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Pharmacology
ADME Toxicity & Drug Screening
ADME toxicity & drug screening
absorbance
New colorimetric cytotoxicity assay for anticancer-drug screening
J. Natl. Cancer Inst. 82: 1107–1112 (1990).
Philip Skehan, Ritsa Storeng, Dominic Scudiero, Anne Monks, James McMahon, David Vistica, Jonathan T. Warren, Heidi Bokesch, Susan Kenney, and Michael R. Boyd.
Division of Cancer Treatment, National Cancer Institute, Frederick, MD 21701 USA.
Abstract. A rapid, sensitive, simple, and inexpensive method is described for measuring the cellular protein content of adherent and suspension cultures in 96-well microplates. Sulforhodamine B (SRB) is used to stain trichloroacetic acid (TCA) fixed cultures. The optical density of the stain is read in a VMax microplate reader (Molecular Devices Corp.) at 564 nm. The computer-interfaced microplate reader enables error free, high throughput of collected data. The SRB assay is linear with cell number ranging in cell densities from sparse subconfluence to multilayered supraconfluence and is very sensitive (a signal-to-noise ratio of 1.5 with 1000 cells per well). The SRB assay provides a colorimetric endpoint that is nondestructive to the cell architecture for the fixed cells, indefinitely stable, and visible to the naked eye. The method is suitable for routine and very large-scale applications.
In vitro toxicity of various classes of test agents using the neutral red assay on a human three-dimensional physiologic skin model
In Vitro Cell. Dev. Biol. 27A: 239–244 (1991).
Dennis Triglia, Sonia Sherard Braa, Christine Yonan, and Gail K. Naughton.
Marrow-Tech, Inc., La Jolla, California 92037 USA.
Authors describe a new three-dimensional human skin model used to assess in vitro toxicity of test agents from various classes. This model consists of human neonatal foreskin-derived fibroblasts and epidermal keratinocytes grown in layers on a nylon mesh. Cells in this new dermal substrate are metabolically viable and actively dividing. The toxicity test is a modified neutral red viability assay. The endpoint assay was read at 540 nm. Authors obtained dose-dependent toxicity curves for test agents from the following classes: detergents (n = 15), alcohols (n = 5), metal chlorides (n = 10), perfumes and colognes (n = 5), shampoos (n = 4), conditioners (n = 3), moisturizers (n = 3), pesticides (n = 3), and antimicrobial preservatives (n = 4). Comparisons are made between the human skin model toxicity and in vivo ocular irritancy data.
Methods: neutral red toxicity assay. 14-mm disks containing fibroblasts and keratinocytes are placed into 24-well plates and treated with 2 mL of various concentrations of test agents overnight at 37 °C/5% CO2 in a humidified incubator. Media is aspirated and replaced with 1 mL per well of DMEM containing 50 µg/mL neutral red (NR). The plates are incubated for 3 hours at 37 °C/5% CO2 after which NR is aspirated and disks are washed twice with 1 mL PBS containing calcium and magnesium for 3–5 minutes followed by another wash for 1–2 minutes with 1 mL of an aqueous solution containing 0.5% formaldehyde and 1% calcium chloride. The formaldehyde is aspirated and the NR absorbed by the cells is solvent-extracted with 2 mL of 1% acetic acid in 50% aqueous ethanol on a shaker platform for 1 hour. Two hundred microliter aliquots of each of the extracted NR solutions are transferred to a 96-well plate, and the optical density at 540 nm (OD540) of each well is determined spectrophotometrically by a VMax microplate reader (Molecular Devices Corp.) making a blank correction to pretreated nylon mesh (without cells) which have been similarly incubated with NR. The mean OD540 of the untreated control wells is set to represent 100% viability. Results for each concentration are plotted as percent of untreated controls vs. concentration of test agent, and an NR-50 value (concentration of test agent that reduces NR uptake by 50% compared to untreated controls) is determined directly from the graph. The lower the NR-50 value, the more toxic the compound because less is required to achieve 50% killing of the cells on the mesh.
A simple colorimetric assay for phenotyping the major human thermostable phenol sulfotransferase (SULT1A1) using platelet cytosols
Drug Metabolism and Disposition 28: 1063-68 (2000).
Lynn T. Frame1, Shogo Ozawa2, Susan A. Nowell5, Hsien-Chang Chou2, Robert R. DeLongchamp3, Daniel R. Doerge4, Nicholas P. Lang1,5, and Fred F. Kadlubar2,5.
1 Department of Surgery, Central Arkansas Veterans’ Health Care System, Little Rock, Arkansas.
2 Division of Molecular Epidemiology, National Center for Toxicological Research, Jefferson, Arkansas.
3 Division of Biometry, National Center for Toxicological Research, Jefferson, Arkansas.
4 Division of Biochemical Toxicology, National Center for Toxicological Research, Jefferson, Arkansas.
5 University of Arkansas for Medical Sciences, Little Rock, Arkansas.
Abstract. A thermostable phenol sulfotransferase, SULT1A1, has been implicated in numerous detoxification
and bioactivation pathways; however, little is known regarding its endogenous function or its putative role in
mediating risk for human environmental disease. A simple endpoint colorimetric assay is described that can be used
for rapid phenotyping of SULT1A1 activity in human populations. The assay utilizes a microtiter-plate format and
relatively small amounts of platelet cytosol-derived enzyme. The enzyme catalyzes the synthesis of 2-naphthylsulfate
from 2-naphthol and 5´-phosphoadenosine 3´-phosphosulfate (PAPS), whereas addition of p-nitrophenyl
sulfate to the assay contributes to an effective PAPS-regenerating system. In contrast to other sulfotransferase
assay methods, 3´-phosphoadenosine 5´-phosphate (PAP) does not accumulate during the incubation to
interfere with enzyme activity, but instead serves as a cofactor to cause the removal of sulfate from p-nitrophenyl
sulfate to regenerate PAPS. This reaction concomitantly results in generation of p-nitrophenol that can be
quantified colorimetrically at 405 nm (S = 18,200 M-1) to give an indirect measure of sulfotransferase activity.
Using platelet enzyme preparations from adult human subjects, sulfation rates of two prototypical thermostable
phenol sulfotransferase substrates (2-naphthol and p-nitrophenol) and one thermolabile phenol sulfotransferase
substrate (dopamine) were determined using standard radiochemical protocols. These data were then compared with
results from the colorimetric assay using 2-naphthol as substrate. There was a good correlation between the
phenotyping assay and radiochemical assays for both 2-naphthol sulfotransferase and p-nitrophenol sulfotransferase
activity (r = 0.85 and 0.69, respectively). However, SULT1A1 activity was approximately 10 to 20 times higher with
the colorimetric determination. As anticipated, there was no correlation between SULT1A1 activity and dopamine
sulfotransferase activity (r = 0.07) in these human platelet preparations. This inexpensive and rapid method for
phenotyping SULT1A1 activity may help investigators assess a role for this enzyme in disease susceptibility.
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