RESUMEN
In vitro assays are widely employed to obtain intrinsic clearance estimates used in toxicokinetic modeling efforts. However, the reliability of these methods is seldom reported. Here we describe the results of an international ring trial designed to evaluate two in vitro assays used to measure intrinsic clearance in rainbow trout. An important application of these assays is to predict the effect of biotransformation on chemical bioaccumulation. Six laboratories performed substrate depletion experiments with cyclohexyl salicylate, fenthion, 4-n-nonylphenol, deltamethrin, methoxychlor, and pyrene using cryopreserved hepatocytes and liver S9 fractions from trout. Variability within and among laboratories was characterized as the percent coefficient of variation (CV) in measured in vitro intrinsic clearance rates (CLIN VITRO, INT; ml/h/mg protein or 106 cells) for each chemical and test system. Mean intralaboratory CVs for each test chemical averaged 18.9% for hepatocytes and 14.1% for S9 fractions, whereas interlaboratory CVs (all chemicals and all tests) averaged 30.1% for hepatocytes and 22.4% for S9 fractions. When CLIN VITRO, INT values were extrapolated to in vivo intrinsic clearance estimates (CLIN VIVO, INT; l/d/kg fish), both assays yielded similar levels of activity (<4-fold difference for all chemicals). Hepatic clearance rates (CLH; l/d/kg fish) calculated using data from both assays exhibited even better agreement. These findings show that both assays are highly reliable and suggest that either may be used to inform chemical bioaccumulation assessments for fish. This study highlights several issues related to the demonstration of assay reliability and may provide a template for evaluating other in vitro biotransformation assays.
Asunto(s)
Técnicas In Vitro/métodos , Oncorhynchus mykiss/metabolismo , Compuestos Orgánicos/farmacocinética , Animales , Biotransformación , Hepatocitos/metabolismo , Interacciones Hidrofóbicas e Hidrofílicas , Hígado/metabolismo , Tasa de Depuración Metabólica , Compuestos Orgánicos/química , Reproducibilidad de los ResultadosRESUMEN
Standard protocols are given for assessing metabolic stability in rainbow trout using the liver S9 fraction. These protocols describe the isolation of S9 fractions from trout livers, evaluation of metabolic stability using a substrate depletion approach, and expression of the result as in vivo intrinsic clearance. Additional guidance is provided on the care and handling of test animals, design and interpretation of preliminary studies, and development of analytical methods. Although initially developed to predict metabolism impacts on chemical accumulation by fish, these procedures can be used to support a broad range of scientific and risk assessment activities including evaluation of emerging chemical contaminants and improved interpretation of toxicity testing results. These protocols have been designed for rainbow trout and can be adapted to other species as long as species-specific considerations are modified accordingly (e.g., fish maintenance and incubation mixture temperature). Rainbow trout is a cold-water species. Protocols for other species (e.g., carp, a warm-water species) can be developed based on these procedures as long as the specific considerations are taken into account.
Asunto(s)
Fase II de la Desintoxicación Metabólica , Fase I de la Desintoxicación Metabólica , Microsomas Hepáticos/efectos de los fármacos , Oncorhynchus mykiss , Pruebas de Toxicidad/métodos , Xenobióticos/toxicidad , Animales , Acuicultura , Bioensayo/métodos , Microsomas Hepáticos/metabolismo , Modelos Animales , Manejo de Especímenes/métodosRESUMEN
Recent regulatory pressures (e.g., REACh, CEPA) requiring bioaccumulation assessments and the need for reduced animal use have increased the necessity for the development of in vitro-based methods to estimate bioaccumulation. Our study explored the potential use of subcellular and cellular hepatic systems to determine the biotransformation potential of two surfactants: octaethylene glycol monohexadecyl ether (C16EO8) and diethylene glycol monotetradecyl ether sulfate (C14EO2S). The subcellular systems tested were liver homogenates and microsomes from the common carp (Cyprinus carpio) and rainbow trout (Oncorhynchus mykiss). Cellular systems consisted of primary hepatocytes from the common carp (C. carpio) and PLHC-1 cells, hepatocarcinoma cells from the desert topminnow (Poeciliopsis lucida) cell line. Each in vitro system was exposed to radiolabeled test compounds and assayed for biotransformation using liquid scintillation and thin layer chromatographic methods. First-order kinetics were used to estimate rates of biotransformation. Bioconcentration of test materials in fish were predicted using an in vitro to in vivo metabolic rate extrapolation model linked to a mass-balance model commonly used to predict bioaccumulation in fish. Both subcellular and cellular tests using microsomes, liver homogenates and hepatocytes respectively showed biotransformation of the parent surfactants. Biotransformation rates were fastest for hepatocytes, followed by microsomes and homogenates. Rates were too low from homogenate tests to extrapolate to in vivo-based biotransformation rates using the extrapolation model. Trout microsomes metabolized C16EO8 faster than carp microsomes, yet rates were approximately the same for C14EO2S. Predicted BCF values incorporating in vitro biotransformation rates from hepatocytes were similar to measured in vivo or USEPA's bioconcentration model (BCFWIN) predicted values. Predicted BCF values using microsomal-based rates from trout and carp studies were only slightly less than default BCF values which assumes a linear logKow to BCF relationship with no biotransformation. However, hepatocyte-based results showed substantially decreased BCFs compared to the default BCF values. These results indicate that BCF estimates based on in vitro metabolic rates can provide reasonable estimates of in vivo BCF values, therefore, supporting the use of in vitro approaches within a tiered approach to assess bioconcentration.
Asunto(s)
Glicoles de Etileno/metabolismo , Peces/metabolismo , Ésteres del Ácido Sulfúrico/metabolismo , Tensoactivos/metabolismo , Contaminantes Químicos del Agua/metabolismo , Animales , Acuicultura , Biotransformación , Carpas/metabolismo , Línea Celular , Glicoles de Etileno/toxicidad , Marcaje Isotópico , Cinética , Microsomas Hepáticos/metabolismo , Oncorhynchus mykiss/metabolismo , Ésteres del Ácido Sulfúrico/toxicidad , Tensoactivos/toxicidad , Contaminantes Químicos del Agua/toxicidadRESUMEN
Developing regulatory activities (e.g., REACh, [DGEE. 2003. Directorates General Enterprise and Environment. The new EU chemicals legislation REACH. DG Enterprise, Brussels, Belgium. (http://www.europa.eu.int/comm/enterprise/reach/index_en.htm)]) will require bioaccumulation to be assessed for thousands of chemicals. Further, there is increasing pressure to reduce, refine or replace animal tests. Given this scenario, there is an urgent need to evaluate the feasibility of in vitro systems to supply data useful for bioaccumulation estimation. Subcellular and cellular hepatic systems were tested to determine the biotransformation of two surfactants: C12-2-LAS (2-phenyl dodecane p-sulfonate) and an alcohol ethoxylate C13EO8 (Octaethylene glycol monotridecyl ether). The subcellular systems tested were liver homogenates and microsomes from the common carp (Cyprinus carpio) and rainbow trout (Oncorhynchus mykiss). Cellular systems consisted of primary hepatocytes from the common carp (Cyprinus carpio) and PLHC-1 cells, hepatocarcinoma cells from the desert topminnow (Poeciliopsis lucida). All in vitro systems were exposed to radiolabeled test compounds and assayed for biotransformation using liquid scintillation and thin layer chromatographic methods. First-order kinetics were used to estimate rates of biotransformation. Bioconcentration of test materials in fish were predicted using an in vitro to in vivo metabolic rate extrapolation model linked to a mass-balance model commonly used to predict bioaccumulation in fish. Subcellular biotransformation rates for each of the surfactants were greatest with microsomes. Cellular loss rates exceeded subcellular rates, leading to lower predicted BCF values. Predicted BCFs corresponded closely to measured values in several fish species, verifying the utility of in vitro systems in refining Kow-only-based BCFs via the inclusion of biotransformation rates.
Asunto(s)
Alcoholes/farmacocinética , Ácidos Alcanesulfónicos/farmacocinética , Carpas/metabolismo , Oncorhynchus mykiss/metabolismo , Tensoactivos/farmacocinética , Animales , Biotransformación , Línea Celular , Supervivencia Celular/efectos de los fármacos , Cromatografía en Capa Delgada , Hepatocitos/metabolismo , Cinética , Microsomas Hepáticos/metabolismo , Fracciones Subcelulares/metabolismoRESUMEN
National and international chemical management programs are assessing thousands of chemicals for their persistence, bioaccumulative and environmental toxic properties; however, data for evaluating the bioaccumulation potential for fish are limited. Computer based models that account for the uptake and elimination processes that contribute to bioaccumulation may help to meet the need for reliable estimates. One critical elimination process of chemicals is metabolic transformation. It has been suggested that in vitro metabolic transformation tests using fish liver hepatocytes or S9 fractions can provide rapid and cost-effective measurements of fish metabolic potential, which could be used to refine bioconcentration factor (BCF) computer model estimates. Therefore, recent activity has focused on developing in vitro methods to measure metabolic transformation in cellular and subcellular fish liver fractions. A method to extrapolate in vitro test data to the whole body metabolic transformation rates is presented that could be used to refine BCF computer model estimates. This extrapolation approach is based on concepts used to determine the fate and distribution of drugs within the human body which have successfully supported the development of new pharmaceuticals for years. In addition, this approach has already been applied in physiologically-based toxicokinetic models for fish. The validity of the in vitro to in vivo extrapolation is illustrated using the rate of loss of parent chemical measured in two independent in vitro test systems: (1) subcellular enzymatic test using the trout liver S9 fraction, and (2) primary hepatocytes isolated from the common carp. The test chemicals evaluated have high quality in vivo BCF values and a range of logK(ow) from 3.5 to 6.7. The results show very good agreement between the measured BCF and estimated BCF values when the extrapolated whole body metabolism rates are included, thus suggesting that in vitro biotransformation data could effectively be used to reduce in vivo BCF testing and refine BCF model estimates. However, additional fish physiological data for parameterization and validation for a wider range of chemicals are needed.
Asunto(s)
Carpas , Modelos Biológicos , Oncorhynchus mykiss , Amidas/metabolismo , Animales , Biotransformación , Células Cultivadas , Cloropirifos/metabolismo , Femenino , Glicolatos/metabolismo , Hepatocitos/metabolismo , Masculino , Plaguicidas/metabolismo , Polietilenglicoles/metabolismoRESUMEN
The feasibility of using in vitro methods to predict in vivo critical body residue (CBR) for single surfactants and mixtures by measuring the critical cell residue (CCR) in a hepatic fish cell line (PLHC-1C) was investigated. The CCR values were determined using radiochemical methods to measure the test compound partitioning between media and cells at varying concentrations for three distinctly different surfactants (anionic, cationic, and nonionic) and their mixture. The cell median effective concentration (EC50) values for hexadecyltrimethylammonium chloride (C16TMAC), dodecyl hexaethoxylate (C12E6), and sodium dodecylbenzene sulfonate (C12LAS) ranged from 2.9 to 163.3 microM, a 54-fold difference. These cell EC50 values indicate that the cells are several-fold less sensitive to surfactants than whole organisms are. However, based on cellular residue levels for each surfactant and their mixture, only an approximately threefold difference was observed with a range of 0.6 to 1.8 mmol/kg. These concentrations correspond closely to in vivo body burdens (0.2-8 mmol/kg) associated with nonpolar organic or narcosis-acting compounds and their mixtures. The CCRs could provide an alternative and rapid technique to predict CBRs.