RESUMEN
To justify investigations on learning and memory (L&M) function in extended one-generation reproductive toxicity studies (EOGRTS; Organization for Economic Co-operation and Development (OECD) test guideline (TG) 443) for registration under Registration, Evaluation, Authorization, and Restriction of Chemical (REACH), the European Chemicals Agency has referred to three publications based on which the Agency concluded that "perturbation of thyroid hormone signaling in offspring affects spatial cognitive abilities (learning and memory)" and "Therefore, it is necessary to conduct spatial learning and memory tests for F1 animals". In this paper, the inclusion of the requested L&M tests in an EOGRTS is challenged. In addition, next to the question on the validity of rodent models in general for testing thyroid hormone-dependent perturbations in brain development, the reliability of the publications specifically relied upon by the agency is questioned as these contain numerous fundamental errors in study methodology, design, and data reporting, provide contradicting results, lack crucial information to validate the results and exclude confounding factors, and finally show no causal relationship. Therefore, in our opinion, these publications cannot be used to substantiate, support, or conclude that decreases in blood thyroid (T4) hormone level on their own would result in impaired L&M in rats and are thus not adequate to use as fundament to ask for L&M testing as part of an EOGRTS.
Asunto(s)
Reproducción , Pruebas de Toxicidad , Ratas , Animales , Pruebas de Toxicidad/métodos , Reproducibilidad de los Resultados , CogniciónRESUMEN
Omics methodologies are widely used in toxicological research to understand modes and mechanisms of toxicity. Increasingly, these methodologies are being applied to questions of regulatory interest such as molecular point-of-departure derivation and chemical grouping/read-across. Despite its value, widespread regulatory acceptance of omics data has not yet occurred. Barriers to the routine application of omics data in regulatory decision making have been: 1) lack of transparency for data processing methods used to convert raw data into an interpretable list of observations; and 2) lack of standardization in reporting to ensure that omics data, associated metadata and the methodologies used to generate results are available for review by stakeholders, including regulators. Thus, in 2017, the Organisation for Economic Co-operation and Development (OECD) Extended Advisory Group on Molecular Screening and Toxicogenomics (EAGMST) launched a project to develop guidance for the reporting of omics data aimed at fostering further regulatory use. Here, we report on the ongoing development of the first formal reporting framework describing the processing and analysis of both transcriptomic and metabolomic data for regulatory toxicology. We introduce the modular structure, content, harmonization and strategy for trialling this reporting framework prior to its publication by the OECD.
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Metabolómica/normas , Organización para la Cooperación y el Desarrollo Económico/normas , Toxicogenética/normas , Toxicología/normas , Transcriptoma/fisiología , Documentación/normas , HumanosRESUMEN
The local lymph node assay (LLNA) is a regulatory accepted test for the identification of skin sensitizing substances by measuring radioactive thymidine incorporation into the lymph node. However, there is evidence that LLNA is overestimating the sensitization potential of certain substance classes in particular those exerting skin irritation. Some reports describe the additional use of flow cytometry-based immunophenotyping to better discriminate irritants from sensitizing irritants in LLNA. In the present study, the 22 performance standards plus 8 surfactants were assessed using the radioactive LLNA method. In addition, lymph node cells were immunophenotyped to evaluate the specificity of the lymph node response using cell surface markers such as B220 or CD19, CD3, CD4, CD8, I-A(κ) and CD69 with the aim to allow a better discrimination above all between irritants and sensitizers, but also non-irritating sensitizers and non-sensitizers. However, the markers assessed in this study do not sufficiently differentiate between irritants and irritant sensitizers and therefore did not improve the predictive capacity of the LLNA.
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Haptenos/toxicidad , Irritantes/toxicidad , Ganglios Linfáticos/efectos de los fármacos , Linfocitos/efectos de los fármacos , Modelos Biológicos , Animales , Antígenos de Superficie/metabolismo , Dermatitis por Contacto/inmunología , Dermatitis por Contacto/metabolismo , Femenino , Inmunofenotipificación , Ensayo del Nódulo Linfático Local , Ganglios Linfáticos/inmunología , Ganglios Linfáticos/metabolismo , Linfocitos/inmunología , Linfocitos/metabolismo , Ratones Endogámicos CBA , Tensoactivos/químicaRESUMEN
Dicyclopentadiene (DCPD) was investigated in a 14-day oral rat toxicity study based on the OECD 407 guideline in combination with plasma metabolomics. Wistar rats received the compound daily via gavage at dose levels of 0, 50 and 150 mg/kg bw. The high dose induced transient clinical signs of toxicity and in males only reduced body weight gain. High dose liver changes were characterized by altered clinical chemistry parameters in both sexes and pathological changes in females. In high dose males an accumulation of alpha-2 u-globulin in the kidney was noted. Comparing the DCPD metabolome with previously established specific metabolome patterns in the MetaMap® Tox data base suggested that the high dose would result in liver enzyme induction leading to increased breakdown of thyroid hormones for males and females. An indication for liver toxicity in males was also noted. Metabolomics also suggested an effect on the functionality of the adrenals in high dose males, which together with published data, is suggestive of a stress related effect in this organ. The results of the present 14-day combined toxicity and metabolome investigations were qualitatively in line with literature data from subchronic oral studies in rats with DCPD. Importantly no other types of organ toxicity, or hormone dysregulation beyond the ones associated with liver enzyme induction and stress were indicated, again in line with results of published 90-day studies. It is therefore suggested that short term "smart" studies, combining classical toxicity with 'omics technologies, could be a 2 R (refine and reduce) new approach method allowing for the reduction of in vivo toxicity testing.
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Indenos , Metaboloma , Masculino , Femenino , Ratas , Animales , Ratas Wistar , Pruebas de ToxicidadRESUMEN
A 14-day rat study with plasma metabolomics was conducted to evaluate the toxicity of Benzene. Wistar rats were orally administered Benzene daily at doses of 0, 300 and 1000â¯mg/kg bw. The study identified liver and kidneys as target organs of Benzene toxicity and found reductions in total white blood cells, absolute lymphocyte and eosinophil cell counts, and increased relative monocyte counts suggesting bone marrow as a target organ. The study also confirmed liver as a target organ using metabolomics, which showed indications of a stress reaction in rats and changes in metabolites suggestive of a metabolic disorder. The metabolomics investigations did not find any other toxicologically relevant modes of action, and the observed metabolite changes were not associated with markers for mitochondrial dysfunction. The study concludes that integration of omics technologies, such as metabolomics, in regulatory toxicity studies is possible, confirms existing knowledge and adds additional information that can be used for mechanistic understanding of observed toxicity.
RESUMEN
An integral part of hazard and safety assessments is the estimation of a chemical's potential to cause skin sensitization. Currently, only animal tests (OECD 406 and 429) are accepted in a regulatory context. Nonanimal test methods are being developed and formally validated. In order to gain more insight into the responses induced by eight exemplary surfactants, a battery of in vivo and in vitro tests were conducted using the same batch of chemicals. In general, the surfactants were negative in the GPMT, KeratinoSens and hCLAT assays and none formed covalent adducts with test peptides. In contrast, all but one was positive in the LLNA. Most were rated as being irritants by the EpiSkin assay with the additional endpoint, IL1-alpha. The weight of evidence based on this comprehensive testing indicates that, with one exception, they are non-sensitizing skin irritants, confirming that the LLNA tends to overestimate the sensitization potential of surfactants. As results obtained from LLNAs are considered as the gold standard for the development of new nonanimal alternative test methods, results such as these highlight the necessity to carefully evaluate the applicability domains of test methods in order to develop reliable nonanimal alternative testing strategies for sensitization testing.
Asunto(s)
Irritantes/farmacología , Ensayo del Nódulo Linfático Local , Piel/efectos de los fármacos , Tensoactivos/farmacología , Animales , Línea Celular , Proliferación Celular/efectos de los fármacos , Glucósidos/metabolismo , Cobayas , Humanos , Interleucina-1alfa/inmunología , Interleucina-1alfa/metabolismo , Irritantes/toxicidad , Ratones , Ratones Endogámicos CBA , Péptidos/química , Relación Estructura-Actividad Cuantitativa , Medición de Riesgo/métodos , Pruebas de Irritación de la Piel/métodos , Estadística como Asunto/métodos , Tensoactivos/toxicidadRESUMEN
The integral membrane lipid phosphatase Sac1p regulates local pools of phosphatidylinositol-4-phosphate (PtdIns(4)P) at endoplasmic reticulum (ER) and Golgi membranes. PtdIns(4)P is important for Golgi trafficking, yet the significance of PtdIns(4)P for ER function is unknown. It also remains unknown how localization of Sac1p to distinct organellar membranes is mediated. Here, we show that a COOH-terminal region in yeast Sac1p is crucial for ER targeting by directly interacting with dolicholphosphate mannose synthase Dpm1p. The interaction with Dpm1p persists during exponential cell division but is rapidly abolished when cell growth slows because of nutrient limitation, causing translocation of Sac1p to Golgi membranes. Cell growth-dependent shuttling of Sac1p between the ER and the Golgi is important for reciprocal control of PtdIns(4)P levels at these organelles. The fraction of Sac1p resident at the ER is also required for efficient dolichol oligosaccharide biosynthesis. Thus, the lipid phosphatase Sac1p may be a key regulator, coordinating the secretory capacity of ER and Golgi membranes in response to growth conditions.
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Retículo Endoplásmico/metabolismo , Aparato de Golgi/metabolismo , Manosiltransferasas/fisiología , Proteínas de la Membrana/fisiología , Transporte de Proteínas/fisiología , Proteínas de Saccharomyces cerevisiae/fisiología , Transducción de Señal/fisiología , Western Blotting , Catepsina A/metabolismo , División Celular , Centrifugación por Gradiente de Densidad , Glucosa/deficiencia , Glicosilación , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Factores de Intercambio de Guanina Nucleótido/análisis , Proteínas de Choque Térmico/análisis , Membranas Intracelulares/química , Manosa/metabolismo , Manosiltransferasas/análisis , Manosiltransferasas/genética , Manosiltransferasas/metabolismo , Proteínas de la Membrana/análisis , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Proteínas de Transporte de Membrana/análisis , Proteínas de Transporte de Membrana/genética , Microscopía Fluorescente , Microsomas/química , Mutación , Oligosacáridos/biosíntesis , Fosfatos de Fosfatidilinositol/metabolismo , Monoéster Fosfórico Hidrolasas , Canales de Translocación SEC , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crecimiento & desarrollo , Saccharomyces cerevisiae/fisiología , Proteínas de Saccharomyces cerevisiae/análisis , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Eliminación de Secuencia , Transformación Genética , Proteínas de Transporte Vesicular/análisisRESUMEN
Understanding the Mode of Action (MOA) for a chemical can help guide decisions in development of Occupational Exposure Limits (OELs). Where sufficient information exists, it can provide the OEL developer the basis for selecting either a health-based or risk-based approach. To support the development of an OEL for benzene, scientific information relevant to MOA assessment for risk-based and health-based OEL approaches was reviewed. Direct-acting mutagenicity was considered as a basis for a risk-based OEL, versus MOAs consistent with a health-based approach: indirect mutagenicity via topoisomerase II inhibition, indirect mutagenicity via reactive oxygen species generation, or an immune-based bone marrow dysfunction. Based on the evidence against direct DNA reactivity, threshold expectations for remaining MOAs, and evidence for dose rate affecting acute myeloid leukemia and myelodysplastic syndrome risk, the weight of evidence favors a health-based OEL approach. In the case of benzene, development of an OEL based on observations of earlier key events (i.e., hematologic changes and genetic toxicity) is anticipated to provide protection from later adverse outcomes such as leukemia.
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Benceno/toxicidad , Mutágenos/toxicidad , Exposición Profesional/efectos adversos , Exposición Profesional/análisis , Valores Limites del Umbral , Humanos , Medición de RiesgoRESUMEN
This paper derives an occupational exposure limit for benzene using quality assessed data. Seventy-seven genotoxicity and 36 haematotoxicity studies in workers were scored for study quality with an adapted tool based on that of Vlaanderen et al., 2008 (Environ Health. Perspect. 116 1700-5). These endpoints were selected as they are the most sensitive and relevant to the proposed mode of action (MOA) and protecting against these will protect against benzene carcinogenicity. Lowest and No- Adverse Effect Concentrations (LOAECs and NOAECs) were derived from the highest quality studies (i.e. those ranked in the top tertile or top half) and further assessed as being "more certain" or "less certain". Several sensitivity analyses were conducted to assess whether alternative "high quality" constructs affected conclusions. The lowest haematotoxicity LOAECs showed effects near 2â¯ppm (8â¯h TWA), and no effects at 0.59â¯ppm. For genotoxicity, studies also showed effects near 2â¯ppm and showed no effects at about 0.69â¯ppm. Several sensitivity analyses supported these observations. These data define a benzene LOAEC of 2â¯ppm (8â¯h TWA) and a NOAEC of 0.5â¯ppm (8â¯h TWA). Allowing for possible subclinical effects in bone marrow not apparent in studies of peripheral blood endpoints, an OEL of 0.25â¯ppm (8â¯h TWA) is proposed.
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Contaminantes Ocupacionales del Aire/toxicidad , Benceno/toxicidad , Mutágenos/toxicidad , Exposición Profesional/análisis , Estudios Epidemiológicos , Humanos , Concentración Máxima Admisible , Nivel sin Efectos Adversos Observados , Exposición Profesional/efectos adversos , Medición de Riesgo , Valores Limites del UmbralRESUMEN
Compartment-specific control of phosphoinositide lipids is essential for cell function. The Sac1 lipid phosphatase regulates endoplasmic reticulum (ER) and Golgi phosphatidylinositol-4-phosphate [PI(4)P] in response to nutrient levels and cell growth stages. During exponential growth, Sac1p interacts with Dpm1p at the ER but shuttles to the Golgi during starvation. Here, we report that a C-terminal region in Sac1p is required for retention in the perinuclear ER, whereas the N-terminal domain is responsible for Golgi localization. We also show that starvation-induced shuttling of Sac1p to the Golgi depends on the coat protein complex II and the Rer1 adaptor protein. Starvation-induced shuttling of Sac1p to the Golgi specifically eliminates a pool of PI(4)P generated by the lipid kinase Pik1p. In addition, absence of nutrients leads to a rapid dissociation of Pik1p, together with its non-catalytical subunit Frq1p, from Golgi membranes. Reciprocal rounds of association/dissociation of the Sac1p lipid phosphatase and the Pik1p/Frq1p lipid kinase complex are responsible for growth-dependent control of Golgi phosphoinositides. Sac1p and Pik1p/Frq1p are therefore elements of a unique machinery that synchronizes ER and Golgi function in response to different growth conditions.
Asunto(s)
1-Fosfatidilinositol 4-Quinasa/fisiología , Regulación Fúngica de la Expresión Génica , Aparato de Golgi/metabolismo , Monoéster Fosfórico Hidrolasas/fisiología , Proteínas de Saccharomyces cerevisiae/fisiología , Secuencias de Aminoácidos , Transporte Biológico , Reactivos de Enlaces Cruzados/química , Retículo Endoplásmico/metabolismo , Genotipo , Proteínas Fluorescentes Verdes/química , Proteínas Fluorescentes Verdes/metabolismo , Microscopía Fluorescente/métodos , Modelos Biológicos , Mutagénesis , Fosfatidilinositoles/metabolismo , Monoéster Fosfórico Hidrolasas/química , Monoéster Fosfórico Hidrolasas/metabolismo , Estructura Terciaria de Proteína , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
The yeast phosphoinositide phosphatase Sac1p localizes to endoplasmic reticulum (ER) and Golgi membranes and has compartment-specific functions in these organelles. In this study we analyzed in detail the topology of Sac1p. Our data show that Sac1p is a type II transmembrane protein with a large N-terminal cytosolic domain, which is anchored in the membrane by the two potential transmembrane helices near the C terminus. Based on this topology, we created a mutation that caused retention of Sac1p in the ER and as a consequence showed specific alterations in cellular phosphoinositide levels. Our results suggest that Sac1p controls a pool of phosphatidylinositol 3-phosphate and phosphatidylinositol 4-phosphate in the ER. Retention of Sac1p in the ER also stimulates ATP transport into the ER lumen but causes the same Golgi-specific defects that are seen in a sac1 null mutant. Taken together this study provides evidence that Sac1p is an important 4-phosphatase in the ER controlling different aspects of ER-based protein processing and secretion.