RESUMO
Next-generation urinary protein biomarkers have been qualified to enable monitoring for drug-induced kidney injury in toxicology studies conducted in rats. However, there is limited literature on the utility of these biomarkers in dogs. To add to the existing body of knowledge on the utility of the next-generation drug-induced kidney injury (DIKI) biomarkers, we evaluated the value of these biomarkers for the early detection of DIKI in Beagle dogs using a differentiated nephrotoxicant, Amphotericin B (AmpB). In dogs with AmpB-induced kidney injury, we monitored the response of urinary albumin, total protein, clusterin, kidney injury molecule 1, neutrophil gelatinase-associated lipocalin and N-acetyl-beta-D-glucosaminidase. We also measured blood urea nitrogen, serum creatinine and cystatin C. The results showed that urinary clusterin (up to â¼ 112x) was much more sensitive to AmpB-induced kidney injury relative to other biomarkers. Moreover, other than urinary clusterin and to a much lesser extent urinary albumin and total protein, none of the other biomarkers analyzed in this study were more sensitive than blood urea nitrogen and serum creatinine. The AmpB related tubular alterations were characterized by minimal to mild, multifocal necrosis, degeneration, regeneration, dilatation and mineralization. The mild nature of these histopathologic findings further attested to the sensitivity of urinary clusterin to AmpB-induced kidney injury in dogs. These results will help drug developers make informed decisions when selecting urinary biomarkers for monitoring DIKI in dogs for toxicology studies.
Assuntos
Injúria Renal Aguda , Nefropatias , Cães , Animais , Ratos , Anfotericina B/toxicidade , Clusterina/urina , Creatinina , Rim/patologia , Biomarcadores , Nefropatias/induzido quimicamente , Albuminas/toxicidade , Injúria Renal Aguda/induzido quimicamenteRESUMO
Novel urinary protein biomarkers have recently been identified and qualified in rats for the early detection of renal injury in drug development studies. However, there are few reports on the utility of these renal biomarkers in mice, another important and widely used preclinical animal species for drug development studies. The purpose of this study was to assess the value of these recently qualified biomarkers for the early detection of drug-induced kidney injury (DIKI) in different strains of mice using multiple assay panels. To this end, we evaluated biomarker response to kidney injury induced by several nephrotoxic agents including amphotericin B, compound X, and compound Y. Several of the biomarkers were shown to be sensitive to DIKI in mice. When measured, urinary albumin and neutrophil gelatinase-associated lipocalin were highly sensitive to renal tubular injury, regardless of the assay platforms, mouse strain, and nephrotoxic agents. Depending on the type of renal tubular injury, kidney injury molecule-1 was also highly sensitive, regardless of the assay platforms and mouse strain. Osteopontin and cystatin C were modestly to highly sensitive to renal tubular injury, but the assay type and/or the mouse strain should be considered before using these biomarkers. Calbindin D28 was highly sensitive to injury to the distal nephron in mice. To our knowledge, this is the first report that demonstrates the utility of novel urinary biomarkers evaluated across multiple assay platforms and nephrotoxicants in different mice strains with DIKI. These results will help drug developers make informed decisions when selecting urinary biomarkers for monitoring DIKI in mice for toxicology studies.
Assuntos
Injúria Renal Aguda/induzido quimicamente , Injúria Renal Aguda/diagnóstico , Anfotericina B/toxicidade , Biomarcadores/urina , Desenvolvimento de Medicamentos/métodos , Animais , Modelos Animais de Doenças , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Valor Preditivo dos TestesRESUMO
Novel urinary protein biomarkers have recently been identified and qualified in rats for the early detection of renal injury in drug development studies. However, there seems to be no standardized normalization method for analyzing these urinary biomarkers, as some users normalize with urinary creatinine (uCr), urine volume (uVol), or leave biomarker un-normalized. More recently, urinary cystatin C is also emerging as a urinary biomarker normalizer, given some of its characteristics as a glomerular filtration marker. The purpose of this study was to identify an optimal drug-induced kidney injury biomarker normalization method that can be adopted more uniformly in the field. To this end, we compared the variability of uVol, urinary cystatin C, and Cr in healthy rats; we evaluated the sensitivity of the renal biomarkers to renal injury after normalization with uVol, uCr, and cystatin C in rats with cisplatin-induced renal injury. We showed that, over time, uCr was less variable than urinary cystatin C and uVol. When the renal biomarkers were normalized with the 3 normalizing end points, the biomarkers showed (1) least variability following normalization with Cr in healthy animals and (2) poor sensitivity when normalized with urinary cystatin C in animals with renal injury. Overall, the results suggested that uCr is better than urinary cystatin C and uVol for normalizing renal biomarkers in rats under controlled preclinical conditions. To our knowledge, this is the first report that compared the variability of uVol, cystatin C, and Cr in the context of renal biomarkers' normalization.
Assuntos
Injúria Renal Aguda/urina , Creatinina/urina , Cistatina C/urina , Desenvolvimento de Medicamentos , Urinálise , Injúria Renal Aguda/patologia , Animais , Animais não Endogâmicos , Biomarcadores/urina , Feminino , Rim/patologia , Masculino , Ratos Sprague-DawleyRESUMO
BACKGROUND: This study was conducted as part of an ILSI-HESI International Life Sciences Institute-Health & Environmental Sciences Institute consortium effort to assess the utility of circulating Inhibin B as an early biomarker of Sertoli cell-specific testicular toxicity in rats. 1, 3-Dinitrobenzene (1,3-DNB) was selected as a testicular toxicant in this study as it is known to target Sertoli cells. METHODS: 1,3-DNB (2 and 6 mg/kg/day) or control (corn oil) was administered orally to male rats for two or five consecutive days. Blood was collected from rats treated for 2 days on days 1 and 2 and from rats treated for 5 days on days 1, 3, and 5. The resulting serum was evaluated for Inhibin B and follicle stimulating hormone. At the end of the treatment periods, the testes were removed, weighed, and examined histopathologically. RESULTS: Daily administration of 1,3-DNB resulted in decreased testis weight only on day 5 and only at the high dose (6 mg/kg/day). There was a time-dependent increase in incidence and severity of testicular findings characterized by degeneration of the germinal epithelium with loss of pachytene spermatocytes and vacuolization of the Sertoli cells in the seminiferous tubules at the high dose. Inhibin B levels in 1,3-DNB-treated animals were decreased with treatment only on day 5 at the high dose; there were no associated changes in follicle stimulating hormone. CONCLUSIONS: Changes in serum Inhibin B levels were detected only in association with moderate or severe testicular toxicity as evidenced by histopathology and is therefore considered to be of limited value as a biomarker for Sertoli cell toxicity.
Assuntos
Dinitrobenzenos/toxicidade , Inibinas/sangue , Testículo/efeitos dos fármacos , Testículo/patologia , Animais , Epididimo/efeitos dos fármacos , Epididimo/patologia , Hormônio Foliculoestimulante/sangue , Masculino , Tamanho do Órgão/efeitos dos fármacos , Ratos , Ratos Sprague-DawleyRESUMO
BACKGROUND: A cross-laboratory analytic evaluation of a commercially available human inhibin B ELISA for measuring inhibin B in rat serum and plasma has been undertaken. METHODS: Dilution linearity, spiked recovery, intra- and inter-assay precision, functional sensitivity, matrix effects, and frozen stability were assessed across five laboratories. Reference ranges were generated for male Sprague Dawley and Han Wistar rats. RESULTS: Acceptable performance was defined as an overall assay coefficient of variation ≤ 20% with an intraday LLOQ ≤ 20 pg/ml. Intra- and inter-assay precision and functional sensitivity (≤6.4 pg/ml) generally met these criteria, but with occasional evidence of greater variability, particularly at lower concentrations. Dilution linearity was acceptable with occasional low recovery. Acceptable recovery of kit calibrators from rat serum confirmed the absence of matrix effects. Matched serum and plasma samples gave comparable results. The signal increased on freezing, remained constant for ≥3 freeze-thaw cycles and was generally stable for at least 8 weeks. Mean inhibin B ranged from 33.5 to 140.6 pg/ml in adult rats across laboratories, with some evidence for a decline from 6 to 9 weeks of age. Power calculations using preliminary reference range data indicated 10 animals/group would generally detect a 40% decrease in inhibin B at AstraZeneca, but laboratories with lower control values would require larger groups. CONCLUSIONS: The assay meets the analytical performance criteria; however, precision at the low end of the standard curve, biological variability, and low control values observed in some laboratories indicate that the utility of the assay may be limited in some laboratories.
Assuntos
Ensaio de Imunoadsorção Enzimática/métodos , Inibinas/sangue , Animais , Bioensaio , Congelamento , Humanos , Masculino , Controle de Qualidade , Ratos , Ratos Sprague-Dawley , Padrões de Referência , Valores de Referência , Soro/metabolismoRESUMO
The Developmental and Reproductive Toxicity Technical Committee of the Health and Environmental Sciences Institute hosted a working consortium of companies to evaluate a new commercially available analytic assay for Inhibin B in rat serum or plasma. After demonstrating that the kit was stable and robust, the group performed a series of independent pathogenesis studies (23 different compound/investigator combinations) designed to examine the correlation between the appearance of lesions in the testis and changes in circulating levels of Inhibin B. These studies were reported individually in the previous articles in this series (this issue), and are discussed in this paper. For roughly half of these exposures, lesions appeared well before Inhibin B changed. A few of the studies showed a good correlation between seminiferous tubule damage and reduced circulating Inhibin B levels, while for seven exposures, circulating Inhibin B was reduced with no detectable alteration in testis histology. Whether this indicates a prodromal response or a false-positive signal will require further investigation. These exceptions could plausibly suggest some value of circulating Inhibin B as a useful biomarker in some circumstances. However, for roughly half of these exposures, Inhibin B appeared to be a lagging biomarker, requiring significant damage to the seminiferous tubules before a consistent and credible reduction in circulating levels of Inhibin B was observed.
Assuntos
Ecologia , Saúde , Inibinas/sangue , Testículo/metabolismo , Testículo/patologia , Animais , Biomarcadores/sangue , Masculino , Ratos , Ratos Sprague-Dawley , Ratos WistarRESUMO
Cisplatin (CDDP) is known to produce renal proximal tubule injury. Various renal biomarkers have been related to CDDP nephrotoxicity in previous research, but the temporal and spatial relationship of these biomarkers to injury reversal has not been well defined. In this study, the progression and reversal of renal histopathology findings relative to serum and urinary biomarker changes were examined during a 4-week postdose period following single intraperitoneal administration of CDDP (1 mg/kg) or 0.9% saline. Degeneration, vacuolation, inflammation, and regeneration of the S3 segment of proximal tubules were evident 72 hours following CDDP administration. Tubular degeneration and regeneration were also observed at 1 and 1.5 weeks but at lower incidences and/or severity indicating partial reversal. Complete histologic reversal was observed by 2 weeks following CDDP administration. Urinary kidney injury molecule 1 (KIM-1), α-glutathione-S-transferase (α-GST), and albumin levels increased at 72 hours postdosing, concurrently with the earliest histologic evidence of tubule injury. Changes in urinary KIM-1 correlated with KIM-1 immunostaining in the proximal tubular epithelial cells. No significant changes in serum biomarkers occurred except for a minimal increase in urea nitrogen at 1.5 weeks postdosing. Of the novel renal biomarkers examined, urinary KIM-1, α-GST, and albumin showed excellent concordance with CDDP-induced renal injury progression and reversal; and these biomarkers were more sensitive than traditional serum biomarkers in detecting early, acute renal tubular damage confirmed by histopathology. Furthermore, urinary KIM-1, α-GST, and albumin outperformed other biomarkers in correlating with the time of maximum histologic injury.
Assuntos
Biomarcadores/sangue , Biomarcadores/urina , Cisplatino/administração & dosagem , Túbulos Renais Proximais/efeitos dos fármacos , Albuminas/metabolismo , Animais , Moléculas de Adesão Celular/metabolismo , Clusterina/urina , Glutationa Transferase/metabolismo , Imuno-Histoquímica , Isoenzimas/metabolismo , Túbulos Renais Proximais/patologia , Masculino , Ratos , Ratos Sprague-DawleyRESUMO
The kidney is one of the main targets of drug toxicity, and early detection of renal damage is critical in preclinical drug development. A model of cisplatin-induced nephrotoxicity in male Sprague Dawley rats treated for 1, 3, 5, 7, or 14 days at 1 mg/kg/day was used to monitor the spatial and temporal expression of various indicators of kidney toxicity during the progression of acute kidney injury (AKI). As early as 1 day after cisplatin treatment, positive kidney injury molecule-1 (Kim-1) immunostaining, observed in the outer medulla of the kidney, and changes in urinary clusterin indicated the onset of proximal tubular injury in the absence of functional effects. After 3 days of treatment, Kim-1 protein levels in urine increased more than 20-fold concomitant with a positive clusterin immunostaining and an increase in urinary osteopontin. Tubular basophilia was also noted, while serum creatinine and blood urea nitrogen levels were elevated only after 5 days, together with tubular degeneration. In conclusion, tissue Kim-1 and urinary clusterin were the most sensitive biomarkers for detection of cisplatin-induced kidney damage. Thereafter, urinary Kim-1 and osteopontin, as well as clusterin immunostaining accurately correlated with the histopathological findings. When AKI is suspected in preclinical rat studies, Kim-1, clusterin, and osteopontin should be part of urinalysis and/or IHC can be performed.
Assuntos
Antineoplásicos/toxicidade , Cisplatino/toxicidade , Clusterina/urina , Nefropatias/induzido quimicamente , Proteínas de Membrana/metabolismo , Testes de Toxicidade/métodos , Animais , Biomarcadores/metabolismo , Análise Química do Sangue , Peso Corporal/efeitos dos fármacos , Modelos Animais de Doenças , Receptor Celular 1 do Vírus da Hepatite A , Rim/efeitos dos fármacos , Rim/metabolismo , Rim/patologia , Nefropatias/metabolismo , Nefropatias/patologia , Túbulos Renais/efeitos dos fármacos , Túbulos Renais/metabolismo , Túbulos Renais/patologia , Masculino , Tamanho do Órgão/efeitos dos fármacos , Osteopontina/urina , Ratos , Ratos Sprague-Dawley , UrináliseRESUMO
Nonclinical rodent and nonrodent toxicity models used to support clinical trials of candidate drugs may produce discordant results or fail to predict complications in humans, contributing to drug failures in the clinic. Here, we applied microengineered Organs-on-Chips technology to design a rat, dog, and human Liver-Chip containing species-specific primary hepatocytes interfaced with liver sinusoidal endothelial cells, with or without Kupffer cells and hepatic stellate cells, cultured under physiological fluid flow. The Liver-Chip detected diverse phenotypes of liver toxicity, including hepatocellular injury, steatosis, cholestasis, and fibrosis, and species-specific toxicities when treated with tool compounds. A multispecies Liver-Chip may provide a useful platform for prediction of liver toxicity and inform human relevance of liver toxicities detected in animal studies to better determine safety and human risk.
Assuntos
Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/patologia , Dispositivos Lab-On-A-Chip , Fígado/patologia , Animais , Biomarcadores/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/patologia , Cães , Humanos , Células de Kupffer/metabolismo , Fígado/lesões , Hepatopatias/patologia , Fenótipo , Ratos , Reprodutibilidade dos Testes , Fatores de Risco , Especificidade da EspécieRESUMO
Amphotericin B (AmpB) nephrotoxicity was used to assess the utility of druginduced kidney injury (DIKI) biomarkers in an exploratory study in male cynomolgus monkeys. All animals had quantifiable levels of AmpB in plasma on days 1 and 4. There were no clinical signs of AmpBinduced toxicity in this study. The gold standard method used to confirm AmpBinduced DIKI was anatomic pathology which revealed microscopic lesions with varying grades of severity. Immunolocalization of alpha1 microglobulin (α1M), kidney injury molecule 1 (KIM1), osteopontin (OPN) and neutrophil gelatinaseassociated lipocalin (NGAL) proteins was evaluated in formalinfixed, paraffinembedded monkey kidney tissue sections. AmpB related immunoreactivities were identified in distinct nephron segments of treated monkeys including α1M in damaged proximal tubule epithelium, KIM1 in damaged medullary tubule epithelium, OPN mostly in the infiltrating cells of cortical tubule interstitium, and NGAL in the granular and cellular cast in dilatated cortical tubules. Variations in α1M, KIM1, OPN and NGAL immunolocalization appear as promising DIKI protein biomarkers when monitoring for AmpBinduced corticomedullary tubule injury in male cynomolgus monkeys.
Assuntos
Anfotericina B/toxicidade , Anti-Infecciosos/toxicidade , Nefropatias/metabolismo , Túbulos Renais/metabolismo , alfa-Globulinas/metabolismo , Anfotericina B/sangue , Anfotericina B/farmacocinética , Animais , Anti-Infecciosos/sangue , Anti-Infecciosos/farmacocinética , Biomarcadores/metabolismo , Receptor Celular 1 do Vírus da Hepatite A/metabolismo , Imuno-Histoquímica , Nefropatias/induzido quimicamente , Nefropatias/patologia , Túbulos Renais/efeitos dos fármacos , Túbulos Renais/patologia , Lipocalina-2/metabolismo , Macaca fascicularis , Masculino , Osteopontina/metabolismoRESUMO
In previous studies, the free radical generating toxin tertiary butylhydroperoxide (t-BuOOH) was found to induce significant cell death in human cortical neuronal cells (HCN2 cells). Pretreatment with the poly (ADP-ribose) polymerase (PARP) inhibitor nicotinamide was able to prevent HCN2 cell death. In this study it is observed that apoptosis is induced following the addition of t-BuOOH at 6 h as indicated by TUNEL-positive cells. When nicotinamide is added prior to t-BuOOH, it is able to prevent neuronal cell death and inhibit apoptosis. DNA microarray studies demonstrate that t-BuOOH administration causes an upregulation of proapoptotic genes like ICH-2 and BimL. On the other hand, nicotinamide-pretreated neurons have higher expression levels of inhibitors of apoptosis (IAP) genes. Therefore, it appears that one mechanism by which nicotinamide acts as neuroprotective agents is by elevating the gene expression levels of IAPs. Moreover, there is an upregulation of the glyceraldehydes-3-phosphate dehydrogenase gene in nicotinamide-pretreated HCN2 cells. Nicotinamide-pretreated cells also had higher expression levels of putative "death domain" genes like p75TNFR, TRAIL2, TNFR1, and HVEM-L. Thus, nicotinamide can regulate multiple apoptotic genes with seemingly opposite roles and through its action on these various genes prevent apoptosis of neuronal cells.
RESUMO
Identification of circulating microRNAs for the diagnosis of liver injury and as an indicator of underlying pathology has been the subject of recent investigations. While several studies have been conducted, with particular emphasis on miR-122, the timing of miRNA release into the circulation and anchoring to tissue pathology has not been systematically evaluated. In this study, miRNA profiling was conducted over a time course of hepatobiliary injury and repair using alpha-naphthylisothiocyanate (ANIT) and a proprietary compound, FP004BA. ANIT administration (50 mg/kg) to rats caused significant biliary epithelial cell and hepatocellular necrosis between 24 and 72 h, followed by resolution and progression to biliary hyperplasia by 120 h which was associated with miRNA release into the blood. FP004BA (100 mg/kg) was used to confirm associations of miRNA along a time course with similar hepatic pathology to ANIT. Treatment with ANIT or FP004BA resulted in significant alterations of overlapping miRNAs during the early and peak injury phases. In addition to well-characterized liver injury markers miR-122-5p and miR-192-5p, multiple members of the 200 family and the 101 family along with miR-802-5p and miR-30d-5p were consistently elevated during hepatobiliary injury caused by both toxicants, suggesting that these species may be potential biomarker candidates for hepatobiliary injury. After 14 days of dosing with 4BA, miR-182-5p remained elevated-while miR-122-5p and miR-192-5p had returned to baseline-suggesting that miR-182-5p may have added utility to monitor for hepatobiliary injury in the repair phases when there remains histological evidence of ongoing cellular injury.
Assuntos
Sistema Biliar/patologia , Doença Hepática Induzida por Substâncias e Drogas/sangue , Fígado/patologia , MicroRNAs/sangue , 1-Naftilisotiocianato/farmacologia , Animais , Biomarcadores/sangue , Doença Hepática Induzida por Substâncias e Drogas/patologia , Modelos Animais de Doenças , Hiperplasia , Masculino , Ratos Sprague-DawleyRESUMO
Traditional kidney biomarkers are insensitive indicators of acute kidney injury, with meaningful changes occurring late in the course of injury. The aim of this work was to demonstrate the diagnostic potential of urinary osteopontin (OPN) and neutrophil gelatinase-associated lipocalin (NGAL) for drug-induced kidney injury (DIKI) in rats using data from a recent regulatory qualification submission of translational DIKI biomarkers and to compare performance of NGAL and OPN to five previously qualified DIKI urinary biomarkers. Data were compiled from 15 studies of 11 different pharmaceuticals contributed by Critical Path Institute's Predictive Safety Testing Consortium (PSTC) Nephrotoxicity Working Group (NWG). Rats were given doses known to cause DIKI or other target organ toxicity, and urinary levels of the candidate biomarkers were assessed relative to kidney histopathology and serum creatinine (sCr) and blood urea nitrogen (BUN).OPN and NGAL outperformed sCr and BUN in identifying DIKI manifested as renal tubular epithelial degeneration or necrosis. In addition, urinary OPN and NGAL, when used with sCr and BUN, increased the ability to detect renal tubular epithelial degeneration or necrosis. NGAL and OPN had comparable or improved performance relative to Kim-1, clusterin, albumin, total protein, and beta-2 microglobulin. Given these data, both urinary OPN and NGAL are appropriate for use with current methods for assessing nephrotoxicity to identify and monitor DIKI in regulatory toxicology studies in rats. These data also support exploratory use of urinary OPN and NGAL in safety monitoring strategies of early clinical trials to aid in the assurance of patient safety.
Assuntos
Injúria Renal Aguda/diagnóstico , Proteínas de Fase Aguda/urina , Lipocalinas/urina , Osteopontina/urina , Proteínas Proto-Oncogênicas/urina , Injúria Renal Aguda/sangue , Injúria Renal Aguda/induzido quimicamente , Injúria Renal Aguda/urina , Animais , Área Sob a Curva , Biomarcadores/sangue , Biomarcadores/urina , Nitrogênio da Ureia Sanguínea , Creatinina/sangue , Modelos Animais de Doenças , Lipocalina-2 , Valor Preditivo dos Testes , Curva ROC , Ratos , Reprodutibilidade dos Testes , UrináliseRESUMO
It is well known that the generation of oxygen radicals can cause neuronal death by both apoptosis and necrosis, which may lead to the onset of neurodegenerative diseases. In previous in vivo studies, nicotinamide was found to prevent both DNA fragmentation and apoptosis that were induced by free radical generating toxins like tertiary butylhydroperoxide (t-BuOOH). Nicotinamide is a precursor for NAD and is an inhibitor of the enzyme poly(ADP-ribose) polymerase (PARP). However, the effect of nicotinamide on the regulation of pro- and anti-apoptotic proteins in neurons is not clear. In our study, the human cortical neuronal cell line HCN1-A has been used to determine the mechanism of action of nicotinamide at the cellular level. Cell viability studies showed that t-BuOOH treatment (both 100 microM and 1mM) caused significant cell death at 24 and 48h compared to control cells. Pretreatment with 1mM nicotinamide before t-BuOOH administration caused significant reduction in cell death. Moreover, the morphology of HCN1-A cells that were treated with both nicotinamide and t-BuOOH appeared to be closer to control cells when compared to HCN1-A cells treated with only t-BuOOH. Also, t-BuOOH treatment caused an elevation in the levels of the pro-apoptotic proteins p53 and p21/WAF-1 and a reduction in the levels of the anti-apoptotic protein bcl-2 compared to their levels in control HCN1-A cells, while pretreatment with nicotinamide reduced p53 and p21/WAF-1 levels even in the presence of t-BuOOH. However nicotinamide did not seem to alter bcl-2 levels. These results indicate that nicotinamide treatment can protect human neuronal cells from the toxic effects of t-BuOOH.
Assuntos
Córtex Cerebral/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Niacinamida/farmacologia , terc-Butil Hidroperóxido/farmacologia , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Córtex Cerebral/citologia , Córtex Cerebral/fisiologia , Humanos , Neurônios/citologia , Neurônios/fisiologiaRESUMO
Genistein, a soy isoflavone, has been shown to mimic the pharmacological actions of the endogenous steroid estrogen with which it has structural similarities. There is now evidence that the genistein can prevent disorders-like heart diseases, cancer and diabetes as well. However, very few studies have looked at the effect of genistein on the central nervous system. Published studies also show conflicting conclusions regarding the effects of genistein in the brain. The current study was conducted in the human cortical cell lines HCN1-A and HCN2 in order to determine the neuroprotective efficacy of genistein. It was observed that pre-treatment with 50 or 10 microM genistein was able to protect HCN1-A and HCN2 cells from the cell death induced by 100 microM or 1 mM tertiary butylhydroperoxide (t-BuOOH; a free radical generating toxin). The morphological disruption caused by t-BuOOH was also prevented by genistein in HCN2 cells. Moreover, genistein was able to prevent the down-regulation of the anti-apoptotic protein bcl-2 that was caused by t-BuOOH treatment. These results indicate that genistein may have neuroprotective effect in cortical cells, which may be mediated by its regulation of the anti-apoptotic protein bcl-2.
Assuntos
Córtex Cerebral/patologia , Genisteína/farmacologia , Neurônios/patologia , Fármacos Neuroprotetores , Estresse Oxidativo/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Linhagem Celular , Córtex Cerebral/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Ensaio de Imunoadsorção Enzimática , Estradiol/farmacologia , Humanos , L-Lactato Desidrogenase/metabolismo , Neurônios/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , terc-Butil Hidroperóxido/toxicidadeRESUMO
The first formal qualification of safety biomarkers for regulatory decision making marks a milestone in the application of biomarkers to drug development. Following submission of drug toxicity studies and analyses of biomarker performance to the Food and Drug Administration (FDA) and European Medicines Agency (EMEA) by the Predictive Safety Testing Consortium's (PSTC) Nephrotoxicity Working Group, seven renal safety biomarkers have been qualified for limited use in nonclinical and clinical drug development to help guide safety assessments. This was a pilot process, and the experience gained will both facilitate better understanding of how the qualification process will probably evolve and clarify the minimal requirements necessary to evaluate the performance of biomarkers of organ injury within specific contexts.
Assuntos
Biomarcadores Farmacológicos , Aprovação de Drogas/legislação & jurisprudência , Rim , Animais , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos , Europa (Continente) , Humanos , Rim/efeitos dos fármacos , Rim/lesões , Preparações Farmacêuticas/normas , Estados Unidos , United States Food and Drug AdministrationRESUMO
In previous studies with mice the oxygen radical generating neurotoxin tertiary butylhydroperoxide (t-BuOOH) was used to mimic the oxidative injury that has been implicated in neurodegenerative diseases. In addition, previous studies have shown that the poly (ADP-ribose) polymerase (PARP) inhibitor nicotinamide is able to prevent DNA fragmentation and apoptosis that is induced by t-BuOOH in mouse brain. However, the molecular mechanism(s) by which nicotinamide is able to protect human brain cells at the cellular level is not clear. Therefore in this study a cell culture model system with human cortical neuronal cells (HCN2 cells) has been employed where the molecular mechanism(s) of nicotinamide action, both in the presence and absence of t-BuOOH has been studied. Human cortical neurons (HCN2 cells) have been shown to differentiate to a neuron-like morphology. In this study, exposure of HCN2 cells to t-BuOOH resulted in altered morphology and disruption of neuronal differentiation leading to cell death. However, in neurons, which were treated with nicotinamide before being exposed to t-BuOOH, neuronal differentiation was preserved; morphological disruption was prevented and cell death was reduced significantly. Moreover, our studies indicate that nicotinamide is able to prevent the up-regulation of the pro-apoptotic proteins p53 and p21/WAF-1, and the down-regulation of the anti-apoptotic protein bcl-2 that is induced by t-BuOOH in HCN2 cells. Thus this study indicates that nicotinamide protects human brain cells from the toxic effects of free radical generating toxins by regulating the levels of various pro- and anti-apoptotic proteins.
RESUMO
Cytoskeleton disruption is a process by which oxidative stress disrupts cellular function. This study compares and contrasts the effect of oxidative stress on the three major cytoskeleton filaments, microfilaments (MFs), microtubule (MT), and vimentin in human cortical neuronal cell line (HCN2). HCN2 cells were treated with 100 microM tertiary butylhydroperoxide (t-BuOOH), a free radical generating neurotoxin for 1, 3, or 6 h. Cell viability studies demonstrated significant cell death although the morphology studies showed that there was a substantial loss in neurites of neurons treated with t-BuOOH for 6 h. Because the cytoskeleton plays a role in neurite outgrowth, the effect of oxidative stress on the cytoskeletal was studied. In neurons subjected to oxidative stress for 30 min or 1 h, there were no major changes in microfilament distribution though there was altered distribution of microtubule and vimentin filaments as compared to controls. However, loss and disruption of all the three cytoskeletal filaments was observed at later times (3 and 6 h), which was confirmed by Western Blot analysis. Further studies were done to measure the gene expression levels of actin, tubulin, and vimentin. Results indicated that the overall loss of the cytoskeletal proteins in neurons treated with free radical generating toxin might not be a direct result of the downregulation of the cytoskeletal genes. This study shows that free radical generation in human neurons leads to the disruption of the cytoskeleton, though there may be a difference in the susceptibility to oxidative stress among the individual components of the cytoskeletal filaments.