Physicochemical substance properties as indicators for unreliable exposure in microplate-based bioassays

Riedl, Janet; Altenburger, Rolf

doi:10.1016/j.chemosphere.2006.12.022

Cited by 73 publications

(91 citation statements)

References 23 publications

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“…Use of different plate formats with different volume to surface ratios may thus introduce confounding bioassay factors such as differences in sorption to and possible interaction with the plastic surface (Schreiber et al, 2008). Such confounding factors may potentially affect the freely dissolved concentration of hydrophobic chemicals (log Kow >3) in the bioassay and result in overestimation of the chemicals actual concentration in the assay (Brown et al, 2001;Mayer et al, 1999;Riedl and Altenburger, 2007). Despite potential for such confounding factors, no significant difference in Vtg protein production was observed in assays run in 6, 24 or 96 well formats with different total surface-volume ratio after 48 and 96 h of exposure.…”

Section: Plate Formatmentioning

confidence: 97%

Evaluation of the sensitivity, responsiveness and reproducibility of primary rainbow trout hepatocyte vitellogenin expression as a screening assay for estrogen mimics

2015

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Vitellogenin (Vtg) induction in primary fish hepatocytes has been proposed as an in vitro screening assay for ER agonists and antagonists, but has not yet been used extensively as a high-throughput screening tool due to poor reproducibility, sensitivity and overall feasibility. The present work has evaluated the role of seasonal variation, normalization, optimal culture and assay conditions on the sensitivity, responsiveness and reproducibility of in vitro vtg gene mRNA and protein expression in rainbow trout (Oncorhynchus mykiss) primary hepatocytes using the xenoestrogen 17α-ethynylestradiol (EE2) as a test chemical. The results show that primary hepatocytes display a relatively high individual and seasonal variation in both Vtg mRNA and protein induction potential, although less variance was observed in assay sensitivity. Data normalization of assay response to maximum (3 nM EE2) and minimum (DMSO) Vtg production dramatically reduced this variance and led to improved assay reproducibility. A time-dependent response was observed both for mRNA and protein expression, reaching maximum Vtg induction after 96 h of exposure, although reproducible concentration response curves for both Vtg mRNA and protein could be obtained already after 48 h. A need for chemical re-exposure of the hepatocytes was identified to be important for sustaining exposure concentrations in extended studies (>48 h), whereas different plate formats (96, 24 or 6 wells) did not affect the bioassay performance. In conclusion, standardization of hepatocyte bioassay and test conditions as well as data-normalization procedures are proposed to be instrumental for more consistent and comparable results in future use of this in vitro assay.

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Section: Plate Formatmentioning

confidence: 97%

Evaluation of the sensitivity, responsiveness and reproducibility of primary rainbow trout hepatocyte vitellogenin expression as a screening assay for estrogen mimics

2015

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“…Traditionally, the effect concentration obtained from a cell assay is based on the nominal concentration of a test chemical added to the culture medium at the start of the exposure period. However, studies have found volatile chemicals to significantly evaporate from microtiter plates, resulting in higher effect concentrations observed in microtiter plates than in sealed flasks (6,7). Studies have also found that serum protein in culture medium, microtiter plate plastic, and cell lipids significantly bind and reduce the free concentration of hydrophobic test chemicals in cell assays (8)(9)(10)(11)(12)(13)(14).…”

Section: Introductionmentioning

confidence: 99%

Development of a Partition-Controlled Dosing System for Cell Assays

Kramer

Busser

Oosterwijk

et al. 2010

Chem. Res. Toxicol.

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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.

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“…Since the 1990s, many researchers have attempted to optimize the procedure, performing tests at microscale using cuvettes, scintillation tubes, or microplates (Arensberg et al, 1995;Nguyen-Ngoc et al, 2009;Paixao et al, 2008;Rojiekova et al, 1998). The 24-or 96-well algal growth inhibition assay performed in a microplate, gave comparable results to standard flask bioassay for the tested algae (Thellen et al, 1990;Rojiekova et al, 1998;Eisentraeger et al, 2003;Horvatic et al, 2007;Riedl and Altenburger, 2007). This simpler method has been used in routine toxicity testing, pollutant phytotoxicity screening, and screening of sensitivity of algae.…”

Section: Introductionmentioning

confidence: 79%

Probabilistic risk assessment of diuron and prometryn in the Gwydir River catchment, Australia, with the input of a novel bioassay based on algal growth

Shi

Burns

Ritchie

et al. 2014

Ecotoxicology and Environmental Safety

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a b s t r a c tA probabilistic risk assessment of the selected herbicides (diuron and prometryn) in the Gwydir River catchment was conducted, with the input of the EC 50 values derived from both literature and a novel bioassay. Laboratory test based on growth of algae exposed to herbicides assayed with a microplate reader was used to examine the toxicity of diuron and prometryn on the growth of Chlorella vulgaris. Both herbicides showed concentration dependent toxicity in inhibiting the growth of Chlorella during the exposure period of 18-72 h. Diuron caused more toxicity as judged by growth rates than prometryn. Thalaba Creek at Merrywinebone was identified as the 'hotspot' for diuron and prometryn risk in the Gwydir catchment. The use of microplate assays coupled with probabilistic risk assessment is recommended for rapid assessment of ecotoxicity of indigenous species, allowing identification of locations in river catchments requiring environmental management.

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Physicochemical substance properties as indicators for unreliable exposure in microplate-based bioassays

Cited by 73 publications

References 23 publications

Evaluation of the sensitivity, responsiveness and reproducibility of primary rainbow trout hepatocyte vitellogenin expression as a screening assay for estrogen mimics

Evaluation of the sensitivity, responsiveness and reproducibility of primary rainbow trout hepatocyte vitellogenin expression as a screening assay for estrogen mimics

Development of a Partition-Controlled Dosing System for Cell Assays

Probabilistic risk assessment of diuron and prometryn in the Gwydir River catchment, Australia, with the input of a novel bioassay based on algal growth

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