Impact of bioavailability on the correlation between in vitro cytotoxic and in vivo acute fish toxic concentrations of chemicals.

The lower sensitivity of in vitro cytotoxicity assays currently restricts their use as alternative to the fish acute toxicity assays for hazard assessment of chemicals in the aquatic environment. In vitro cytotoxic potencies mostly refer to nominal concentrations. The main objective of the present study was to investigate, whether a reduced availability of chemicals in vitro can account for the lower sensitivity of in vitro toxicity test systems. For this purpose, the bioavailable free fractions of the nominal cytotoxic concentrations (EC50) of chemicals determined with a cytotoxicity test system using Balb/c 3T3 cells and the corresponding free cytotoxic concentrations (ECu50) were calculated. The algorithm applied is based on a previously developed simple equilibrium distribution model for chemicals in cell cultures with serum-supplemented culture media. This model considers the distribution of chemicals between water, lipids and serum albumin. The algorithm requires the relative lipid volume of the test system, the octanol-water partition coefficient (K(ow)) and the in vitro albumin-bound fraction of the chemicals. The latter was determined from EC50-measurements in the presence of different albumin concentrations with the Balb/c 3T3 test system. Organic chemicals covering a wide range of cytotoxic potency (EC50: 0.16-527000 microM) and lipophilicity (logK(ow): -5.0-6.96) were selected, for which fish acute toxicity data (LC50-values) from at least one of the three fish species, medaka, rainbow trout and fathead minnow, respectively, were available. The availability of several chemicals was shown to be extensively reduced either by partitioning into lipids or by serum albumin binding, or due to both mechanisms. Reduction of bioavailability became more important with increasing cytotoxic potency. The sensitivity of the Balb/c 3T3 cytotoxicity assay and the correspondence between in vivo and in vitro toxic potencies were increased when the free cytotoxic concentrations instead of the nominal cytotoxic concentrations were used as measure of cytotoxic potency. The few remaining prominent differences between cytotoxic and acute toxic concentrations can be explained by a more specific mechanism of acute toxic action than basal cytotoxicity. It is concluded that the frequently observed low sensitivity of in vitro cytotoxicity test systems, compared to fish acute toxicity assays, at least in part, can be explained by differences in the availability of chemicals in vitro and in vivo. Moreover, neglecting these differences systematically causes a bias of the correlation between in vivo and in vitro toxic potencies of chemicals. Taking them into account, however, increases the predictivity of the in vitro assays.

[1]  A. Leo,et al.  Substituent constants for correlation analysis in chemistry and biology , 1979 .

[2]  A. Castaño,et al.  Correlations between the RTG-2 cytotoxicity test EC50 and in vivo LC50 rainbow trout bioassay , 1996 .

[3]  H. Babich,et al.  Cytotoxicity and genotoxicity assays with cultured fish cells: A review. , 1991, Toxicology in vitro : an international journal published in association with BIBRA.

[4]  H Seibert,et al.  Factors influencing nominal effective concentrations of chemical compounds in vitro: cell concentration. , 2001, Toxicology in vitro : an international journal published in association with BIBRA.

[5]  H. Segner.  Cytotoxicity Assays with Fish Cells as an Alternative to the Acute Lethality Test with Fish , 2004, Alternatives to laboratory animals : ATLA.

[6]  Philip H. Howard,et al.  Handbook of environmental fate and exposure data for organic chemicals. Volume II: Solvents , 1989 .

[7]  B. Isomaa,et al.  A comparison of the toxicity of 50 reference chemicals to freshly isolated rainbow trout hepatocytes and Daphnia magna , 1994 .

[8]  H. Segner,et al.  Cytotoxicity assays with the rainbow trout R(1) cell line. , 1993, Toxicology in vitro : an international journal published in association with BIBRA.

[9]  H Seibert,et al.  Factors influencing nominal effective concentrations of chemical compounds in vitro: medium protein concentration. , 2002, Toxicology in vitro : an international journal published in association with BIBRA.

[10]  K. Fent,et al.  Ecotoxicology of organotin compounds. , 1996, Critical reviews in toxicology.

[11]  Robert I. Krieger,et al.  HANDBOOK OF PESTICIDE TOXICOLOGY , 2001 .

[12]  Joop L. M. Hermens,et al.  Solid phase microextraction as a tool to determine membrane/water partition coefficients and bioavailable concentrations in in vitro systems. , 1997, Chemical research in toxicology.

[13]  W. E. Lindup.  Plasma protein binding of drugs: some basic and clinical aspects , 1987 .

[14]  K. Netter.  Progress in drug metabolism , 1985 .

[15]  Hasso Seibert,et al.  In vitro-in vivo extrapolation: estimation of human serum concentrations of chemicals equivalent to cytotoxic concentrations in vitro. , 2003, Toxicology.

[16]  W. Giger,et al.  Partitioning of alkylphenols and alkylphenol polyethoxylates between water and organic solvents , 1993 .

[17]  Hasso Seibert,et al.  Impact of protein binding on the availability and cytotoxic potency of organochlorine pesticides and chlorophenols in vitro. , 2002, Toxicology.

[18]  H. Babich,et al.  Structure-activity relationship (SAR) models established in vitro with the neutral red cytotoxicity assay. , 1987, Toxicology in vitro : an international journal published in association with BIBRA.

[19]  K. Fent.  Fish cell lines as versatile tools in ecotoxicology: assessment of cytotoxicity, cytochrome P4501A induction potential and estrogenic activity of chemicals and environmental samples. , 2001, Toxicology in vitro : an international journal published in association with BIBRA.

[20]  O. Wassermann,et al.  Bovine spermatozoa as anin vitro model for studies on the cytotoxicity of chemicals: effects of chlorophenols , 1989, Cell Biology and Toxicology.

[21]  M. Gülden,et al.  Comparison of mammalian and fish cell line cytotoxicity: impact of endpoint and exposure duration. , 2005, Aquatic toxicology.

[22]  Hasso Seibert,et al.  Serum albumin binding at cytotoxic concentrations of chemicals as determined with a cell proliferation assay. , 2003, Toxicology letters.

[23]  F. Yamauchi,et al.  In vitro cytotoxicity of chlorophenols to goldfish GF‐scale (GFS) cells and quantitative structure‐activity relationships , 1991 .

[24]  B. Isomaa,et al.  Aquatic Toxicology in Vitro: A Brief Review , 1994 .

[25]  Minne B Heringa,et al.  Toward more useful in vitro toxicity data with measured free concentrations. , 2004, Environmental science & technology.

[26]  H. Segner.  Fish cell lines as a tool in aquatic toxicology. , 1998, EXS.

[27]  O. H. Lowry,et al.  Protein measurement with the Folin phenol reagent. , 1951, The Journal of biological chemistry.

[28]  K. Fent,et al.  Cytotoxicity of organic environmental chemicals to fish liver cells (PLHC-1) , 1996 .