RESUMEN
The detection of N-nitrosamines in drug products has raised global regulatory interest in recent years due to the carcinogenic potential of some nitrosamines in animals and a need to identify a testing strategy has emerged. Ideally, methods used would allow for the use of quantitative analysis of dose-response data from in vivo genotoxicity assays to determine a compound-specific acceptable intake for novel nitrosamines without sufficient carcinogenicity data. In a previous study we compared the dose-response relationships of N-nitrosodiethylamine (NDEA) in three in vivo genotoxicity endpoints in rats. Here we report a comparison of NDEA's genotoxicity profile in mice. Big Blue® mice were administered NDEA at doses of 0.001, 0.01, 0.1, 1 and 3 mg/kg/day by oral gavage for 28 days followed by 3 days of expression. Statistically significant increases in the NDEA induced mutations were detected by both the transgenic rodent mutation assay (TGR) using the cII endpoint and by duplex sequencing in the liver but not bone marrow of mice. In addition, administration of NDEA for two consecutive days in male C57BL/6N mice caused elevated DNA damage levels in the liver as measured by % tail DNA in comet assay. The benchmark dose (BMD) analysis shows a BMDL50 of 0.03, 0.04 and 0.72 mg/kg/day for TGR, duplex sequencing and comet endpoints, respectively. Overall, this study demonstrated a similar genotoxicity profile of NDEA between mice and rats and provides a reference that can be used to compare the potential potency of other novel nitrosamines for the induction of gene mutations.
RESUMEN
Risk management of in vitro aneugens for topically applied compounds is not clearly defined because there is no validated methodology to accurately measure compound concentration in proliferating stratum basale keratinocytes of the skin. Here, we experimentally tested several known aneugens in the EpiDerm reconstructed human skin in vitro micronucleus assay and compared the results to flow cytometric mechanistic biomarkers (phospho-H3; MPM2, DNA content). We then evaluated similar biomarkers (Ki-67, nuclear area) using immunohistochemistry in skin sections of minipigs following topical exposure the potent aneugens, colchicine, and hesperadin. Data from the EpiDerm model showed positive micronucleus responses for all aneugens tested following topical or direct media dosing with similar sensitivity when adjusted for applied dose. Quantitative benchmark dose-response analysis exhibited increases in the mitotic index biomarkers phospho-H3 and MPM2 for tubulin binders and polyploidy for aurora kinase inhibitors are at least as sensitive as the micronucleus endpoint. By comparison, the aneugens tested did not induce histopathological changes, increases in Ki-67 immunolabeling or nuclear area in skin sections from the in vivo minipig study at doses in significant excess of those eliciting a response in vitro. Results indicate the EpiDerm in vitro micronucleus assay is suitable for the hazard identification of aneugens. The lack of response in the minipig studies indicates that the barrier function of the minipig skin, which is comparable to human skin, protects from the effects of aneugens in vivo. These results provide a basis for conducting additional studies in the future to further refine this understanding.
Asunto(s)
Aneugénicos , Mutágenos , Animales , Epidermis , Humanos , Pruebas de Micronúcleos , Porcinos , Porcinos EnanosRESUMEN
2-Hydroxypyridine N-oxide (HOPO) is an important coupling reagent used in pharmaceutical synthesis. Our laboratory previously reported HOPO as equivocal in the Ames assay following extensive testing of multiple lots of material. Given the lack of reproducibility between lots of material and the weak increase in revertants observed, it was concluded that it would be highly unlikely that HOPO would pose a mutagenic risk in vivo. The purpose of the current investigation was to assess experimentally in rats the mutagenic (Pig-a mutation induction) and more broadly genotoxic (micronucleus and comet induction) potential of HOPO. Rats were administered HOPO (0, 50, 150, 300, and 500 mg/kg/day) by oral gavage for 28 days. At the end of study, the following parameters were assessed: frequency of Pig-a mutant red blood cells and reticulocytes, frequency of peripheral blood micronuclei, and the incidence of comet formation in liver. Toxicokinetic data collected on study Days 1 and 28 demonstrated systemic exposure to HOPO. Although there were no overt clinical signs, animals treated with HOPO showed a dose-related decrease in body weight gain. There were no increases observed in any of the genotoxicity endpoints assessed. The results from this study further support the conclusion that in the context of pharmaceutical synthesis, HOPO should not be considered a mutagenic impurity but rather controlled as a normal process-related impurity. Environ. Mol. Mutagen. 2019. © 2019 Wiley Periodicals, Inc.
Asunto(s)
Óxidos N-Cíclicos/efectos adversos , Mutagénesis/efectos de los fármacos , Mutágenos/efectos adversos , Piridinas/efectos adversos , Animales , Eritrocitos/efectos de los fármacos , Hígado/efectos de los fármacos , Masculino , Pruebas de Mutagenicidad/métodos , Mutación/efectos de los fármacos , Ratas , Ratas Wistar , Reproducibilidad de los Resultados , Reticulocitos/efectos de los fármacosRESUMEN
The Pig-a assay has rapidly gained international interest as a useful tool for assessing the mutagenic potential of compounds in vivo. Although a large number of compounds, including both mutagens and non-mutagens, have been tested in the rat Pig-a assay in haematopoietic cells, there is limited understanding of how perturbations in haematopoiesis affect assay performance. Of particular concern is the possibility that regenerative haematopoiesis alone, without exposure to a genotoxic agent, could result in elevated Pig-a mutant cell frequencies. To address this concern, Wistar-Han rats were dosed by oral gavage with a non-genotoxic haemolytic agent, 2-butoxyethanol (2-BE). Dose levels ranging from 0 to 450 mg/kg were tested using both single administration and 28-day treatment regimens. Haematology parameters were assessed at minimum within the first 24h of treatment and 8 days after the final administration. Pig-a mutant frequencies were assessed on Days 15 and ~30 for both treatment protocols and also on Days 43 and 57 for the 28-day protocol. Even at doses of 2-BE that induced marked intravascular lysis and strong compensatory erythropoiesis, the average Pig-a mutant phenotype red blood cell and reticulocyte frequencies were within the historical vehicle control distribution. 2-BE therefore showed no evidence of in vivo mutagenicity in these studies. The data suggest that perturbations in haematopoiesis alone do not lead to an observation of increased mutant frequency in the Pig-a assay.
Asunto(s)
Eritropoyesis/efectos de los fármacos , Glicoles de Etileno/toxicidad , Hemolíticos/toxicidad , Proteínas de la Membrana/genética , Mutágenos/toxicidad , Animales , Análisis Mutacional de ADN , Genes Reporteros , Masculino , Mutagénesis , Pruebas de Mutagenicidad , Mutación , Ratas Wistar , Reticulocitos/efectos de los fármacosRESUMEN
Understanding the mutagenic dose response could prove beneficial in the management of pharmaceutically relevant impurities. For most alkyl ester impurities, such as isopropyl methanesulfonate (IPMS), little in vivo mutagenicity data exist for dose analysis. The likelihood of a sublinear dose response for IPMS was assessed by comparing the Swain Scott constant, the SN 1/SN 2 reaction mechanism and the O(6) :N(7) guanine adduct ratio to that of more well-known alkyl esters. Based on available information, IPMS was predicted to have a mutagenic profile most like ethyl nitrosourea. To test this hypothesis, mature male Wistar Han rats were administered IPMS using acute (single administration at 3.5 to 56 mg/kg) or subchronic (28 days at 0.125 to 2 mg/kg/day) exposures. The in vivo Pig-a mutation assay was used to identify mutant phenotype reticulocyte (Ret) and red blood cell (RBC) populations. The maximum mutant response occurred approximately 15 and 28 days after the last dose administration in the mutant Ret and RBC populations respectively in the acute study and on Day 29 and 56 in the mutant Ret and RBC populations, respectively, in the subchronic study. A comparison of RBC mutant frequencies from acute and subchronic protocols suggests a sublinear response; however, this was not substantiated by statistical analysis. A No Observed Effect Level (NOEL) of 0.25 mg/kg/day resulted in a Permitted Daily Exposure equivalent to the Threshold of Toxicological Concern. An estimate of the NOEL based on the previously mentioned factors, in practice, would have pre-empted further investigation of the potent mutagen IPMS.
Asunto(s)
Eritrocitos/efectos de los fármacos , Proteínas de la Membrana/genética , Mesilatos/toxicidad , Pruebas de Mutagenicidad , Mutágenos/toxicidad , Reticulocitos/efectos de los fármacos , Animales , Antígenos CD59/análisis , Eritrocitos/metabolismo , Masculino , Mesilatos/administración & dosificación , Pruebas de Micronúcleos , Pruebas de Mutagenicidad/métodos , Mutágenos/administración & dosificación , Nivel sin Efectos Adversos Observados , Ratas , Ratas Wistar , Reticulocitos/metabolismoRESUMEN
Ethyl methanesulfonate (EMS) was evaluated as part of the validation effort for the rat Pig-a mutation assay and compared with other well-established in vivo genotoxicity endpoints. Male Sprague-Dawley (SD) rats were given a daily dose of 0, 6.25, 12.5, 25, 50, or 100 mg/kg/day EMS for 28 days, and evaluated for a variety of genotoxicity endpoints in peripheral blood, liver, and colon. Blood was sampled pre-dose (Day 1) and at various time points up to Day 105. Pig-a mutant frequencies were determined in total red blood cells (RBCs) and reticulocytes (RETs) as RBC(CD59-) and RET(CD59-) frequencies. The first statistically significant increases in mutant frequencies were seen in RETs on Day 15 and in RBCs on Day 29 with the maximum RET(CD59-) on Day 29 and of RBC(CD59-) on Day 55. The lowest dose producing a statistically significant increase of RET(CD59-) was 12.5 mg/kg on Day 55 and 25 mg/kg for RBC(CD59-) on Day 55. EMS also induced significant increases in % micronucleated RETs (MN-RETs) in peripheral blood on Days 3, 15, and 28. No statistically significant increases in micronuclei were seen in liver or colon. Results from the in vivo Comet assay on Day 29 showed generally weak increases in DNA damage in all tissues evaluated with little evidence for accumulation of damage seen over time. The results with EMS indicate that the assessment of RBC(CD59-) and/or RET(CD59-) in the Pig-a assay could be a useful and sensitive endpoint for a repeat dose protocol and complements other genotoxicity endpoints.
Asunto(s)
Ensayo Cometa/métodos , Metanosulfonato de Etilo/toxicidad , Proteínas de la Membrana/genética , Pruebas de Micronúcleos/métodos , Animales , Colon/citología , Colon/efectos de los fármacos , Daño del ADN/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Determinación de Punto Final , Eritrocitos/efectos de los fármacos , Hígado/citología , Hígado/efectos de los fármacos , Masculino , Proteínas de la Membrana/efectos de los fármacos , Tasa de Mutación , Ratas , Ratas Sprague-Dawley , Reticulocitos/efectos de los fármacosRESUMEN
The Organization for Economic Co-operation and Development (OECD) has recently adopted Test Guideline 487 (TG487) for conducting the in vitro micronucleus (MNvit) assay. The purpose of this study is to evaluate and validate treatment conditions for the use of p53 competent TK6 human lymphoblastoid cells in a TG487 compliant MNvit assay. The ten reference compounds suggested in TG487 (mitomycin C, cytosine arabinoside, cyclophosphamide, benzo-a-pyrene, vinblastine sulphate, colchicine, sodium chloride, nalidixic acid and di(2-ethylhexyl)phthalate and pyrene) and noscapine hydrochloride were chosen for this study. In order to optimize the micronucleus response after treatment with some positive substances, we extended the recovery time after pulse treatment from 2 cell cycles recommended in TG487 to 3 cell cycles for untreated cells (40h). Each compound was tested in at least one of four exposure conditions: a 4h exposure followed by a 40h recovery, a 4h exposure followed by a 24h recovery, a 4h exposure in the presence of an exogenous metabolic activation system followed by a 40h recovery period, and a 27h continuous direct treatment. Results show that the direct acting clastogens, clastogens requiring metabolic activation and aneugens caused a robust increase in micronuclei in at least one test condition whereas the negative compounds did not induce micronuclei. The negative control cultures exhibited reproducibly low and consistent micronucleus frequencies ranging from 0.4 to 1.8% (0.8±0.3% average and standard deviation). Furthermore, extending the recovery period from 24h to 40h produced a 2-fold higher micronucleus frequency after a 4h pulse treatment with mitomycin C. In summary, the protocol described in this study in TK6 cells produced the expected result with model compounds and should be suitable for performing the MNvit assay in accordance with guideline TG487.
Asunto(s)
Antineoplásicos/toxicidad , Pruebas de Micronúcleos/métodos , Mutágenos/toxicidad , Aneugénicos/toxicidad , Biotransformación , Línea Celular , Guías como Asunto , HumanosRESUMEN
N-Ethyl-N-nitrosourea (ENU) was evaluated as part of the Stage III trial for the rat Pig-a gene mutation assay. Groups of six- to eight-week-old male Sprague Dawley (SD) or Fischer 344 (F344) rats were given 28 daily doses of the phosphate buffered saline vehicle, or 2.5, 5, or 10 mg/kg ENU, and evaluated for a variety of genotoxicity endpoints in peripheral blood, spleen, liver, and colon. Blood was sampled predose (Day-1) and at various time points up to Day 57. Pig-a mutant frequencies were determined in total red blood cells (RBCs) and reticulocytes (RETs) as RBC(CD592-) and RET(CD592-) frequencies. Consistent with the results from a reference laboratory, RBC(CD592-) and RET(CD592-) frequencies increased in a dose and time-dependent manner, producing significant increases at all doses by Day 15, with similar frequencies seen in both rat strains. ENU also induced small but significant increases in % micronucleated RETs on Days 4 and 29. No significant increases in micronuclei were seen in the liver or colon of the ENU-treated SD rats. Hprt and Pig-a lymphocyte mutation assays conducted on splenocytes from Day 56 F344 rats detected two- to fourfold stronger responses for Hprt than Pig-a mutations. Results from the in vivo Comet assay in SD rats at Day 29 showed generally weak increases in DNA damage in all tissues evaluated. The results with ENU indicate that the Pig-a RET and RBC assays are reproducible, transferable, and complement other genotoxicity endpoints that could potentially be integrated into 28-day repeat dose rat studies.
Asunto(s)
Dietilnitrosamina/toxicidad , Proteínas de la Membrana/genética , Pruebas de Mutagenicidad , Mutágenos/toxicidad , Mutación , Animales , Antígenos CD59/genética , Calibración , Colon/efectos de los fármacos , Colon/ultraestructura , Ensayo Cometa/métodos , Ensayo Cometa/normas , Interpretación Estadística de Datos , Relación Dosis-Respuesta a Droga , Determinación de Punto Final , Eritrocitos/efectos de los fármacos , Eritrocitos/ultraestructura , Citometría de Flujo , Hígado/efectos de los fármacos , Hígado/ultraestructura , Masculino , Pruebas de Micronúcleos/métodos , Pruebas de Micronúcleos/normas , Pruebas de Mutagenicidad/métodos , Pruebas de Mutagenicidad/normas , Especificidad de Órganos , Ratas , Ratas Endogámicas F344 , Ratas Sprague-Dawley , Estándares de Referencia , Reproducibilidad de los Resultados , Reticulocitos/efectos de los fármacos , Reticulocitos/ultraestructura , Especificidad de la Especie , Bazo/efectos de los fármacos , Bazo/ultraestructura , Factores de TiempoRESUMEN
In recent years, experimental evidence has accumulated that supports the existence of sublinear dose-response relationships at low doses of DNA reactive mutagens. However, creating the in vivo data necessary to allow for a more detailed dose-response modeling with the currently available tools might not always be practical. The purpose of the current work was to evaluate the utility of the Pig-a gene mutation assay to rapidly identify dose-response relationships for direct acting genotoxicants. The induction of mutations in the peripheral blood of rats was evaluated following 28 days of exposure down to low doses of the direct acting alkylating agents ethyl methane sulfonate (EMS) and ethylnitrosourea (ENU). Using statistical modeling based on the 28-day studies, a threshold for mutation induction for EMS was estimated to be 21.9mg/kg, whereas for the more potent ENU, the threshold was estimated to be 0.88mg/kg. Comparing mutation frequencies from acute and sub-chronic dosing indicated less than additive dose-response relationships, further confirming the possibility of a threshold dose-response relationship for both compounds. In conclusion, the work presented provides evidence that the Pig-a assay might be a practical alternative to other in vivo mutation assays when assessing dose-response relationships for direct acting mutagens and that an experimental approach using fractionated dosing could be used to substantiate a biological mechanism responsible for the observation of a sublinear dose-response relationship.
Asunto(s)
Relación Dosis-Respuesta a Droga , Metanosulfonato de Etilo/toxicidad , Etilnitrosourea/toxicidad , Proteínas de la Membrana/genética , Pruebas de Mutagenicidad/métodos , Mutágenos/toxicidad , Animales , RatasRESUMEN
The in vivo bone marrow (BM) micronucleus assay is one of the three tests in the standard test battery to assess the genotoxic potential of a pharmaceutical candidate. In some cases, depending on results of in vitro studies, the route of administration or the degree of systemic exposure, in vivo assessment of genotoxicity in the BM alone may not be sufficient. Based on the potential for high gut exposures to orally administered compounds with low systemic exposures as well as the potential susceptibility of rapidly dividing cells of the intestinal tissues, we have developed a modified technique for evaluating micronuclei formation in both the duodenum and colon of rats based on earlier publications. Adult male Sprague Dawley rats were treated once daily for 2 days with either vehicle control or with the test articles acetyl salicylic acid (ASA), carbendazim (CAR), cyclophosphamide (CP), dimethylhydrazine (DMH), mitomycin C (MMC) or vinblastine sulfate (VIN). The duodenum, colon, and BM were harvested, processed, and analyzed for micronucleus induction. Results from these studies demonstrated differences in the susceptibility for different test compounds in the three tissues tested. When MMC and VIN were dosed by different routes at the same dose levels both compounds produced positive results in all three tissues by intraperitoneal injection but not oral administration. These studies suggest that overall the GI micronucleus assay might be a useful tool for clastogenic and aneugenic compounds that are expected to produce high sustained concentrations in the gastrointestinal tract with little systemic exposure.
Asunto(s)
Colon/efectos de los fármacos , Duodeno/efectos de los fármacos , Pruebas de Micronúcleos/métodos , Animales , Aspirina/efectos adversos , Aspirina/farmacología , Bencimidazoles/efectos adversos , Bencimidazoles/farmacología , Carbamatos/efectos adversos , Carbamatos/farmacología , Ciclofosfamida/efectos adversos , Ciclofosfamida/farmacología , Daño del ADN/efectos de los fármacos , Dimetilhidrazinas/efectos adversos , Dimetilhidrazinas/farmacología , Masculino , Mitomicina/efectos adversos , Mitomicina/farmacología , Pruebas de Mutagenicidad/métodos , Mutágenos/efectos adversos , Mutágenos/farmacología , Ratas , Ratas Sprague-Dawley , Vinblastina/efectos adversos , Vinblastina/farmacologíaRESUMEN
2-(3-Chlorobenzyloxy)-6-(piperazin-1-yl)pyrazine (3) is a potent and selective 5-HT(2C) agonist that exhibits dose-dependent inhibition of food intake and reduction in body weight in rats, making it an attractive candidate for treatment of obesity. However, examination of the genotoxicity potential of 3 in the Salmonella Ames assay using tester strains TA98, TA100, TA1535, and TA1537 revealed a metabolism (rat S9/NADPH)- and dose-dependent increase of reverse mutations in strains TA100 and TA1537. The increase in reverse mutations was attenuated upon coincubation with methoxylamine and glutathione. The irreversible and concentration-dependent incorporation of radioactivity in calf thymus DNA after incubations with [14C]3 in the presence of rat S9/NADPH suggested that 3 was bioactivated to a reactive intermediate that covalently bound DNA. In vitro metabolism studies on 3 with rat S9/NADPH in the presence of methoxylamine and cyanide led to the detection of amine and cyano conjugates of 3. The mass spectrum of the amine conjugate was consistent with condensation of amine with an aldehyde metabolite derived from hydroxylation of the secondary piperazine nitrogen-alpha-carbon bond. The mass spectrum of the cyano conjugate suggested a bioactivation pathway involving N-hydroxylation of the secondary piperazine nitrogen followed by two-electron oxidation to generate an electrophilic nitrone, which reacted with cyanide. The 3-chlorobenzyl motif in 3 was also bioactivated via initial aromatic ring hydroxylation followed by elimination to a quinone-methide species that reacted with glutathione or with the secondary piperazine ring nitrogen in 3 and its monohydroxylated metabolite(s). The metabolism studies described herein provide a mechanistic basis for the mutagenicity of 3.
Asunto(s)
Piperazinas/toxicidad , Pirazinas/toxicidad , Salmonella typhimurium/efectos de los fármacos , Agonistas del Receptor de Serotonina 5-HT2 , Biotransformación , Pruebas de Mutagenicidad , Obesidad , Piperazinas/síntesis química , Piperazinas/metabolismo , Pirazinas/síntesis química , Pirazinas/metabolismo , Salmonella typhimurium/genética , Salmonella typhimurium/metabolismoRESUMEN
The epidermal growth factor receptor (EGFR), a long-standing drug development target, is also a desirable target for imaging. Sixteen dialkoxyquinazoline analogues, suitable for labeling with positron-emitting isotopes, have been synthesized and evaluated in a battery of in vitro assays to ascertain their chemical and biological properties. These characteristics provided the basis for the adoption of a selection schema to identify lead molecules for labeling and in vivo evaluation. A new EGFR tyrosine kinase radiometric binding assay revealed that all of the compounds possessed suitable affinity (IC50 = 0.4-51 nM) for the EGFR tyrosine kinase. All of the analogues inhibited ligand-induced EGFR tyrosine phosphorylation (IC50 = 0.8-20 nM). The HPLC-estimated octanol/water partition coefficients ranged from 2 to 5.5. Four compounds, 4-(2'-fluoroanilino)- and 4-(3'-fluoroanilino)-6,7-diethoxyquinazoline as well as 4-(3'-chloroanilino)- and 4-(3'-bromoanilino)-6,7-dimethoxyquinazoline, possess the best combination of characteristics that warrant radioisotope labeling and further evaluation in tumor-bearing mice.
Asunto(s)
Compuestos de Anilina/síntesis química , Receptores ErbB/antagonistas & inhibidores , Quinazolinas/síntesis química , Radiofármacos/síntesis química , Compuestos de Anilina/química , Compuestos de Anilina/farmacología , Animales , Unión Competitiva , Radioisótopos de Carbono , Línea Celular , ADN/antagonistas & inhibidores , ADN/biosíntesis , Receptores ErbB/metabolismo , Radioisótopos de Flúor , Humanos , Marcaje Isotópico , Ligandos , Ratones , Neoplasias/diagnóstico por imagen , Fosforilación , Quinazolinas/química , Quinazolinas/farmacología , Ensayo de Unión Radioligante , Cintigrafía , Radiofármacos/química , Radiofármacos/farmacología , Receptor ErbB-2/metabolismo , Receptor ErbB-4 , Relación Estructura-Actividad , Tirosina/metabolismoRESUMEN
The neuregulins (NRGs) are members of the epidermal growth factor (EGF) family of peptide growth factors. These hormones are agonists for the ErbB family of receptor tyrosine kinases, a family that includes the epidermal growth factor receptor (EGFR/ErbB1), ErbB2/Neu/HER2, ErbB3/HER3, and ErbB4/HER4. We recently observed that the EGF family hormone NRG2beta is a potent agonist for ErbB4. In contrast, NRG2alpha, a splicing isoform of the same gene that encodes NRG2beta, is a poor ErbB4 agonist. We hypothesized that carboxyl-terminal residues of NRG2beta are critical for stimulation of ErbB4 tyrosine phosphorylation and coupling to downstream signaling events. Here, we demonstrate that the substitution of a lysine residue for Phe45 in NRG2beta results in reduced ligand potency. We also demonstrate that substitution of a phenylalanine for Lys45 in NRG2alpha results in increased ligand potency. Finally, analyses of the gain-of-function NRG2alpha Chg5 mutant demonstrate that Gln43, Met47, Asn49, and Phe50 regulate ligand efficacy. Thus, these data indicate that carboxyl-terminal residues of NRG2beta are critical for activation of ErbB4 signaling. Moreover, these NRG2alpha and NRG2beta mutants reveal new insights into models for ligand-induced ErbB family receptor tyrosine phosphorylation and coupling to downstream signaling events.
Asunto(s)
Receptores ErbB/fisiología , Factores de Crecimiento Nervioso/fisiología , Transducción de Señal/fisiología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Línea Celular , Cartilla de ADN , Dimerización , Datos de Secuencia Molecular , Factores de Crecimiento Nervioso/química , Receptor ErbB-4RESUMEN
During the last decade, several novel members of the Epidermal Growth Factor family of peptide growth factors have been identified. Most prominent among these are the Neuregulins or Heregulins. To date, four different Neuregulin genes have been identified (Neuregulin1-4) and several different splicing isoforms have been identified for at least two of these genes (Neuregulin1 and Neuregulin2). While Neuregulin1 isoforms have been extensively studied, comparatively little is known about Neuregulin3, Neuregulin4, or the Neuregulin2 isoforms. Indeed, there has been no systematic comparison of the activities of these molecules. Here we demonstrate that Neuregulin2alpha and Neuregulin2beta stimulate ErbB3 tyrosine phosphorylation and coupling to biological responses. In contrast, Neuregulin3 and Neuregulin4 fail to activate ErbB3 signaling. Furthermore, Neuregulin2beta, but not Neuregulin2alpha, stimulates ErbB4 tyrosine phosphorylation and coupling to biological responses. Finally, both Neuregulin3 and Neuregulin4 stimulate modest amounts of ErbB4 tyrosine phosphorylation. However, whereas Neuregulin3 stimulates a modest amount of ErbB4 coupling to biological responses, Neuregulin4 fails to stimulate ErbB4 coupling to biological responses. This suggests that there are qualitative as well as quantitative differences in ErbB family receptor activation by Neuregulin isoforms.