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1.
Toxicol Sci ; 198(1): 31-39, 2024 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-38175793

RESUMO

In order to evaluate the role of the placenta in the etiology of ethylene glycol (EG) developmental toxicity, the distribution of EG and its main metabolites, glycolic acid (GA) and oxalic acid (OX), into the conceptus was determined at the beginning and completion of placentation in the rat and rabbit. Two groups (n = 28) of timed-pregnant Wistar rats were administered EG (1000 mg/kg bw/day, oral gavage) from gestation day (GD) 6 to either GD 11 or GD 16; similarly, two groups (n = 28) of timed-pregnant New Zealand White rabbits were administered EG from GD 6 to either GD 10 or GD 19. Four animals from each group were sacrificed at 1, 3, 6, 9, 12, 18, or 24 h after the final administration, and maternal blood, extraembryonic fluid, and embryonic tissue were removed for analysis of EG, GA, and OX. The three analytes were predominantly cleared from all compartments in both species within 24 h. Neither EG nor OX preferentially accumulated into the conceptus compartments, compared with the maternal blood, in either species. Critically, GA was preferentially accumulated from the maternal blood only into the rat embryo at GD 11, but not at GD 16 and not into the rabbit embryo at either GD 10 or GD 19. The accumulation of GA into the rat embryo, and its decline over the course of placentation, is discussed in relation to the expression of monocarboxylate transporter isoforms across the syncytiotrophoblast.


Assuntos
Etilenoglicol , Glicolatos , Placentação , Gravidez , Feminino , Ratos , Coelhos , Animais , Etilenoglicol/toxicidade , Ratos Wistar , Administração Oral
2.
J Med Chem ; 61(24): 11074-11100, 2018 12 27.
Artigo em Inglês | MEDLINE | ID: mdl-30384606

RESUMO

A HTS campaign identified compound 1, an excellent hit-like molecule to initiate medicinal chemistry efforts to optimize a dual ROCK1 and ROCK2 inhibitor. Substitution (2-Cl, 2-NH2, 2-F, 3-F) of the pyridine hinge binding motif or replacement with pyrimidine afforded compounds with a clean CYP inhibition profile. Cocrystal structures of an early lead compound were obtained in PKA, ROCK1, and ROCK2. This provided critical structural information for medicinal chemistry to drive compound design. The structural data indicated the preferred configuration at the central benzylic carbon would be ( R), and application of this information to compound design resulted in compound 16. This compound was shown to be a potent and selective dual ROCK inhibitor in both enzyme and cell assays and efficacious in the retinal nerve fiber layer model after oral dosing. This tool compound has been made available through the AbbVie Compound Toolbox. Finally, the cocrystal structures also identified that aspartic acid residues 176 and 218 in ROCK2, which are glutamic acids in PKA, could be targeted as residues to drive both potency and kinome selectivity. Introduction of a piperidin-3-ylmethanamine group to the compound series resulted in compound 58, a potent and selective dual ROCK inhibitor with excellent predicted drug-like properties.


Assuntos
Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Quinases Associadas a rho/antagonistas & inibidores , Administração Oral , Animais , Disponibilidade Biológica , Cristalografia por Raios X , Inibidores do Citocromo P-450 CYP2C9/química , Inibidores do Citocromo P-450 CYP2C9/farmacologia , Inibidores do Citocromo P-450 CYP3A/química , Inibidores do Citocromo P-450 CYP3A/farmacologia , Desenho de Fármacos , Avaliação Pré-Clínica de Medicamentos/métodos , Meia-Vida , Humanos , Camundongos Endogâmicos C57BL , Traumatismos do Nervo Óptico/tratamento farmacológico , Traumatismos do Nervo Óptico/patologia , Ratos Sprague-Dawley , Relação Estrutura-Atividade , Quinases Associadas a rho/química
3.
Reprod Toxicol ; 78: 102-110, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29635045

RESUMO

Ethanolamine (EA) reduced implantation success in a two-generation reproduction toxicity study; the aim of this work was to explore the underlying basis for this response. When administered to pregnant rats during gestation days (GD) 1-3, 4-5, or 6-7, EA had no effect upon implantation success. In a second experiment, EA was administered either in the diet or by oral gavage from two weeks prior to mating through to GD 8. Parallel groups also received a diet supplemented with choline. In the absence of supplementary choline, EA induced early resorptions, statistically significant only when administered in the diet. A slight reduction in implantation success was ameliorated by supplementary choline. We conclude that implantation is affected by EA only when exposure starts before mating; that dietary administration is more effective than gavage dosing; and that interference with choline homeostasis may play a role in the aetiology of this lesion.


Assuntos
Colina/farmacologia , Suplementos Nutricionais , Implantação do Embrião/efeitos dos fármacos , Etanolamina/toxicidade , Animais , Feminino , Masculino , Gravidez , Ratos Wistar
4.
Toxicol Sci ; 161(2): 421-430, 2018 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-29069465

RESUMO

Ethylene glycol (EG) is a developmental toxicant in pregnant rats and mice. A suggested mechanism for this toxicity is that the EG metabolite, glycolic acid (GA), causes acidosis which may affect the embryonic heart rate (HR). This inhibition would cause periods of embryonic bradycardia and arrhythmia resulting in increased embryonic death and malformation in surviving embryos. This hypothesis was investigated using gestational day (GD) 11 and 13 rat embryos in vitro. Increasing concentrations of GA or lactic acid in the incubation medium caused a decrease in external pH (pHe) and a concentration-dependent decrease in embryonic HR. Increased concentrations of GA or lactic acid with pHe corrected to normal levels did not affect HR. Severely decreased pHe, caused by reduced NaHCO3 in the incubation medium, had little effect on the HR of GD 13 embryos but substantially reduced the HR of GD 11 embryos. These results suggest that increased monocarboxylate concentration (glycolate or lactate) needs to be in combination with increased H+ concentration (low pHe) to influence the embryonic HR. These results implicate the monocarboxylate transporter reported to be present in the early postnatal rat heart, the chick embryonic heart throughout development, and the chorioallantoic placenta. The results showed some evidence that the adverse effect of GA and reduced pHe on the embryonic HR increased with duration of exposure and hence lends support to the suggested mechanism of embryotoxicity for EG.


Assuntos
Acidose/induzido quimicamente , Desenvolvimento Embrionário/efeitos dos fármacos , Etilenoglicol/toxicidade , Glicolatos/toxicidade , Frequência Cardíaca/efeitos dos fármacos , Teratogênese/efeitos dos fármacos , Acidose/embriologia , Acidose/fisiopatologia , Animais , Meios de Cultura/química , Idade Gestacional , Concentração de Íons de Hidrogênio , Técnicas In Vitro , Ratos , Ratos Sprague-Dawley
5.
Environ Mol Mutagen ; 58(3): 122-134, 2017 04.
Artigo em Inglês | MEDLINE | ID: mdl-28326610

RESUMO

Ethylene oxide (EO) is a direct acting alkylating agent; in vitro and in vivo studies indicate that it is both a mutagen and a carcinogen. However, it remains unclear whether the mode of action (MOA) for cancer for EO is a mutagenic MOA, specifically via point mutation. To investigate the MOA for EO-induced mouse lung tumors, male Big Blue (BB) B6C3F1 mice (10/group) were exposed to EO by inhalation, 6 hr/day, 5 days/week for 4 (0, 10, 50, 100, or 200 ppm EO), 8, or 12 weeks (0, 100, or 200 ppm EO). Lung DNA samples were analyzed for cII mutant frequency (MF) at 4, 8 and 12 weeks of exposure; the mutation spectrum was analyzed for mutants from control and 200 ppm EO treatments. Although EO-induced cII MFs were 1.5- to 2.7-fold higher than the concurrent controls at 4 weeks, statistically significant increases in the cII MF were found only after 8 and 12 weeks of exposure and only at 200 ppm EO (P ≤ 0.05), which is twice the highest concentration used in the cancer bioassay. Consistent with the positive response, DNA sequencing of cII mutants showed a significant shift in the mutational spectra between control and 200 ppm EO following 8 and 12 week exposures (P ≤ 0.035), but not at 4 weeks. Thus, EO mutagenic activity in vivo was relatively weak and required higher than tumorigenic concentrations and longer than 4 weeks exposure durations. These data do not follow the classical patterns for a MOA mediated by point mutations. Environ. Mol. Mutagen. 58:122-134, 2017. © 2017 Wiley Periodicals, Inc.


Assuntos
Carcinógenos/toxicidade , Óxido de Etileno/toxicidade , Exposição por Inalação/efeitos adversos , Pulmão/efeitos dos fármacos , Mutagênicos/toxicidade , Mutação Puntual , Animais , Relação Dose-Resposta a Droga , Pulmão/patologia , Neoplasias Pulmonares/induzido quimicamente , Neoplasias Pulmonares/genética , Masculino , Camundongos Endogâmicos , Fatores de Tempo
7.
Int J Cell Biol ; 2016: 2084252, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27843454

RESUMO

The distribution of monocarboxylate transporter (MCT) isoforms 1 and 4, which mediate the plasmalemmal transport of l-lactic and pyruvic acids, has been identified in the placentae of rats and rabbits at different ages of gestation. Groups of three pregnant Sprague-Dawley rats and New Zealand White rabbits were sacrificed on gestation days (GD) 11, 14, 18, or 20 and on GD 13, 18, or 28, respectively. Placentae were removed and processed for immunohistochemical detection of MCT1 and MCT4. In the rat, staining for MCT1 was associated with lakes and blood vessels containing enucleated red blood cells (maternal vessels) while staining for MCT4 was associated with vessels containing nucleated red blood cells (embryofoetal vessels). In the rabbit, staining for MCT1 was associated with blood vessels containing nucleated red blood cells while staining for MCT4 was associated with vessels containing enucleated red blood cells. Strength of staining for MCT1 decreased during gestation in both species, but that for MCT4 was stronger than that for MCT1 and was consistent between gestation days. The results imply an opposite polarity of MCT1 and MCT4 across the trophoblast between rat and rabbit.

9.
Regul Toxicol Pharmacol ; 80: 32-40, 2016 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-27245879

RESUMO

The extended one-generation reproduction toxicity study (EOGRTS; OECD test guideline 433) is a new and technically complex design to evaluate the putative effects of chemicals on fertility and development, including effects upon the developing nervous and immune systems. In addition to offering a more comprehensive assessment of developmental toxicity, the EOGRTS offers important improvements in animal welfare through reduction and refinement in a modular study design. The challenge to the practitioner is to know how the modular aspects of the study should be triggered on the basis of prior knowledge of a particular chemical, or on earlier findings in the EOGRTS itself, requirements of specific regulatory frameworks notwithstanding. The purpose of this document is to offer guidance on science-based triggers for these extended evaluations.


Assuntos
Fertilidade/efeitos dos fármacos , Organização para a Cooperação e Desenvolvimento Econômico , Reprodução/efeitos dos fármacos , Testes de Toxicidade/métodos , Animais , Feminino , Humanos , Sistema Imunitário/efeitos dos fármacos , Sistema Imunitário/crescimento & desenvolvimento , Masculino , Modelos Animais , Sistema Nervoso/efeitos dos fármacos , Sistema Nervoso/crescimento & desenvolvimento , Organização para a Cooperação e Desenvolvimento Econômico/normas , Ratos , Medição de Risco , Testes de Toxicidade/normas
10.
J Med Chem ; 58(23): 9154-70, 2015 Dec 10.
Artigo em Inglês | MEDLINE | ID: mdl-26509640

RESUMO

S1P5 is one of 5 receptors for sphingosine-1-phosphate and is highly expressed on endothelial cells within the blood-brain barrier, where it maintains barrier integrity in in vitro models (J. Neuroinflamm. 2012, 9, 133). Little more is known about the effects of S1P5 modulation due to the absence of tool molecules with suitable selectivity and drug-like properties. We recently reported that molecule A-971432 (Harris, 2010) (29 in this paper) is highly efficacious in reversing lipid accumulation and age-related cognitive decline in rats (Van der Kam , , AAIC 2014). Herein we describe the development of a series of selective S1P5 agonists that led to the identification of compound 29, which is highly selective for S1P5 and has excellent plasma and CNS exposure after oral dosing in preclinical species. To further support its suitability for in vivo studies of S1P5 biology, we extensively characterized 29, including confirmation of its selectivity in pharmacodynamic assays of S1P1 and S1P3 function in rats. In addition, we found that 29 improves blood-brain barrier integrity in an in vitro model and reverses age-related cognitive decline in mice. These results suggest that S1P5 agonism is an innovative approach with potential benefit in neurodegenerative disorders involving lipid imbalance and/or compromised blood-brain barrier such as Alzheimer's disease or multiple sclerosis.


Assuntos
Ácido Azetidinocarboxílico/análogos & derivados , Derivados de Benzeno/química , Derivados de Benzeno/farmacologia , Doenças Neurodegenerativas/tratamento farmacológico , Receptores de Lisoesfingolipídeo/agonistas , Administração Oral , Animais , Ácido Azetidinocarboxílico/administração & dosagem , Ácido Azetidinocarboxílico/química , Ácido Azetidinocarboxílico/farmacocinética , Ácido Azetidinocarboxílico/farmacologia , Derivados de Benzeno/administração & dosagem , Derivados de Benzeno/farmacocinética , Barreira Hematoencefálica/efeitos dos fármacos , Barreira Hematoencefálica/metabolismo , Barreira Hematoencefálica/patologia , Linhagem Celular , Envelhecimento Cognitivo , Cães , Feminino , Humanos , Macaca fascicularis , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Doenças Neurodegenerativas/metabolismo , Doenças Neurodegenerativas/patologia , Ratos , Ratos Sprague-Dawley , Receptores de Lisoesfingolipídeo/metabolismo
11.
Chem Biol Interact ; 241: 59-65, 2015 Nov 05.
Artigo em Inglês | MEDLINE | ID: mdl-25861726

RESUMO

REACH (Registration, Evaluation, Authorisation and Restriction of Chemicals) is the European Union's chemical regulation for the management of risk to human health and the environment (European Chemicals Agency, 2006). This regulation entered into force in June 2007 and required manufacturers and importers to register substances produced in annual quantities of 1000 tonnes or more by December 2010, with further deadlines for lower tonnages in 2013 and 2018. Depending on the type of registration, required information included the substance's identification, the hazards of the substance, the potential exposure arising from the manufacture or import, the identified uses of the substance, and the operational conditions and risk management measures applied or recommended to downstream users. Among the content developed to support this information were Derived No-Effect Levels or Derived Minimal Effect Levels (DNELs/DMELs) for human health hazard assessment, Predicted No Effect Concentrations (PNECs) for environmental hazard assessment, and exposure scenarios for exposure and risk assessment. Once registered, substances may undergo evaluation by the European Chemicals Agency (ECHA) or Member State authorities and be subject to requests for additional information or testing as well as additional risk reduction measures. To manage the REACH registration and related activities for the European olefins and aromatics industry, the Lower Olefins and Aromatics REACH Consortium was formed in 2008 with administrative and technical support provided by Penman Consulting. A total of 135 substances are managed by this group including 26 individual chemical registrations (e.g. benzene, 1,3-butadiene) and 13 categories consisting of 5-26 substances. This presentation will describe the content of selected registrations prepared for 2010 in addition to the significant post-2010 activities. Beyond REACH, content of the registrations may also be relevant to other European activities, for example consideration of worker DNELs/DMELs for occupational exposure level setting, discussion of this aspect will be presented for 1,3-butadiene.


Assuntos
Alcenos/toxicidade , Substâncias Perigosas/toxicidade , Exposição Ocupacional/legislação & jurisprudência , Benzeno/toxicidade , Butadienos/toxicidade , Meio Ambiente , União Europeia , Humanos , Medição de Risco/métodos
12.
Biomed Chromatogr ; 29(9): 1364-74, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-25641649

RESUMO

Glutathione (GSH), glutathione disulfide (GSSG) and 2-hydroxyethylated glutathione (HESG) are important biomarkers for exploring the genotoxicity mechanism of ethylene oxide (EO) or ethylene in vivo. A liquid chromatography-tandem mass spectrometry method was developed for simultaneous determination of GSH, GSSG and HESG in mouse lung tissues after inhalation exposure to EO. The lower limit of quantitation for all these biomarkers was 0.002 µg/mL. The linearity of the calibration curves for all analytes was >0.998. The intra-day assay precision relative standard deviation (RSD) values for quality control samples for all analytes were ≤12.8% with accuracy values ranging from 87.2 to 113%. The inter-day assay precision (RSD) values for all analytes were ≤13.1% with accuracy values ranging from 86.9 to 103%. This method was applied to concurrently determine the levels of GSH, GSSG and HESG in lung samples isolated from mouse after 4-week inhalation exposure to EO at 0, 10, 50, 100 and 200 ppm.


Assuntos
Cromatografia Líquida/métodos , Óxido de Etileno/toxicidade , Dissulfeto de Glutationa/análise , Glutationa/análise , Pulmão/química , Espectrometria de Massas em Tandem/métodos , Animais , Glutationa/análogos & derivados , Pulmão/efeitos dos fármacos , Masculino , Camundongos , Sensibilidade e Especificidade , Espectrometria de Massas por Ionização por Electrospray/métodos
13.
Artigo em Inglês | MEDLINE | ID: mdl-25652268

RESUMO

Histiotrophic nutrition via the visceral yolk sac is an essential nutritional pathway of the rodent conceptus, and inhibition of this pathway may cause growth retardation, malformations, and death in rodent embryos. Morphologic differences among species during early development indicate that the visceral yolk sac histiotrophic nutrition pathway may be of lesser importance in nonrodent species, including humans. Here, comparative studies were conducted with inhibitors of different steps in the visceral yolk sac histiotrophic nutrition pathway to determine whether the rabbit is similarly responsive to the rat. Early somite stage New Zealand White rabbit and Crl:CD(SD) rat conceptuses (gestation day 9, rabbits; gestation day 10, rats) were exposed for 48 hr to three different histiotrophic nutrition pathway inhibitors using whole embryo culture techniques, after which they were evaluated for growth and malformations. Cubilin antibody, an inhibitor of endocytosis, reduced growth and development and increased malformations in both rat and rabbit embryos, although the rabbit appeared more sensitive. Leupeptin, a lysosomal cysteine protease inhibitor, also impaired growth and development and increased malformations in rat embryos, while in the rabbit it induced malformations and a slight decrease in morphology score but had no effect upon growth. Trypan blue, an inhibitor of endocytosis and endosome maturation, affected all measures in both species to a similar degree at the highest concentration (2500 µg/ml), but rat embryos responded to a greater extent at lower concentrations. Although the specific adverse outcomes appear to be different, these results demonstrate that rabbits, like rats, are sensitive to inhibitors of the histiotrophic nutrition pathway.


Assuntos
Fenômenos Fisiológicos da Nutrição Animal/efeitos dos fármacos , Feto/efeitos dos fármacos , Feto/fisiologia , Animais , Anticorpos/farmacologia , Embrião de Mamíferos/efeitos dos fármacos , Desenvolvimento Embrionário/efeitos dos fármacos , Feminino , Leupeptinas/farmacologia , Coelhos , Ratos Sprague-Dawley , Receptores de Superfície Celular/imunologia , Coloração e Rotulagem , Azul Tripano/farmacologia
14.
Artigo em Inglês | MEDLINE | ID: mdl-25529473

RESUMO

2-Hydroxyethylated and oxidative DNA nucleosides (DNA adduct biomarkers), such as O6-(2-hydroxyethyl)-2'-deoxyguanosine (O6HEdG), N6-(2-hydroxyethyl)-2'-deoxyadenosine (N6HEdA), 1-(2-hydroxyethyl)-2'-deoxyadenosine (N1HEdA), and 8-hydroxy-2'-deoxyguanosine (8-OHdG), N2,3-etheno-2'-deoxyguanosine (N2,3-ethenodG), α-methyl-γ-hydroxy-1,N2-propano-2'-deoxyguanosine (CrotondG), are important proposed biomarkers for exploring the genotoxicity mechanism of ethylene oxide (EO) in vivo. A liquid chromatography-tandem mass spectrometric method was developed for the simultaneous determination of O6HEdG, N6HEdA, N1HEdA, 8-OHdG, CrotondG, and N2,3-ethenodG together with regular 2'-deoxyguanosine (dG), and 2'-deoxyadenosine (dA) nucleosides in the DNA extracted from mouse lung tissues for the assessment of exposure to EO after inhalation. The lower limits of quantitation for 8-OHdG, CrotondG, N2,3-EthenodG, O6HEdG, N1HEdA, N6HEdA, dG, and dA were 0.025, 0.00125, 0.025, 0.00125, 0.025, 0.01, 2342, and 2500ng/mL, respectively. The linearity of the calibration curves for all analytes were >0.989. The intra-day assay precision relative standard deviation (RSD) values for quality control (QC) samples for all analytes were ≤13.5% with accuracy values ranging from 86.5% to 111%. The inter-day assay precision (RSD) values for all analytes were ≤18.8% with accuracy values ranging from 87.9% to 119%. This method was used for simultaneous determination of the levels of 8-OHdG, CrotondG, N2,3-EthenodG, O6HEdG, dG, N1HEdA, N6HEdA, and dA in DNA enzymatic hydrolysates from DNA extracted from mouse lung after 12 weeks' inhalation exposure to EO at atmospheric concentrations of 0, 100, and 200ppm. Overall, N2,3-ethenodG was not detected in any samples. 8-OHdG, CrotondG, dG, and dA were all quantifiable in all samples. O6HEdG, N1HEdA, and N6HEdA were quantifiable in most samples and the ratio of the corresponding adduct versus their corresponding DNA base (dG or dA) [×10 (e6)] was increased as the EO exposure concentration increased.


Assuntos
DNA/análise , DNA/química , Óxido de Etileno/farmacologia , Nucleosídeos/análise , Nucleosídeos/química , Animais , Cromatografia Líquida , Masculino , Camundongos , Espectrometria de Massas em Tandem
15.
Reprod Toxicol ; 46: 46-55, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24598581

RESUMO

High dose gavage administration of ethylene glycol (EG) induces teratogenicity in rodents, but not in rabbits, resulting from saturation of intermediate EG metabolism and glycolic acid (GA) accumulation. In vivo, rat embryos sequester GA 2-4-fold higher than maternal blood, a phenomenon absent in rabbits and proposed not to occur in humans. This research explored the mechanisms of GA disposition into rat and rabbit conceptuses using whole embryo culture (WEC). Rat and rabbit embryos concentrated GA from the culture medium. In vitro to in vivo discordance in the rabbit plausibly stemmed from anatomical differences between these models. GA sequestration was attenuated at 4°C in both species. Rat embryos further demonstrated pH-dependence of GA sequestration and inhibition by D-lactic acid. These data suggest GA disposition into rat and rabbit embryos is energy- and pH-dependent, and carrier-mediated. Anatomical and maternal-to-conceptal pH gradient differences likely underlie the lack of enhanced GA disposition in non-rodent species.


Assuntos
Embrião de Mamíferos/metabolismo , Glicolatos/metabolismo , Animais , Ligação Competitiva , Técnicas de Cultura Embrionária , Feminino , Concentração de Íons de Hidrogênio , Ácido Láctico/metabolismo , Gravidez , Proteínas/metabolismo , Coelhos , Ratos , Ratos Sprague-Dawley , Temperatura
16.
Crit Rev Toxicol ; 43(10): 850-91, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-24274377

RESUMO

The Globally Harmonised System of Classification (GHS) is a framework within which the intrinsic hazards of substances may be determined and communicated. It is not a legislative instrument per se, but is enacted into national legislation with the appropriate legislative instruments. GHS covers many aspects of effects upon health and the environment, including adverse effects upon sexual function and fertility or on development. Classification for these effects is based upon observations in humans or from properly designed experiments in animals, although only the latter is covered herein. The decision to classify a substance based upon experimental data, and the category of classification ascribed, is determined by the level of evidence that is available for an adverse effect on sexual function and fertility or on development that does not arise as a secondary non-specific consequence of other toxic effect. This document offers guidance on the determination of level of concern as a measure of adversity, and the level of evidence to ascribe classification based on data from tests in laboratory animals.


Assuntos
Substâncias Perigosas/classificação , Substâncias Perigosas/toxicidade , Reprodução/efeitos dos fármacos , Animais , Butadienos/classificação , Butadienos/toxicidade , Etanolaminas/classificação , Etanolaminas/toxicidade , Feminino , Guias como Assunto , Internacionalidade , Masculino , Nitrobenzenos/classificação , Nitrobenzenos/toxicidade , Ácidos Ftálicos/classificação , Ácidos Ftálicos/toxicidade , Rotulagem de Produtos , Testes de Toxicidade
17.
Toxicol Sci ; 136(1): 26-38, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-24029818

RESUMO

Ethylene oxide (EO) is a genotoxicant and a mouse lung carcinogen, but whether EO is carcinogenic through a mutagenic mode of action remains unclear. To investigate this question, 8-week-old male Big Blue B6C3F1 mice (10 mice/group) were exposed to EO by inhalation-6 h/day, 5 days/week for 4 weeks (0, 10, 50, 100, or 200 ppm EO) and 8 or 12 weeks (0, 100, or 200 ppm EO). Lung DNA samples were analyzed for levels of 3 K-ras codon 12 mutations (GGT→GAT, GGT→GTT, and GGT→TGT) using ACB-PCR. No measureable level of K-ras codon 12 TGT mutation was detected (ie, all lung mutant fractions [MFs] ≤ 10⁻5). Four weeks of inhalation of 100 ppm EO caused a significant increase in K-ras codon 12 GGT→GTT MF relative to controls, whereas 50, 100, and 200 ppm EO caused significant increases in K-ras codon 12 GGT→GAT MF. In addition, significant inverse correlations were observed between K-ras codon 12 GGT→GTT MF and cII mutant frequency in the lungs of the same mice exposed to 50, 100, or 200 ppm EO for 4 weeks. Surprisingly, 8 weeks of exposure to 100 and 200 ppm EO caused significant decreases in K-ras MFs relative to controls. Thus, the changes in K-ras MF as a function of cumulative EO dose were nonmonotonic and were consistent with EO causing early amplification of preexisting K-ras mutations, rather than induction of K-ras mutation through genotoxicity at codon 12. The possibility that these changes reflect K-ras mutant cell selection under varying degrees of oxidative stress is discussed.


Assuntos
Carcinógenos/toxicidade , Transformação Celular Neoplásica/induzido quimicamente , Óxido de Etileno/toxicidade , Neoplasias Pulmonares/induzido quimicamente , Pulmão/efeitos dos fármacos , Mutação , Proteínas Proto-Oncogênicas p21(ras)/genética , Animais , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Códon , Relação Dose-Resposta a Droga , Exposição por Inalação , Pulmão/metabolismo , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Masculino , Camundongos , Estresse Oxidativo/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Fatores de Tempo
18.
Exp Toxicol Pathol ; 65(7-8): 1109-15, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-23726758

RESUMO

The National Toxicology Program (NTP) database of technical reports on carcinogenicity bioassays has been interrogated for the incidence of primary pulmonary neoplasms in B6C3F1 mice. A total of 170 study reports were selected, from studies that completed the in-life phase during 1983-2007, which included neoplasm incidence data for 180 control groups comprising both male and female mice. The incidence (median and inter-quartile range) of males with alveolar/bronchiolar adenoma was 16% (12-20%), and for females it was 5% (2-8%); the incidence of males with alveolar/bronchiolar carcinoma was 8% (4-12%), and for females it was 2% (0-4%); and the incidence of males with combined alveolar/bronchiolar adenoma or carcinoma was 24% (18-30%), and for females it was 8% (6-12%). Comparing the incidence of animals bearing these lesions on a per study basis showed the median incidence in males to be 3.0-fold, 2.0-fold, and 2.8-fold higher than in females. The incidence of other primary pulmonary neoplasms was <10% of the alveolar/bronchiolar neoplasms. Comparison of gender-specific response to lung tumorigens showed that the increase in incidence of tumors above control levels was greater in females than in males.


Assuntos
Neoplasias Pulmonares/epidemiologia , Caracteres Sexuais , Animais , Bioensaio , Testes de Carcinogenicidade , Feminino , Incidência , Neoplasias Pulmonares/induzido quimicamente , Neoplasias Pulmonares/patologia , Masculino , Camundongos , Estudos Retrospectivos
19.
Regul Toxicol Pharmacol ; 66(1): 30-7, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23461858

RESUMO

Acute systemic toxicity data (LD50 values) and hazard classifications derived in the rat following oral administration and dermal application have been analysed to examine whether or not orally-derived hazard classification or LD50 values can be used to determine dermal hazard classification. Comparing the oral and dermal classifications for 335 substances derived from oral and dermal LD50 values respectively revealed 17% concordance, and indicated that 7% of substances would be classified less severely while 76% would be classified more severely if oral classifications were applied directly to the dermal route. In contrast, applying the oral LD50 values within the dermal classification criteria to determine the dermal classification reduced the concordance to 15% and the relative 'under-classification' to 1%, but increased the relative 'over-classification' to 84%. Both under- and over-classification are undesirable, and mitigation strategies are discussed. Finally, no substance with an oral LD50 of >2000mg/kg was classified for acute systemic toxicity by the dermal route, suggesting that dermal testing for acute systemic toxicity of such substances adds nothing to the hazard characterisation and should be removed from routine regulatory data requirements.


Assuntos
Substâncias Perigosas/toxicidade , Testes Cutâneos/métodos , Testes de Toxicidade Aguda/métodos , Administração Cutânea , Administração Oral , Animais , Bases de Dados Factuais , Substâncias Perigosas/administração & dosagem , Substâncias Perigosas/classificação , Humanos , Dose Letal Mediana , Ratos
20.
BMC Struct Biol ; 12: 22, 2012 Sep 20.
Artigo em Inglês | MEDLINE | ID: mdl-22995073

RESUMO

BACKGROUND: Structure-based drug design (SBDD) can accelerate inhibitor lead design and optimization, and efficient methods including protein purification, characterization, crystallization, and high-resolution diffraction are all needed for rapid, iterative structure determination. Janus kinases are important targets that are amenable to structure-based drug design. Here we present the first mouse Tyk2 crystal structures, which are complexed to 3-aminoindazole compounds. RESULTS: A comprehensive construct design effort included N- and C-terminal variations, kinase-inactive mutations, and multiple species orthologs. High-throughput cloning and expression methods were coupled with an abbreviated purification protocol to optimize protein solubility and stability. In total, 50 Tyk2 constructs were generated. Many displayed poor expression, inadequate solubility, or incomplete affinity tag processing. One kinase-inactive murine Tyk2 construct, complexed with an ATP-competitive 3-aminoindazole inhibitor, provided crystals that diffracted to 2.5-2.6 Å resolution. This structure revealed initial "hot-spot" regions for SBDD, and provided a robust platform for ligand soaking experiments. Compared to previously reported human Tyk2 inhibitor crystal structures (Chrencik et al. (2010) J Mol Biol 400:413), our structures revealed a key difference in the glycine-rich loop conformation that is induced by the inhibitor. Ligand binding also conferred resistance to proteolytic degradation by thermolysin. As crystals could not be obtained with the unliganded enzyme, this enhanced stability is likely important for successful crystallization and inhibitor soaking methods. CONCLUSIONS: Practical criteria for construct performance and prioritization, the optimization of purification protocols to enhance protein yields and stability, and use of high-throughput construct exploration enable structure determination methods early in the drug discovery process. Additionally, specific ligands stabilize Tyk2 protein and may thereby enable crystallization.


Assuntos
Desenho de Fármacos , Indazóis/química , Indazóis/farmacologia , TYK2 Quinase/antagonistas & inibidores , TYK2 Quinase/química , Sequência de Aminoácidos , Animais , Cristalização , Cristalografia por Raios X , Estabilidade Enzimática/efeitos dos fármacos , Humanos , Janus Quinases/antagonistas & inibidores , Janus Quinases/metabolismo , Camundongos , Dados de Sequência Molecular , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Estrutura Secundária de Proteína , Proteólise/efeitos dos fármacos , Relação Estrutura-Atividade , TYK2 Quinase/isolamento & purificação
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