RESUMEN
Toxicologic/veterinary pathologists are working remotely from Good Laboratory Practice (GLP) test facilities (TFs) in increasing numbers, most commonly in home-office settings. A study pathologist (SP) generating data on GLP-compliant nonclinical studies must be keenly aware of applicable national GLP regulations and comply with TF and protocol requirements. This Toxicological Pathology Forum Opinion Piece will summarize primary areas of emphasis for the SP generating GLP data using glass slides. Peer review and digital review of whole slide images are out of scope for this opinion piece. Key GLP considerations for primary pathology on glass slides are discussed with respect to SP location and employment status, including pathologist qualifications, specimen management, facilities, equipment, archive, and quality assurance. Notable differences between national GLP regulations of the United States, the United Kingdom, Germany, the Netherlands, France, Ireland, Switzerland, Italy, and Israel are presented. With the understanding that each combination of location and employment is unique, the authors provide a general overview of considerations for successful remote GLP work.
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Patología , Revisión por Pares , Humanos , Laboratorios , Patólogos , Patología/métodos , Estados UnidosRESUMEN
Chronic granulomatous disease (CGD) is a rare inherited disorder due to loss-of-function mutations in genes encoding the NADPH oxidase subunits. Hematopoietic stem and progenitor cell (HSPC) gene therapy (GT) using regulated lentiviral vectors (LVs) has emerged as a promising therapeutic option for CGD patients. We performed non-clinical Good Laboratory Practice (GLP) and laboratory-grade studies to assess the safety and genotoxicity of LV targeting myeloid-specific Gp91phox expression in X-linked chronic granulomatous disease (XCGD) mice. We found persistence of gene-corrected cells for up to 1 year, restoration of Gp91phox expression and NADPH oxidase activity in XCGD phagocytes, and reduced tissue inflammation after LV-mediated HSPC GT. Although most of the mice showed no hematological or biochemical toxicity, a small subset of XCGD GT mice developed T cell lymphoblastic lymphoma (2.94%) and myeloid leukemia (5.88%). No hematological malignancies were identified in C57BL/6 mice transplanted with transduced XCGD HSPCs. Integration pattern analysis revealed an oligoclonal composition with rare dominant clones harboring vector insertions near oncogenes in mice with tumors. Collectively, our data support the long-term efficacy of LV-mediated HSPC GT in XCGD mice and provide a safety warning because the chronic inflammatory XCGD background may contribute to oncogenesis.
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Terapia Genética , Vectores Genéticos/genética , Enfermedad Granulomatosa Crónica/complicaciones , Enfermedad Granulomatosa Crónica/terapia , Neoplasias Hematológicas/etiología , Lentivirus/genética , Animales , Modelos Animales de Enfermedad , Terapia Genética/efectos adversos , Terapia Genética/métodos , Vectores Genéticos/administración & dosificación , Enfermedad Granulomatosa Crónica/genética , Humanos , Ratones , NADPH Oxidasa 2/genética , NADPH Oxidasa 2/metabolismo , Factores de Tiempo , Resultado del TratamientoRESUMEN
A therapeutic option for monogenic disorders is gene therapy with ex vivo-transduced autologous hematopoietic stem cells (HSCs). Safety or efficacy studies of ex vivo-modified HSCs are conducted in humanized mouse models after ablation of the murine bone marrow and transfer of human CD34+ HSCs. Engrafted human CD34+ cells migrate to bone marrow and differentiate into various human hematopoietic lineages. A 12-week study was conducted in NSG™ mice to evaluate engraftment, differentiation, and safety of human CD34+ cells that were transduced (ex vivo) with a proprietary lentiviral vector encoding a human gene (BMRN-1) or a mock (green fluorescent protein) vector. Several mice intravenously injected with naive CD34+ cells or transduced CD34+ cells had variable lymphohistiocytic inflammatory cell infiltrates and microgranulomas in the liver and lungs consistent with graft-versus-host disease (GVHD). Spleen, bone marrow, stomach, reproductive tract, but not the skin had similar inflammatory changes. Ex vivo viral transduction of CD34+ cells did not impact engraftment or predispose to xenogeneic GVHD.
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Antígenos CD34/genética , Terapia Genética/métodos , Enfermedad Injerto contra Huésped , Trasplante de Células Madre Hematopoyéticas , Células Madre Hematopoyéticas/citología , Xenoinjertos , Animales , Antígenos CD34/inmunología , Técnicas de Transferencia de Gen , Enfermedad Injerto contra Huésped/inmunología , Proteínas Fluorescentes Verdes/genética , Células Madre Hematopoyéticas/inmunología , Humanos , Ratones Endogámicos NOD , Ratones SCID , Investigación Biomédica TraslacionalRESUMEN
To test the diagnostic approach described in part 1 of this article, 2 exercises were completed by pathologists from multiple companies/agencies. Pathologist's examination of whole slide image (WSI) heart sections from rats using personal diagnostic approaches (exercise #1) corroborated conclusions from study #1. Using the diagnostic approach described in part 1, these pathologists examined the same WSI heart sections (exercise #2) to determine whether that approach increased consistency of diagnosis of rodent progressive cardiomyopathy (PCM) lesions. In exercise #2, there was improved consistency of categorization of small borderline morphologies and mild lesions, but a decrement in consistency of categorizing minimal lesions. Exercises 1 and 2 suggest the described diagnostic approach is representative of that in use by the majority of toxicologic pathologists across companies/agencies and that application by all may improve diagnostic consistency of PCM/like lesions. Additionally, a criterion of approximately 5% heart section involvement is suggested for separating mild from moderate or greater severity. While evidence is not absolute, until further investigation shows otherwise, microscopic changes resembling PCM, but located in the epicardial and subepicardial region of the right ventricle, may be considered as part of the spectrum of PCM.
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Cardiomiopatías/patología , Diagnóstico por Imagen/métodos , Ventrículos Cardíacos/patología , Ratas Sprague-Dawley , Enfermedades de los Roedores/patología , Pruebas de Toxicidad/métodos , Animales , Cardiomiopatías/veterinaria , Cardiotoxicidad/patología , Cardiotoxicidad/veterinaria , Simulación por Computador , Diagnóstico por Imagen/normas , Diagnóstico por Imagen/veterinaria , Progresión de la Enfermedad , Masculino , Pruebas de Toxicidad/veterinariaRESUMEN
Spontaneous rodent progressive cardiomyopathy (PCM) in the Sprague Dawley rat may confound identification and/or interpretation of potential test article (TA)-related cardiotoxicity. Pathologists apply diagnostic term(s) and thresholds for diagnosing and assigning severity grades for PCM and/or PCM-like (PCM/like) lesions consistently within a study, which is necessary to identify and interpret TA-related findings. Due to differences in training and/or experiences, diagnostic terms and thresholds may vary between pathologists. Harmonized terminology and thresholds across studies will generate better historical control data, will likely enhance interpretation of study data, and may further enhance our understanding of the spontaneous change. An assessment of the diagnostic approaches of a group of 37 pathologists identified an approach that is relatively easily applied; and if adopted, it could enhance diagnostic consistency across studies. This approach uses the single "slash" term "necrosis/inflammatory cell infiltrate (NICI)" as the diagnosis for the spectrum of lesions seen in younger rats, uses no threshold for diagnosis (e.g., diagnose all lesions clearly identifiable as PCM/like), and uses aggregate lesion size of approximately ≥45% of the field of view (FOV) using a 10×/22 eyepiece and the 40× objective or approximately ≥100% of the FOV using the 60× objective as the criterion separating minimal from mild severities.
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Cardiomiopatías/patología , Diagnóstico por Imagen/métodos , Ratas Sprague-Dawley , Enfermedades de los Roedores/patología , Pruebas de Toxicidad/veterinaria , Animales , Cardiomiopatías/veterinaria , Cardiotoxicidad/patología , Cardiotoxicidad/veterinaria , Simulación por Computador , Diagnóstico por Imagen/normas , Diagnóstico por Imagen/veterinaria , Progresión de la Enfermedad , Masculino , Necrosis , Índice de Severidad de la EnfermedadRESUMEN
Glutathione-dependent bioactivation is a common pathway in nephrotoxicity caused by haloalkanes and haloalkenes. Glutathione conjugation forms the link between halogenated hydrocarbons, based on the formation of an episulfonium ion (vicinal halomethanes) or a cysteine conjugate (haloalkenes). Herein, we review the metabolic pathways underlying the nephrotoxic effects of the three well-known haloalkenes trichloroethylene, tetrachloroethylene, and hexachloro-1:3-butadiene to emphasize the role of cysteine-conjugate ß-lyase and the oxidative metabolism in renal toxicity. Activation by cysteine-conjugate ß-lyase is the best-characterized mechanism causing toxicity due to haloalkene treatment in experimental models. However, the severity of toxicity differs considerably, with S-(1,2,2-trichlorovinyl)-L-cysteine being more toxic than S-(1,2-dichlorovinyl)-L-cysteine, which is in turn more toxic than S-(1,2,3,4,4-pentachloro-1:3-butadienyl)-L-cysteine. Moreover, two oxidative pathways involving cysteine S-conjugates (mediated by flavin-containing monooxigenase 3) and N-acetyl-L-cysteine conjugates (mediated by cytochrome P-450 3A) form derived sulfoxides, which represent alternative metabolites with toxic effects. In vitro and in vivo studies showed that sulfoxide metabolites are more toxic than cysteine-conjugate derivates. The cytochrome P-450 3A family, on the other hand, is sex specific, and its expression has only been reported in adult male rats and rabbits. In summary, haloalkenes are highly nephrotoxic in vivo and in vitro and their toxicity mechanisms are well documented experimentally. However, little information is available on their toxicity in humans, except for the carcinogenic effects established for high exposure levels of trichloroethylene and tetrachloroethylene.
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Butadienos/toxicidad , Contaminantes Ambientales/toxicidad , Enfermedades Renales/inducido químicamente , Tetracloroetileno/toxicidad , Tricloroetileno/toxicidad , Animales , Butadienos/metabolismo , Contaminantes Ambientales/metabolismo , Humanos , Inactivación Metabólica , Exposición Profesional , Tetracloroetileno/metabolismo , Tricloroetileno/metabolismoRESUMEN
Phospholipidosis (PLD) is characterized by an intracellular accumulation of phospholipids in lysosomes and concurrent development of concentric lamellar bodies. It is induced in humans and in animals by drugs with a cationic amphiphilic structure. The purpose of the present study was to identify a set of molecular biomarkers of PLD in rat blood and heart, hypotheticallya pplicable in preclinical screens within the drug development process. A toxicological study was set up in rats orally treated up to 11 days with 300 mg kg(1) per day(1) amiodarone (AMD). Light and transmission electron microscopy investigations were performed to confirm the presence of lamellar bodies indicative of phospholipid accumulation. The effects of AMD upon the transcriptome of these tissues were estimated using DNA microarray technology. Microarray data analysis showed that a total of 545 and 8218 genes were modulated by AMD treatment in heart and blood, respectively. Some genes implicated in the phospholipid accumulation in cells, such as phospholipase A2, showed similar alterations of gene expression. After transcriptome criteria of analysis and target selection, including also the involvement in the onset of PLD, 7 genes (Pla2g2a, Pla2g7, Gal, Il1b, Cebpb, Fcgr2b, Acer 2) were selected as candidate biomarkers of PLD in heart and blood tissues, and their potential usefulness as a sensitive screening test was screened and confirmed by quantitative Real-Time PCR analysis. Collectively, these data underscore the importance of transcriptional profiling in drug discovery and development, and suggest blood as a surrogate tissue for possible phospholipid accumulation in cardiomyocytes.
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Amiodarona/toxicidad , Lipidosis/inducido químicamente , Miocardio/metabolismo , Fosfolípidos/sangre , Fosfolípidos/metabolismo , Animales , Biomarcadores/sangre , Cardiotoxicidad , Evaluación Preclínica de Medicamentos , Ontología de Genes , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/metabolismo , Lipidosis/sangre , Lipidosis/genética , Lipidosis/metabolismo , Linfocitos/efectos de los fármacos , Linfocitos/metabolismo , Lisosomas/metabolismo , Masculino , Microscopía Electrónica , Miocardio/ultraestructura , Ratas Sprague-Dawley , Reacción en Cadena en Tiempo Real de la Polimerasa , Transcriptoma/efectos de los fármacosRESUMEN
Cardiovascular safety signals in nonclinical studies remain among the main reasons for drug attrition during pharmaceutical research and development. Drug-induced changes can be functional and/or associated with morphological alterations in the normal heart histology. It is therefore crucial to understand the normal variations in histology to discriminate test article-related changes from background lesions. Rodent progressive cardiomyopathy is probably the most commonly encountered change in control animals of nonclinical toxicity studies. A multisite study mimicking standard short-term toxicity studies using young male Sprague-Dawley rats was performed to better characterize this finding. Using an enhanced sectioning method for this research study, it was observed that the incidence of background cardiomyopathy was 100%. The vast majority of the microscopic findings were inflammatory in nature, with associated necrotic changes (defined as necrosis/inflammatory cell infiltrate) and these changes were mainly located in the myocardium of the mid region of the ventricles (the left side being predominantly affected). The monitored environmental factors in this study (multiple facilities, study duration, handling) did not have an effect on the incidence or severity of the spontaneous cardiomyopathy. In addition, cardiac-specific serum troponin levels were measured and were within the published control range.
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Cardiomiopatías/veterinaria , Corazón/anatomía & histología , Miocardio/patología , Enfermedades de los Roedores/patología , Pruebas de Toxicidad/normas , Animales , Investigación Biomédica , Cardiomiopatías/patología , Histocitoquímica , Masculino , Necrosis/patología , Ratas , Ratas Sprague-Dawley , Enfermedades de los Roedores/sangre , Troponina I/sangreRESUMEN
The present research investigates the occurrence of hyaline droplet (HD) accumulation related to age, dose and time after treatment in male Wistar rats given a single i.p. injection of hexachloro-1:3-butadiene (HCBD). In the study on age, rats from 1 to 12 months of age were treated with 100 mg kg(-1) body weight (b.w.) HCBD dose. Rats treated at 2 months of age showed a greater accumulation of HD than the other age groups; HD accumulation was not observed in 1-month-old rats. In the dose-response study, the treatment with 25, 50 and 100 mg kg(-1) b.w. at 2 months of age caused HD accumulation in the proximal convoluted tubule at all doses, with the 100 mg kg(-1) b.w. group slightly more affected. Finally, in the time-course study, rats treated with a 100 mg kg(-1) b.w. dose at 2 months of age and sacrificed at 6, 12, 24, 48, 72 and 96 h post-dosing showed a time-related HD accumulation in terms of incidence and severity, after 6 h, with a peak at 24 and 48 h and decreasing at 72 and 96 h. The present results show that HD accumulation is an early finding, and is unrelated to dose level and particularly evident in rats of 2 month of age. These findings in male rats treated with HCBD emphasize the importance of considering the age of rats at the start of a study. The more sensitive model was used in the detection of nephrotoxic effects of chemicals.
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Butadienos/toxicidad , Fungicidas Industriales/toxicidad , Hialina/metabolismo , Riñón/efectos de los fármacos , Factores de Edad , Animales , Citoplasma/efectos de los fármacos , Citoplasma/metabolismo , Citoplasma/patología , Relación Dosis-Respuesta a Droga , Riñón/metabolismo , Riñón/patología , Túbulos Renales Proximales/efectos de los fármacos , Túbulos Renales Proximales/metabolismo , Túbulos Renales Proximales/patología , Masculino , Ratas , Ratas Wistar , Factores de TiempoRESUMEN
The atrial natriuretic peptide (ANP) and its precursor (N-terminal fragment of atrial natriuretic peptide, NT-proANP) are natriuretic peptides released into the circulation as a consequence of an acute atrial stretch. As for the brain natriuretic peptide and its N-terminal fragment, the biological significance of ANP and NT-proANP has been widely studied in humans, but the literature is lacking information about the determination of these biomarkers in veterinary medicine and, in particular, in the toxicological species used in preclinical pharmaceutical drug development. This paper describes the evaluation of ANP and NT-proANP levels in a healthy population of Han Wistar and Sprague-Dawley rats, as well as in a rodent model of hypertension (Spontaneously Hypertensive rats). Both biomarkers were measured by mean of two commercially available enzyme immunoassays and serum levels were correlated with heart weight and histopathological findings in the heart, with the aim of building an integrated assessment of the significance of these biomarkers. Results obtained demonstrated that NT-proANP and ANP can be accurately measured in the different rat strains, with NT-proANP concentrations higher than those of ANP, as expected because of its longer half-life. In addition, both correlated well with cardiac hypertrophy evaluated by means of heart weight and histopathological examination. NT-proANP and ANP represent reliable markers of cardiac hypertrophy in the rat.
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Factor Natriurético Atrial/sangre , Biomarcadores/sangre , Animales , Presión Sanguínea , Cardiomegalia/sangre , Semivida , Atrios Cardíacos/metabolismo , Masculino , Tamaño de los Órganos , Radioinmunoensayo , Ratas , Ratas Endogámicas SHR , Ratas Sprague-Dawley , Ratas WistarRESUMEN
Gold(III)-dithiocarbamato complexes have recently gained increasing attention as potential anticancer agents because of their strong tumor cell growth--inhibitory effects, generally achieved by exploiting non-cisplatin-like mechanisms of action. The rationale of our research work is to combine the antitumor properties of the gold(III) metal center with the potential chemoprotective function of coordinated dithiocarbamates in order to reduce toxic side effects (in particular nephrotoxicity) induced by clinically established platinum-based drugs. In this context, [Au(III) Br(2) (ESDT)] (AUL12) was proved to exert promising and outstanding antitumor activity in vitro and to overcome both acquired and intrinsic resistance showed by some types of tumors toward cisplatin. As a subsequent extension of our previous work, we here report on detailed in vivo studies in rodents, including antitumor activity toward three transplantable murine tumor models, toxicity, nephrotoxicity and histopathological investigations. Remarkably, the gold(III) complex AUL12 stands out for higher anticancer activity than cisplatin toward all the murine tumor models examined, inducing up to 80% inhibition of tumor growth. In addition, it shows low acute toxicity levels (lethal dose, LD(50) = 30 mg kg(-1) ) and reduced nephrotoxicity. Altogether, these results confirm the reliability of our drug design strategy and support the validation of this gold(III)-dithiocarbamato derivative as a suitable candidate for clinical trials.
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Antineoplásicos/farmacología , Compuestos de Oro/farmacología , Neoplasias/prevención & control , Tiocarbamatos/farmacología , Animales , Antineoplásicos/efectos adversos , Compuestos de Oro/efectos adversos , Masculino , Ratones , Neoplasias/patología , Distribución Aleatoria , Ratas , Ratas Wistar , Tiocarbamatos/efectos adversosRESUMEN
In dogs Helicobacter spp. are found in all gastric regions usually localized in the surface mucus, gastric glands and parietal cells. The aim of this study was to detail the distribution of Helicobacter spp. in the fundic mucosa of asymptomatic Beagle dogs and their intracellular localization within parietal cells, in order to evaluate species-specific pathogenetic effects on gastric cells. The presence of Helicobacter spp. was investigated by immunohistochemistry, TEM, and PCR in the fundic mucosa of six Beagle dogs. Helicobacter spp. were found in all dogs examined, and H. bizzozeronii and H. felis were identified by PCR and confirmed by TEM. In the lumen of the fundic glands, co-localization was common. H. bizzozeronii was present in larger numbers than H. felis in both intraluminal and intraparietal localization. The amounts of H. bizzozeronii were similar in superficial and basal portions of the glands. H. felis was predominantly localized in the superficial portions of gastric glands but almost absent from the base. Within parietal cells, most Helicobacter organisms were intracanalicular, but intact and degenerate Helicobacter organisms were also visualized free in the cytoplasm or in secondary lysosomes. No specific degenerative lesions were found in infected parietal cells. Helicobacter organisms were also observed within macrophages in the lamina propria. In conclusion, there is a differential distribution of H. bizzozeronii and H. felis in the fundic mucosa of Beagle dogs, and their intracellular localization in parietal cells and macrophages suggests novel pathogenic scenarios for the development of immune response and maintenance of chronic gastritis in dogs.
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Enfermedades de los Perros/microbiología , Mucosa Gástrica/microbiología , Infecciones por Helicobacter/veterinaria , Helicobacter/fisiología , Animales , Enfermedades de los Perros/inmunología , Enfermedades de los Perros/patología , Perros , Femenino , Mucosa Gástrica/patología , Mucosa Gástrica/ultraestructura , Gastritis/inmunología , Gastritis/microbiología , Gastritis/patología , Gastritis/veterinaria , Helicobacter/genética , Helicobacter/aislamiento & purificación , Infecciones por Helicobacter/inmunología , Infecciones por Helicobacter/microbiología , Infecciones por Helicobacter/patología , Inmunohistoquímica/veterinaria , Masculino , Microscopía Electrónica de Transmisión/veterinaria , Células Parietales Gástricas/microbiología , Células Parietales Gástricas/patología , Células Parietales Gástricas/ultraestructura , Reacción en Cadena de la Polimerasa/veterinaria , Especificidad de la EspecieRESUMEN
The amino-terminal pro-brain natriuretic peptide (NT-proBNP) is released into the plasma predominantly from ventricular cardiomyocytes, particularly in patients with chronic cardiac diseases, although small amounts are detectable in the plasma of healthy subjects. While NT-proBNP has been widely exploited in human medicine, limited literature is available related to its characterization in veterinary medicine (e.g., correlation with damage and specificity) and, particularly, in the context of preclinical drug safety assessment. This paper describes the analytical performance characteristics and the biological variability of NT-proBNP in male beagle dogs by using a commercially available enzyme-linked immunosorbent assay. Male beagle dogs were treated with Casopitant, an NK1 receptor antagonist under development for depression and anxiety, which, when administered chronically to dogs, caused cardiac toxicity. Heart weight increase, myocardial necrosis, degeneration, and inflammation associated with high serum levels of cardiac troponin I characterized the end stage pathology observed in dogs treated orally at 40 mg/kg for 39 weeks. Based on these data, ad hoc studies were designed in order to evaluate the possible relationship between NT-proBNP serum levels and both standard toxicology endpoints, such as the organ weight and histology, as well as nonstandard endpoints such as macroscopic morphometry and echocardiography. Early changes of NT-proBNP serum levels were observed following 2 weeks of treatment onward, preceding most, if not all of the anatomical and functional changes. The results obtained demonstrate that NT-proBNP acts as an early biomarker of cardiac changes, representing a sensitive and predictive marker of drug-induced cardiac liability.
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Antidepresivos/efectos adversos , Biomarcadores/sangre , Cardiopatías/sangre , Péptido Natriurético Encefálico/sangre , Fragmentos de Péptidos/sangre , Piperazinas/efectos adversos , Piperidinas/efectos adversos , Administración Oral , Animales , Antidepresivos/administración & dosificación , Trastornos de Ansiedad/tratamiento farmacológico , Depresión/tratamiento farmacológico , Perros , Esquema de Medicación , Ecocardiografía , Ensayo de Inmunoadsorción Enzimática , Cardiopatías/inducido químicamente , Cardiopatías/fisiopatología , Humanos , Masculino , Péptido Natriurético Encefálico/metabolismo , Antagonistas del Receptor de Neuroquinina-1 , Tamaño de los Órganos/efectos de los fármacos , Fragmentos de Péptidos/metabolismo , Piperazinas/administración & dosificación , Piperidinas/administración & dosificación , Valor Predictivo de las Pruebas , Factores de Riesgo , Troponina I/sangreRESUMEN
Cardiovascular toxicity represents one of the major reasons for the termination of the development of drugs, even in late development phases. This growing issue is often not restricted to specific therapeutic areas, and it is gaining critical importance, in particular for chronically administered drugs, highlighting the limitations in terms of sensitivity of the current investigational paradigms. Furthermore, drug-related changes may become evident after long-term administration for different reasons, including accumulation of the drug in the heart. This article describes how the integrated use of investigational tools represents a powerful approach for the early identification and characterization of cardiotoxicity in preclinical development. Cardiac changes were observed in the dog after long-term oral administration of casopitant, a neurokinin 1 receptor antagonist, developed for the treatment of depression and anxiety. Different approaches and sensitive biomarkers were used in a time-course study to investigate the onset, progression, and reversibility of the lesion. The integrated evaluation of cardiovascular parameters, electron microscopy, troponin I, and natriuretic peptide results highlighted any minimal early changes, allowing the full and deep characterization of the lesion. The outcome of this study was the driver for drug development decision making on casopitant and backup drugs.
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Cardiopatías/inducido químicamente , Antagonistas del Receptor de Neuroquinina-1 , Piperazinas/administración & dosificación , Piperazinas/toxicidad , Piperidinas/administración & dosificación , Piperidinas/toxicidad , Administración Oral , Animales , Biomarcadores , Forma MB de la Creatina-Quinasa/análisis , Perros , Evaluación Preclínica de Medicamentos , Cardiopatías/patología , Masculino , Microscopía Electrónica de Transmisión , Modelos Animales , Miocardio/patología , Miocardio/ultraestructura , Péptido Natriurético Encefálico/análisis , Fragmentos de Péptidos/análisis , Troponina I/análisisRESUMEN
Hematopoietic stem cell gene therapy has become a successful therapeutic strategy for some inherited genetic disorders. Pre-clinical toxicity studies performed to support the human clinical trials using viral-mediated gene transfer and autologous hematopoietic stem and progenitor cell (HSPC) transplantation are complex and the use of mouse models of human diseases makes interpretation of the results challenging. In addition, they rely on the use of conditioning agents that must induce enough myeloablation to allow engraftment of transduced and transplanted HSPC. Busulfan and total body irradiation (TBI) are the most commonly used conditioning regimens in the mouse. Lenticular degeneration and atrophy of reproductive organs are expected histopathological changes. Proliferative and nonproliferative lesions can be observed with different incidence and distribution across strains and mouse models of diseases. The occurrence of these lesions can interfere with the interpretation of pre-clinical toxicity and tumorigenicity studies performed to support the human clinical studies. As such, it is important to be aware of the background incidence of lesions induced by different conditioning regimens. We review the histopathology results from seven long-term studies, five using TBI and two using busulfan.
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Trasplante de Células Madre Hematopoyéticas , Acondicionamiento Pretrasplante , Animales , Busulfano , Ciclofosfamida , Terapia Genética , Ratones , Irradiación Corporal TotalRESUMEN
Dose-response expression of kidney injury molecule-1 (KIM-1) gene in kidney cortex and its correlation with morphology and traditional biomarkers of nephrotoxicity (plasma creatinine and blood urea nitrogen, BUN) or segment-specific marker of proximal tubule injury (kidney glutamine synthetase, GSK) were studied in male rats treated with proximal tubule segment-specific nephrotoxicants. These included hexachloro-1:3-butadiene (HCBD, S(3) segment-specific), potassium dichromate (chromate, S(1)-S(2) segment-specific), and cephaloridine (Cph, S(2) segment-specific). Rats were treated with a single intraperitoneal (ip) injection of HCBD 25, 50, and 100 mg/kg, subcutaneous (sc) injection of chromate 8, 12.5, and 25 mg/kg; or ip injection of Cph 250, 500, and 1,000 mg/kg. KIM-1 gene showed a dose-dependent up-regulation induced by all segment-specific nephrotoxicants. Interestingly, magnitude of the up-regulation reflected the severity of microscopic tubular changes (degeneration, necrosis, and regeneration). Even low-severity microscopic observations were evidenced by significant gene expression changes. Furthermore, KIM-1 showed significant up-regulation even in the absence of morphological changes. In contrast, traditional and specific markers demonstrated low sensitivity or specificity. In conclusion, this study suggested KIM-1 as a sensitive molecular marker of different levels of tubular injury, and it is likely to represent a potential tool for early screening of nephrotoxicants.
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Moléculas de Adhesión Celular/biosíntesis , Expresión Génica/efectos de los fármacos , Enfermedades Renales/inducido químicamente , Túbulos Renales Proximales/lesiones , Túbulos Renales Proximales/metabolismo , Animales , Antibacterianos/toxicidad , Biomarcadores/análisis , Butadienos/toxicidad , Cáusticos/toxicidad , Cefaloridina/toxicidad , Fungicidas Industriales/toxicidad , Enfermedades Renales/metabolismo , Masculino , Dicromato de Potasio/toxicidad , Ratas , Ratas WistarRESUMEN
Little is known concerning the sensitivity of aged rats to xenobiotics inducing kidney damage. To increase this knowledge, the age-dependent response of the kidney to hexachloro-1 : 3-butadiene (HCBD) or potassium dichromate (chromate) was investigated. Rats were treated at different ages with a single dose of segment-specific nephrotoxicants of the proximal tubule, chosen on the basis of their specificity for S(3) and for S(1)-S(2) segments, respectively. The toxicological impact of these xenobiotics has been evaluated through biochemical and genomic markers, and histopathological investigation of kidney samples. HCBD treatment induced tubular necrosis of the S(3) segment of the proximal tubule associated with changes of toxicological markers unrelated to the age. In contrast, chromate treatment induced an increased kidney damage related to the rat age. In fact, histopathological investigation revealed that at 1 month of age tubular vacuolar degeneration was seen affecting S(1)-S(2) segments of the proximal tubule, whereas at 3 months of age tubular necrosis occurred in the same segments associated with tubular dilation of the distal portions. Consistently, biochemical analysis confirmed a direct correlation among genomic and biochemical marker variability and animal age. Altogether, the results show that during aging there is an increased sensitivity of kidney to chromate but not to HCBD-induced damage and evidence differential age-related selectivity of rats for nephrotoxic compounds. Significance for human risk assessment is discussed.
Asunto(s)
Envejecimiento/fisiología , Butadienos/toxicidad , Necrosis Tubular Aguda/inducido químicamente , Túbulos Renales Proximales/efectos de los fármacos , Dicromato de Potasio/toxicidad , Animales , Biomarcadores/análisis , Riñón/patología , Necrosis Tubular Aguda/patología , Túbulos Renales Proximales/patología , Masculino , Ratas , Ratas Wistar , Xenobióticos/toxicidadRESUMEN
A flow cytometry method, to monitor peripheral lymphocytes phospholipidosis, has been set up using a single staining with Nile red and double staining with Nile red and anti-CD3 monoclonal antibody. Blood has been collected from rats treated with amiodarone (phospholipidogenic antiarrhythmic drug). By flow cytometer, it is possible to detect phospholipids, using Nile red, a probe for intracellular lipids staining, changing its fluorescence on the stained lipid basis. CD3 antigen has been selected to focus on T cells, to evaluate whether these cells are the target of phospholipidosis amiodarone-dependent. In the study A, Sprague-Dawley rats were treated with three different doses (75, 150, and 300 mg kg(-1) day(-1)) of amiodarone or vehicle alone, for 14 days, followed by 14 days of recovery: Data obtained show that by flow cytometry, with Nile red alone, it is possible to detect a dose- and time-related response of phospholipidosis-positive lymphocytes; a partial recovery is also assessed. In the study B, Sprague-Dawley rats were treated with a single dose (300 mg kg(-1) day(-1)) of amiodarone, for 14 days: Data obtained show that animals treated with amiodarone have a significant increase of phospholipidosis-positive lymphocytes (p = 0.008), in particular of CD3+ cells (p = 0.0056). Transmission electron microscopy analysis confirmed data obtained by flow cytometry. This work shows that flow cytometry with Nile red could be a good tool to monitor ex vivo phospholipidosis in lymphocyte cells of animals treated with amiodarone: The phospholipidogenic effect is more evident focusing on CD3+ T lymphocytes, thus suggesting that these cells are probably the target of phospholipidosis.
Asunto(s)
Complejo CD3/inmunología , Citometría de Flujo/métodos , Oxazinas , Fosfolípidos/metabolismo , Linfocitos T/metabolismo , Amiodarona/farmacología , Animales , Antiarrítmicos/farmacología , Anticuerpos Monoclonales , Relación Dosis-Respuesta a Droga , Metabolismo de los Lípidos/efectos de los fármacos , Fosfolípidos/análisis , Ratas , Linfocitos T/citología , Factores de TiempoRESUMEN
BACKGROUND: The rat is used often to assess the toxicity of new chemical entities in preclinical drug development. Bilirubin concentration in rat serum is routinely determined by colorimetric methods, but false positive results due to hemolyzed serum or direct interferences by test compounds may occur. Capillary electrophoresis (CE) is an automated method that requires small sample volume and facilitates the direct detection of bilirubin. OBJECTIVE: The purpose of this study was to evaluate a CE method for detecting bilirubin and albumin-bound bilirubin in rat serum and to measure potential interference by hemolysis and specific test compounds. METHODS: Serum samples from male Sprague Dawley rats (n=20) were used in the study. Results obtained on a Beckman P/ACE MDQ CE instrument equipped with a UV-detector were compared with those obtained using a colorimetric method on a Hitachi 912 analyzer. Bilirubin standards were used to evaluate the detection and stability of bilirubin in rat serum, and vials with ultrafiltration membranes were used to separate albumin-bound bilirubin. Intraday and interday coefficients of variation (CV), linearity, and the effects of added hemoglobin and a test compound on CE results were determined. RESULTS: The CE method was capable of detecting bilirubin and albumin-bound bilirubin in rat serum samples with reproducible results and good accuracy. CVs were <3% and linearity of the CE assay was high (R(2)=0.9951). Abnormally high bilirubin peaks due to the presence of hemoglobin or the test compound were easily distinguished by means of CE. CONCLUSION: CE is a good alternative to the colorimetric methods currently used for the determination of bilirubin in rat serum.
Asunto(s)
Bilirrubina/sangre , Electroforesis Capilar/métodos , Animales , Bilirrubina/análisis , Masculino , Ratas , Ratas Sprague-Dawley , Sensibilidad y EspecificidadRESUMEN
Liver-directed gene therapy for the coagulation disorder hemophilia showed safe and effective results in clinical trials using adeno-associated viral vectors to replace a functional coagulation factor, although some unmet needs remain. Lentiviral vectors (LVs) may address some of these hurdles because of their potential for stable expression and the low prevalence of preexisting viral immunity in humans. However, systemic LV administration to hemophilic dogs was associated to mild acute toxicity and low efficacy at the administered doses. Here, exploiting intravital microscopy and LV surface engineering, we report a major role of the human phagocytosis inhibitor CD47, incorporated into LV cell membrane, in protecting LVs from uptake by professional phagocytes and innate immune sensing, thus favoring biodistribution to hepatocytes after systemic administration. By enforcing high CD47 surface content, we generated phagocytosis-shielded LVs which, upon intravenous administration to nonhuman primates, showed selective liver and spleen targeting and enhanced hepatocyte gene transfer compared to parental LV, reaching supraphysiological activity of human coagulation factor IX, the protein encoded by the transgene, without signs of toxicity or clonal expansion of transduced cells.