Development of a Partition-Controlled Dosing System for Cell Assays

Abstract

Hydrophobic and volatile chemicals have proven to be difficult to dose in cell assays. Cosolvents are often needed to dissolve these chemicals in cell culture medium. Moreover, the free concentration of these chemicals in culture medium may diminish over time due to metabolism, evaporation, and nonspecific binding to well plate surfaces and serum constituents. The aim of this study was to develop a partitioncontrolled dosing system to maintain constant concentrations of benzo(a)pyrene, 1,2-dichlorobenzene, and 1,2,4-trichlorobenzene in an ethoxyresorufin-O-deethylase (EROD) assay and a cytotoxicity assay with the rainbow trout (Oncorhynchus mykiss) cell lines RTL-W1 and RTgill-W1. Polydimethylsiloxane (PDMS) sheets were loaded with test chemicals in a spiked methanol/water solution and placed in the wells, filled with culture medium, of a 24-well culture plate. Cells were grown on inserts and were subsequently added to the wells with the PDMS sheets. The system reached equilibrium within 24 h, even for the very hydrophobic chemical benzo(a)pyrene. The reservoir of test chemical in PDMS was large enough to compensate for the loss of >95% of the test chemical from the culture medium. The PDMS sheets maintained medium concentrations constant for >72 h. Nominal median effect concentrations (EC 50 ) were 1.3-7.0 times lower in the partition-controlled dosing systems than in conventional assays spiked using dimethyl sulfoxide (DMSO) as a carrier solvent, thus indicating that the apparent sensitivity of the bioassay increased when controlled and constant exposure conditions could be assured. The EC 50 values of the test chemicals based on free concentrations were estimated in the partition-controlled dosing systems using measured PDMS-bare culture medium partition coefficients. Results indicated that 61, 70, and 99.8% of 1,2-diclorobenzene, 1,2,4-trichlorobenzene, and benzo(a)pyrene were bound to serum constituents in the culture medium.

A high throughput passive dosing format for the Fish Embryo Acute Toxicity test

“…Smith et al (2010a) used glass test vessels cast with a layer of PDMS as a passive dosing reservoir for exposure of Daphnia magna. Kramer et al (2010) placed PDMS sheets on the bottoms of the wells of a 24-well plate, for exposure of fish cell lines that were grown on membranes of well plate inserts, and modelled free medium concentrations. Recently, two studies applied passive dosing for exposing zebrafish early life stages to HOCs.…”

The Control of Hydrophobic Compound Exposure in In Vitro Tests for Genotoxicity

“…These include general toxicity in the Microtox bacterial assay [ 18 ], immunotoxic effects in human cell lines [ 9 ], cytotoxicity and EROD activity in rainbow trout cell lines [ 24 ], dioxin-like activity in the DR-Calux ® assay [ 25 ], as well as mutagenicity in the Ames II assay [ 23 , 26 ]. These studies have all shown the utility of passive dosing for providing rigorously defi ned and stable C Free exposure concentrations, leading to an increase in the apparent test sensitivity when compared to conventional solvent spiking [ 23 -26 ].…”

“…Kramer et al [ 24 ] used 0.177 mL silicone disks to passively dose 1.7 mL medium, and Booij et al [ 25 ] cast 0.01-0.1 mL PDMS silicone into the base of culture wells for dosing 2 mL medium. For compounds with K Silicone/Free values below 1,000, the selection of a suitable ratio is more critical since the HOC mass distribution is shifted towards the aqueous phase, and reduced V Water / V Silicone ratios are needed to avoid depletion (see ( 2 )).…”

The use of biomarkers of toxicity for integrating in vitro hazard estimates into risk assessment for humans

“…Since the freely available concentration usually is the driving force for kinetic processes, as well as toxic reactions on the (sub-) cellular level, these processes will influence the free concentration and thus the effect . It is therefore necessary to estimate or measure this free concentration, especially when it is expected that the free concentration will differ from the nominal concentration (on the basis of known physico-chemical properties such as lipophilicity) (Gülden and Seibert, 2003;Heringa et al, 2004;Kramer et al, 2010Kramer et al, , 2012.…”