RESUMEN
Pharmacokinetics of ethyl methanesulfonate (EMS) were characterized in mice, rats and in cynomolgus monkeys with unlabelled and (14)C-radiolabelled EMS by either quantification of unchanged EMS or the N-ethyl-valine haemoglobin (Hb) adduct. EMS was well absorbed and exhibited close to 100% oral bioavailability. EMS showed some species differences in systemic clearance (intermediate in mice, low in rats and monkeys) representing only 1-4% of the cardiac output. The volume of distribution (0.5-0.8L/kg) was constant across species and corresponded to extracellular water. As a result of the species differences in clearance, the half-life ranged from 10 min in mouse (at low dose) to 5h in monkey. The systemic exposure of free EMS and the levels of its Hb adduct increased nearly dose proportionately from 1 to 5 and 0.5 to 80 mg/kg, respectively. The persistence of the N-ethyl-valine Hb adduct was much longer than EMS itself, consistent with the long life span of haemoglobin. No species differences were evident for the binding of EMS to Hb in whole blood ex vivo as determined by the second order rate constants. Following administration of Viracept tablets of the contaminated production batches (to monkeys leading to EMS doses of 0.08-27 microg/kg, concentrations of ethyl-valine Hb adducts) were near or below the detection limit of the assay (0.043 nmol/g Hb).
Asunto(s)
Alquilantes/farmacocinética , Contaminación de Medicamentos , Metanosulfonato de Etilo/farmacocinética , Mutágenos/farmacocinética , Animales , Relación Dosis-Respuesta a Droga , Inhibidores de la Proteasa del VIH/química , Semivida , Hemoglobinas/química , Hemoglobinas/metabolismo , Humanos , Macaca fascicularis , Masculino , Ratones , Microsomas Hepáticos/efectos de los fármacos , Microsomas Hepáticos/metabolismo , Nelfinavir/química , Ratas , Medición de Riesgo , Especificidad de la Especie , Comprimidos , Valina/análogos & derivados , Valina/química , Valina/metabolismoRESUMEN
In order to support the toxicological risk assessment for the ethyl methanesulfonate (EMS) exposure of patients ingesting contaminated Viracept tablets (Müller and Singer, 2009), there was a need to correlate the effects observed in in vivo genotoxicity studies with mice to EMS exposure and to estimate human exposure to EMS at the level of contamination of Viracept tablets. The species differences in volume of distribution of EMS, a key factor for determination of its C(max), were small in the species investigated (mouse, rat, monkey), the species differences in clearance, the key factor involved in AUC assessment, were large (Lavé et al., 2009). Because of this uncertainty in extrapolation of clearance across species we used a conservative approach for human exposure predictions in terms of AUC where clearance was assumed to solely reflect the chemical stability of EMS neglecting additional clearance pathways such as metabolism and exhalation. This approach was compared to the estimates obtained from allometric scaling based on rat clearance, the species leading to the lowest clearance predicted in man. We found that both approaches led to nearly identical predictions of the human AUC. Thus, we predict a human AUC of 13 microM h for patients ingesting the most contaminated Viracept tablets, corresponding to a maximal daily intake of 0.055 mg/kg of EMS. The C(max) of EMS in these patients is predicted to be 0.85 microM. In order to provide a basis for toxicological risk assessment, these maximal human AUC and C(max) values are to be compared to the AUC and C(max) values in mice at the EMS dose of 25mg/kg which was found to be the threshold dose for induction of mutagenic effects, i.e. the dose at which no mutagenic effects were observed (Gocke et al., 2009-a). We calculate AUC and C(max) in mice at the threshold dose to be 350 microM h and 315 microM, respectively. Thus we conclude that a large safety factor can be deduced, whatever the basis of comparison, as is discussed in detail by Müller et al. (2009).
Asunto(s)
Alquilantes/farmacocinética , Contaminación de Medicamentos , Metanosulfonato de Etilo/farmacocinética , Mutágenos/farmacocinética , Animales , Área Bajo la Curva , Simulación por Computador , Relación Dosis-Respuesta a Droga , Inhibidores de la Proteasa del VIH/química , Hemoglobinas/química , Hemoglobinas/efectos de los fármacos , Hemoglobinas/metabolismo , Humanos , Técnicas In Vitro , Macaca fascicularis , Ratones , Microsomas Hepáticos/efectos de los fármacos , Microsomas Hepáticos/metabolismo , Modelos Biológicos , Nelfinavir/química , Ratas , Medición de Riesgo , Especificidad de la Especie , Valina/análogos & derivados , Valina/química , Valina/metabolismoRESUMEN
Based on a production accident Viracept (nelfinavir mesilate) tablets, an HIV protease inhibitor supplied by Roche outside the US, Canada and Japan was contaminated with relatively high levels of ethyl methanesulfonate (EMS) for at most 3 months in spring of 2007. On the basis of a wide variety of toxicological data including critical experiments for mutation induction under chronic exposure conditions and cross-species exposure scaling experiments to extrapolate to humans, we estimate the added risk of adverse effects (cancer, birth abnormalities, heritable defects) in any individual patient accidentally exposed to EMS via contaminated Viracept tablets in the context of this production accident as essentially zero. Of critical important for this risk assessment are pivotal in vivo genotoxicity studies (MNT, MutaMouse) providing evidence for 'hockey-stick', like dose-response relationships for the risk defining induction of gene mutations and chromosomal damage by EMS [Gocke, E., Müller, L., Pfister, T., Buergin, H., 2009a. Literature review on the genotoxicity, reproductive toxicity, and carcinogenicity of ethyl methanesulfonate. Toxicol. Lett.; Gocke, E., Müller, L., Pfister, T., 2009b. EMS in Viracept-initial ('traditional') assessment of risk to patients based on linear dose response relations. Toxicol. Lett.; Gocke, E., Müller, L., Ballantyne, M., Whitwell, J., Müller, L., 2009c. MNT and MutaMouse studies to definde the in vivo dose-response relations of the genotoxicity of EMS and ENU. Toxicol. Lett.]. As outlined in Gocke and Wall [Gocke, E., Wall, M., 2009. In vivo genotoxicity of EMS: Statistical assessment of the dose response curves. Toxicol. Lett.], several statistical approaches are in support of a threshold model to best fit the data. The presence of clear no effect levels in bone marrow, liver and GI-tract tissue with several dose levels tested below the NOEL permits the calculation of safety factors with considerable confidence. In calculating the ratio of the NOEL dose in the animal studies (25mg/kg/day) divided by the calculated maximal daily dose of the patients (1068ppm EMS in 2.92g Viracept tablets=2.75mg EMS or 0.055mg/kg for a 50kg person) we derive a safety factor of 454 based on oral intake. Detailed absorption, distribution and metabolism studies in mice, rats and monkeys and with human surrogates in vitro enable us to estimate the safety factors also for the calculated likely highest exposure (AUC and C(max)) of patients to EMS [Lave, T., Birnböck, H., Götschi, A., Ramp, T., Pähler, A., 2009a. In vivo and in vitro characterization of ethyl methanesulfonate pharmacokinetics in animals and in human. Toxicol. Lett.; Lave, T., Paehler, A., Grimm, H.P., 2009b. Modelling of patient EMS exposure: translating pharmacokinetics of EMS in vitro and in animals into patients. Toxicol. Lett.]. We calculate the total exposure (AUC) based safety factor to amount to at least 28. This lower value is due to the conservative prediction of a longer half-life of EMS in man versus mouse, rat and monkey. Based on the estimated human C(max) the safety factor for affected Viracept patients is calculated to be 370, as C(max) is mainly dependent on volume of distribution, which is not much different for EMS in different species. We consider that the total exposure based safety factor constitutes a minimal value since the considerations regarding evidence of error-free repair at sub-threshold concentrations argues in favor of using the highest EMS concentration (C(max)) rather than the AUC as basis for risk assessment. The 'true value' very likely lies somewhere between these two numbers as aspects such as repair enzyme availability and status of the cell cycle relative to the insult are important parameters that may not fully support safety factors based solely on C(max) estimates. Potential adverse effects of EMS such as cancer, birth abnormalities and heritable effects are considered to be sequelae of its genotoxic activity. Hence, the thresholded dose-response relationships should also apply to these endpoints. We also provide a comprehensive discussion of the specific disease situation of the HIV infected target population and potential influences of co-medications on the susceptibilities and repair capacities of EMS induced DNA lesions.
Asunto(s)
Alquilantes/toxicidad , Contaminación de Medicamentos , Metanosulfonato de Etilo/toxicidad , Inhibidores de la Proteasa del VIH/química , Mutágenos/toxicidad , Nelfinavir/química , Pruebas de Toxicidad , Alquilantes/análisis , Alquilantes/farmacocinética , Animales , Relación Dosis-Respuesta a Droga , Exposición a Riesgos Ambientales , Metanosulfonato de Etilo/análisis , Metanosulfonato de Etilo/farmacocinética , Humanos , Ratones , Mutágenos/análisis , Mutágenos/farmacocinética , Mutación/efectos de los fármacos , Neoplasias/inducido químicamente , Nivel sin Efectos Adversos Observados , Ratas , Reproducción/efectos de los fármacos , Medición de RiesgoRESUMEN
Cyclic nucleotide-gated (CNG) channels belong to the P-loop-containing family of ion channels that also includes KcsA, MthK, and Shaker channels. In this study, we investigated the structure and rearrangement of the CNGA1 channel pore using cysteine mutations and cysteine-specific modification. We constructed 16 mutant channels, each one containing a cysteine mutation at one of the positions between 384 and 399 in the S6 region of the pore. By measuring currents activated by saturating concentrations of the full agonist cGMP and the partial agonists cIMP and cAMP, we show that mutating S6 residues to cysteine caused both favorable and unfavorable changes in the free energy of channel opening. The time course of cysteine modification with 2-aminoethylmethane thiosulfonate hydrochloride (MTSEA) was complex. For many positions we observed decreases in current activated by cGMP and concomitant increases in current activated by cIMP and cAMP. A model where modification affected both gating and permeation successfully reproduced the complex time course of modification for most of the mutant channels. From the model fits to the time course of modification for each mutant channel, we quantified the following: (a) the bimolecular rate constant of modification in the open state, (b) the change in conductance, and (c) the change in the free energy of channel opening for modification of each cysteine. At many S6 cysteines, modification by MTSEA caused a decrease in conductance and a favorable change in the free energy of channel opening. Our results are interpreted within the structural framework of the known structures of KcsA and MthK. We conclude that: (a) MTSEA modification affects both gating and permeation, (b) the open configuration of the pore of CNGA1 channels is consistent with the structure of MthK, and (c) the modification of S6 residues disrupts the helical packing of the closed channel, making it easier for channels to open.
Asunto(s)
Metanosulfonato de Etilo/análogos & derivados , Activación del Canal Iónico/fisiología , Canales Iónicos/fisiología , Secuencia de Aminoácidos , AMP Cíclico/metabolismo , Canales Catiónicos Regulados por Nucleótidos Cíclicos , Cisteína/genética , Metanosulfonato de Etilo/farmacocinética , Humanos , Indicadores y Reactivos/farmacocinética , Canales Iónicos/química , Datos de Secuencia Molecular , Oocitos , Técnicas de Placa-Clamp , Permeabilidad , Mutación Puntual , Estructura Secundaria de ProteínaRESUMEN
In AMPA receptor channels, a single amino acid residue (Q/R site) of the M2 segment controls permeation of calcium ions, single-channel conductance, blockade by intracellular polyamines, and permeation of anions. The structural environment of the Q/R site and its positioning with regard to a narrow constriction were probed with the accessibility of substituted cysteines to positively and negatively charged methanethiosulfonate reagents, applied from the extracellular and cytoplasmic sides of the channel. The accessibility patterns confirm that the M2 segment forms a pore loop with the Q/R site positioned at the tip of the loop (position 0) facing the extracellular vestibule. Cytoplasmically accessible residues on the N- and C-terminal sides of position 0 form the ascending alpha-helical (-8 to -1) and descending random coil (+1 to +6) components of the loop, respectively. Substitution of a glycine residue at position +2 with alanine strongly decreased the permeability of organic cations, indicating that position +2 contributes to the narrow constriction. The anionic 2-sulfonatoethyl-methanethiosufonate reacted with a cysteine at position 0 only from the external side and with cysteines at positions +1 to +4 only from the cytoplasmic side. These results suggest that charge selectivity occurs external to the constriction (+2) and possibly involves interactions of ions with the negative electrostatic potential created by the dipole of the alpha-helix formed by the ascending limb of the loop.
Asunto(s)
Activación del Canal Iónico/fisiología , Receptores AMPA/metabolismo , Sustitución de Aminoácidos , Animales , Calcio/metabolismo , Cisteína/química , Cisteína/efectos de los fármacos , Cisteína/genética , Citoplasma/metabolismo , Relación Dosis-Respuesta a Droga , Metanosulfonato de Etilo/análogos & derivados , Metanosulfonato de Etilo/química , Metanosulfonato de Etilo/farmacocinética , Ácido Glutámico/farmacología , Activación del Canal Iónico/efectos de los fármacos , Ácido Kaínico/farmacología , Cinética , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Mesilatos/química , Mesilatos/farmacocinética , Microinyecciones , Mutagénesis Sitio-Dirigida , Oocitos/efectos de los fármacos , Oocitos/metabolismo , Técnicas de Placa-Clamp , Permeabilidad/efectos de los fármacos , Receptores AMPA/genética , Relación Estructura-Actividad , Reactivos de Sulfhidrilo/química , Reactivos de Sulfhidrilo/farmacocinética , Xenopus laevisRESUMEN
The substituted cysteine-accessibility method and two sulfhydryl-specific reagents, the methane-thiosulfonate derivative 2-aminoethyl methanethiosulfonate (MTSEA) and the alpha(2)-adrenergic receptor (alpha(2)-AR) agonist chloroethylclonidine (CEC), were used to determine the relative accessibility of engineered cysteines in the fifth transmembrane domain of the human alpha(2A)-AR (Halpha2A). The second-order rate constants for the reaction of the receptor with MTSEA and CEC were determined with the wild type Halpha2A (cysteine at position 201) and receptor mutants containing accessible cysteines at other positions within the binding-site crevice (positions 197, 200, and 204). The rate of reaction of CEC was similar to that of MTSEA at residues Cys-197, Cys-201, and Cys-204. The rate of reaction of CEC with Cys-200, however, was more than 5 times that of MTSEA, suggesting that these compounds may interact with two different receptor conformations. MTSEA, having no recognition specificity for the receptor, likely reacts with the predominant inactive receptor conformation (R), whereas the agonist CEC may stabilize and react preferentially with the active receptor conformation (R*). This hypothesis was consistent with three-dimensional receptor-ligand models, which further suggest that alpha(2A)-AR activation may involve the clockwise rotation of transmembrane domain 5.
Asunto(s)
Antagonistas Adrenérgicos alfa/farmacocinética , Clonidina/análogos & derivados , Metanosulfonato de Etilo/análogos & derivados , Receptores Adrenérgicos alfa 2/química , Receptores Adrenérgicos alfa 2/metabolismo , Sustitución de Aminoácidos , Animales , Sitios de Unión , Células CHO , Clonidina/farmacocinética , Clonidina/farmacología , Cricetinae , Cisteína , Metanosulfonato de Etilo/farmacocinética , Metanosulfonato de Etilo/farmacología , Humanos , Concentración de Iones de Hidrógeno , Cinética , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Conformación Proteica , Estructura Secundaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Termodinámica , TransfecciónRESUMEN
EDB significantly depressed GSH in caput and cauda epididymis, but not in testis, 2 hours following injection. This depression was dose-related. EDB enhanced EMS-induced dominant lethal mutations at mating weeks 2 and 3 (of 6). At mating week 2 the fetal death rate was increased two-fold, while at week 3, the fetal death rate had increased to nearly three-fold greater than the EMS-only controls. Enhancement of fetal death rate was confined to postimplantation loss. As with EMS alone, the EDB potentiation of EMS-induced mutations was limited to postmeiotic stages of spermatogenesis. EDB also enhanced alkylation of rat spermatozoa by labeled EMS. Depression of GSH in reproductive tissues is correlated with a potentiation of dominant lethal mutations, as well as an increase in the binding of EMS to sperm heads.
Asunto(s)
Metanosulfonato de Etilo/toxicidad , Dibromuro de Etileno/toxicidad , Genitales Masculinos/metabolismo , Glutatión/metabolismo , Mutagénesis , Mutágenos , Animales , Sinergismo Farmacológico , Implantación del Embrión/efectos de los fármacos , Metanosulfonato de Etilo/farmacocinética , Femenino , Muerte Fetal , Reabsorción del Feto , Genes Dominantes , Genes Letales , Genitales Masculinos/efectos de los fármacos , Masculino , Embarazo , Ratas , Ratas Endogámicas F344 , Espermatozoides/metabolismoRESUMEN
A simple method is described for the determination of the lethal effects of chemicals assayed with the L-arabinose resistance test of Salmonella typhimurium. The method uses a mixed culture of 2 isogenic bacterial strains which are distinguished on the basis of their different nutritional requirements: sensitivity or resistance to L-arabinose, auxotrophy or prototrophy to histidine and leucine. BA13 (the mutation indicator strain) is the strain for routine screening of mutagens and allows the selection of forward mutation from L-arabinose sensitivity to L-arabinose resistance. BAL13 (the survival indicator strain) is a derivative of BA13. Both bacterial strains are found to be equally sensitive to the lethal effects of mutagens. The method described permits the measurement of cell survival at the same high cell concentration as used in the measurement of the mutant yield and in the same type of minimal medium with L-arabinose and glycerol, except for the histidine supplement in the mutant plates or the leucine in the survival plates.
Asunto(s)
Arabinosa , Pruebas de Mutagenicidad , Mutágenos , Salmonella typhimurium/genética , 2-Naftilamina/farmacocinética , 2-Naftilamina/toxicidad , Biotransformación , Farmacorresistencia Microbiana , Metanosulfonato de Etilo/farmacocinética , Metanosulfonato de Etilo/toxicidad , Metilmetanosulfonato/farmacocinética , Metilmetanosulfonato/toxicidad , Metilnitronitrosoguanidina/toxicidad , Mutágenos/farmacocinéticaRESUMEN
Buthionine sulfoximine (BSO) treatment significantly reduced testicular epididymal and vas deferens glutathione (GSH) levels in rats. Testicular levels of GSH were reduced by 20%, while epididymal GSH levels were reduced by more than 50%. BSO treatment correspondingly enhanced ethyl methanesulfonate (EMS)-induced dominant lethal mutations. EMS-induced resorption rates were doubled following BSO treatment. This effect was observed in mating wk 2 and 3 (d 8-19 following treatment), indicating effects on those germ cells which were in late testicular stages or were caput epididymal spermatozoa at the time of EMS treatment. The enhancement of the mutagenic action of EMS by BSO is restricted to the same time period (spermatid-spermatozoa transition, early epididymal maturation) as maximum sensitivity to the clastogenic action of EMS on male germ cells. The temporal pattern of EMS alkylation of rat spermatozoa correlated with the incidence of EMS-induced dominant lethal mutations. BSO depresses GSH in the male reproductive tract in a dose- and time-dependent manner. Perturbation of GSH in the male reproductive tract appears to influence chemical-induced germ cell mutations.