiogénesis/química , ARN/administración & dosificación , Aptámeros de Nucleótidos/administración & dosificación , Aptámeros de Nucleótidos/química , Humanos , Angiopoyetina 2 , Masculino , Ratones , Conjuntiva/metabolismo , Inyecciones Intraoculares , Proliferación Celular/efectos de los fármacos , Neovascularización Patológica/tratamiento farmacológico , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Retina/metabolismo , Retina/efectos de los fármacos , Sistemas de Liberación de Medicamentos/métodos , Ratones Endogámicos C57BL , AngiogénesisRESUMEN
BACKGROUND: Irinotecan is widely used to treat various types of solid and metastatic cancer. It is an ester prodrug and its hydrolytic metabolite (SN-38) exerts potent anticancer activity. Irinotecan is hydrolyzed primarily by carboxylesterase-2 (CES2), a hydrolase abundantly present in the intestine such as the duodenum. We have identified several potent and covalent CES2 inhibi¬tors such as remdesivir and sofosbuvir. Remdesivir is the first small molecule drug approved for COVID-19, whereas sofosbuvir is a paradigm-shift medicine for hepatitis C viral infection. Irinotecan is generally well-tolerated but associated with severe/life-threatening diarrhea due to intestinal accu¬¬mula¬tion of SN-38. OBJECTIVE: This study was to test the hypothesis that remdesivir and sofosbuvir protect against irinotecan-induced epithelial injury associated with gastrointestinal toxicity. METHODS: To test this hypothesis, formation of organoids derived from mouse duodenal crypts, a robust cellular model for intestinal regeneration, was induced in the presence or absence of irinotecan +/- pretreatment with a CES2 drug inhibitor. RESULTS: Irinotecan profoundly inhibited the formation of intestinal organoids and the magnitude of the inhibition was greater with female crypts than their male counterparts. Consistently, crypts from female mice had significantly higher hydrolytic activity toward irinotecan. Critically, remdesivir and sofosbuvir both reduced irinotecan hydrolysis and reversed irinotecan-reduced formation of organoids. Human duodenal samples robustly hydrolyzed irinotecan, stable CES2 transfection induced cytotoxicity and the cytotoxicity was reduced by CES2 drug inhibitor. CONCLUSION: These findings establish a therapeutic rationale to reduce irinotecan-gastrointestinal injury and serve as a cellular foundation to develop oral formulations of irinotecan with high safety.
RESUMEN
In vitro models are available as alternatives for the Draize eye irritation test. However, most of the alternative models are not quantitative nor designed to evaluate the effects of chemicals on the corneal barrier such as those encountered in pharmaceutical and cosmetic products. The objective of the present study was to investigate tissue electrical resistance to provide sensitive in vitro testing of tissue alteration caused by chemicals in pharmaceutical and cosmetic formulations as a potential eye irritation testing approach. The experimental protocols for effective tissue resistance measurements were examined using two in vitro eye models: porcine cornea and EpiCorneal. In these models, a test chemical was applied to the cornea or EpiCorneal tissue for 1 min, and tissue resistances/conductances were measured at 1-60 min after the application. The changes in conductance of the tissues after exposure to the chemicals were shown to provide quantitative evaluations to the influence of the chemicals. A correlation was found between the two in vitro models. The results suggest that these models can provide quantitative in vitro assessments of eye-irritating chemicals.
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Alternativas a las Pruebas en Animales , Cosméticos , Animales , Porcinos , Alternativas a las Pruebas en Animales/métodos , Irritantes/toxicidad , Córnea , Técnicas In Vitro , Preparaciones Farmacéuticas , OjoRESUMEN
The pregnane X receptor (PXR) has been established to induce chemoresistance and metabolic diseases. Ochratoxin A (OTA), a mycotoxin, decreases the expression of PXR protein in human primary hepatocytes. OTA is chlorinated and has a methylated lactone ring. Both structures are associated with OTA toxicity. The study was to test the hypothesis that structural modifications differentially impact PXR blocking activity over cytotoxicity. To test this hypothesis, OTA-M and OTA-Cl/M were synthesized. OTA-M lacked the methyl group of the lactone-ring, whereas OTA-Cl/M had neither the methyl group nor the chlorine atom. The blocking activity of PXR activation was determined in a stable cell line, harboring both PXR (coding sequence) and its luciferase element reporter. OTA-Cl/M showed the highest blocking activity, followed by OTA-M and OTA. OTA-Cl/M was 60 times as potent as the common PXR blocker ketoconazole based on calculated IC50 values. OTA-Cl/M decreased by 90 % the expression of PXR protein and was the least cytotoxic among the tested compounds. Molecular docking identified that OTA and its derivatives interacted with different sets of residues in PXR, providing a molecular basis for selectivity. Excessive activation of PXR has been implicated in chemoresistance and metabolic diseases. Downregulation of PXR protein expression likely delivers an effective mechanism against structurally diverse PXR agonists.
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Carcinógenos/química , Carcinógenos/toxicidad , Ocratoxinas/química , Ocratoxinas/toxicidad , Receptor X de Pregnano/antagonistas & inhibidores , Supervivencia Celular , Desmetilación , Expresión Génica/efectos de los fármacos , Células HEK293 , Halogenación , Humanos , Cetoconazol/farmacología , Simulación del Acoplamiento Molecular , Receptor X de Pregnano/biosíntesisRESUMEN
Melphalan is used in selective ophthalmic artery infusion chemotherapy. In the procedure to catheterize the ophthalmic artery in selective ophthalmic artery infusion chemotherapy, the microcatheter is usually filled with an iodinated contrast medium. It is not known whether mixing of iodinated contrast medium with melphalan solutions promotes precipitation of melphalan during selective ophthalmic artery infusion chemotherapy. The objective of the present study was to examine the compatibility of melphalan and iodinated contrast medium. In the experiments, melphalan solutions were prepared with and without iodinated contrast medium (iohexol 300 mg Iodine/mL or ioversol 74%). Melphalan solution of clinically relevant concentration (0.45 mg/mL) in the absence of iodinated contrast medium was evaluated as a reference. The 0.45-mg/mL melphalan solution was diluted with iodinated contrast medium at 1:9 and 1:1 ratios to form solutions comprising iodinated contrast medium at 10% or 50% (v/v), and each was evaluated. The formation of particles was examined using filtration (pore size 0.45 µm) followed by high-performance liquid chromatography at four quarterly time intervals (15 min, 30 min, 45 min, and 60 min) over a 1-hour period after solution preparation. High-performance liquid chromatography analysis of microfiltered solutions did not reveal evidence of melphalan crystal formation over a 60-minute period for the solutions studied. The filtration studies suggest that mixing of the iodinated contrast medium with melphalan solutions does not result in significant melphalan precipitation.
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Medios de Contraste/administración & dosificación , Yodo , Yohexol/administración & dosificación , Melfalán , Yodo/química , Arteria OftálmicaRESUMEN
Petrolatum is a mixture of hydrocarbons that is widely used as a moisturizer. It is incorporated in bodywash formulations to help hydrate and maintain healthy skin appearance. The aim of this study was to investigate skin deposition and penetration of petrolatum from an experimental bodywash system consisting of petrolatum in vitro. Experiments were performed using cadaver split-thickness skin and Franz diffusion cells. Radiolabeled 14C-dotriacontane (C32-alkane) was used as a model permeant for petrolatum. The bodywash was applied on the skin and subsequently rinsed. At predetermined time points, the skin was wiped to remove the residual material on the surface, and tape-stripping was performed. Petrolatum was observed to deposit from the bodywash when applied on split-thickness skin with simulated rinsing. Petrolatum then penetrated into the stratum corneum and was detected at the depth of 12 tape-stripping and in the epidermis. The bodywash formulation could provide significant deposition and penetration of petrolatum into the stratum corneum at 1-72 hours postapplication.
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Epidermis , Vaselina , Células Epidérmicas , Absorción CutáneaRESUMEN
In a previous study investigating the relationships between solute physicochemical properties and solvent effects on skin permeation of solutes under finite dose conditions, urea was found to have the highest percent dose absorbed among the model solutes studied. The objective of this study is to probe the mechanism of the observed high skin permeation of urea at finite dose, in contrast to its permeability coefficient obtained under infinite dose condition. Skin permeation experiments were performed with Franz diffusion cells and human epidermal membrane. Dose-dependence and penetration enhancing effects of urea permeation were investigated. Tape stripping was performed. A small hydrophilic solute ethylene glycol with molecular weight similar to urea was studied for comparison. The results suggest that urea did not have penetration enhancing effect to enhance solute permeation across skin under the finite dose conditions. Tape stripping data are consistent with skin permeation mechanism of solute deposition and diffusion. The skin permeation behavior of urea could be attributed to its small molecular size. This suggests that, under the finite dose conditions examined in this study, solutes with molecular sizes similar to or less than urea and ethylene glycol could lead to high percent of skin absorption despite their hydrophilicities.
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Absorción Cutánea/efectos de los fármacos , Urea/farmacología , Urea/farmacocinética , Administración Cutánea , Anciano , Epidermis/efectos de los fármacos , Epidermis/metabolismo , Glicol de Etileno/administración & dosificación , Glicol de Etileno/farmacocinética , Glicerol/administración & dosificación , Glicerol/farmacocinética , Humanos , Persona de Mediana Edad , Urea/administración & dosificaciónRESUMEN
Thermodynamically and chemically stable RNA nanoparticles derived from the three-way junction (3WJ) of the pRNA from bacteriophage phi29 DNA packaging motor were examined previously for ocular delivery. It was reported that, after subconjunctival injection, RNA nanoparticles with tri-way shape entered the corneal cells but not the retinal cells, whereas particle with four-way shape entered both corneal and retinal cells. The present study evaluated ocular delivery of RNA nanoparticles with various shapes and sizes, and assessed the effect of thermosensitive hydrogels (poly(lactic-co-glycolic acid)-b-poly(ethylene glycol)-b-poly(lactic-co-glycolic acid); PLGA-PEG-PLGA) for increasing the retention of RNA nanoparticles in the eye. Fluorescence imaging of mouse eyes and fluorescence microscopy of dissected eye tissues from the conjunctiva, cornea, retina, and sclera were performed to determine the distribution and clearance of the nanoparticles in the eyes after subconjunctival injection in vivo. RNA nanoparticles entered the cells of the conjunctiva, cornea, retina, and sclera after subconjunctival delivery. The clearance of RNA pentagon was slower than both RNA square and triangle of the same designed edge length (10nm) in the eye, and the clearance of RNA squares of the longer edge lengths (10 and 20nm) was slower than RNA square of the shorter edge length (5nm), thus indicating that the size could affect ocular pharmacokinetics of the nanoparticles. At 24h after the injection, approximately 6-10% of the fluorescence signal from the larger nanoparticles in the study (RNA square of 20nm edge length and RNA pentagon of 10nm edge length) remained in the eye, and up to 70% of the retinal cells contained the nanoparticles. The results suggest that the larger nanoparticles were "gulped" in conjunctival, corneal, retinal, and scleral cells, similar to the behavior observed in macrophages. Additionally, the combination of RNA nanoparticles with the thermosensitive polymers increased the retention of the nanoparticles in the eye.
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Ojo/metabolismo , Hidrogeles/administración & dosificación , Nanopartículas/administración & dosificación , ARN/administración & dosificación , Animales , Inyecciones , Ratones Pelados , Ratones Endogámicos C57BL , Polietilenglicoles/administración & dosificación , Poliglactina 910/administración & dosificación , ARN/farmacocinéticaRESUMEN
A novel drug delivery vehicle using nanodroplets activated by light irradiation for drug release in a controlled manner has been developed. The drug encapsulated in the nanodroplets was released upon phase transition from a liquid droplet to microbubbles (vaporization) by plasmonic photothermal heat from gold nanorods adsorbed on the surface of the nanodroplets. The nanodroplets were stable against aggregation and dissolution at 4 °C over 3 months to date. The phase transition was quantitatively analyzed by ultrasound imaging to examine the amount of drug release noninvasively. In vitro studies showed that cell death occurred only when light irradiation was performed on the drug-encapsulated nanodroplets. Ex vivo studies demonstrated a potential application of the nanodroplets for treating posterior eye diseases. Thus, it has been demonstrated that our gold-nanorod-coated light-activatable nanodroplets can be a candidate for a controlled release and a dosage-monitored drug delivery system.
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Sistemas de Liberación de Medicamentos , Liberación de Fármacos , Oro , Nanotubos , Nanomedicina TeranósticaRESUMEN
The objective of this research was to evaluate the optimal passage number according to the biological characteristics of mouse skin melanocytes from different passages. Skin punch biopsies harvested from the dorsal region of 2-day old mice were used to establish melanocyte cultures. The cells from passage 4, 7, 10 and 13 were collected and evaluated for their melanogenic activity. Histochemical staining for tyrosinase (TYR) activity and immunostaining for the melanocyte specific markers including S-100 antigen, TYR, tyrosinase related protein 1 (TYRP1), tyrosinase related protein 2 (TYRP2) and micropthalmia associated transcription factor (MITF) confirmed purity and melanogenic capacity of melanocytes from different passages, with better melanogenic activity of passage 10 and 13 cells being observed. Treatment of passage 13 melanocytes with α-melanocyte stimulating hormone (α-MSH) showed increased expression of MITF, TYR and TYRP2 mRNA. However, considering the TYR mRNA dramatically high expression which is the characteristics of melanoma cells, melanocytes from passage 10 was the optimal passage number for the further research. Our results demonstrate culture of pure populations of mouse melanocytes to at least 10 passages and illustrate the potential utility of passage 10 cells for studies of intrinsic and extrinsic regulation of genes controlling pigmentation and coat color in mouse.
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Técnicas de Cultivo de Célula/métodos , Melanocitos/citología , Pigmentación/genética , Piel/citología , Animales , Regulación del Desarrollo de la Expresión Génica/genética , Oxidorreductasas Intramoleculares/biosíntesis , Melanocitos/metabolismo , Glicoproteínas de Membrana/biosíntesis , Ratones , Factor de Transcripción Asociado a Microftalmía/biosíntesis , Monofenol Monooxigenasa/biosíntesis , Oxidorreductasas/biosíntesis , Proteínas S100/biosíntesis , Piel/metabolismoRESUMEN
The detachment of epithelial cells, but not cancer cells, causes anoikis due to reduced energy production. Invasive tumor cells generate three splice variants of the metastasis gene osteopontin, the shortest of which (osteopontin-c) supports anchorage-independence. Osteopontin-c signaling upregulates three interdependent pathways of the energy metabolism. Glutathione, glutamine and glutamate support the hexose monophosphate shunt and glycolysis and can feed into the tricarboxylic acid cycle, leading to mitochondrial ATP production. Activation of the glycerol phosphate shuttle also supports the mitochondrial respiratory chain. Drawing substrates from glutamine and glycolysis, the elevated creatine may be synthesized from serine via glycine and supports the energy metabolism by increasing the formation of ATP. Metabolic probing with N-acetyl-L-cysteine, L-glutamate, or glycerol identified differential regulation of the pathway components, with mitochondrial activity being redox dependent and the creatine pathway depending on glutamine. The multiple skewed components in the cellular metabolism synergize in a flow toward two mechanisms of ATP generation, via creatine and the respiratory chain. It is consistent with a stimulation of the energy metabolism that supports anti-anoikis. Our findings imply a coalescence in cancer cells between osteopontin-a, which increases the cellular glucose levels, and osteopontin-c, which utilizes this glucose to generate energy.
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Neoplasias de la Mama/metabolismo , Mama/patología , Metabolismo Energético , Osteopontina/metabolismo , Adenosina Trifosfato/metabolismo , Secuencia de Aminoácidos , Mama/metabolismo , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Femenino , Regulación Neoplásica de la Expresión Génica , Glutamina/metabolismo , Glicerol/metabolismo , Glucólisis , Humanos , Células MCF-7 , Datos de Secuencia Molecular , Mutación , Invasividad Neoplásica/genética , Invasividad Neoplásica/patología , Osteopontina/química , Osteopontina/genética , Oxidación-Reducción , Oxidorreductasas/genética , Peróxidos/metabolismo , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Transducción de SeñalRESUMEN
Invasive breast tumor cells generate three splice variants of the metastasis gene osteopontin, while non-invasive breast cells express only the unspliced form or no osteopontin at all. One role for osteopontin in tumor progression is the support of anchorage-independence. Here we show that the full-length gene product, osteopontin-a, induces a gene expression profile that is associated with tissue remodeling and directed movement/sprouting. This occurs via signals through STAT1 and STAT3 to sn-glycero-3-phosphocholine. Osteopontin-a upregulates the levels of glucose in breast cancer cells, likely through STAT3 and its transcriptional targets apolipoprotein D and IGFBP5. The splice variants osteopontin-a and osteopontin-c may synergize, with each form activating signal transduction pathways that are distinct from the other. The elevated glucose is used by osteopontin-c dependent signals to generate chemical energy (Shi et al. submitted for publication). The splice variant-specific metabolic effects of osteopontin add a novel aspect to the pro-metastatic functions of this molecule.
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Neoplasias de la Mama/metabolismo , Glucosa/metabolismo , Homeostasis/fisiología , Invasividad Neoplásica , Osteopontina/metabolismo , Neoplasias de la Mama/patología , Adhesión Celular , Humanos , Immunoblotting , Células MCF-7 , Metabolómica , Isoformas de Proteínas , Transducción de Señal/fisiología , TransfecciónRESUMEN
Nuclear magnetic resonance (NMR) spectroscopy-based metabonomics is of growing importance for discovery of human disease biomarkers. Identification and validation of disease biomarkers using statistical significance analysis (SSA) is critical for translation to clinical practice. SSA is performed by assessing a null hypothesis test using a derivative of the Student's t test, e.g., a Welch's t test. Choosing how to correct the significance level for rejecting null hypotheses in the case of multiple testing to maintain a constant family-wise type I error rate is a common problem in such tests. The multiple testing problem arises because the likelihood of falsely rejecting the null hypothesis, i.e., a false positive, grows as the number of tests applied to the same data set increases. Several methods have been introduced to address this problem. Bonferroni correction (BC) assumes all variables are independent and therefore sacrifices sensitivity for detecting true positives in partially dependent data sets. False discovery rate (FDR) methods are more sensitive than BC but uniformly ascribe highest stringency to lowest p value variables. Here, we introduce standard deviation step down (SDSD), which is more sensitive and appropriate than BC for partially dependent data sets. Sensitivity and type I error rate of SDSD can be adjusted based on the degree of variable dependency. SDSD generates fundamentally different profiles of critical p values compared with FDR methods potentially leading to reduced type II error rates. SDSD is increasingly sensitive for more concentrated metabolites. SDSD is demonstrated using NMR-based metabonomics data collected on three different breast cancer cell line extracts.
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Neoplasias de la Mama/metabolismo , Metabolómica/métodos , Mama/metabolismo , Línea Celular Tumoral , Reacciones Falso Positivas , Femenino , Humanos , Análisis de los Mínimos Cuadrados , Modelos Estadísticos , Análisis de Componente Principal , ProbabilidadRESUMEN
Benzo[a]pyrene (BaP) is a prototypical polycyclic aromatic hydrocarbon (PAH); this ubiquitous environmental carcinogenic agent is found in tobacco smoke, charcoal-grilled foods, and PAH-contaminated surfaces of roofs, playgrounds, and highways. Cytochrome P450 1 wild-type, Cyp1a2(-/-), Cyp1b1(-/-), or Cyp1a2/1b1(-/-) knockouts, and mice with Cyp1a1 expression deleted in hepatocytes can ingest large oral BaP doses (125 mg/kg/d) without apparent toxicity. Cyp1a1(-/-) and Cyp1a1/1a2(-/-) knockouts and mice with Cyp1a1 expression deleted in gastrointestinal (GI) tract epithelial cells develop immunotoxicity and die within 32 days, indicating that GI tract inducible CYP1A1 is absolutely required for detoxication of oral BaP. Cyp1a1/1b1(-/-) and Cyp1a1/1a2/1b1(-/-) mice are rescued from immunosuppression and early death due to absent metabolic activation of BaP by CYP1B1 in immune cells. Ten-fold lower oral BaP doses result in adenocarcinoma of the proximal small intestine (PSI) in Cyp1a1(-/-) mice; Cyp1a1/1b1(-/-) double-knockout mice show no PSI cancer but develop squamous cell carcinoma of the preputial gland duct (PGD). BaP-metabolizing CYP1B1 in the PSI and CYP3A59 in the PGD are the most likely candidates to participate in tumor initiation in the epithelial cells of these two tissues; oncogenes and tumor-suppressor genes upregulated and downregulated during tumorigenesis are completely different between these tissues. This "oral BaP Cyp1" mouse paradigm represents a powerful teaching tool, showing that gene-environment interactions depend on route-of-administration: the same oral, but not intraperitoneal, BaP exposure leads to dramatic differences in target-organ toxicity and tumor type as a function of dose and Cyp1 genotype.
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Benzo(a)pireno/toxicidad , Carcinógenos Ambientales/toxicidad , Citocromo P-450 CYP1A1/genética , Neoplasias Experimentales/enzimología , Administración Oral , Animales , Hidrocarburo de Aril Hidroxilasas/genética , Hidrocarburo de Aril Hidroxilasas/metabolismo , Benzo(a)pireno/administración & dosificación , Benzo(a)pireno/farmacocinética , Carcinógenos Ambientales/administración & dosificación , Carcinógenos Ambientales/farmacocinética , Citocromo P-450 CYP1A1/metabolismo , Citocromo P-450 CYP1A2/genética , Citocromo P-450 CYP1A2/metabolismo , Citocromo P-450 CYP1B1 , Relación Dosis-Respuesta a Droga , Interacción Gen-Ambiente , Neoplasias Intestinales/inducido químicamente , Neoplasias Intestinales/enzimología , Neoplasias Intestinales/patología , Fase II de la Desintoxicación Metabólica , Ratones , Ratones Noqueados , Neoplasias Experimentales/inducido químicamente , Neoplasias Experimentales/patología , Especificidad de Órganos , Glándulas Odoríferas/enzimología , Glándulas Odoríferas/patología , Especificidad de la EspecieRESUMEN
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.
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Adenocarcinoma/genética , Hidrocarburo de Aril Hidroxilasas/fisiología , Biomarcadores de Tumor/genética , Carcinoma de Células Escamosas/genética , Citocromo P-450 CYP1A1/fisiología , Perfilación de la Expresión Génica , Glándulas Odoríferas/patología , Adenocarcinoma/tratamiento farmacológico , Adenocarcinoma/patología , Animales , Carcinoma de Células Escamosas/tratamiento farmacológico , Carcinoma de Células Escamosas/patología , Citocromo P-450 CYP1B1 , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Análisis de Secuencia por Matrices de Oligonucleótidos , Glándulas Odoríferas/efectos de los fármacos , Glándulas Odoríferas/metabolismoRESUMEN
Exposure to aristolochic acid (AA) is associated with human nephropathy and urothelial cancer. Individual susceptibility to AA-induced disease likely reflects individual differences in enzymes that metabolize AA. Herein, we evaluated AAI metabolism by human cytochrome P450 (CYP) 1A1 and 1A2 in two CYP1A-humanized mouse lines that carry functional human CYP1A1 and CYP1A2 genes in the absence of the mouse Cyp1a1/1a2 orthologs. Human and mouse hepatic microsomes and human CYPs were also studied. Human CYP1A1 and 1A2 were found to be principally responsible for reductive activation of AAI to form AAI-DNA adducts and for oxidative detoxication to 8-hydroxyaristolochic acid (AAIa), both in the intact mouse and in microsomes. Overall, AAI-DNA adduct levels were higher in CYP1A-humanized mice relative to wild-type mice, indicating that expression of human CYP1A1 and 1A2 in mice leads to higher AAI bioactivation than in mice containing the mouse CYP1A1 and 1A2 orthologs. Furthermore, an exclusive role of human CYP1A1 and 1A2 in AAI oxidation to AAIa was observed in human liver microsomes under the aerobic (i.e., oxidative) conditions. Because CYP1A2 levels in human liver are at least 100-fold greater than those of CYP1A1 and there exists a > 60-fold genetic variation in CYP1A2 levels in human populations, the role of CYP1A2 in AAI metabolism is clinically relevant. The results suggest that, in addition to CYP1A1 and 1A2 expression levels, in vivo oxygen concentration in specific tissues might affect the balance between AAI nitroreduction and demethylation, which in turn would influence tissue-specific toxicity or carcinogenicity.
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Ácidos Aristolóquicos/metabolismo , Carcinógenos/metabolismo , Citocromo P-450 CYP1A1/metabolismo , Citocromo P-450 CYP1A2/metabolismo , Hígado/enzimología , Animales , Ácidos Aristolóquicos/toxicidad , Ácidos Aristolóquicos/orina , Carcinógenos/toxicidad , Cromatografía Líquida de Alta Presión , Citocromo P-450 CYP1A1/antagonistas & inhibidores , Citocromo P-450 CYP1A1/genética , Citocromo P-450 CYP1A2/genética , Inhibidores del Citocromo P-450 CYP1A2 , Citosol/enzimología , Aductos de ADN/metabolismo , Remoción de Radical Alquila , Inhibidores Enzimáticos/farmacología , Femenino , Humanos , Inactivación Metabólica , Hígado/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Microsomas Hepáticos/enzimología , Oxidación-Reducción , Proteínas Recombinantes/metabolismo , Neoplasias Urológicas/inducido químicamente , Urotelio/efectos de los fármacosRESUMEN
Exposure to aristolochic acid I (AAI) is associated with aristolochic acid nephropathy, Balkan endemic nephropathy, and urothelial cancer. Individual differences in xenobiotic-metabolizing enzyme activities are likely to be a reason for interindividual susceptibility to AA-induced disease. We evaluated the reductive activation and oxidative detoxication of AAI by cytochrome P450 (P450) 1A1 and 1A2 using the Cyp1a1(-/-) and Cyp1a2(-/-) single-knockout and Cyp1a1/1a2(-/-) double-knockout mouse lines. Incubations with hepatic microsomes were also carried out in vitro. P450 1A1 and 1A2 were found to (i) activate AAI to form DNA adducts and (ii) detoxicate it to 8-hydroxyaristolochic acid I (AAIa). AAI-DNA adduct formation was significantly higher in all tissues of Cyp1a1/1a2(-/-) than Cyp1a(+/+) wild-type (WT) mice. AAI-DNA adduct levels were elevated only in selected tissues from Cyp1a1(-/-) versus Cyp1a2(-/-) mice, compared with those in WT mice. In hepatic microsomes, those from WT as well as Cyp1a1(-/-) and Cyp1a2(-/-) mice were able to detoxicate AAI to AAIa, whereas Cyp1a1/1a2(-/-) microsomes were less effective in catalyzing this reaction, confirming that both mouse P450 1A1 and 1A2 are both involved in AAI detoxication. Under hypoxic conditions, mouse P450 1A1 and 1A2 were capable of reducing AAI to form DNA adducts in hepatic microsomes; the major roles of P450 1A1 and 1A2 in AAI-DNA adduct formation were further confirmed using selective inhibitors. Our results suggest that, in addition to P450 1A1 and 1A2 expression levels in liver, in vivo oxygen concentration in specific tissues might affect the balance between AAI nitroreduction and demethylation, which in turn would influence tissue-specific toxicity or carcinogenicity.
Asunto(s)
Ácidos Aristolóquicos/farmacocinética , Carcinógenos/farmacocinética , Citocromo P-450 CYP1A1/metabolismo , Citocromo P-450 CYP1A2/metabolismo , Medicamentos Herbarios Chinos/farmacocinética , Animales , Ácidos Aristolóquicos/orina , Nefropatía de los Balcanes/enzimología , Biotransformación , Citocromo P-450 CYP1A1/deficiencia , Citocromo P-450 CYP1A1/genética , Citocromo P-450 CYP1A2/deficiencia , Citocromo P-450 CYP1A2/genética , Aductos de ADN , Susceptibilidad a Enfermedades , Femenino , Riñón/enzimología , Hígado/enzimología , Pulmón/enzimología , Ratones , Ratones Noqueados , Microsomas/enzimología , Neoplasias Urológicas/enzimologíaRESUMEN
Polycyclic aromatic hydrocarbons (PAHs) are widely distributed environmental toxicants derived from sources that include cigarette smoke, petroleum distillation, gas- and diesel-engine exhaust, and charcoal-grilled food. The gastrointestinal tract is the principal route of PAH exposures, even when inhaled. The most thoroughly studied prototype of PAHs is benzo[a]pyrene (BaP), well known to be toxic, mutagenic, and carcinogenic in various tissues and cell types. This lab has previously shown that Cyp1a1(-/-) global knockout mice treated by oral administration of BaP die at 28 to 32 days with immunosuppression, whereas wild-type mice remain healthy for 1 year on high BaP doses (125 mg/kg/day). Thus, for oral BaP, CYP1A1 is more important in detoxication than in metabolic activation. After several days of oral BaP, we found surprisingly low CYP1A1 levels in liver, compared with that in small intestine; we postulated that this finding might reflect efficient detoxication of oral BaP in proximal small intestine such that significant amounts of the inducer BaP no longer reach the liver. In the present study, many parameters were therefore compared in wild-type, Cyp1a1(-/-) global knockout, intestinal epithelial cell-specific Cyp1a1 knockout, and hepatocyte-specific Cyp1a1 knockout mice as a function of long-term oral exposure to BaP. The peak of CYP1A1 (mRNA, protein) expression in liver occurred at 12 h, whereas highly induced CYP1A1 in small intestine persisted throughout the 30-day experiment. Hepatocyte-specific Cyp1a1 knockout mice remained as healthy as wild-type mice; intestinal epithelial cell-specific Cyp1a1 knockout mice behaved like Cyp1a1(-/-) mice, dying with immunosuppression approximately 30 days on oral BaP. We conclude that small intestine CYP1A1, and not liver CYP1A1, is critically important in oral BaP detoxication.
Asunto(s)
Benzo(a)pireno/farmacocinética , Citocromo P-450 CYP1A1/metabolismo , Dieta , Inactivación Metabólica , Animales , Secuencia de Bases , Benzo(a)pireno/administración & dosificación , Benzo(a)pireno/farmacología , Peso Corporal/efectos de los fármacos , Citocromo P-450 CYP1A1/genética , Cartilla de ADN , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Tamaño de los Órganos/efectos de los fármacos , ARN Mensajero/genéticaRESUMEN
Benzo[a]pyrene (BaP) is a prototypical polycyclic aromatic hydrocarbon (PAH) found in combustion processes. Cytochrome P450 1A1 and 1B1 enzymes (CYP1A1 and CYP1B1) can both detoxify PAHs and activate them to cancer-causing reactive intermediates. Following high dosage of oral BaP (125 mg/kg/day), ablation of the mouse Cyp1a1 gene causes immunosuppression and death within â¼28 days, whereas Cyp1(+/+) wild-type mice remain healthy for >12 months on this regimen. In this study, male Cyp1(+/+) wild-type, Cyp1a1(-/-) and Cyp1b1(-/-) single-knockout and Cyp1a1/1b1(-/-) double-knockout mice received a lower dose (12.5 mg/kg/day) of oral BaP. Tissues from 16 different organs-including proximal small intestine (PSI), liver and preputial gland duct (PGD)-were evaluated; microarray cDNA expression and >30 mRNA levels were measured. Cyp1a1(-/-) mice revealed markedly increased CYP1B1 mRNA levels in the PSI, and between 8 and 12 weeks developed unique PSI adenomas and adenocarcinomas. Cyp1a1/1b1(-/-) mice showed no PSI tumors but instead developed squamous cell carcinoma of the PGD. Cyp1(+/+) and Cyp1b1(-/-) mice remained healthy with no remarkable abnormalities in any tissue examined. PSI adenocarcinomas exhibited striking upregulation of the Xist gene, suggesting epigenetic silencing of specific genes on the Y-chromosome; the Rab30 oncogene was upregulated; the Nr0b2 tumor suppressor gene was downregulated; paradoxical overexpression of numerous immunoglobulin kappa- and heavy-chain variable genes was found-although the adenocarcinoma showed no immunohistochemical evidence of being lymphatic in origin. This oral BaP mouse paradigm represents an example of "gene-environment interactions" in which the same exposure of carcinogen results in altered target organ and tumor type, as a function of just 1 or 2 globally absent genes.