Solid phase microextraction (SPME) is an extraction technique that uses a polymer-coated fiber as the extraction device. After extraction, the compound of interest can be desorbed from the fiber and subsequently analyzed by GC or HPLC. One of the properties of SPME is that only the freely dissolved fraction of a chemical is available for partitioning to the extraction device. The method can be applied in a way that small amounts are extracted from the sample, which allows negligible depletion extraction. These two properties make SPME devices particularly suitable for measurements of free concentrations. In toxicological studies the free concentration is considered to be a more relevant parameter, concerning toxic effects, than the nominal concentration that is used most frequently. In the current study, the usefulness of this method to measure phospholipid/water partition coefficients and free concentrations in three different in vitro test systems (rat hepatocytes in primary culture, 9000 g and 100,000 g homogenate fractions of rainbow trout liver) was demonstrated. Results show separate relationships between phospholipid/water and n-octanol/water partition coefficients for a set of polar and nonpolar organic chemicals, respectively. These observations suggest that phospholipid/water partition coefficients may be a more suitable parameter in modeling the kinetic behavior of organic chemicals. Additionally, differences between the nominal and the actual free concentration in in vitro systems are more pronounced for more hydrophobic compounds, as was expected based on theoretical considerations. To our knowledge, the approach presented here is the first analytical method to measure toxicologically relevant concentrations in in vitro test systems in a fast and efficient way.

Estrogenic potencies of several xenoestrogens were determined in vitro, using cultured hepatocytes from a genetically uniform male carp strain (Cyprinus carpio). Estrogenicity was measured as induction of the yolk protein precursor vitellogenin (Vtg), and compared to Vtg induction by 17beta-estradiol (E2). The order of estrogenic potency was: methoxychlor (MXCL) > o,p-DDT > chlordecone approximately/= bisphenol-A approximately/= 4-t-pentylphenol. Estrogenic potencies of these compounds varied from 1 x 10(-3) to 1 x 10(-4) relative to E2. The synthetic estrogen DES had a relative estrogenic potency of 0.5, whereas dieldrin, beta-endosulfan, o,p-DDE, and toxaphene (technical mixture) did not induce vitellogenesis at concentrations up to 100 microM. Experiments in which cells were simultaneously exposed to E2 and these xenoestrogens showed that the Vtg-inducing activities of E2 and 4-t-pentylphenol or bisphenol-A were (partially) additive, whereas E2 antagonized the estrogenic effects of MXCL and o,p-DDT. The effect of cytochrome P4501A (CYP1A)-induction on the estrogenicity of MXCL was studied by co-exposing cells to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). TCDD (10 pM) caused a greater than 50-fold induction of CYP1A, measured as ethoxyresorufin O-deethylase (EROD) activity, but Vtg induction by MXCL was not significantly affected. This indicates that CYP1A is not involved in the bioactivation of MXCL to more potent estrogenic metabolites in carp. The CARP-HEP (hepatocyte) assay can detect xenoestrogens with a potency > or = 2 x 10(-5) relative to E2. It allows simultaneous testing of more than 10 compounds for both estrogenic and antiestrogenic effects, which makes it a promising tool for the screening of suspected xenoestrogens.

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Vitellogenin (vtg) is a precursor of the yolk proteins lipovitelline and phosvitin and is synthesized as a consequence of estrogen-dependent gene expression in female and male hepatocytes of egg-laying vertebrates. Freshly isolated carp hepatocytes of a genetically uniform strain of adult male carp (Cyprinus carpio) were used to investigate the effects of primary exposure to estrogenic compounds on the vitellogenic response to xenoestrogens. Isolated carp hepatocytes were first exposed (primary exposure) to 50 or 100 microM of either methoxychlor (MXCL) or bisphenol A (BPA), different concentrations of estrone (E1; 1 or 10 nM) or 17beta-estradiol (E2; 0.1 or 1 nM) for 2 days. Hepatocytes were exposed to xenoestrogens (secondary exposure) at both 2 and 5 days after isolation. Hepatocytes were cultured for a total period of 8 days. A competitive indirect ELISA was used to determine the level of vtg after 8 days. The concentration of chemicals used for the primary exposure induced vtg to a level that was less than 10% of the response elicited by E2 (1000 nM). A cytotoxic response, measured by MTT, was not observed after primary exposure to any of the xenoestrogens. After primary exposure to MXCL, vtg production in response to E2 was increased by 4-fold, and vitellogenesis in response to E1 treatment was doubled compared with vitellogenesis without pretreatment. No significant differences were observed between primary exposure to 50 and 100 microM MXCL. Primary exposure to 50 and 100 microM BPA increased the maximum vtg production in response to secondary E2 exposure by about 5- and 7-fold, respectively. Primary exposure to BPA (50 and 100 microM) followed by secondary exposure to E1 showed a 4- and 5-fold increase of the vtg synthesis in comparison to E1 exposure alone. Primary exposure to the endogenous estrogens had no significant influence on the vtg synthesis in response to secondary exposure to E1 or E2. Compared to hepatocytes exposed only to MXCL or BPA, primary exposure to E2 increased the vtg synthesis in hepatocytes induced by MXCL or BPA by almost a factor of 2. Primary exposure to E1 increased vitellogenesis after secondary exposure to MXCL only marginally. The present results indicate that weakly estrogenic environmental chemicals such as MXCL and BPA can increase the sensitivity of carp hepatocytes towards endogenous estrogens with respect to VTG synthesis.

The cytotoxic events in freshly isolated rat hepatocytes following exposure over 2 h to menadione (2-methyl-1,4-naphthoquinone) and two closely related quinones, 2,3-dimethyl-1,4-naphthoquinone (DMNQ) and 1,4-naphthoquinone (NQ), were examined. These quinones differ in their arylation capacity (NQ > menadione >> DMNQ) and in their potential to induce redox cycling (NQ approximately menadione >> DMNQ) The glutathione status (reduced and oxidized glutathione) of the hepatocytes was determined using HPLC after derivatization with monobromobimane. Protein thiols were measured spectrophotometrically and the energy charge of the cells was determined with HPLC using ion pair chromatography. The leakage of lactate dehydrogenase was used as a marker for cell viability. All three quinones caused alterations of the glutathione status of the exposed cells but the effects were markedly different. Exposure to DMNQ resulted in a slow decrease of reduced glutathione and an increase of mixed disulfides. The other two quinones caused an almost complete depletion of reduced glutathione within 5 min. Hepatocytes exposed to NQ accumulated oxidized glutathione whereas menadione-exposed hepatocytes showed increased levels of mixed disulfides. We did not find any effects of DMNQ (200 microM) on protein thiols, energy charge or cell viability. There was a clear difference in the effects of menadione and NQ on protein thiols, energy charge and cell viability; exposure to NQ resulted in a more extensive decrease of protein thiols and energy charge and an earlier onset of lactate dehydrogenase leakage.(ABSTRACT TRUNCATED AT 250 WORDS)