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Cytotoxic effect of poly-dispersed single walled carbon nanotubes on erythrocytes in vitro and in vivo.

Sachar S, Saxena RK - PLoS ONE (2011)

Bottom Line: Single wall Carbon Nanotubes (SWCNTs) are hydrophobic and do not disperse in aqueous solvents.Administration of AF-SWCNTs through intravenous route resulted in a transient anemia as seen by a sharp decline in blood erythrocyte count accompanied with a significant drop in blood haemoglobin level.Administration of AF-SWCNTs through intratracheal administration also showed significant decline in RBC count while administration through other routes (gavage and intra-peritoneal) was not effective.

View Article: PubMed Central - PubMed

Affiliation: School of Life Sciences, Jawaharlal Nehru University, New Delhi, India.

ABSTRACT
Single wall Carbon Nanotubes (SWCNTs) are hydrophobic and do not disperse in aqueous solvents. Acid functionalization of SWCNTs results in attachment of carboxy and sulfonate groups to carbon atoms and the resulting acid functionalized product (AF-SWCNTs) is negatively charged and disperses easily in water and buffers. In the present study, effect of AF-SWCNTs on blood erythrocytes was examined. Incubation of mouse erythrocytes with AF-SWCNTs and not with control SWCNTs, resulted in a dose and time dependent lysis of erythrocyte. Using fluorescence tagged AF-SWCNTs, binding of AF-SWCNTs with erythrocytes could be demonstrated. Confocal microscopy results indicated that AF-SWCNTs could enter the erythrocytes. Treatment with AF-SWCNTs resulted in exposure of hydrophobic patches on erythrocyte membrane that is indicative of membrane damage. A time and dose dependent increase in externalization of phosphatidylserine on erythrocyte membrane bilayer was also found. Administration of AF-SWCNTs through intravenous route resulted in a transient anemia as seen by a sharp decline in blood erythrocyte count accompanied with a significant drop in blood haemoglobin level. Administration of AF-SWCNTs through intratracheal administration also showed significant decline in RBC count while administration through other routes (gavage and intra-peritoneal) was not effective. By using a recently developed technique of a two step in vivo biotinylation of erythrocytes that enables simultaneous enumeration of young (age <10 days) and old (age>40 days) erythrocytes in mouse blood, it was found that the in vivo toxic effect of AF-SWCNTs was more pronounced on older subpopulation of erythrocytes. Subpopulation of old erythrocytes fell after treatment with AF-SWCNTs but recovered by third day after the intravenous administration of AF-SWCNTs. Taken together our results indicate that treatment with AF-SWCNTs results in acute membrane damage and eventual lysis of erythrocytes. Intravenous administration of AF-SWCNTs resulted in a transient anemia in which older erythrocytes are preferably lysed.

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Effect of single intravenous dose of AF-SWCNTs on the proportions of erythrocyte cohorts of different age groups in mouse blood.Mice were prepared by DIB technique as described in materials and methods. AF-SWCNTs (100 µg) was administered i.v. and proportions of erythrocyte cohorts of different ages in blood circulation were monitored. Erythrocyte cohorts were identified by biotin levels on erythrocytes as per the DIB technique [9]. Results of a representative experiment in panel A show the distribution of the three erythrocytes cohorts [Old erythrocytes, (biotinhigh, age>40 days, top boxes); intermediate age erythrocytes(biotinlow age10–40 days, middle boxes); young erythrocytes (biotinnegative age<10 days, lower boxes)] before and 3h after intravenous administration of AF-SWCNT (100 µg) in 6–8 weeks old Swiss mice. Blood samples were also taken from these mice at different intervals of time and effect of AF-SWCNT administration on the distribution of the three erythrocyte cohorts was assessed at each time points (panel B). Proportion of young, intermediate and old erythrocytes at zero time point were taken as hundred and changes induced by AF-SWCNTs have been depicted as percent of control. Each data point in panel B represents mean ± SEM of data from swiss mice. *p<0.05 as compared to control groups by ANOVA.
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pone-0022032-g008: Effect of single intravenous dose of AF-SWCNTs on the proportions of erythrocyte cohorts of different age groups in mouse blood.Mice were prepared by DIB technique as described in materials and methods. AF-SWCNTs (100 µg) was administered i.v. and proportions of erythrocyte cohorts of different ages in blood circulation were monitored. Erythrocyte cohorts were identified by biotin levels on erythrocytes as per the DIB technique [9]. Results of a representative experiment in panel A show the distribution of the three erythrocytes cohorts [Old erythrocytes, (biotinhigh, age>40 days, top boxes); intermediate age erythrocytes(biotinlow age10–40 days, middle boxes); young erythrocytes (biotinnegative age<10 days, lower boxes)] before and 3h after intravenous administration of AF-SWCNT (100 µg) in 6–8 weeks old Swiss mice. Blood samples were also taken from these mice at different intervals of time and effect of AF-SWCNT administration on the distribution of the three erythrocyte cohorts was assessed at each time points (panel B). Proportion of young, intermediate and old erythrocytes at zero time point were taken as hundred and changes induced by AF-SWCNTs have been depicted as percent of control. Each data point in panel B represents mean ± SEM of data from swiss mice. *p<0.05 as compared to control groups by ANOVA.

Mentions: Results so far indicate that the intravenous administration of AF-SWCNTs caused a marked and transient anemia in mice. It was of interest to determine if the toxic effect of AF-SWCNTs was generalize or was selective for erythrocytes of specific age groups. To determine the susceptibility of erythrocytes of different age groups, we used a technique that we recently developed to enumerate circulating erythrocyte cohorts of different age groups [9]. In this technique that involves a two step in vivo biotinylation of circulating erythrocytes, erythrocytes of different age groups can be identified as a biotin negative population (young erythrocytes, age <10 days), a biotin high population (Old erythrocytes, age>40 days) and a biotin low population (erythrocytes of age between 10 to 40 days). Results in Figure 8A show that just 3 h after a single dose of AF-SWCNTs, the proportion of old erythrocyte (age>40 days) in blood fell from 7.3% to 5.8%, whereas the proportion of young erythrocytes (age<10days) increased from 36.19% to 38.95%. No significant change occurred in the erythrocytes of intermediate age group (age 10–40 days). Time kinetics of changes in the proportion of old and young erythrocytes is shown in Figure 8B. These results suggest that the older erythrocytes in blood circulation may be most susceptible to the toxic effect of AF-SWCNTs.


Cytotoxic effect of poly-dispersed single walled carbon nanotubes on erythrocytes in vitro and in vivo.

Sachar S, Saxena RK - PLoS ONE (2011)

Effect of single intravenous dose of AF-SWCNTs on the proportions of erythrocyte cohorts of different age groups in mouse blood.Mice were prepared by DIB technique as described in materials and methods. AF-SWCNTs (100 µg) was administered i.v. and proportions of erythrocyte cohorts of different ages in blood circulation were monitored. Erythrocyte cohorts were identified by biotin levels on erythrocytes as per the DIB technique [9]. Results of a representative experiment in panel A show the distribution of the three erythrocytes cohorts [Old erythrocytes, (biotinhigh, age>40 days, top boxes); intermediate age erythrocytes(biotinlow age10–40 days, middle boxes); young erythrocytes (biotinnegative age<10 days, lower boxes)] before and 3h after intravenous administration of AF-SWCNT (100 µg) in 6–8 weeks old Swiss mice. Blood samples were also taken from these mice at different intervals of time and effect of AF-SWCNT administration on the distribution of the three erythrocyte cohorts was assessed at each time points (panel B). Proportion of young, intermediate and old erythrocytes at zero time point were taken as hundred and changes induced by AF-SWCNTs have been depicted as percent of control. Each data point in panel B represents mean ± SEM of data from swiss mice. *p<0.05 as compared to control groups by ANOVA.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3139600&req=5

pone-0022032-g008: Effect of single intravenous dose of AF-SWCNTs on the proportions of erythrocyte cohorts of different age groups in mouse blood.Mice were prepared by DIB technique as described in materials and methods. AF-SWCNTs (100 µg) was administered i.v. and proportions of erythrocyte cohorts of different ages in blood circulation were monitored. Erythrocyte cohorts were identified by biotin levels on erythrocytes as per the DIB technique [9]. Results of a representative experiment in panel A show the distribution of the three erythrocytes cohorts [Old erythrocytes, (biotinhigh, age>40 days, top boxes); intermediate age erythrocytes(biotinlow age10–40 days, middle boxes); young erythrocytes (biotinnegative age<10 days, lower boxes)] before and 3h after intravenous administration of AF-SWCNT (100 µg) in 6–8 weeks old Swiss mice. Blood samples were also taken from these mice at different intervals of time and effect of AF-SWCNT administration on the distribution of the three erythrocyte cohorts was assessed at each time points (panel B). Proportion of young, intermediate and old erythrocytes at zero time point were taken as hundred and changes induced by AF-SWCNTs have been depicted as percent of control. Each data point in panel B represents mean ± SEM of data from swiss mice. *p<0.05 as compared to control groups by ANOVA.
Mentions: Results so far indicate that the intravenous administration of AF-SWCNTs caused a marked and transient anemia in mice. It was of interest to determine if the toxic effect of AF-SWCNTs was generalize or was selective for erythrocytes of specific age groups. To determine the susceptibility of erythrocytes of different age groups, we used a technique that we recently developed to enumerate circulating erythrocyte cohorts of different age groups [9]. In this technique that involves a two step in vivo biotinylation of circulating erythrocytes, erythrocytes of different age groups can be identified as a biotin negative population (young erythrocytes, age <10 days), a biotin high population (Old erythrocytes, age>40 days) and a biotin low population (erythrocytes of age between 10 to 40 days). Results in Figure 8A show that just 3 h after a single dose of AF-SWCNTs, the proportion of old erythrocyte (age>40 days) in blood fell from 7.3% to 5.8%, whereas the proportion of young erythrocytes (age<10days) increased from 36.19% to 38.95%. No significant change occurred in the erythrocytes of intermediate age group (age 10–40 days). Time kinetics of changes in the proportion of old and young erythrocytes is shown in Figure 8B. These results suggest that the older erythrocytes in blood circulation may be most susceptible to the toxic effect of AF-SWCNTs.

Bottom Line: Single wall Carbon Nanotubes (SWCNTs) are hydrophobic and do not disperse in aqueous solvents.Administration of AF-SWCNTs through intravenous route resulted in a transient anemia as seen by a sharp decline in blood erythrocyte count accompanied with a significant drop in blood haemoglobin level.Administration of AF-SWCNTs through intratracheal administration also showed significant decline in RBC count while administration through other routes (gavage and intra-peritoneal) was not effective.

View Article: PubMed Central - PubMed

Affiliation: School of Life Sciences, Jawaharlal Nehru University, New Delhi, India.

ABSTRACT
Single wall Carbon Nanotubes (SWCNTs) are hydrophobic and do not disperse in aqueous solvents. Acid functionalization of SWCNTs results in attachment of carboxy and sulfonate groups to carbon atoms and the resulting acid functionalized product (AF-SWCNTs) is negatively charged and disperses easily in water and buffers. In the present study, effect of AF-SWCNTs on blood erythrocytes was examined. Incubation of mouse erythrocytes with AF-SWCNTs and not with control SWCNTs, resulted in a dose and time dependent lysis of erythrocyte. Using fluorescence tagged AF-SWCNTs, binding of AF-SWCNTs with erythrocytes could be demonstrated. Confocal microscopy results indicated that AF-SWCNTs could enter the erythrocytes. Treatment with AF-SWCNTs resulted in exposure of hydrophobic patches on erythrocyte membrane that is indicative of membrane damage. A time and dose dependent increase in externalization of phosphatidylserine on erythrocyte membrane bilayer was also found. Administration of AF-SWCNTs through intravenous route resulted in a transient anemia as seen by a sharp decline in blood erythrocyte count accompanied with a significant drop in blood haemoglobin level. Administration of AF-SWCNTs through intratracheal administration also showed significant decline in RBC count while administration through other routes (gavage and intra-peritoneal) was not effective. By using a recently developed technique of a two step in vivo biotinylation of erythrocytes that enables simultaneous enumeration of young (age <10 days) and old (age>40 days) erythrocytes in mouse blood, it was found that the in vivo toxic effect of AF-SWCNTs was more pronounced on older subpopulation of erythrocytes. Subpopulation of old erythrocytes fell after treatment with AF-SWCNTs but recovered by third day after the intravenous administration of AF-SWCNTs. Taken together our results indicate that treatment with AF-SWCNTs results in acute membrane damage and eventual lysis of erythrocytes. Intravenous administration of AF-SWCNTs resulted in a transient anemia in which older erythrocytes are preferably lysed.

Show MeSH
Related in: MedlinePlus