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Research paper thumbnail of miR-34a suppresses mutagenesis by inducing apoptosis in human lymphoblastoid TK6 cells

a b s t r a c t miR-34a, a tumor suppressor miRNA, has been identified as a direct transcriptiona... more a b s t r a c t miR-34a, a tumor suppressor miRNA, has been identified as a direct transcriptional target of P53. miRNA precursors and inhibitors have been used to modulate the expression of their targeted mRNA and thereby study miRNA functions. We indicated in our previous work that X-ray induces miR-34a expression in a time and dose dependent manner. The objective of this study was to elucidate the role of miR-34a in X-ray-induced mutations in human lymphoblast TK6 cells. Neither over-expression of miR-34a by lipid transfection of miR-34a precursor nor down regulation of endogenous miR-34a by miR-34a inhibitor had any effect on X-ray-induced micronucleus frequency in TK6 cells. Over-expression of miR-34a in TK6 cells significantly reduced X-ray induced mutant frequency (MF) in the Thymidine Kinase (TK) locus while suppression of endogenous miR-34a can increase the background level MF in TK6 cells. Furthermore, over-expression of miR-34a promoted and down-regulation of miR-34a inhibited background and Xray-induced apoptosis in TK6 cells. Our study suggests miR-34a is an important negative regulator of mutagenesis and the mechanism is possibly mediated through apoptosis.

Research paper thumbnail of In vitro toxicological assessment of iron oxide, aluminium oxide and copper nanoparticles in prokaryotic and eukaryotic cell types

Drug and Chemical Toxicology, 2014

ABSTRACT Metallic nanoparticles (NPs) have a variety of applications in different industries incl... more ABSTRACT Metallic nanoparticles (NPs) have a variety of applications in different industries including pharmaceutical industry where these NPs are used mainly for image analysis and drug delivery. The increasing interest in nanotechnology is largely associated with undefined risks to the human health and to the environment. Therefore, in the present study cytotoxic and genotoxic effects of iron oxide, aluminium oxide and copper nanoparticles were evaluated using most commonly used assays i.e. Ames assay, in vitro cytotoxicity assay, micronucleus assay and comet assay. Cytotoxicity to bacterial cells was assessed in terms of colony forming units by using Escherichia coli (gram negative) and Bacillus subtilis (gram positive). Ames assay was carried out using two bacterial strains of Salmonella typhimurium TA98 and TA100. Genotoxicity of these NPs was evaluated following exposure to monkey kidney cell line, CHS-20. No cytotoxic and genotoxic effects were observed for iron oxide, and aluminium oxide NPs. Copper NPs were found mutagenic in TA98 and in TA100 and also found cytotoxic in dose dependent manner. Copper NPs induced significant (p < 0.01) increase in number of binucleated cells with micronuclei (96.6 ± 5.40) at the highest concentration (25 µg/mL). Copper NPs also induced DNA strand breaks at 10 µg/mL and oxidative DNA damage at 5 and 10 µg/mL. We consider these findings very useful in evaluating the genotoxic potential of NPs especially because of their increasing applications in human health and environment with limited knowledge of their toxicity and genotoxicity.

Research paper thumbnail of Genotoxicity of TiO2 anatase nanoparticles in B6C3F1 male mice evaluated using Pig-a and flow cytometric micronucleus assays

Mutation Research/Genetic Toxicology and Environmental Mutagenesis, 2012

In vivo micronucleus and Pig-a (phosphatidylinositol glycan, class A gene) mutation assays were c... more In vivo micronucleus and Pig-a (phosphatidylinositol glycan, class A gene) mutation assays were conducted to evaluate the genotoxicity of 10 nm titanium dioxide anatase nanoparticles (TiO 2 -NPs) in mice. Groups of five 6-7-week-old male B6C3F1 mice were treated intravenously for three consecutive days with 0.5, 5.0, and 50 mg/kg TiO 2 -NPs for the two assays; mouse blood was sampled one day before the treatment and on Day 4, and Weeks 1, 2, 4, and 6 after the beginning of the treatment; Pig-a mutant frequencies were determined at Day −1 and Weeks 1, 2, 4 and 6, while percent micronucleated-reticulocyte (%MN-RET) frequencies were measured on Day 4 only. Additional animals were treated intravenously with three daily doses of 50 mg/kg TiO 2 -NPs for the measurement of titanium levels in bone marrow after 4, 24, and 48 h of the last treatment. The measurement indicated that the accumulation of the nanoparticles reached the peak in the tissue 4 h after the administration and the levels were maintained for a few days. No increase in either Pig-a mutant frequency or the frequency of %MN-RETs was detected, although the %RETs was reduced in the treated animals on Day 4 in a dose-dependent manner indicating cytotoxicity of TiO 2 -NPs in the bone marrow. These results suggest that although TiO 2 -NPs can reach the mouse bone marrow and are capable of inducing cytotoxicity, the nanoparticles are not genotoxic when assessed with in vivo micronucleus and Pig-a gene mutation tests.

Research paper thumbnail of miR-34a suppresses mutagenesis by inducing apoptosis in human lymphoblastoid TK6 cells

Mutation Research/Genetic Toxicology and Environmental Mutagenesis, 2013

a b s t r a c t miR-34a, a tumor suppressor miRNA, has been identified as a direct transcriptiona... more a b s t r a c t miR-34a, a tumor suppressor miRNA, has been identified as a direct transcriptional target of P53. miRNA precursors and inhibitors have been used to modulate the expression of their targeted mRNA and thereby study miRNA functions. We indicated in our previous work that X-ray induces miR-34a expression in a time and dose dependent manner. The objective of this study was to elucidate the role of miR-34a in X-ray-induced mutations in human lymphoblast TK6 cells. Neither over-expression of miR-34a by lipid transfection of miR-34a precursor nor down regulation of endogenous miR-34a by miR-34a inhibitor had any effect on X-ray-induced micronucleus frequency in TK6 cells. Over-expression of miR-34a in TK6 cells significantly reduced X-ray induced mutant frequency (MF) in the Thymidine Kinase (TK) locus while suppression of endogenous miR-34a can increase the background level MF in TK6 cells. Furthermore, over-expression of miR-34a promoted and down-regulation of miR-34a inhibited background and Xray-induced apoptosis in TK6 cells. Our study suggests miR-34a is an important negative regulator of mutagenesis and the mechanism is possibly mediated through apoptosis.

Research paper thumbnail of Cytotoxicity and genotoxicity assessment of silver nanoparticles in mouse

Nanotoxicology, 2013

Silver nanoparticles (AgNPs) are among the most commercially used nanomaterials and their toxicit... more Silver nanoparticles (AgNPs) are among the most commercially used nanomaterials and their toxicity and genotoxicity are controversial. Although many in vitro studies have been conducted to evaluate the genotoxicity of AgNPs, in vivo genotoxicity studies on the nanomaterials are limited. Given the unique physicochemical properties and complex pharmacokinetics behavior of nanoparticles (NPs), in vivo genotoxicity assessment of AgNPs is badly needed. In this study, the clastogenicity and mutagenicity of AgNPs with different sizes and coatings were evaluated using mouse micronucleus (MN) assay, Pig-a assay and Comet assay. Five 7-week-old male B6C3F1 mice per group were treated with 5 nm polyvinylpyrrolidone (PVP)-coated AgNPs at a single dose of 0.5, 1.0, 2.5, 5.0, 10.0 or 20.0 mg/kg body weight (bw) via intravenous injection for both the MN and Pig-a assays; or with 15-100 nm PVP-or 10-80 nm silicon-coated AgNPs at a single or 3-day repeated dose of 25.0 mg/kg bw for the MN assay and Comet assay in mouse liver. Inductively coupled plasma mass spectrometry (ICP-MS) and transmission electron microscopy (TEM) analyses indicated that AgNPs reached the testing tissues (bone marrow for the MN and Pig-a assays and liver for the Comet assay). Although there was a reduction of reticulocytes in the PVP-coated AgNPs-treated animals, indicating cytotoxicity of the AgNPs, none of the treatments resulted in a significant increase of either mutant frequencies in the Pig-a gene or the percent of micronucleated reticulocyte over the concurrent controls. However, both the PVP-and silicon-coated AgNPs induced oxidative DNA damage in mouse liver. These results demonstrate that the AgNPs can reach mouse bone marrow and liver, and generate cytotoxicity to the reticulocytes and oxidative DNA damage to the liver.

Research paper thumbnail of Withdrawn

Research paper thumbnail of miR-34a suppresses mutagenesis by inducing apoptosis in human lymphoblastoid TK6 cells

a b s t r a c t miR-34a, a tumor suppressor miRNA, has been identified as a direct transcriptiona... more a b s t r a c t miR-34a, a tumor suppressor miRNA, has been identified as a direct transcriptional target of P53. miRNA precursors and inhibitors have been used to modulate the expression of their targeted mRNA and thereby study miRNA functions. We indicated in our previous work that X-ray induces miR-34a expression in a time and dose dependent manner. The objective of this study was to elucidate the role of miR-34a in X-ray-induced mutations in human lymphoblast TK6 cells. Neither over-expression of miR-34a by lipid transfection of miR-34a precursor nor down regulation of endogenous miR-34a by miR-34a inhibitor had any effect on X-ray-induced micronucleus frequency in TK6 cells. Over-expression of miR-34a in TK6 cells significantly reduced X-ray induced mutant frequency (MF) in the Thymidine Kinase (TK) locus while suppression of endogenous miR-34a can increase the background level MF in TK6 cells. Furthermore, over-expression of miR-34a promoted and down-regulation of miR-34a inhibited background and Xray-induced apoptosis in TK6 cells. Our study suggests miR-34a is an important negative regulator of mutagenesis and the mechanism is possibly mediated through apoptosis.

Research paper thumbnail of In vitro toxicological assessment of iron oxide, aluminium oxide and copper nanoparticles in prokaryotic and eukaryotic cell types

Drug and Chemical Toxicology, 2014

ABSTRACT Metallic nanoparticles (NPs) have a variety of applications in different industries incl... more ABSTRACT Metallic nanoparticles (NPs) have a variety of applications in different industries including pharmaceutical industry where these NPs are used mainly for image analysis and drug delivery. The increasing interest in nanotechnology is largely associated with undefined risks to the human health and to the environment. Therefore, in the present study cytotoxic and genotoxic effects of iron oxide, aluminium oxide and copper nanoparticles were evaluated using most commonly used assays i.e. Ames assay, in vitro cytotoxicity assay, micronucleus assay and comet assay. Cytotoxicity to bacterial cells was assessed in terms of colony forming units by using Escherichia coli (gram negative) and Bacillus subtilis (gram positive). Ames assay was carried out using two bacterial strains of Salmonella typhimurium TA98 and TA100. Genotoxicity of these NPs was evaluated following exposure to monkey kidney cell line, CHS-20. No cytotoxic and genotoxic effects were observed for iron oxide, and aluminium oxide NPs. Copper NPs were found mutagenic in TA98 and in TA100 and also found cytotoxic in dose dependent manner. Copper NPs induced significant (p < 0.01) increase in number of binucleated cells with micronuclei (96.6 ± 5.40) at the highest concentration (25 µg/mL). Copper NPs also induced DNA strand breaks at 10 µg/mL and oxidative DNA damage at 5 and 10 µg/mL. We consider these findings very useful in evaluating the genotoxic potential of NPs especially because of their increasing applications in human health and environment with limited knowledge of their toxicity and genotoxicity.

Research paper thumbnail of Genotoxicity of TiO2 anatase nanoparticles in B6C3F1 male mice evaluated using Pig-a and flow cytometric micronucleus assays

Mutation Research/Genetic Toxicology and Environmental Mutagenesis, 2012

In vivo micronucleus and Pig-a (phosphatidylinositol glycan, class A gene) mutation assays were c... more In vivo micronucleus and Pig-a (phosphatidylinositol glycan, class A gene) mutation assays were conducted to evaluate the genotoxicity of 10 nm titanium dioxide anatase nanoparticles (TiO 2 -NPs) in mice. Groups of five 6-7-week-old male B6C3F1 mice were treated intravenously for three consecutive days with 0.5, 5.0, and 50 mg/kg TiO 2 -NPs for the two assays; mouse blood was sampled one day before the treatment and on Day 4, and Weeks 1, 2, 4, and 6 after the beginning of the treatment; Pig-a mutant frequencies were determined at Day −1 and Weeks 1, 2, 4 and 6, while percent micronucleated-reticulocyte (%MN-RET) frequencies were measured on Day 4 only. Additional animals were treated intravenously with three daily doses of 50 mg/kg TiO 2 -NPs for the measurement of titanium levels in bone marrow after 4, 24, and 48 h of the last treatment. The measurement indicated that the accumulation of the nanoparticles reached the peak in the tissue 4 h after the administration and the levels were maintained for a few days. No increase in either Pig-a mutant frequency or the frequency of %MN-RETs was detected, although the %RETs was reduced in the treated animals on Day 4 in a dose-dependent manner indicating cytotoxicity of TiO 2 -NPs in the bone marrow. These results suggest that although TiO 2 -NPs can reach the mouse bone marrow and are capable of inducing cytotoxicity, the nanoparticles are not genotoxic when assessed with in vivo micronucleus and Pig-a gene mutation tests.

Research paper thumbnail of miR-34a suppresses mutagenesis by inducing apoptosis in human lymphoblastoid TK6 cells

Mutation Research/Genetic Toxicology and Environmental Mutagenesis, 2013

a b s t r a c t miR-34a, a tumor suppressor miRNA, has been identified as a direct transcriptiona... more a b s t r a c t miR-34a, a tumor suppressor miRNA, has been identified as a direct transcriptional target of P53. miRNA precursors and inhibitors have been used to modulate the expression of their targeted mRNA and thereby study miRNA functions. We indicated in our previous work that X-ray induces miR-34a expression in a time and dose dependent manner. The objective of this study was to elucidate the role of miR-34a in X-ray-induced mutations in human lymphoblast TK6 cells. Neither over-expression of miR-34a by lipid transfection of miR-34a precursor nor down regulation of endogenous miR-34a by miR-34a inhibitor had any effect on X-ray-induced micronucleus frequency in TK6 cells. Over-expression of miR-34a in TK6 cells significantly reduced X-ray induced mutant frequency (MF) in the Thymidine Kinase (TK) locus while suppression of endogenous miR-34a can increase the background level MF in TK6 cells. Furthermore, over-expression of miR-34a promoted and down-regulation of miR-34a inhibited background and Xray-induced apoptosis in TK6 cells. Our study suggests miR-34a is an important negative regulator of mutagenesis and the mechanism is possibly mediated through apoptosis.

Research paper thumbnail of Cytotoxicity and genotoxicity assessment of silver nanoparticles in mouse

Nanotoxicology, 2013

Silver nanoparticles (AgNPs) are among the most commercially used nanomaterials and their toxicit... more Silver nanoparticles (AgNPs) are among the most commercially used nanomaterials and their toxicity and genotoxicity are controversial. Although many in vitro studies have been conducted to evaluate the genotoxicity of AgNPs, in vivo genotoxicity studies on the nanomaterials are limited. Given the unique physicochemical properties and complex pharmacokinetics behavior of nanoparticles (NPs), in vivo genotoxicity assessment of AgNPs is badly needed. In this study, the clastogenicity and mutagenicity of AgNPs with different sizes and coatings were evaluated using mouse micronucleus (MN) assay, Pig-a assay and Comet assay. Five 7-week-old male B6C3F1 mice per group were treated with 5 nm polyvinylpyrrolidone (PVP)-coated AgNPs at a single dose of 0.5, 1.0, 2.5, 5.0, 10.0 or 20.0 mg/kg body weight (bw) via intravenous injection for both the MN and Pig-a assays; or with 15-100 nm PVP-or 10-80 nm silicon-coated AgNPs at a single or 3-day repeated dose of 25.0 mg/kg bw for the MN assay and Comet assay in mouse liver. Inductively coupled plasma mass spectrometry (ICP-MS) and transmission electron microscopy (TEM) analyses indicated that AgNPs reached the testing tissues (bone marrow for the MN and Pig-a assays and liver for the Comet assay). Although there was a reduction of reticulocytes in the PVP-coated AgNPs-treated animals, indicating cytotoxicity of the AgNPs, none of the treatments resulted in a significant increase of either mutant frequencies in the Pig-a gene or the percent of micronucleated reticulocyte over the concurrent controls. However, both the PVP-and silicon-coated AgNPs induced oxidative DNA damage in mouse liver. These results demonstrate that the AgNPs can reach mouse bone marrow and liver, and generate cytotoxicity to the reticulocytes and oxidative DNA damage to the liver.