RESUMEN
Current global efforts are aiming to increase use of mechanistic information in regulatory testing. In tiered testing paradigms, in vitro, in silico, and in vivo studies are employed progressively to identify and classify health hazards, which are then compared against human equivalent doses. We used data from three companion papers on the brominated flame retardant hexabromocyclododecane (HBCD) to conduct a case study on tiered testing. We included ToxCast™ and in vitro-in vivo extrapolation (Tier 1), rat liver transcriptomic (Tier 2), and conventional rat (Tier 3) data. Bioactivity-exposure ratios (BERs) were derived by comparing human administered dose equivalents of the measured effects to Canadian exposure levels. Biological perturbations were highly aligned between Tiers 1/2, and consistent with apical effects in Tier 3. Tier 1 had the smallest BERs, and Tiers 2/3 were similar. The study demonstrates the promise of using physiologically-based pharmacokinetic modeling and mechanistic analyses in a tiered framework to identify pathways through which chemicals exert toxicological effects; however, they also point to some shortcomings associated with in vitro and in silico approaches. Additional case studies of chemicals from multiple classes are required to define optimal tiered screening procedures to reduce future in vivo requirements in health hazard assessments.
Asunto(s)
Retardadores de Llama/toxicidad , Hidrocarburos Bromados/toxicidad , Animales , Apoptosis/efectos de los fármacos , Femenino , Retardadores de Llama/administración & dosificación , Expresión Génica/efectos de los fármacos , Humanos , Hidrocarburos Bromados/administración & dosificación , Masculino , Ratas Endogámicas F344 , Ratas Sprague-Dawley , Ratas Wistar , Medición de Riesgo , Pruebas de Toxicidad/métodosRESUMEN
Hexabromocyclododecane (HBCD) is a brominated flame retardant found in the environment and human tissues. The toxicological effects of HBCD exposure are not clearly understood. We employed whole-genome RNA-sequencing on liver samples from male and female Fischer rats exposed to 0, 250, 1250, and 5000â¯mg technical mixture of HBCD/kg diet for 28 days to gain further insight into HBCD toxicity. HBCD altered 428 and 250 gene transcripts in males and females, respectively, which were involved in metabolism of xenobiotics, oxidative stress, immune response, metabolism of glucose and lipids, circadian regulation, cell cycle, fibrotic activity, and hormonal balance. Signature analysis supported that HBCD operates through the constitutive androstane and pregnane X receptors. The median transcriptomic benchmark dose (BMD) for the lowest statistically significant pathway was within 1.5-fold of the BMD for increased liver weight, while the BMD for the lowest pathway with at least three modeled genes (minimum 5% of pathway) was similar to the lowest apical endpoint BMD. The results show how transcriptional analyses can inform mechanisms underlying chemical toxicity and the doses at which potentially adverse effects occur. This experiment is part of a larger study exploring the use of toxicogenomics and high-throughput screening for human health risk assessment.
Asunto(s)
Retardadores de Llama/toxicidad , Hidrocarburos Bromados/toxicidad , Hígado/efectos de los fármacos , Transcripción Genética/efectos de los fármacos , Transcriptoma/efectos de los fármacos , Animales , Relación Dosis-Respuesta a Droga , Femenino , Masculino , ARN Mensajero/genética , Ratas Endogámicas F344 , Análisis de Secuencia de ARN , Toxicogenética/métodosRESUMEN
The presence of the mycotoxin ochratoxin A (OTA) in cereal grains is due to the growth of toxigenic Penicillium mold on stored crops. Human exposure to OTA is higher in infants, toddlers, and children than in adolescents and adults, based on exposure assessments of ng OTA consumed/kg body weight/day. Ochratoxin A is nephrotoxic and teratogenic in animals, but its effects on juveniles exposed during the reproduction and development period have not been studied. To address this, Fischer rats were exposed to 0, 0.16, 0.4, 1.0, or 2.5 mg OTA/kg diet throughout breeding, gestation, and lactation and its adverse effects were assessed in adult rats and their offspring on postnatal day (PND) 21. There were no effects on implantation but post-implantation fetotoxicity was observed in the 2.5 mg/kg dose group, corresponding to a calculated dose of 167.0 µg/kg bw/day in dams. Adverse effects on body and kidney weights and on clinical parameters indicative of renal toxicity were significant in adult rats exposed to 1.0 mg OTA/kg diet (55.2 and 73.3 µg/kg bw/day in adult males and females, respectively) and in PND21 rats at the 0.4 mg/kg dose (33.9 µg/kg bw/day in dams), suggesting that weanling rats were more sensitive to OTA than adults. Overall, nephrotoxicity was the primary effect of OTA in weanling rats exposed throughout gestation and lactation at sub-fetotoxic concentrations in diet.
Asunto(s)
Ocratoxinas/toxicidad , Intoxicación/patología , Complicaciones del Embarazo/patología , Insuficiencia Renal/patología , Teratógenos/toxicidad , Anomalías Inducidas por Medicamentos/epidemiología , Anomalías Inducidas por Medicamentos/patología , Administración Oral , Animales , Modelos Animales de Enfermedad , Femenino , Ocratoxinas/administración & dosificación , Embarazo , Ratas Endogámicas F344 , Insuficiencia Renal/inducido químicamenteRESUMEN
Cigarette smoking is a lifestyle behavior associated with significant adverse health effects, including subfertility and premature ovarian failure. Cigarette smoke contains a number of chemicals, many of which are involved in the generation of reactive oxygen species, which can lead to apoptosis and autophagy. Autophagy is a fundamental process that removes damaged organelles and proteins through lysosomal degradation. The relevance of autophagy to toxicant-induced changes in ovarian function is largely unexplored. Previously, we reported that exposure to cigarette smoke causes follicle loss, oxidative stress, activation of the autophagy pathway, and decreased expression of manganese superoxide dismutase, which points to altered mitochondrial function. Therefore, our objective here was to test whether exposure to cigarette smoke results in the dysregulation of mitochondrial repair mechanisms leading to loss of follicles via autophagy-mediated granulosa cell death. In this study, mice were exposed to cigarette smoke or room air for 8 wk. The expression of genes and proteins of autophagy and mitochondrial repair factors was measured using quantitative real-time PCR and Western blot analysis, immunohistochemistry, and enzyme-linked immunosorbent assay. Increased expression of parkin and decreased expression of the mitofusins suggest that exposure to cigarette smoke triggers mitochondrial damage. Moreover, the autophagy cascade proteins, BECN1 and LC3, were upregulated, whereas the antagonist BCL2 was downregulated, following treatment. Taken together, our results suggest exposure to cigarette smoke induces dysfunction of mitochondrial repair mechanisms, leading to autophagy-mediated follicle death.
Asunto(s)
Autofagia/efectos de los fármacos , Células de la Granulosa/efectos de los fármacos , Dinámicas Mitocondriales/efectos de los fármacos , Fumar/efectos adversos , Contaminación por Humo de Tabaco/efectos adversos , Animales , Proteínas Reguladoras de la Apoptosis/metabolismo , Beclina-1 , Femenino , Ratones , Ratones Endogámicos C57BL , Regulación hacia ArribaRESUMEN
Trinucleotide repeat (TNR) expansions cause at least 17 heritable neurological diseases, including Huntington's disease. Expansions are thought to arise from abnormal processing of TNR DNA by specific trans-acting proteins. For example, the DNA repair complex MutSß (MSH2-MSH3 heterodimer) is required in mice for on-going expansions of long, disease-causing alleles. A distinctive feature of TNR expansions is a threshold effect, a narrow range of repeat units (â¼30-40 in humans) at which mutation frequency rises dramatically and disease can initiate. The goal of this study was to identify factors that promote expansion of threshold-length CTGâ¢CAG repeats in a human astrocytic cell line. siRNA knockdown of the MutSß subunits MSH2 or MSH3 impeded expansions of threshold-length repeats, while knockdown of the MutSα subunit MSH6 had no effect. Chromatin immunoprecipitation experiments indicated that MutSß, but not MutSα, was enriched at the TNR. These findings imply a direct role for MutSß in promoting expansion of threshold-length CTGâ¢CAG tracts. We identified the class II deacetylase HDAC5 as a novel promoting factor for expansions, joining the class I deacetylase HDAC3 that was previously identified. Double knockdowns were consistent with the possibility that MutSß, HDAC3 and HDAC5 act through a common pathway to promote expansions of threshold-length TNRs.
Asunto(s)
Proteínas de Unión al ADN/fisiología , Histona Desacetilasas/fisiología , Proteína 2 Homóloga a MutS/fisiología , Expansión de Repetición de Trinucleótido , Línea Celular , Proteínas de Unión al ADN/antagonistas & inhibidores , Proteínas de Unión al ADN/genética , Técnicas de Silenciamiento del Gen , Histona Desacetilasas/genética , Humanos , Proteína 2 Homóloga a MutS/antagonistas & inhibidores , Proteína 2 Homóloga a MutS/genética , Proteína 3 Homóloga de MutSRESUMEN
Cigarette smoking among reproductive-aged women is increasing worldwide. Cigarette smoking is a lifestyle behavior associated with important adverse health effects including subfertility and premature ovarian failure. We previously demonstrated that cigarette smoke (CS) exposure in mice decreases the primordial follicle pool; however, the mechanism of action is largely unknown. Therefore, the present study was designed to elucidate the mechanisms underlying CS exposure-induced ovarian follicle loss. CS exposure induced a significant decrease in the relative ovarian weight and the number of primordial and growing follicles. Oxidative stress, as shown by increased Hsp25 and decreased superoxide dismutase 2 protein expression, was found in mice exposed to CS for 8 weeks. Exposure decreased Bcl-2 but failed to induce apoptosis. An increased number of autophagosomes in granulosa cells of ovarian follicles together with increased expression of Beclin-1 and microtubule-associated protein light chain 3, key regulatory proteins in the autophagy (Atg) pathway, was found in CS-exposed mice compared with the control group. Taken together, our results suggest that CS exposure does not induce apoptosis but rather activates the Atg pathway ultimately leading to ovarian follicle loss. We further postulate that Atg is a novel mechanism of toxicant-induced ovarian follicle loss.