RESUMEN

OBJECTIVE: The ROR1 and ROR2 receptor tyrosine kinases have both been implicated in ovarian cancer progression and have been shown to drive migration and invasion. There is an increasing importance of the role of stroma in ovarian cancer metastasis; however, neither ROR1 nor ROR2 expression in tumor or stromal cells has been analyzed in the same clinical cohort. AIM: To determine ROR1 and ROR2 expression in ovarian cancer and surrounding microenvironment and examine associations with clinicopathological characteristics. METHODS: Immunohistochemistry for ROR1 and ROR2 was used to assess receptor expression in a cohort of epithelial ovarian cancer patients (n=178). Results were analyzed in relation to clinical and histopathological characteristics and survival. Matched patient sample case studies of normal, primary, and metastatic lesions were used to examine ROR expression in relation to ovarian cancer progression. RESULTS: ROR1 and ROR2 are abnormally expressed in malignant ovarian epithelium and stroma. Higher ROR2 tumor expression was found in early-stage, low-grade endometrioid carcinomas. ROR2 stromal expression was highest in the serous subtype. In matched patient case studies, metastatic samples had higher expression of ROR2 in the stroma, and a recurrent sample had the highest expression of ROR2 in both tumor and stroma. CONCLUSION: ROR1 and ROR2 are expressed in tumor-associated stroma in all histological subtypes of ovarian cancer and hold potential as therapeutic targets which may disrupt tumor and stroma interactions.

RESUMEN

Juvenile rainbow trout Oncorhynchus mykiss were exposed to two concentrations each of 17ß-oestradiol (E2; natural oestrogen hormone) or 17α-ethinyl oestradiol (EE2; a potent synthetic oestrogen hormone) to evaluate their potential effects on burst-swimming performance. In each of six successive burst-swimming assays, burst-swimming speed (Uburst ) was lower in fish exposed to 0.5 and 1 µg l(-1) E2 and EE2 for four days compared with control fish. A practice swim (2 days prior to exposure initiation) in control fish elevated initial Uburst values, but this training effect was not evident in the 1 µg l(-1) EE2-exposed fish. Several potential oestrogen-mediated mechanisms for Uburst reductions were investigated, including effects on metabolic products, osmoregulation and blood oxygen-carrying capacity. Prior to burst-swimming trials, fish exposed to E2 and EE2 for 4 days had significantly reduced erythrocyte numbers and lower plasma glucose concentrations. After six repeated burst-swimming trials, plasma glucose, lactate and creatinine concentrations were not significantly different among treatment groups; however, plasma Cl(-) concentrations were significantly reduced in E2- and EE2-treated fish. In summary, E2 and EE2 exposure altered oxygen-carrying capacity ([erythrocytes]) and an osmoregulatory-related variable ([Cl(-) ]), effects that may underlie reductions in burst-swimming speed, which will have implications for fish performance in the wild.

Asunto(s)

Estradiol/análisis , Etinilestradiol/análisis , Oncorhynchus mykiss/fisiología , Natación , Contaminantes Químicos del Agua/análisis , Animales , Glucemia/análisis , Cloruros/análisis , Exposición a Riesgos Ambientales , Eritrocitos/efectos de los fármacos , Hematócrito , Hidrocortisona/sangre , Esfuerzo Físico , Vitelogeninas/sangre

RESUMEN

Estrogenic compounds found in the aquatic environment include natural and synthetic estrogen hormones as well as other less potent estrogenic xenobiotics. In this study, a comprehensive approach was used to examine effects on fish endocrine system endpoints during a short-term xenoestrogen exposure as well as after post-exposure recovery. Rainbow trout (Oncorhynchus mykiss) were exposed to an aqueous 17ß-estradiol (E2) concentration of 0.473 µg l(-1) for 2 and 7 days (d) followed by a 14-d recovery period. At d2 and d7, plasma E2 concentrations in treated fish were 458- and 205-fold higher than in control fish and 23- and 16-fold higher than the exposure water concentration. E2 treatment resulted in significant increases in hepatosomatic index (HSI), plasma vitellogenin (VTG) protein concentrations, and liver VTG and estrogen receptor alpha mRNA levels. All of these parameters, with the exception of plasma VTG protein, returned to baseline values during the recovery period. Plasma cortisol concentrations were unaffected by treatment. This research shows varied time frames of the estrogen-responsive molecular-, biochemical-, and tissue-level alterations, as well as their persistence, in juvenile rainbow trout treated with aqueous E2. These results have implications for feral rainbow trout exposed to xenoestrogens and indicate the importance of evaluating a comprehensive suite of endpoints in assessing the impact of this type of environmental contaminant.

Asunto(s)

Estradiol/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Oncorhynchus mykiss , Animales , Estradiol/administración & dosificación , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores de Estrógenos/genética , Receptores de Estrógenos/metabolismo , Maduración Sexual , Agua/química

RESUMEN

Changes in liver gene expression were examined in juvenile Chinook salmon (Oncorhynchus tshawytscha) exposed in vivo for 8d to seawater (control) or one of 5 concentrations of sewage (environmentally-relevant dilutions of 0.05%, 0.1%, and 0.7%; 2%, 5% or 10%) and subsequently transferred to clean seawater for an 8-d recovery period. Livers were sampled on days 1, 4, 8 (sewage-exposed) and 16 (8d of sewage exposure plus 8d of recovery). A custom cDNA microarray using a universal DNA reference design was used to examine trends of altered gene expression across sewage concentrations, across timepoints, and at the end of the recovery period. Alterations in gene expression followed four distinct concentration-dependent patterns: (1) concentration response (e.g. estrogen receptor alpha), (2) inverse-concentration response (e.g. insulin receptor beta), U-shaped (e.g. mineralocorticoid receptor), (3) inverse U-shaped (e.g. benzodiazepine receptor), and (4) concentration-independent responses (e.g. ubiquitin). Temporal trends included: (1) peak gene expression at one of the sewage exposure timepoints with recovery to baseline levels after the depuration phase (e.g. vitelline envelope protein beta), (2) gene expression alterations that did not recover (e.g. glucose transporter 3), and (3) delayed gene expression alterations initiated only at the recovery timepoint (e.g. insulin-like growth factor 2). In summary, patterns in gene expression changes were found across sewage concentrations and exposure timepoints. This study is the first to show gene expression trends of this nature.

Asunto(s)

Hígado/efectos de los fármacos , Salmón/genética , Aguas del Alcantarillado , Transcriptoma/efectos de los fármacos , Contaminantes Químicos del Agua/toxicidad , Animales , Proteínas de Peces/análisis , Proteínas de Peces/genética , Proteínas de Peces/metabolismo , Expresión Génica/efectos de los fármacos , Perfilación de la Expresión Génica , Hígado/química , Hígado/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Oncorhynchus mykiss/genética , Proyectos de Investigación , Salmón/metabolismo , Agua de Mar

RESUMEN

Eggs were taken from adult sockeye salmon Oncorhynchus nerka that had reached their journey's end in spawn-ready and moribund condition, and fertilized by healthy males. Egg number, size, hatching success and offspring growth did not differ with maternal condition, which suggests the absence of any persisting physiological maternal effects. Differences were noted in the swimming behaviour and physiology of the offspring at parr stage. In a 30 min schooling test conducted using groups of five in a flume, parr from moribund females were more likely to fatigue, were not as tightly schooled, and had a diminished startle response, both in the per cent responding and the burst distance. In individual, confined swimming tests conducted within a tube, post-exercise plasma lactate concentration, which is an indicator of white muscle use, was greater for parr from moribund adult females. The moribund females also had elevated lactate following exercise (their migration), which suggests heritable differences may exist in muscle use. This study shows that juvenile O. nerka artificially propagated from females exhausted by their return migration can exhibit swimming performance differences, indicating that maternal condition may need to be considered in breeding programmes.

Asunto(s)

Constitución Corporal/fisiología , Salmón/fisiología , Animales , Tamaño Corporal/fisiología , Femenino , Hidrocortisona/sangre , Masculino , Óvulo/citología , Reflejo de Sobresalto/fisiología , Natación/fisiología

RESUMEN

Parchment, a biologically based material obtained from the processed hides of animals such as cattle and sheep, has been used for millennia as a writing medium. Although numerous studies have concentrated on the structure and degradation of collagen within parchment, little attention has been paid to noncollagenous components, such as lipids. In this study, we present the results of biochemical and structural analyses of historical and newly manufactured parchment to examine the potential role that lipid plays in parchment stability. The lipid fraction extracted from the parchments displayed different fatty acid compositions between historical and reference materials. Gas chromatography, small-angle X-ray scattering, and solid-state NMR were used to identify and investigate the lipid fraction from parchment samples and to study its contribution to collagen structure and degradation. We hypothesize that the origin of this lipid fraction is either intrinsic, attributable to incomplete fat removal in the manufacturing process, or extrinsic, attributable to microbiological attack on the proteinaceous component of parchments. Furthermore, we consider that the possible formation of protein-lipid complexes in parchment over the course of oxidative degradation may be mediated by reactive oxygen species formed by lipid peroxidation.

Asunto(s)

Lípidos/análisis , Lípidos/química , Papel/historia , Animales , Cromatografía Líquida de Alta Presión , Colágeno Tipo I/química , Fluorometría , Historia Medieval , Espectroscopía de Resonancia Magnética , Difracción de Rayos X

RESUMEN

Juvenile rainbow trout (Oncorhynchus mykiss) were fasted or fed one of three isoenergetic diets varying in protein and lipid content at full satiation levels or half rations for up to 9 weeks. At 3, 6, and 9 weeks, fish in each treatment group were dosed intraperitoneally with 10 mg tritiated benzo[a]pyrene [3H]-B[a]P/kg (B[a]P) to examine the effects of diet composition and energy intake on xenobiotic biotransformation and excretion. The percent dose eliminated during the experiment did not differ among fish receiving the different diet compositions or rations (range 73% to 84%). However, it was significantly decreased (to 53%) in the group that was fasted for 9 weeks. Examination of fish fasted for 6 and 9 weeks showed a significant increase in the proportion of phase I metabolites and a concomitant decrease in the proportion of phase II metabolites found in bile compared with all other groups. Also, fish that were fasted for 9 weeks produced proportionately less 9,10-dihydroxy-benzo[a]pyrene-trans-9,10-diol, more 3-hydroxybenzo[a]pyrene and 9-hydroxybenzo[a]pyrene, and more glucuronic acid conjugates compared with all other groups. Thus, dietary protein and lipid concentration did not appear to affect either the rate of B[a]P metabolism or its excretion; however, prolonged fasting resulted in a shift in metabolite profiles and decreased excretion.

Asunto(s)

Benzo(a)pireno/metabolismo , Benzo(a)pireno/farmacocinética , Carcinógenos/metabolismo , Carcinógenos/farmacocinética , Grasas de la Dieta/farmacología , Proteínas en la Dieta/farmacología , Privación de Alimentos , Oncorhynchus mykiss/fisiología , Alimentación Animal , Animales , Estado Nutricional

RESUMEN

Many freshwater aquatic environments in the Pacific Northwest of North America contain neurotoxic pesticides, an issue of concern given the use of many of these habitats by Pacific salmon (Oncorhynchus sp.). Pesticides such as carbamates are known to affect fundamental physiological systems (such as the enzyme acetylcholinesterase (AChE)), and have been shown to affect salmonid olfactory-mediated behaviors. A neurophysiological measure of olfactory function, the electro-olfactogram (EOG), was used in this study to examine the impacts of acute localized exposure to three carbamates (the insecticide carbofuran, the antisapstain IPBC, and the fungicide mancozeb) on olfactory function in the coho salmon (Oncorhynchus kisutch). We also examine the potential for these pesticides to alter AChE levels in the primary olfactory system and brain with brief exposures (30 min to only the olfactory rosette (OR)). In results, we find that the EOG in coho salmon is highly sensitive to brief localized exposures of two of these three carbamate pesticides. The effective nominal concentration required to cause a 50% reduction in EOG amplitude (EC50) for carbofuran was 10.4 microg/l and for IPBC was 1.28 microg/l. For mancozeb, the EC50 was higher at 2.05 mg/l. All three carbamates also affected AChE activity levels in the OR and brain (BR): carbofuran exposure at 200 microg/l significantly inhibited AChE activity in the OR, and both IPBC and mancozeb significantly increased AChE activity in BR at multiple concentrations with acute localized exposure. These carbamate effects highlight the sensitivity of salmon olfactory neurophysiology to pesticides acting not only potentially via AChE-inhibition, but also by other currently unknown modes of action.

Asunto(s)

Colinesterasas/metabolismo , Inhibidores Enzimáticos/toxicidad , Herbicidas/toxicidad , Oncorhynchus kisutch/metabolismo , Olfato/efectos de los fármacos , Análisis de Varianza , Animales , Carbofurano , Relación Dosis-Respuesta a Droga , Electrofisiología , Maneb , Oncorhynchus kisutch/fisiología , Receptores Odorantes/toxicidad , Pruebas de Toxicidad Aguda , Zineb

RESUMEN

Molecular interactions between collagen and chitosan (CC) have the potential to produce biocomposites with novel properties. We have characterised the molecular interactions in CC complexes by viscometry, wide angle X-ray scattering and Fourier transform infrared spectroscopy. It was found that CC are miscible at the molecular level and exhibit interactions between the components; X-ray diffraction of CC blends indicate that the collagen helix structure is lost in CC films with increasing chitosan content. Non-linear viscometic behaviour with decreasing chitosan content is interpreted as evidence of a third structural phase formed as a complex of CC. The blending of collagen with chitosan gives the possibility of producing new bespoke materials for potential biomedical applications.

Asunto(s)

Materiales Biocompatibles/química , Quitina/análogos & derivados , Quitina/química , Colágeno/química , Mezclas Complejas/química , Materiales Manufacturados/análisis , Ensayo de Materiales/métodos , Materiales Biocompatibles/síntesis química , Quitosano , Mezclas Complejas/síntesis química , Sustancias Macromoleculares , Conformación Molecular , Transición de Fase , Viscosidad

RESUMEN

The purpose of this study was to characterize the association between hepatic heat shock protein 70 (hsp70) and the glucocorticoid receptor in rainbow trout that were exposed to heat stress, cortisol, and beta-naphthoflavone. This study is the first to document that the glucocorticoid receptor complex in rainbow trout hepatic tissues contains hsp70. Heat stress significantly increased levels of total cellular hsp70, and by discerning the association of hsp70 with the glucocorticoid receptor, we demonstrated that heat stress significantly increased the amount of hsp70 not bound to the glucocorticoid receptor, while significantly decreasing the amount of hsp70 bound to the glucocorticoid receptor. By calculating the ratio of hsp70 bound to the glucocorticoid receptor, to the total number of glucocorticoid receptors, stress (heat stress and cortisol-treatment) promoted the association of hsp70 with the glucocorticoid receptor. These findings demonstrate a functional and structural link between hsp70 and the glucocorticoid receptor in rainbow trout, and raise questions regarding the existence of a complex, interrelated stress response that spans all levels of biological organization within the whole animal.

Asunto(s)

Proteínas HSP70 de Choque Térmico/metabolismo , Oncorhynchus mykiss/metabolismo , Receptores de Glucocorticoides/metabolismo , Estrés Fisiológico/fisiopatología , Estrés Fisiológico/veterinaria , Animales , Electroforesis en Gel de Poliacrilamida , Enfermedades de los Peces/metabolismo , Enfermedades de los Peces/fisiopatología , Respuesta al Choque Térmico/fisiología , Hidrocortisona/sangre , Hidrocortisona/farmacología , Immunoblotting , Hígado/efectos de los fármacos , Hígado/metabolismo , Unión Proteica , beta-naftoflavona/farmacología

RESUMEN

To determine if elevated concentrations of waterborne selenium (Se), caused by coal mining, in the Elk River in southeastern British Columbia, may be causing reproductive or teratogenic effects in wild cutthroat trout (Oncorhynchus clarki lewisi), fertilized eggs from exposed and reference fish were raised in the laboratory. Eggs from each female were reared separately and the percent mortalities and deformities were related to the selenium content of the eggs. Selenium concentrations in females from the exposed site were highest in the liver (36.6 +/- 22.5 microg/g dry weight, range: 18.3 to 114), followed by the eggs (21.0 +/- 18.3 microg/g, range: 8.7 to 81.3) and the muscle (12.5 +/- 7.7 microg/g, range: 6.7 to 41). Despite these elevated egg Se concentrations, there was no significant effect on fertilization; time to hatch; percent hatch; or egg, larvae, and fry deformities or mortalities. Reproductive failure and embryonic terata have been reported at much lower egg Se concentrations in other fish species. The lack of any toxic response in this study may be due to an evolved tolerance to higher tissue Se concentrations in a population of fish living in a seleniferous river system.

Asunto(s)

Anomalías Inducidas por Medicamentos/patología , Oncorhynchus , Selenio/toxicidad , Animales , Animales Recién Nacidos , Colombia Británica , Femenino , Larva , Oncorhynchus/embriología , Oncorhynchus/metabolismo , Óvulo/patología , Selenio/metabolismo

RESUMEN

The effects of environmental salinity on the distribution, metabolism, and elimination of benzo[a]pyrene (B[a]P) were examined in mature rainbow trout. Trout acclimated to either fresh water (0 ppt, FW) or sea water (20 ppt, SW) for 3 weeks received a single 10 mg/kg intra-arterial injection of [(3)H]-benzo[a]pyrene (B[a]P) at their acclimation salinity or when subjected to an acute salinity change. Statistically significant differences in the percent body burden of B[a]P-derived radioactivity in various tissues were seen between fish in FW versus SW. Significant differences in the distribution of B[a]P and its metabolites were also noted when fish were subjected to an acute salinity change after chemical injection. Modulation of B[a]P metabolism by environmental salinity included: (1) significant differences in the proportions of Phase I metabolites in the bile of FW- (2.3%) versus SW-acclimated (14.1%) fish, and (2) alterations in the accumulations of specific metabolites (predominantly t-9, 10-dihydrodiol-B[a]P in FW fish, and 3-hydroxy-B[a]P in SW fish). The percentages of the [(3)H]-B[a]P dose eliminated by 48 h was similar in FW and SW fish, but decreased in fish subjected to an acute salinity change (FW 98.8% eliminated, FW:SW 90.4%, SW 98.1%, and SW:FW 93.1%). Pharmacokinetic modeling confirmed that acute salinity changes can result in longer terminal half-lives and slower total body clearances of B[a]P.

Asunto(s)

Benzo(a)pireno/farmacocinética , Carcinógenos/farmacocinética , Oncorhynchus mykiss/fisiología , Adaptación Fisiológica , Animales , Benzo(a)pireno/toxicidad , Carcinógenos/toxicidad , Semivida , Cloruro de Sodio , Agua/química

RESUMEN

Hepatocytes from sablefish (Anoplopoma fimbria), black rockfish (Sebastes melanops) and chub mackerel (Scomber japonicus) were isolated from 11 degrees C acclimated animals. The uptake, metabolism, and excretion of benzo[a]pyrene (B[a]P) in hepatocytes was measured at 6, 11 and 19 degrees C. Chub mackerel hepatocyte uptake rates were significantly lower (0.012 +/- 0.003 microg/s per g cell) at 11 degrees C than black rockfish (0.028 +/- 0.009 microg/s per g cell) or sablefish (0.032 +/- 0.012 microg/s per g cell) hepatocytes at all temperatures. Hepatocytes metabolized B[a]P to phase I (1-8%) and phase II (92-99%) metabolites. Accumulation of phase II metabolites was lower in chub mackerel hepatocytes (0.016 +/- 0.004 microg/h per g cell), than black rockfish (0.052 +/- 0.012 microg/h per g cell), or sablefish hepatocytes (0.060 +/- 0.015 microg/h per g cell). Phase II metabolite accumulation increased greatest with temperature in chub mackerel hepatocytes (Q10 = 1.94 +/- 0.30), followed by sablefish (Q10 = 1.65 +/- 0.30), and rockfish (Q10 = 1.38 +/- 0.30). Sablefish hepatocytes had higher excretion rates of phase II metabolites (0.010 +/- 0.0023 microg/h per g cell), than mackerel (0.0046 +/- 0.0009 microg/h per g cell) or rockfish hepatocytes (0.0029 +/- 0.0008 microg/h per g cell). Phase II metabolite excretion rates increased with temperature only in sablefish hepatocytes (Q1O = 1.67 +/- 0.76). These differences in toxicokinetics may indicate distinct consequences for various species exposed to xenobiotics.

Asunto(s)

Benzo(a)pireno/farmacocinética , Peces/metabolismo , Hígado/metabolismo , Animales , Benzo(a)pireno/toxicidad , Células Cultivadas , Hígado/citología , Hígado/enzimología , Temperatura

RESUMEN

A cell line, PHL, has been successfully established from newly hatched herring larvae. The cells are maintained in growth medium consisting of Leibovitz's L-15 supplemented with 15% fetal bovine serum (FBS), and have been cryopreserved and maintain viability after thawing. These cells retain a diploid karotype after 65 population doublings. PHL are susceptible to infection by the North American strain of viral hemorrhagic septicemia (VHS) virus, and are sensitive to the cytotoxic effects of naphthalene, a common environmental contaminant. Naphthalene is a component of crude and refined oil, and may be found in the marine environment following acute events such as oil spills. In addition, chronic sources of naphthalene contamination include offshore drilling and petroleum contamination from areas such as docks and marinas that have creosote-treated docks and pilings and also receive constant small inputs of petroleum products. This cell line should be useful for investigations of the toxicity of naphthalene and other petroleum components to juvenile herring. In addition, studies of the VHS virus will be facilitated by the availability of a susceptible cell line from an alternative species.

Asunto(s)

Técnicas de Cultivo de Célula/métodos , Enfermedades de los Peces/virología , Naftalenos/toxicidad , Infecciones por Rhabdoviridae/fisiopatología , Rhabdoviridae , Animales , Apoptosis/efectos de los fármacos , Adhesión Celular/efectos de los fármacos , División Celular/efectos de los fármacos , Cromosomas , Citocromo P-450 CYP1A1/metabolismo , Sistema Enzimático del Citocromo P-450/metabolismo , Proteínas Fetales/farmacología , Enfermedades de los Peces/inducido químicamente , Peces , Aceites Combustibles , Hemorragia/virología , Larva/citología , Larva/enzimología , Oxidorreductasas/metabolismo , Sepsis/virología , Temperatura , Contaminación del Agua

RESUMEN

The acute toxicity of Polyphase P-100, an antisapstain wood preservative that contains 97% 3-iodo-2-propynyl butyl carbamate (IPBC), was determined for three species of fish (coho salmon, rainbow trout, and starry flounder) and three species of aquatic invertebrates (Daphnia magna, Hyalella azteca, and Neomysis mercedis). The 96-h LC50 values for the various fish species exposed to Polyphase P-100 ranged from 95 ppb for coho smolts (Oncorhynchus kisutch) to 370 ppm for juvenile starry flounder (Platichthys stellatus). The sensitivity of coho to Polyphase P-100 was altered by their developmental stage. Coho embryos were six to nine times more tolerant of Polyphase P-100 than coho alevins, which were twice as tolerant as coho smolts. The 48-h LC50 values for the invertebrates D. magna, H. azteca, and N. mercedis were 40 ppb, 500 ppb, and 2,920 ppb, respectively. In addition to a wider range of sensitivity to Polyphase P-100 compared with the fish species, the invertebrate species were characterized by a shallower concentration-response. In acute, 24-h sublethal tests with juvenile starry flounder and rainbow trout, there was no primary or secondary stress response (changes in hematocrit, leucocrit, hemoglobin concentration, plasma lactate concentration, and plasma cortisol concentration) at concentrations up to 50% of the 96-h LC50 value. The acute toxicity of a 1:8 mixture of Polyphase P-100 and Bardac 2280 (another antisapstain compound that contains didecyldimethylammonium chloride [DDAC] as the active ingredient) was close to additive for fish, but not for invertebrate species. The acute toxicity of the mixture was seven to eight times more than additive for H. azteca, but two to three times less than additive for D. magna. Some sublethal stress responses were revealed with the mixture that were not observed with the test chemicals alone.

Asunto(s)

Carbamatos/toxicidad , Lenguado , Fungicidas Industriales/toxicidad , Oncorhynchus kisutch , Oncorhynchus mykiss , Animales , Crustáceos/fisiología , Daphnia , Dosificación Letal Mediana

RESUMEN

We report for the first time that beta-naphthoflavone (BNF) abolishes ACTH stimulation of cortisol production in rainbow trout (Oncorhynchus mykiss). There was significantly higher hepatic cytochrome P450 content and ethoxyresorufin O-de-ethylase and uridine-5'-diphosphoglucuronic acid transferase activities in BNF-treated fish than in sham-treated controls. BNF did not significantly affect either plasma turnover or tissue distribution of [3H]cortisol-derived radioactivity. Hepatic membrane fluidity and hepatocyte capacity for cortisol uptake were not altered by BNF as compared with the sham-treated fish. These results taken together suggest that BNF does not affect cortisol-clearance mechanisms in trout. A 3 min handling disturbance period elicited a plasma cortisol response in the sham-treated fish; however, the response in the BNF-treated fish was muted and significantly lower than in the sham fish. This in vivo response corroborates the lack of interrenal sensitivity to ACTH in vitro in the BNF-treated fish, suggesting that BNF affects the ACTH pathway in trout. Our results suggest the possibility that cytochrome P450-inducing compounds may affect cortisol dynamics by decreasing interrenal responsiveness to ACTH stimulation in fish, thereby impairing the physiological responses that are necessary for the animal to cope with the stressor.

Asunto(s)

Hormona Adrenocorticotrópica/farmacología , Sistema Enzimático del Citocromo P-450/metabolismo , Glándula Interrenal/metabolismo , Oncorhynchus mykiss/metabolismo , beta-naftoflavona/farmacología , Animales , Hidrocortisona/sangre , Hidrocortisona/metabolismo , Glándula Interrenal/efectos de los fármacos , Hígado/metabolismo , Fluidez de la Membrana , Estimulación Química , Estrés Psicológico , Factores de Tiempo

RESUMEN

PURPOSE: To determine the characteristics of patients developing retinal detachment secondary to retinal dialysis in Western Australia and to confirm the clinical impression that these patients had a low rate of proliferative vitreoretinopathy (PVR). METHODS: A retrospective analysis of the records of 1601 consecutive patients with rhegmatogenous retinal detachment identified 71 patients in whom the retinal detachment was caused by a retinal dialysis. RESULTS: The majority of these patients were young adults (mean age of 30 years) and the male to female ratio was 1.3:1. Seventy per cent of patients provided a history of significant trauma to the affected eye. Sporting injuries, assault, and motor vehicle injuries together accounted for 72% of identifiable trauma. Examination revealed a dialysis of the inferotemporal quadrant in 75% of cases and despite obvious signs of chronicity of the associated retinal detachment (such as intraretinal macrocysts and demarcation lines) in approximately one-third of the eyes, only 5.6% developed grade CI PVR either pre- or postoperatively. CONCLUSION: The present study supports the view that it is the low rate of PVR that explains the good prognosis and high surgical success rate for retinal detachments caused by retinal dialysis. It is postulated that a major reason for the low rate of PVR is that the vitreous base attachment to the posterior margin of a retinal dialysis acts as a significant barrier to the migration of potentially proliferative retinal pigment epithelial cells. This may lead to containment of the responsible proliferative cells within the loculated subretinal space.

Asunto(s)

Desprendimiento de Retina/etiología , Perforaciones de la Retina/complicaciones , Adolescente , Adulto , Anciano , Niño , Lesiones Oculares/complicaciones , Lesiones Oculares/diagnóstico , Lesiones Oculares/cirugía , Femenino , Estudios de Seguimiento , Humanos , Masculino , Persona de Mediana Edad , Retina/lesiones , Retina/patología , Retina/cirugía , Desprendimiento de Retina/diagnóstico , Desprendimiento de Retina/cirugía , Perforaciones de la Retina/diagnóstico , Perforaciones de la Retina/cirugía , Estudios Retrospectivos , Agudeza Visual , Campos Visuales , Vitreorretinopatía Proliferativa/etiología , Heridas no Penetrantes/complicaciones , Heridas no Penetrantes/diagnóstico , Heridas no Penetrantes/cirugía

RESUMEN

A response in heat shock protein 70 (hsp 70) expression in the beta-naphthoflavone (BNF) treated rainbow trout (Oncorhynchus mykiss) corresponded to altered metabolic status of the liver as evidenced by the lower phosphoenolpyruvate carboxykinase (PEPCK), lactate dehydrogenase and 3-hydroxyacylcoA dehydrogenase activities. The BNF-induced increase in hsp70 levels and conjugation enzyme activities (phase I and phase II) were not modified by handling stress. Indeed handling stress did not affect either hsp 70 levels or conjugation enzyme activities in trout liver. The decrease in hepatic PEPCK activity in the BNF group may be responsible for the attenuation of the increase in liver glucose concentration after a 3 min handling stress in this species, suggesting that BNF affects liver gluconeogenic capacity in this species. Handling stress elicited a plasma cortisol and glucose response in both the sham and BNF group, however, the cortisol response with BNF was erratic compared with the sham, implying alterations in the cortisol dynamics post-stress. These results show for the first time that BNF affects cellular metabolic responses to stress and suggests the possibility of using hsp 70 as a biomarker for toxic effects in trout.

Asunto(s)

Proteínas de Arabidopsis , Proteínas HSP70 de Choque Térmico/biosíntesis , Hígado/metabolismo , Estrés Fisiológico/metabolismo , beta-naftoflavona/farmacología , 3-Hidroxiacil-CoA Deshidrogenasas/metabolismo , Animales , Glucemia/metabolismo , Citocromo P-450 CYP1A1/metabolismo , Sistema Enzimático del Citocromo P-450/metabolismo , Glucosa/metabolismo , Glucosiltransferasas/metabolismo , Manejo Psicológico , Hidrocortisona/sangre , L-Lactato Deshidrogenasa/metabolismo , Hígado/efectos de los fármacos , Hígado/enzimología , Oncorhynchus mykiss , Fosfoenolpiruvato Carboxiquinasa (GTP)/metabolismo , Piruvato Quinasa/metabolismo

RESUMEN

PURPOSE: The primary aim of this study was to develop and characterize a simple flow cytometric method of quantifying rod outer segment (ROS) phagocytosis in cultured retinal pigment epithelial (RPE) cells. A secondary aim was to compare the kinetics of ROS phagocytosis in an immortal human RPE cell line with untransformed human RPE cells. METHODS: Flow cytometry was performed on RPE cells that had been challenged with fluorescein isothiocyanate-labeled ROS (FITC-ROS) and phagocytosis was calculated by subtracting background cellular autofluorescence. RESULTS: Non-specific uptake of fluorescent label was negligible and RPE cells phagocytosed FITC-ROS and unlabeled ROS with equal efficacy. The kinetics of FITC-ROS phagocytosis in the D407 RPE cell line differed from early passage untransformed human RPE cultures. FITC-ROS phagocytosis proceeded at a fairly linear rate for the first 12 h in the 3 human cell cultures studied, but was rapid for the first 3 h before slowing in the D407 cells. Within all cell populations, there was a heterogeneity of phagocytic activity which varied with time. CONCLUSIONS: This automated technique for measuring phagocytosis is rapid, simple, highly accurate, avoids radiation hazards, and permits study of heterogeneity within cell populations. The biochemistry, physiology and pathophysiology of the interactions between retinal pigment epithelial (RPE) cells and photoreceptors continue to be areas of considerable research interest (1, 2, 3). Vital to such work is the ability to accurately quantify rod outer segment (ROS) phagocytosis by RPE cells. Current in vitro techniques of measuring ROS phagocytosis use either automated or manual methods to count phagosomes. While manual counting techniques offer the advantage of visual quality control, they are highly labor intensive, there is a practical limitation to the number of phagosomes that can be counted, and measurements suffer from relatively large standard errors (3). Automated methods include scintillation counting and flow cytometry. Problems with radiolabels include radiation hazards, nonspecific radiolabel uptake, and limited visual control (3). Flow cytometry, on the other hand, circumvents nearly all of these problems and may prove to be the optimal phagocytosis assay.

Asunto(s)

Citometría de Flujo/métodos , Fagocitosis/fisiología , Epitelio Pigmentado Ocular/fisiología , Segmento Externo de la Célula en Bastón/fisiología , Adulto , Anciano , Línea Celular , Células Cultivadas , Fluoresceína-5-Isotiocianato , Colorantes Fluorescentes , Humanos , Cinética , Persona de Mediana Edad

RESUMEN

The purpose of the present study was to investigate the accumulation of rod outer segment (ROS)-derived debris in cultured human retinal pigment epithelial cells (RPE). The RPE cell layer is responsible for the phagocytosis and digestion of photoreceptor outer segments. Due to the immense volume of photoreceptor-derived material processed by the RPE cells, even minor changes in the efficiency of ROS processing may cause the accumulation of lipofuscin and photoreceptor derived debris. In this work, 17 RPE cultures were established from the globes of eye bank donors whose ages ranged from 18 to 79 years. Third passage cultures were challenged with bovine ROS and the accumulation of an autofluorescent debris was quantified using a flow cytometer. It was demonstrated that ROS challenge greatly increased the rate of autofluorescent debris accumulation. The accumulation of autofluorescent debris varied significantly from culture to culture. This variation was independent of the phagocytosing capacity of individual cultures and was not age dependent. To further investigate the factors which may be responsible for these differences, the presence of cathepsin D, an aspartic protease responsible for 80% of proteolysis of rhodopsin, was analysed by Western blot. Although the 34 kDa active form of cathepsin D was found in all cultures, in 41% of the cultures higher-molecular-weight forms of cathepsin D were additionally present, thus providing a multimer form of cathepsin D in these cultures. The rate of autofluorescent debris accumulation in cultures possessing a multimer form of cathepsin D was significantly greater (mean 42.3, S.D. +/- 19.8) than those in cultures having a singlet active form (mean 18.8, S.D. +/- 5.5) at 34 kDa (Student's t-test, DF = 15, t = 6.834, P < 0.001). The former cultures included one from a donor with age related macular degeneration, the latter cultures included one from a donor with diabetic retinopathy. This study demonstrates that the rate of autofluorescent debris accumulation in cultured RPE cells is not age dependent, but is an intrinsic property of the donor RPE cells that is possibly related to the presence of a multimer form of the lysosomal enzyme cathepsin D.

Asunto(s)

Catepsina D/metabolismo , Epitelio Pigmentado Ocular/citología , Segmento Externo de la Célula en Bastón/metabolismo , Animales , Western Blotting , Catepsina D/análisis , Bovinos , Células Cultivadas , Humanos , Lipofuscina/análisis , Microscopía Fluorescente , Peso Molecular , Fagocitosis/fisiología , Epitelio Pigmentado Ocular/fisiología