Genotoxicity of unmodified and organo-modified montmorillonite

Anoop Kumar Sharma, Bjørn Schmidt, Henrik Lauritz Frandsen, N.R. Jacobsen, Erik Huusfeldt Larsen, Mona-Lise Binderup

Research output: Contribution to journalJournal articleResearchpeer-review

Abstract

The natural clay mineral montmorillonite (Cloisite (R) Na+) and an organo-modified montmorillonite (Cloisite (R) 30B) were investigated for genotoxic potential as crude suspensions and as suspensions filtrated through a 0.2-mu m pore-size filter to remove particles above the nanometre range. Filtered and unfiltered water suspensions of both clays did not induce mutations in the Salmonella/microsome assay at concentrations up to 141 mu g/ml of the crude clay, using the tester strains TA98 and TA100. Filtered and unfiltered Cloisite (R) Na+ suspensions in culture medium did not induce DNA strand-breaks in Caco-2 cells after 24 h of exposure, as tested in the alkaline comet assay. However, both the filtered and the unfiltered samples of Cloisite (R) 30B induced DNA strand-breaks in a concentration-dependent manner and the two highest test concentrations produced statistically significantly different results from those seen with control samples (p <0.01 and p <0.001) and (p <0.05 and p <0.01), respectively. The unfiltered samples were tested up to concentrations of 170 mu g/ml and the filtered samples up to 216 mu g/ml before filtration. When tested in the same concentration range as used in the comet assay, none of the clays produced ROS in a cell-free test system (the DCFH-DA assay). Inductively coupled plasma mass-spectrometry (ICP-MS) was used to detect clay particles in the filtered samples using aluminium as a tracer element characteristic to clay. The results indicated that clay particles were absent in the filtered samples, which was independently confirmed by dynamic light-scattering measurements. Detection and identification of free quaternary ammonium modifier in the filtered sample was carried out by HPLC-Q-TOF/MS and revealed a total concentration of a mixture of quaternary ammonium analogues of 1.57 mu g/ml. These findings suggest that the genotoxicity of organo-modified montmorillonite was caused by the organo-modifier. The detected organo-modifier mixture was synthesized and comet-assay results showed that the genotoxic potency of this synthesized organo-modifier was in the same order of magnitude at equimolar concentrations of organo-modifier in filtrated Cloisite (R) 30B suspensions, and could therefore at least partly explain the genotoxic effect of Cloisite (R) 30B.
Original languageEnglish
JournalMutation Research - Genetic Toxicology and Environmental Mutagenesis
Volume700
Issue number1-2
Pages (from-to)18-25
ISSN1383-5718
DOIs
Publication statusPublished - 2010

Keywords

  • Salmonella/microsome assay
  • Cytotoxicity
  • ICP-MS
  • ROS
  • Nanoclays
  • Quaternary ammonium compound
  • Alkaline comet assay

Cite this

Sharma, Anoop Kumar ; Schmidt, Bjørn ; Frandsen, Henrik Lauritz ; Jacobsen, N.R. ; Larsen, Erik Huusfeldt ; Binderup, Mona-Lise. / Genotoxicity of unmodified and organo-modified montmorillonite. In: Mutation Research - Genetic Toxicology and Environmental Mutagenesis. 2010 ; Vol. 700, No. 1-2. pp. 18-25.
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abstract = "The natural clay mineral montmorillonite (Cloisite (R) Na+) and an organo-modified montmorillonite (Cloisite (R) 30B) were investigated for genotoxic potential as crude suspensions and as suspensions filtrated through a 0.2-mu m pore-size filter to remove particles above the nanometre range. Filtered and unfiltered water suspensions of both clays did not induce mutations in the Salmonella/microsome assay at concentrations up to 141 mu g/ml of the crude clay, using the tester strains TA98 and TA100. Filtered and unfiltered Cloisite (R) Na+ suspensions in culture medium did not induce DNA strand-breaks in Caco-2 cells after 24 h of exposure, as tested in the alkaline comet assay. However, both the filtered and the unfiltered samples of Cloisite (R) 30B induced DNA strand-breaks in a concentration-dependent manner and the two highest test concentrations produced statistically significantly different results from those seen with control samples (p <0.01 and p <0.001) and (p <0.05 and p <0.01), respectively. The unfiltered samples were tested up to concentrations of 170 mu g/ml and the filtered samples up to 216 mu g/ml before filtration. When tested in the same concentration range as used in the comet assay, none of the clays produced ROS in a cell-free test system (the DCFH-DA assay). Inductively coupled plasma mass-spectrometry (ICP-MS) was used to detect clay particles in the filtered samples using aluminium as a tracer element characteristic to clay. The results indicated that clay particles were absent in the filtered samples, which was independently confirmed by dynamic light-scattering measurements. Detection and identification of free quaternary ammonium modifier in the filtered sample was carried out by HPLC-Q-TOF/MS and revealed a total concentration of a mixture of quaternary ammonium analogues of 1.57 mu g/ml. These findings suggest that the genotoxicity of organo-modified montmorillonite was caused by the organo-modifier. The detected organo-modifier mixture was synthesized and comet-assay results showed that the genotoxic potency of this synthesized organo-modifier was in the same order of magnitude at equimolar concentrations of organo-modifier in filtrated Cloisite (R) 30B suspensions, and could therefore at least partly explain the genotoxic effect of Cloisite (R) 30B.",
keywords = "Salmonella/microsome assay, Cytotoxicity, ICP-MS, ROS, Nanoclays, Quaternary ammonium compound, Alkaline comet assay",
author = "Sharma, {Anoop Kumar} and Bj{\o}rn Schmidt and Frandsen, {Henrik Lauritz} and N.R. Jacobsen and Larsen, {Erik Huusfeldt} and Mona-Lise Binderup",
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Genotoxicity of unmodified and organo-modified montmorillonite. / Sharma, Anoop Kumar; Schmidt, Bjørn; Frandsen, Henrik Lauritz; Jacobsen, N.R.; Larsen, Erik Huusfeldt; Binderup, Mona-Lise.

In: Mutation Research - Genetic Toxicology and Environmental Mutagenesis, Vol. 700, No. 1-2, 2010, p. 18-25.

Research output: Contribution to journalJournal articleResearchpeer-review

TY - JOUR

T1 - Genotoxicity of unmodified and organo-modified montmorillonite

AU - Sharma, Anoop Kumar

AU - Schmidt, Bjørn

AU - Frandsen, Henrik Lauritz

AU - Jacobsen, N.R.

AU - Larsen, Erik Huusfeldt

AU - Binderup, Mona-Lise

PY - 2010

Y1 - 2010

N2 - The natural clay mineral montmorillonite (Cloisite (R) Na+) and an organo-modified montmorillonite (Cloisite (R) 30B) were investigated for genotoxic potential as crude suspensions and as suspensions filtrated through a 0.2-mu m pore-size filter to remove particles above the nanometre range. Filtered and unfiltered water suspensions of both clays did not induce mutations in the Salmonella/microsome assay at concentrations up to 141 mu g/ml of the crude clay, using the tester strains TA98 and TA100. Filtered and unfiltered Cloisite (R) Na+ suspensions in culture medium did not induce DNA strand-breaks in Caco-2 cells after 24 h of exposure, as tested in the alkaline comet assay. However, both the filtered and the unfiltered samples of Cloisite (R) 30B induced DNA strand-breaks in a concentration-dependent manner and the two highest test concentrations produced statistically significantly different results from those seen with control samples (p <0.01 and p <0.001) and (p <0.05 and p <0.01), respectively. The unfiltered samples were tested up to concentrations of 170 mu g/ml and the filtered samples up to 216 mu g/ml before filtration. When tested in the same concentration range as used in the comet assay, none of the clays produced ROS in a cell-free test system (the DCFH-DA assay). Inductively coupled plasma mass-spectrometry (ICP-MS) was used to detect clay particles in the filtered samples using aluminium as a tracer element characteristic to clay. The results indicated that clay particles were absent in the filtered samples, which was independently confirmed by dynamic light-scattering measurements. Detection and identification of free quaternary ammonium modifier in the filtered sample was carried out by HPLC-Q-TOF/MS and revealed a total concentration of a mixture of quaternary ammonium analogues of 1.57 mu g/ml. These findings suggest that the genotoxicity of organo-modified montmorillonite was caused by the organo-modifier. The detected organo-modifier mixture was synthesized and comet-assay results showed that the genotoxic potency of this synthesized organo-modifier was in the same order of magnitude at equimolar concentrations of organo-modifier in filtrated Cloisite (R) 30B suspensions, and could therefore at least partly explain the genotoxic effect of Cloisite (R) 30B.

AB - The natural clay mineral montmorillonite (Cloisite (R) Na+) and an organo-modified montmorillonite (Cloisite (R) 30B) were investigated for genotoxic potential as crude suspensions and as suspensions filtrated through a 0.2-mu m pore-size filter to remove particles above the nanometre range. Filtered and unfiltered water suspensions of both clays did not induce mutations in the Salmonella/microsome assay at concentrations up to 141 mu g/ml of the crude clay, using the tester strains TA98 and TA100. Filtered and unfiltered Cloisite (R) Na+ suspensions in culture medium did not induce DNA strand-breaks in Caco-2 cells after 24 h of exposure, as tested in the alkaline comet assay. However, both the filtered and the unfiltered samples of Cloisite (R) 30B induced DNA strand-breaks in a concentration-dependent manner and the two highest test concentrations produced statistically significantly different results from those seen with control samples (p <0.01 and p <0.001) and (p <0.05 and p <0.01), respectively. The unfiltered samples were tested up to concentrations of 170 mu g/ml and the filtered samples up to 216 mu g/ml before filtration. When tested in the same concentration range as used in the comet assay, none of the clays produced ROS in a cell-free test system (the DCFH-DA assay). Inductively coupled plasma mass-spectrometry (ICP-MS) was used to detect clay particles in the filtered samples using aluminium as a tracer element characteristic to clay. The results indicated that clay particles were absent in the filtered samples, which was independently confirmed by dynamic light-scattering measurements. Detection and identification of free quaternary ammonium modifier in the filtered sample was carried out by HPLC-Q-TOF/MS and revealed a total concentration of a mixture of quaternary ammonium analogues of 1.57 mu g/ml. These findings suggest that the genotoxicity of organo-modified montmorillonite was caused by the organo-modifier. The detected organo-modifier mixture was synthesized and comet-assay results showed that the genotoxic potency of this synthesized organo-modifier was in the same order of magnitude at equimolar concentrations of organo-modifier in filtrated Cloisite (R) 30B suspensions, and could therefore at least partly explain the genotoxic effect of Cloisite (R) 30B.

KW - Salmonella/microsome assay

KW - Cytotoxicity

KW - ICP-MS

KW - ROS

KW - Nanoclays

KW - Quaternary ammonium compound

KW - Alkaline comet assay

U2 - 10.1016/j.mrgentox.2010.04.021

DO - 10.1016/j.mrgentox.2010.04.021

M3 - Journal article

VL - 700

SP - 18

EP - 25

JO - Mutation Research - Genetic Toxicology and Environmental Mutagenesis

JF - Mutation Research - Genetic Toxicology and Environmental Mutagenesis

SN - 1383-5718

IS - 1-2

ER -