Impact of protein binding on the availability and cytotoxic potency of organochlorine pesticides and chlorophenols in vitro

Gülden, Michael; Mörchel, Sabine; Tahan, Sail; Seibert, Hasso

doi:10.1016/s0300-483x(02)00085-9

Cited by 107 publications

(87 citation statements)

References 24 publications

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“…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). Changes in cell assay setup, such as changes in serum levels and cell concentrations, may therefore alter the observed nominal effect concentration (10)(11)(12)(13)(14). Moreover, cosolvents are normally used to dose poorly soluble test chemicals in cell assays.…”

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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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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“…The chronic reduction of the GABA A receptor function was afforded by the use of a nominal concentration of 3 lM dieldrin during the long-term exposure of the culture. Dieldrin binds to serum proteins and lipids, and the free concentration of dieldrin would be reduced by this effect (Gulden et al, 2002(Gulden et al, , 2006. For this reason, although the potency of dieldrin to inhibit the GABA-induced chloride influx in cerebellar granule cells was previously reported (IC 50 5 0.2 lM; Vale et al 2003), we had to test which concentration of Long-term exposure Control 4.5 6 2.0 108 6 7 3 lM dieldrin 6.6 6 1.6 67 6 5* Acute exposure Control 12.4 6 4.9 110 6 4 3 lM dieldrin 13.6 6 3.0 110 6 24 y The values of intracellular calcium concentration for each individual experiment were normalized to the maximum response induced by 100 lM NMDA (Babot et al, 2005) in control cultures.…”

Section: Discussionmentioning

confidence: 99%

“…5). Because dieldrin is a highly lipophilic compound, some dieldrin may remain bound to the cell surface after removing the culture medium (Gulden et al, 2002). Therefore, we rinsed the cells with HBSS containing 2% BSA to wash out dieldrin completely (Llorens et al, 1990) before exposing the cultures to the excitotoxic stimulus.…”

Section: Long-term Exposure To Dieldrin Prevents the Excitotoxic Effementioning

confidence: 99%

Long‐term exposure to dieldrin reduces γ‐aminobutyric acid type A and N‐methyl‐D‐aspartate receptor function in primary cultures of mouse cerebellar granule cells

Babot

Vilaró

Suñol

2007

J of Neuroscience Research

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The organochlorine pesticide dieldrin is a persistent organic pollutant that accumulates in the fatty tissue of living organisms. In mammals, it antagonizes the GABAA receptor, producing convulsions after acute exposure. Although accumulation in human brain has been reported, little is known about the effects of long‐term exposure to dieldrin in the nervous system. Homeostatic control of the balance between excitation and inhibition has been reported when neuronal activity is chronically altered. We hypothesized that noncytotoxic concentrations of dieldrin could decrease glutamatergic neurotransmission as a consequence of a prolonged reduction in GABAA receptor function. Long‐term exposure of primary cerebellar granule cell cultures to 3 μM dieldrin reduced the GABAA receptor function to 55% of control, as measured by the GABA‐induced 36Cl− uptake. This exposure produced a significant reduction (∼35%) of the NMDA‐induced increase in [Ca2+]i and of the [3H]MK‐801 binding, which was not accompanied by a reduction in the NMDA receptor subunit NR1, as determined by Western blot. Consistent with the decreased NMDA receptor function, dieldrin‐treated cultures were insensitive to an excitotoxic stimulus induced by exposure to high potassium. In summary, we report that the chronic reduction of GABAA receptor function induced by dieldrin decreases the number of functional NMDA receptors, which may be attributable to a mechanism of synaptic scaling. These effects could underlie neural mechanisms involved in cognitive impairment produced by low‐level exposure to dieldrin. © 2007 Wiley‐Liss, Inc.

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“…Our major conclusion is that in the case of SR, the structure of biomembrane and the presence of membrane proteins do not alter partitioning of TeCP into the biomembrane and that TeCP resides entirely within the lipid matrix of SR. Our data suggest that TeCP does not partition into SR proteins. This observation is important since chlorinated phenols, including TeCP, are expected to bind to bovine serum albumin (Gulden, et al 2003). …”

Section: Partitioning Of Tecp Between Water and Sarco-plasmic Reticulmentioning

confidence: 99%

Partitioning of Tetrachlorophenol into Lipid Bilayers and Sarcoplasmic Reticulum: Effect of Length of Acyl Chains, Carbonyl Group of Lipids and Biomembrane Structure

Word

Smejtek

2005

J Membrane Biol

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Abstract. We report results of a partitioning study of 2, 3,4,. In the study we explored (1) the effect of the length of acyl chains of lipids (C16:1 -C24:1) and alkanes (C6-C16), (2) the role of the carbonyl group of lipids, and (3) the effect of molecular structure of the sarcoplasmic reticulum membrane on TeCP partitioning. Mole fraction partition coefficients have been measured using equilibrium dialysis for un-ionized (HA), and ionized (A) species, Kp x HA , Kp x A . Their values are concentrationdependent. Partition coefficients were analyzed in terms of a model that accounts for saturation of membrane associated with the finite area of partition site, and electrostatic interactions of (A-) species with charged membrane. Limiting values of partition coefficients, corresponding to infinite dilution of solute, Kp x0 HA , Kp x0 A were obtained. Kp x0 HA and Kp x0 A measure the strength of solute-membrane interactions. Studies were done with single-layered vesicles of lipids with variable chain length: 1,2-dipalmitoleoyl-sn-glycero-3-phosphocholine (C16:1), 1,2-dioleoyl-sn-glycero-3-phosphocholine (C18:1), 1,2-dierucoyl-sn-glycero-3-phosphocholine (C22:1), and 1 ,2-dinervonoyl-sn-glycero-3-phosphocholine (C24:1), and egg-PC. Kp x0 for transfer of TeCP from water into lipid membranes was found to be independent of the length of acyl chains, whereas Kp x0 for transfer from water into alkanes increased with the length of alkane. The effect of the carbonyl CO group of lipids on partitioning was measured using 1,2-di-o-octadecenyl-sn-glycero-3-phosphocholine (CO absent) and 1,2-dioleoyl-sn-glycero-3-phosphocholine (CO present) liposomes. Carbonyl groups, known to change dipolar potential, had no effect on partitioning. Partition coefficients of unionized and ionized forms of TeCP were invariant to the presence of proteins and other membrane components of sarcoplasmic reticulum (SR) membrane.

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Impact of protein binding on the availability and cytotoxic potency of organochlorine pesticides and chlorophenols in vitro

Cited by 107 publications

References 24 publications

Development of a Partition-Controlled Dosing System for Cell Assays

Development of a Partition-Controlled Dosing System for Cell Assays

Long‐term exposure to dieldrin reduces γ‐aminobutyric acid type A and N‐methyl‐D‐aspartate receptor function in primary cultures of mouse cerebellar granule cells

Partitioning of Tetrachlorophenol into Lipid Bilayers and Sarcoplasmic Reticulum: Effect of Length of Acyl Chains, Carbonyl Group of Lipids and Biomembrane Structure

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