RESUMO
Fluorescence tumor imaging using exogenous fluorescent tumor-targeting agents has potential to improve early tumor detection. The fluorescent contrast agent indocyanine green (ICG) is used in medical diagnostics. The aim of the present study is to investigate the tumor imaging capability and the imaging mechanism of i.v. administered ICG in a mouse model of colitis-associated colon cancer. To do this, an azoxymethane/dextran sodium sulfate-induced colon cancer mouse model was used. Ex vivo imaging experiments were carried out 1 hour after i.v. injection of ICG. The ICG fluorescence was observed in the colon tumor tissues, with sufficient tumor to normal tissue ratio, correlating with tumor malignancy. In the tumor tissues, ICG fluorescence was localized in the vascular interstitial tissue. Immunofluorescence microscopy revealed that tumor cells formed tight junctions normally, suggesting an inability of tumor cellular uptake of ICG. In contrast, tumor tissues increased the CD31-immunoreactive endothelial cell area, and accumulated stromal cells immunoreactive for COX-2 and tumor cell population immunoreactive for inducible nitric oxide synthase. In vivo vascular permeability assay revealed that prostaglandin E2 promoted the endothelial cell permeability of ICG. In conclusion, our data indicated that fluorescence contrast-enhanced imaging following i.v. administered ICG can be applied to the detection of colon tumors in a mouse colitis-associated colon cancer model. The tumor tissue preference of ICG in the present model can be attributed to the enhanced vascular leakage of ICG involving inflammatory mediators, such as COX-2 and inducible nitric oxide synthase, in conjunction with increased tumor vascularity.
Assuntos
Colite/complicações , Neoplasias do Colo/diagnóstico por imagem , Verde de Indocianina/administração & dosagem , Animais , Permeabilidade Capilar , Neoplasias do Colo/irrigação sanguínea , Modelos Animais de Doenças , Feminino , Fluorescência , Injeções Intravenosas , Camundongos , Camundongos Endogâmicos ICR , Junções ÍntimasRESUMO
Indocyanine green (ICG) is a fluorescent agent approved for clinical applications by the Food and Drug Administration and European Medicines Agency. This study examined the mechanism of tumor imaging using intravenously administered ICG. The in vivo kinetics of intravenously administered ICG were determined in tumor xenografts using microscopic approaches that enabled both spatio-temporal and high-magnification analyses. The mechanism of ICG-based tumor imaging was examined at the cellular level in six phenotypically different human colon cancer cell lines exhibiting different grades of epithelioid organization. ICG fluorescence imaging detected xenograft tumors, even those < 1 mm in size, based on their preferential cellular uptake and retention of the dye following its rapid tissue-non-specific delivery, in contrast to its rapid clearance by normal tissue. Live-cell imaging revealed that cellular ICG uptake is temperature-dependent and occurs after ICG binding to the cellular membrane, a pattern suggesting endocytic uptake as the mechanism. Cellular ICG uptake correlated inversely with the formation of tight junctions. Intracellular ICG was entrapped in the membrane traffic system, resulting in its slow turnover and prolonged retention by tumor cells. Our results suggest that tumor-specific imaging by ICG involves non-specific delivery of the dye to tissues followed by preferential tumor cellular uptake and retention. The tumor cell-preference of ICG is driven by passive tumor cell-targeting, the inherent ability of ICG to bind to cell membranes, and the high endocytic activity of tumor cells in association with the disruption of their tight junctions.
Assuntos
Verde de Indocianina/metabolismo , Neoplasias/diagnóstico por imagem , Neoplasias/metabolismo , Imagem Óptica/métodos , Animais , Linhagem Celular Tumoral , Modelos Animais de Doenças , Fluorescência , Xenoenxertos , Humanos , Verde de Indocianina/administração & dosagem , Verde de Indocianina/farmacocinética , Camundongos , Camundongos Nus , Distribuição TecidualRESUMO
To determine the developmental exposure effects of T-2 toxin on postnatal hippocampal neurogenesis, pregnant ICR mice were provided a diet containing T-2 toxin at 0, 1, 3, or 9 ppm from gestation day 6 to day 21 on weaning after delivery. Offspring were maintained through postnatal day (PND) 77 without T-2 toxin exposure. In the hippocampal dentate gyrus of male PND 21 offspring, GFAP(+) and BLBP(+) type-1 stem cells and PAX6(+) and TBR2(+) type-2 progenitor cells decreased in the subgranular zone (SGZ) at 9 and ≥3 ppm, respectively, in parallel with increased apoptosis at ≥3 ppm. In the dentate hilus, reelin(+) γ-aminobutyric acid (GABA)-ergic interneurons increased at 9 ppm, suggesting reflection of neuronal mismigration. T-2 toxin decreased transcript levels of cholinergic and glutamate receptor subunits (Chrna4, Chrnb2 and Gria2) and glutamate transporter (Slc17a6) in the dentate gyrus, suggesting decreased cholinergic signals on hilar GABAergic interneurons innervating type-2 cells and decreased glutamatergic signals on type-1 and type-2 cells. T-2 toxin decreased SGZ cells expressing stem cell factor (SCF) and increased cells accumulating malondialdehydes. Neurogenesis-related changes disappeared on PND 77, suggesting that T-2 toxin reversibly affects neurogenesis by inducing apoptosis of type-1 and type-2 cells with different threshold levels. Decreased cholinergic and glutamatergic signals may decrease type-2 cells at ≥3 ppm. Additionally, decreased SCF/c-Kit interactions and increased oxidative stress may decrease type-1 and type-2 cells at 9 ppm. The no-observed-adverse-effect level for offspring neurogenesis was determined to be 1 ppm (0.14-0.49 mg/kg body weight/day).
Assuntos
Hipocampo/efeitos dos fármacos , Células-Tronco Neurais/efeitos dos fármacos , Neurogênese/efeitos dos fármacos , Efeitos Tardios da Exposição Pré-Natal/induzido quimicamente , Toxina T-2/toxicidade , Animais , Animais Recém-Nascidos , Apoptose/efeitos dos fármacos , Feminino , Idade Gestacional , Hipocampo/crescimento & desenvolvimento , Hipocampo/metabolismo , Hipocampo/patologia , Masculino , Camundongos Endogâmicos ICR , Células-Tronco Neurais/metabolismo , Células-Tronco Neurais/patologia , Nível de Efeito Adverso não Observado , Estresse Oxidativo/efeitos dos fármacos , Gravidez , Efeitos Tardios da Exposição Pré-Natal/metabolismo , Efeitos Tardios da Exposição Pré-Natal/patologia , Receptores Colinérgicos/metabolismo , Proteína Reelina , Fator de Células-Tronco/metabolismoRESUMO
We have previously reported that a 28-day treatment of carcinogens evoking target cell proliferation activates G1 /S checkpoint function and apoptosis, as well as induction of aberrant ubiquitin D (Ubd) expression, suggesting disruptive spindle checkpoint function, in rats. The present study aimed to determine the onset time of rat liver cells to undergo carcinogen-specific cell cycle aberration and proliferation. Animals were treated orally with a hepatocarcinogenic dose of methyleugenol or thioacetamide for 3, 7 or 28 days. For comparison, some animals were subjected to partial hepatectomy or treated with noncarcinogenic hepatotoxicants (acetaminophen, α-naphthyl isothiocyanate or promethazine). Carcinogen-specific liver cell kinetics appeared at day 28 as evident by increases of cell proliferation, p21(Cip1+) cells, phosphorylated-Mdm2(+) cells and cleaved caspase 3(+) cells, and upregulation of DNA damage-related genes. Hepatocarcinogens also downregulated Rbl2 and upregulated Cdkn1a and Mdm2, and decreased Ubd(+) cells co-expressing phosphorylated-histone H3 (p-Histone H3) and p-Histone H3(+) cell ratio within the Ki-67(+) proliferating population. These results suggest that it takes 28 days to induce hepatocarcinogen-specific early withdrawal of proliferating cells from M phase due to disruptive spindle checkpoint function as evidenced by reduction of Ubd(+) cells staying at M phase. Disruption of G1 /S checkpoint function reflected by downregulation of Rbl2 as well as upregulation of Mdm2 suggestive of sequestration of retinoblastoma protein is responsible for the facilitation of carcinogen-induced cell proliferation at day 28. Accumulation of DNA damage probably in association with facilitation of p53 degradation by activation of Mdm2 may be a prerequisite for aberrant p21(Cip1) activation, which is responsible for apoptosis.
Assuntos
Apoptose/efeitos dos fármacos , Carcinógenos/toxicidade , Ciclo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Hepatectomia/efeitos adversos , Fígado/efeitos dos fármacos , Fígado/crescimento & desenvolvimento , Animais , Carcinogênese/efeitos dos fármacos , Eugenol/análogos & derivados , Eugenol/toxicidade , Masculino , Ratos , Ratos Endogâmicos F344 , Tioacetamida/toxicidadeRESUMO
Near-infrared photoimmunotherapy (NIR-PIT) is a new phototherapy that utilizes a monoclonal antibody (mAb) against cancer antigens and a phthalocyanine dye, IRDye700DX (IR700) conjugate (mAb-IR700). Photodynamic therapy (PDT) is a combination therapy that utilizes photoreactive agents and light irradiation as well as NIR-PIT. In the present study, we compared these therapies in vitro. The characterization of cellular binding/uptake specificity and cytotoxicity were examined using two mAb-IR700 forms and a conventional PDT agent, talaporfin sodium, in three cell lines. As designed, mAb-IR700 had high molecular selectivity and visualized target molecule-positive cells at the lowest concentration examined. NIR-PIT induced necrosis and damage-associated molecular patterns (DAMPs), a surrogate maker of immunogenic cell death. In contrast, talaporfin sodium was taken up by cells regardless of cell type, and its uptake was enhanced in a concentration-dependent manner. PDT induced cell death, with the pattern of cell death shifting from apoptosis to necrosis depending on the concentration of the photosensitizer. Induction of DAMPs was observed at the highest concentration, but their sensitivity differed among cell lines. Overall, our data suggest that molecule-specific NIR-PIT may have potential advantages compared with PDT in terms of the efficiency of tumor visualization and induction of DAMPs.
RESUMO
Near-infrared photoimmunotherapy (NIR-PIT) is a novel form of cancer treatment using conjugates of antibody against overexpressed antigens in cancers and photoabsorber IRDye700DX. HER2 is overexpressed in various cancers, for which molecular targeted therapy such as trastuzumab has been developed. The present study investigated the efficacy potential of HER2-targeted NIR-PIT using trastuzumab-IRDye700DX conjugate (Tra-IR700) in HER2-positive breast cancer. We first examined the reactivity of Tra-IR700 and the cytotoxicity of NIR-PIT in vitro. HER2-positive BT-474 and SK-BR-3 cells and HER2-negative BT-20 cells were used. Tra-IR700 fluorescence was only observed in HER2-positive breast cancer cell lines, and the fluorescence was localized to the cell surface. Furthermore, HER2-positive breast cancer cell lines treated with NIR-PIT showed swelling and blebbing shortly after irradiation, and eventually increased PI-positive dead cells. Next, tumor accumulation of Tra-IR700 and tumor damage by NIR-PIT were examined in vivo. Tra-IR700 was administered intravenously to a xenograft model in which BT-474 cells were implanted subcutaneously in BALB/c nude mice. Tra-IR700 fluorescence was the highest in tumor tissue 1 day after administration, and the fluorescence was localized to the cell membrane of tumor cells. At this time point, NIR-PIT resulted in diffuse necrosis of tumor tissues 1 day after irradiation. These results suggest that NIR-PIT with Tra-IR700 induces a highly selective therapeutic effect in a HER2-positive breast cancer model. NIR-PIT using Tra-IR700 is expected to be a novel treatment for HER2-positive cancers, including breast cancer.
Assuntos
Neoplasias da Mama , Fototerapia , Humanos , Animais , Camundongos , Feminino , Trastuzumab/farmacologia , Trastuzumab/uso terapêutico , Xenoenxertos , Camundongos Nus , Linhagem Celular Tumoral , Fototerapia/métodos , Imunoterapia/métodos , Neoplasias da Mama/tratamento farmacológico , Ensaios Antitumorais Modelo de Xenoenxerto , Fármacos FotossensibilizantesRESUMO
Dimethyltin dichloride (DMTC) is widely used as a heat stabilizer in manufacturing the polyvinyl chloride. We previously reported a case of acute DMTC poisoning with neurological manifestations very similar to trimethylated tin (TMT) encephalopathy, based on results of speciation analysis of methylated tins in the patient's urine with use of a combination of high performance liquid chromatography and inductively coupled plasma-mass spectrometry (HPLC-ICP-MS), which yielded peaks corresponding to DMT and TMT. In this study, we developed an analytical method to confirm TMT in urine using high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS), and found TMT molecular ion in the patient's urine.
Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Espectrometria de Massas em Tandem/métodos , Compostos de Trimetilestanho/urina , Humanos , Compostos de Trimetilestanho/toxicidadeRESUMO
In order to determine low levels of Cd in urine samples, we tried to remove Mo interference using ICP-MS with a dynamic reaction-cell technique, but failed due to the low sensitivity and the variance with the standards. We then performed solid-phase extraction (SPE) before the ICP-MS measurement. A commercially available chelating resin, NOBIAS PA-1, was used for SPE, and could effectively remove Mo from urine samples, permitting the accurate determination of Cd by ICP-MS. This SPE-ICP-MS method gave 0.012 microg Cd L(-1) as the method limit of quantification, and the mean recovery of Cd spiked with 0.0505 and 5.05 microg L(-1) was 93.1 and 97.6%, respectively.
Assuntos
Cádmio/urina , Espectrometria de Massas/métodos , Extração em Fase Sólida/métodos , Molibdênio/química , Molibdênio/isolamento & purificação , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
Aluminum (Al) is neurotoxic to adults and also to infants. In this study, we investigated the developmental exposure effect of AlCl3 on postnatal hippocampal neurogenesis. Pregnant mice were administered 0-, 900-, or 1800-ppm AlCl3 via drinking water from gestational day 6 to postnatal day (PND) 21, with their offspring examined on PND 21 and PND 77. On PND 21, GFAP-immunoreactive (+) neural stem cells (NSCs) and p21Cip1/Waf1+ cells were decreased in number in the subgranular zone at 900 and ≥900 ppm, respectively. Pcna transcript level examined at 1800 ppm was decreased in the dentate gyrus. These results suggest induction of compromised cell quiescence that caused impaired self-renewal capacity of NSCs accompanying slowing down of cell cycling, which ultimately resulted in exhaustion of the NSC pool. At 1800 ppm, Reelin+ hilar GABAergic interneurons were also decreased, suggesting a contribution to the NSC reduction. At this dose, TBR2+ or DCX+ progenitor and immature granule cells and PVALB+ interneurons were increased. Moreover, COX-2+ granule cells were increased at ≥900 ppm. These results suggest facilitation of transient progenitor cell proliferation and differentiation during exposure. Moreover, TUNEL+ or Morin-stained granule cells were increased, together with Casp12 transcript upregulation, suggesting induction of Al accumulation-related endoplasmic reticulum stress-mediated granule cell apoptosis. Transcript expression changes on cholinergic and glutamatergic signals and synaptic plasticity suggested contribution to disruptive neurogenesis. The NSC-targeting effects sustained through the adult stage despite no sustained Al-accumulation. These results suggest that developmental AlCl3-exposure irreversibly affects postnatal hippocampal neurogenesis involving multiple functions in mice.
Assuntos
Cloreto de Alumínio/toxicidade , Hipocampo/efeitos dos fármacos , Neurogênese/efeitos dos fármacos , Efeitos Tardios da Exposição Pré-Natal/induzido quimicamente , Animais , Relação Dose-Resposta a Droga , Proteína Duplacortina , Feminino , Idade Gestacional , Hipocampo/crescimento & desenvolvimento , Hipocampo/patologia , Masculino , Camundongos , Camundongos Endogâmicos ICR , Células-Tronco Neurais/citologia , Células-Tronco Neurais/efeitos dos fármacos , Gravidez , Efeitos Tardios da Exposição Pré-Natal/patologia , Efeitos Tardios da Exposição Pré-Natal/fisiopatologia , Proteína ReelinaRESUMO
An analytical method using a combination of solid-phase extraction (SPE) and gas chromatography with a flame thermionic detector (GC/FTD) was developed for determination of N-methyl-2-pyrrolidone (NMP), N-methylsuccinimide (MSI), and 2-hydroxy-N-methylsuccinimide (2-HMSI) in human urine. The SPE cartridge of poly(divinylbenzene/hydroxymethacrylate) used was directly loaded with urine sample, followed by elution with methyl isobutyl ketone (MIBK) and subsequent centrifugation, and the supernatant was injected into the capillary GC using a DB1701. This method allowed efficient separation of NMP, MSI, and 2-HMSI, which were nearly free of interference by other GC peaks arising from urine. Recoveries of NMP, MSI, and 2-HMSI from the SPE cartridge were about 98, 101, and 67%, respectively, with limits of detection of 0.04, 0.02, and 0.06 mg/L, respectively, which met the regulatory requirements. The present method was used for assay in biological monitoring of workers exposed to NMP in their occupational environment.
Assuntos
Cromatografia Gasosa/métodos , Pirrolidinonas/urina , Calibragem , Monitoramento Ambiental , Concentração de Íons de Hidrogênio , Sensibilidade e EspecificidadeRESUMO
The fluorescent contrast agent indocyanine green (ICG) is approved by the Food and Drug Administration for clinical applications. We previously reported that cultured human colon tumor cells preferentially take up ICG by endocytic activity in association with disruption of their tight junctions. The present study explored ICG availability in fluorescence imaging of the colon to identify proliferative lesions during colonoscopy. The cellular uptake of ICG in cultured rat colon tumor cells was examined using live-cell imaging. Colon lesions in rats administered an ICG-containing enema were further assessed in rats with azoxymethane-induced colon carcinogenesis, using in vivo endoscopy, ex vivo microscopy, and immunofluorescence microscopy. The uptake of ICG by the cultured cells was temperature-dependent. The intracellular retention of the dye in the membrane trafficking system suggested endocytosis as the uptake mechanism. ICG administered via enema accumulated in colon proliferative lesions ranging from tiny aberrant crypt foci to adenomas and localized in proliferating cells. Fluorescence endoscopy detected these ICG-positive colonic proliferative lesions in vivo. The immunoreactivity of the tight-junction molecule occludin was altered in the proliferative lesions, suggesting the disruption of the integrity of tight junctions. These results suggest that fluorescence contrast-enhanced imaging following the administration of an ICG-containing enema can enhance the detection of mucosal proliferative lesions of the colon during colonoscopy. The tissue preference of ICG in the rat model evaluated in this study can be attributed to the disruption of tight junctions, which in turn promotes endocytosis by proliferative cells and the cellular uptake of ICG.
RESUMO
The present study aimed to determine the onset time of hepatocarcinogen/hepatocarcinogenic tumor promoter-specific cell proliferation, apoptosis and aberrant cell cycle regulation after post-initiation treatment. Six-week-old rats were treated with the genotoxic hepatocarcinogen, carbadox (CRB), the marginally hepatocarcinogenic leucomalachite green (LMG), the tumor promoter, ß-naphthoflavone (BNF) or the non-carcinogenic hepatotoxicant, acetaminophen, for 2, 4 or 6 weeks during the post-initiation phase using a medium-term liver bioassay. Cell proliferation activity, expression of G2 to M phase- and spindle checkpoint-related molecules, and apoptosis were immunohistochemically analyzed at week 2 and 4, and tumor promotion activity was assessed at week 6. At week 2, hepatocarcinogen/tumor promoter-specific aberrant cell cycle regulation was not observed. At week 4, BNF and LMG increased cell proliferation together with hepatotoxicity, while CRB did not. Additionally, BNF and CRB reduced the number of cells expressing phosphorylated-histone H3 in both ubiquitin D (UBD)(+) cells and Ki-67(+) proliferating cells, suggesting development of spindle checkpoint dysfunction, regardless of cell proliferation activity. At week 6, examined hepatocarcinogens/tumor promoters increased preneoplastic hepatic foci expressing glutathione S-transferase placental form. These results suggest that some hepatocarcinogens/tumor promoters increase their toxicity after post-initiation treatment, causing regenerative cell proliferation. In contrast, some genotoxic hepatocarcinogens may disrupt the spindle checkpoint without facilitating cell proliferation at the early stage of tumor promotion. This suggests that facilitation of cell proliferation and disruption of spindle checkpoint function are induced by different mechanisms during hepatocarcinogenesis. Four weeks of post-initiation treatment may be sufficient to induce hepatocarcinogen/tumor promoter-specific cellular responses.
Assuntos
Apoptose/efeitos dos fármacos , Carcinógenos/toxicidade , Proliferação de Células/efeitos dos fármacos , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , Fígado/efeitos dos fármacos , Pontos de Checagem da Fase M do Ciclo Celular/efeitos dos fármacos , Acetaminofen/toxicidade , Animais , Carbadox/toxicidade , Cocarcinogênese , Dietilnitrosamina/toxicidade , Imuno-Histoquímica , Antígeno Ki-67/metabolismo , Fígado/metabolismo , Fígado/patologia , Masculino , Ratos Endogâmicos F344 , Corantes de Rosanilina/toxicidade , Fatores de Tempo , beta-Naftoflavona/toxicidadeRESUMO
We previously reported that 28-day exposure to hepatocarcinogens that facilitate cell proliferation specifically alters the expression of G1/S checkpoint-related genes and proteins, induces aberrant early expression of ubiquitin D (UBD) at the G2 phase, and increases apoptosis in the rat liver, indicating G1/S and spindle checkpoint dysfunction. The present study aimed to determine the time of onset of carcinogen-specific cell-cycle disruption after repeated administration of renal carcinogens for up to 28 days. Rats were orally administered the renal carcinogens nitrofurantoin (NFT), 1-amino-2,4-dibromoantraquinone (ADAQ), and 1,2,3-trichloropropane (TCP) or the non-carcinogenic renal toxicants 1-chloro-2-propanol, triamterene, and carboxin for 3, 7 or 28 days. Both immunohistochemical single-molecule analysis and real-time RT-PCR analysis revealed that carcinogen-specific expression changes were not observed after 28 days of administration. However, the renal carcinogens ADAQ and TCP specifically reduced the number of cells expressing phosphorylated-histone H3 at Ser10 in both UBD(+) cells and proliferating cells, suggestive of insufficient UBD expression at the M phase and early transition of proliferating cells from the M phase, without increasing apoptosis, after 28 days of administration. In contrast, NFT, which has marginal carcinogenic potential, did not induce such cellular responses. These results suggest that it may take 28 days to induce spindle checkpoint dysfunction by renal carcinogens; however, induction of apoptosis may not be essential. Thus, induction of spindle checkpoint dysfunction may be dependent on carcinogenic potential of carcinogen examined, and marginal carcinogens may not exert sufficient responses even after 28 days of administration.
Assuntos
Antraquinonas/administração & dosagem , Antraquinonas/toxicidade , Rim/citologia , Pontos de Checagem da Fase M do Ciclo Celular/efeitos dos fármacos , Nitrofurantoína/administração & dosagem , Nitrofurantoína/toxicidade , Propano/análogos & derivados , Animais , Peso Corporal/efeitos dos fármacos , Carboxina/administração & dosagem , Carboxina/toxicidade , Proliferação de Células/efeitos dos fármacos , Cloridrinas/administração & dosagem , Cloridrinas/toxicidade , Histonas/metabolismo , Rim/efeitos dos fármacos , Masculino , Tamanho do Órgão/efeitos dos fármacos , Propano/administração & dosagem , Propano/toxicidade , Ratos Endogâmicos F344 , Fatores de Tempo , Triantereno/administração & dosagem , Triantereno/toxicidade , Ubiquitinas/metabolismoRESUMO
We aimed to clarify the hepatocarcinogen-specific disruption of cell cycle checkpoint functions and its time course after repeated administration of hepatocarcinogens. Thus, rats were repeatedly administered with hepatocarcinogens (methapyrilene, carbadox and thioacetamide), a marginal hepatocarcinogen (leucomalachite green), hepatocarcinogenic promoters (oxfendazole and ß-naphthoflavone) or non-carcinogenic hepatotoxicants (promethazine and acetaminophen) for 7, 28 or 90 days, and the temporal changes in cell proliferation, expression of G1/S and spindle checkpoint-related molecules, and apoptosis were examined using immunohistochemistry and/or real-time RT-PCR analysis. Hepatocarcinogens facilitating cell proliferation at day 28 of administration also facilitated cell proliferation and apoptosis at day 90. Hepatocarcinogen- or hepatocarcinogenic promoter-specific cellular responses were not detected by immunohistochemical single molecule analysis even after 90 days. Expression of Cdkn1a, Mad2l1, Chek1 and Rbl2 mRNA also lacked specificity to hepatocarcinogens or hepatocarcinogenic promoters. In contrast, all hepatocarcinogens and the marginally hepatocarcinogenic leucomalachite green induced Mdm2 upregulation or increase in the number of phosphorylated MDM2(+) cells from day 28, irrespective of the lack of cell proliferation facilitation by some compounds. However, different Tp53 expression levels suggest different mechanisms of induction or activation of MDM2 among hepatocarcinogens. On the other hand, hepatocarcinogenic methapyrilene and carbadox downregulated the number of both ubiquitin D(+) cells and proliferating cells remaining in M phase at day 28 and/or day 90, irrespective of the lack of cell proliferation facilitation in the latter. These results suggest that hepatocarcinogens disrupt spindle checkpoint function after 28 or 90 days of administration, which may be induced ahead of cell proliferation facilitation.
Assuntos
Carbadox/toxicidade , Carcinógenos/administração & dosagem , Carcinógenos/toxicidade , Proliferação de Células/efeitos dos fármacos , Fígado/citologia , Pontos de Checagem da Fase M do Ciclo Celular/efeitos dos fármacos , Metapirileno/toxicidade , Tioacetamida/toxicidade , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Benzimidazóis/administração & dosagem , Benzimidazóis/toxicidade , Carbadox/administração & dosagem , Proliferação de Células/genética , Quinase 1 do Ponto de Checagem , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Expressão Gênica/efeitos dos fármacos , Masculino , Metapirileno/administração & dosagem , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Ratos Endogâmicos F344 , Corantes de Rosanilina/toxicidade , Fatores de Tempo , Ubiquitinas/genética , Ubiquitinas/metabolismoRESUMO
Azoxymethane (AOM) is a potent carcinogen used for induction of colon tumors in rats and mice. It is also known that AOM treatment induces small bowel tumors in addition to colorectal tumors in rats. The present study examined the histogenesis of AOM-induced rat duodenal tumors in comparison with concurrently induced colorectal tumors by histochemical and immunohistochemical approaches. Duodenal and colorectal tumors were positive for both periodic acid-Schiff reaction and Alcian blue staining. Immunohistochemically, duodenal tumors were positive for intestinal epithelial markers such as cytokeratin (CK) 20 (100%) and mucin (MUC) 2 (91.7%) but negative for pancreaticobiliary markers such as CK7 (100%) and MUC1 (100%). All colorectal tumors were also negative for CK7 and MUC1 but positive for CK20. Eighty percent of colorectal tumors were positive for MUC2. In addition, nuclear accumulation of ß-catenin was found in duodenal tumors (70.8%), which was similar to colorectal tumors (90.0%). These results indicate that duodenal tumors induced by AOM treatment of rats were derived from intestinal epithelium. Similar to colorectal tumors, nuclear accumulation of ß-catenin indicates activation of Wnt signaling as a driving force for tumor progression in AOM-induced duodenal tumors.
Assuntos
Azoximetano/toxicidade , Carcinógenos/toxicidade , Neoplasias Colorretais/induzido quimicamente , Neoplasias Duodenais/induzido quimicamente , Mucosa Intestinal/efeitos dos fármacos , beta Catenina/metabolismo , Adenocarcinoma/induzido quimicamente , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Animais , Carcinogênese/induzido quimicamente , Neoplasias Colorretais/patologia , Neoplasias Duodenais/metabolismo , Neoplasias Duodenais/patologia , Duodeno/efeitos dos fármacos , Duodeno/metabolismo , Duodeno/patologia , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Masculino , Ratos , Ratos Endogâmicos F344RESUMO
To elucidate the maternal exposure effects of aflatoxin B1 (AFB1) and its metabolite aflatoxin M1, which is transferred into milk, on postnatal hippocampal neurogenesis, pregnant Sprague-Dawley rats were provided a diet containing AFB1 at 0, 0.1, 0.3, or 1.0 ppm from gestational day 6 to day 21 after delivery on weaning. Offspring were maintained through postnatal day (PND) 77 without AFB1 exposure. Following exposure to 1.0 ppm AFB1, offspring showed no apparent systemic toxicity at weaning, whereas dams showed increased liver weight and DNA repair gene upregulation in the liver. In the hippocampal dentate gyrus of male PND 21 offspring, the number of doublecortin(+) progenitor cells were decreased, which was associated with decreased proliferative cell population in the subgranular zone at ≥ 0.3 ppm, although T-box brain 2(+) cells, tubulin beta III(+) cells, gamma-H2A histone family, member X(+) cells, and cyclin-dependent kinase inhibitor 1A(+) cells did not fluctuate in number. AFB1 exposure examined at 1.0 ppm also resulted in transcript downregulation of the cholinergic receptor subunit Chrna7 and dopaminergic receptor Drd2 in the dentate gyrus, although there was no change in transcript levels of DNA repair genes. In the hippocampal dentate hilus, interneurons expressing CHRNA7 or phosphorylated tropomyosin receptor kinase B (TRKB) decreased at ≥ 0.3 ppm. On PND 77, there were no changes in neurogenesis-related parameters. These results suggested that maternal AFB1 exposure reversibly affects hippocampal neurogenesis targeting type-3 progenitor cells. This mechanism likely involves suppression of cholinergic signals on hilar GABAergic interneurons and brain-derived neurotrophic factor-TRKB signaling from granule cells. The no-observed-adverse-effect level for offspring neurogenesis was determined to be 0.1 ppm (7.1-13.6 mg/kg body weight/day).
Assuntos
Aflatoxina B1/toxicidade , Hipocampo/efeitos dos fármacos , Células-Tronco Neurais/efeitos dos fármacos , Neurogênese/efeitos dos fármacos , Animais , Animais Recém-Nascidos/crescimento & desenvolvimento , Neurônios Colinérgicos/efeitos dos fármacos , Giro Denteado/efeitos dos fármacos , Giro Denteado/crescimento & desenvolvimento , Relação Dose-Resposta a Droga , Proteína Duplacortina , Feminino , Hipocampo/crescimento & desenvolvimento , Masculino , Gravidez , Efeitos Tardios da Exposição Pré-Natal/etiologia , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Carbonyl compounds (aldehydes and ketones) are suspected to be among the chemical compounds responsible for Sick Building Syndrome and Multiple Chemical Sensitivities. A headspace gas chromatography/mass spectrometry (GC/MS) analysis for these compounds was developed using derivatization of the compounds into volatile derivatives with o-(2,3,4,5,6-pentafluorobenzyl)hydroxylamine (PFBOA). For GC/MS detection, two ionization modes including electron impact ionization (EI) and negative chemical ionization (NCI) were compared. The NCI mode seemed to be better because of its higher selectivity and sensitivity. This headspace GC/MS (NCI mode) was employed as analysis for aldehydes and ketones in materials (fiber products, adhesives, and printed materials). Formaldehyde was detected in the range of N.D. (not detected) to 39 microg/g; acetaldehyde, N.D. to 4.1 microg/g; propionaldehyde, N.D. to 1.0 microg/g; n-butyraldehyde, N.D. to 0.10 microg/g; and acetone, N.D. to 3.1 microg/g in the samples analyzed.
RESUMO
This study evaluated the detection of tumors using in vivo imaging with a commercially available and systemically administered protease-activatable fluorescent probe, ProSense. To this end, we analyzed the delivery and uptake of ProSense as well as the target protease and its cellular source in a mouse xenograft tumor model. In vivo and ex vivo multi wavelength imaging revealed that ProSense signals accumulated within tumors, with preferential distribution in the vascular leakage area that correlates with vasculature development at the tumor periphery. Immunohistochemically, cathepsin B, which is targeted by ProSense, was specifically localized in macrophages. The codistribution of tenascin C immunoreactivity and gelatinase activity provided evidence of tissue-remodeling at the tumor periphery. Furthermore, in situ zymography revealed extracellular ProSense cleavage in such areas. Colocalization of cathepsin B expression and ProSense signals showing reduction by addition of cathepsin B inhibitor was confirmed in cultured macrophage-derived RAW264.7 cells. These results suggest that increased tissue-remodeling activity involving infiltration of macrophages is a mechanism that may be responsible for the tumor accumulation of ProSense signals in our xenograft model. We further confirmed ProSense signals at the tumor margin showing cathepsin B(+) macrophage infiltration in a rat colon carcinogenesis model. Together, these data demonstrate that systemically administered protease-activatable probes can effectively detect cancer invasive fronts, where tissue-remodeling activity is high to facilitate neoplastic cell invasion.
RESUMO
Cochineal extracts (CE) is a coccid-derived natural food colorant containing carminic acid (CA) as an active ingredient that potentiates inhibition of tissue proteolysis mediated by activation of plasma hyaluronan-binding protein (PHBP). In our previous study, dietary administered CE (CA: 28.5% in CE) has shown to promote the macroscopic development of capsular invasive carcinomas (CICs) associated with up-regulation of angiogenesis-related genes in an intracapsular invasion model of experimental thyroid cancers using rats. However, the promoting effect of CE could not be confirmed histopathologically. The purpose of the present study was to confirm the promoting effect of CE through direct injections to animals on the development of CICs using this cancer invasion model. One week after initiation with N-bis(hydroxypropyl)nitrosamine, male F344/NSlc rats were administered CA-enriched CE (CA: 52.6% in CE) by intraperitoneal injections every other day (10 mg/kg body weight) during the promotion with 0.15% sulfadimethoxine in the drinking water for 8 weeks. The multiplicities of macroscopical CICs and the mean area of early capsular invasive foci estimated by Tenascin (TN)-C-immunoreactivity in the thyroid significantly increased with CE-treatment, while the number of TN-C-positive foci did not change with CE. Transcript level of Phbp and downstream genes unchanged; however, transcript level of angiogenesis-related genes, i.e, Vegfb and its transcription factor gene, Hif1a, those being downstream of phosphatase and tensin homolog (PTEN)/Akt signaling, up-regulated in the thyroid tissue with CE-administration. These results suggest that CE potentiates promotion activity by facilitating angiogenesis through activation of PTEN/Akt signaling without accompanying modification of PHBP-related proteolysis.
Assuntos
Carmim/análogos & derivados , Extratos Vegetais/farmacologia , Neoplasias da Glândula Tireoide/tratamento farmacológico , Indutores da Angiogênese/uso terapêutico , Animais , Carmim/farmacologia , Modelos Animais de Doenças , Água Potável/administração & dosagem , Água Potável/química , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Masculino , Nitrosaminas/toxicidade , PTEN Fosfo-Hidrolase/genética , PTEN Fosfo-Hidrolase/metabolismo , Proteólise/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Ratos Endogâmicos F344 , Transdução de Sinais , Sulfadimetoxina/metabolismo , Glândula Tireoide/efeitos dos fármacos , Glândula Tireoide/metabolismo , Glândula Tireoide/patologia , Neoplasias da Glândula Tireoide/induzido quimicamente , Neoplasias da Glândula Tireoide/patologia , Regulação para CimaRESUMO
Coccid-derived natural food colorants contain active ingredients that potentiate inhibition of tissue proteolysis mediated by activation of plasma hyaluronan-binding protein (PHBP). In the present study, we examined the effect of lac color (LC) and cochineal extract (CE), representative coccid-derived colorants containing laccaic acid and carminic acid as active ingredients, in an intracapsular invasion model of experimental thyroid cancers using rats. One week after initiation with N-bis(hydroxypropyl)nitrosamine, male F344/NSIc rats were fed a powdered diet containing 5.0% LC or 3.0% CE during promotion with 0.15% sulfadimethoxine (SDM) in the drinking water for 13 weeks. Capsular invasive carcinomas (CICs) and lung metastases were decreased by LC treatment and accompanied by transcript downregulation on angiogenesis and PHBP-related tissue proteolysis in CICs. In contrast, CE upregulated angiogenesis-related genes in CICs. PHBP was expressed in capsular macrophages and thyroid proliferative lesions with increased intensity in CICs, and LC decreased PHBP-expressing CICs. The size of CICs and their proliferation activity, however, were unchanged compared with those treated with SDM alone. Suppression of cancer by invasion by LC was more evident after an eight-week treatment, exhibiting a profound decrease in tenascin-C-positive early invasive foci and marked reductions in capsular inflammation and fibrosis. These results suggest that LC and CE exerted dissimilar effects on CIC development, the former suppressing the initial step of neoplastic cell invasion into the capsule by targeting PHBP activity of macrophages and neoplastic cells on tissue proteolysis involving inflammatory responses and angiogenesis, and the latter promoting angiogenesis of developed CICs at later stages.