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Mechanistic investigation of ROS-induced DNA damage by oestrogenic compounds in lymphocytes and sperm using the comet assay.

Cemeli E, Anderson D - Int J Mol Sci (2011)

Bottom Line: No effects were observed for sperm.The successful detection of oxidized bases in the negative control (untreated) of sperm provides an opportunity for its application in biomonitoring studies.Rapid rejoining of DNA, in a matter of hours, is a characteristic of DNA damaged by ROS.

View Article: PubMed Central - PubMed

Affiliation: Genetic and Reproductive Toxicology Group, Division of Biomedical Sciences, University of Bradford, Bradford, West Yorkshire, BD7 1DP, UK; E-Mail: Eduardo_Cemeli@bat.com.

ABSTRACT
Past research has demonstrated that oestrogenic compounds produce strand breaks in the DNA of sperm and lymphocytes via reactive oxygen species (ROS). In the current investigation, sperm and lymphocytes were treated in vitro with oestrogenic compounds (diethylstilboestrol, progesterone, 17β-oestradiol, noradrenaline and triiodotyronine) and several aspects of DNA damage were investigated. Firstly, mediation of DNA damage by lipid peroxidation was investigated in the presence of BHA (a lipid peroxidation blocker). BHA reduced the DNA damage generated by 17β-oestradiol and diethylstilboestrol in a statistically significant manner. No effects were observed for sperm. Secondly, the presence of oxidized bases employing FPG and EndoIII were detected for lymphocytes and sperm in the negative control and after 24 h recovery in lymphocytes but not immediately after treatment for both cell types. The successful detection of oxidized bases in the negative control (untreated) of sperm provides an opportunity for its application in biomonitoring studies. DNA repair at 24 h after exposure was also studied. A nearly complete recovery to negative control levels was shown in lymphocytes 24 h recovery after oestrogenic exposure and this was statistically significant in all cases. Rapid rejoining of DNA, in a matter of hours, is a characteristic of DNA damaged by ROS.

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The Olive Tail Moment values for lymphocytes (a) and sperm (b) treated with oestrogenic compounds or H2O2 for 30 min at 37 °C in the presence and absence of BHA (500 μM). The figures express the mean of the values obtained from 7 independent experiments (n = 7 different donors) in (a) and 5 independent experiments (n = 5 different donors) in (b). For (a) the average age was 27.57 ± 2.41 and for (b) the average age was 30.80 ± 2.35. Normality of the data was evaluated with the Shapiro-Wilks test. Data were normally distributed. The Student’s T-test was applied. For statistical interpretation of the data, treatment in the absence of BHA was compared to treatment with the same compound with its presence. The levels of significance are included: n.s. not significant; *p ≤ 0.05; **p ≤ 0.01 and ***p ≤ 0.001.
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f1-ijms-12-02783: The Olive Tail Moment values for lymphocytes (a) and sperm (b) treated with oestrogenic compounds or H2O2 for 30 min at 37 °C in the presence and absence of BHA (500 μM). The figures express the mean of the values obtained from 7 independent experiments (n = 7 different donors) in (a) and 5 independent experiments (n = 5 different donors) in (b). For (a) the average age was 27.57 ± 2.41 and for (b) the average age was 30.80 ± 2.35. Normality of the data was evaluated with the Shapiro-Wilks test. Data were normally distributed. The Student’s T-test was applied. For statistical interpretation of the data, treatment in the absence of BHA was compared to treatment with the same compound with its presence. The levels of significance are included: n.s. not significant; *p ≤ 0.05; **p ≤ 0.01 and ***p ≤ 0.001.

Mentions: In Figure 1a and b, the effect of BHA on the genotoxicity of oestrogenic compounds on lymphocytes and sperm is displayed. The negative control for lymphocytes and sperm in the absence of BHA provided an Olive tail moment value which was within the historical records in our laboratory as observed in publications by this group. BHA at a concentration of 500 μM exhibited a pro-oxidant effect which generated DNA damage in a statistically significant manner in both cell types. All oestrogenic compounds, the solvent (DMSO) and the positive control (H2O2) induced an increase in the values of Olive tail moment when compared to the negative control in both cell types. In lymphocytes (Figure 1a), BHA reduced the DNA damage generated by the oestrogenic compounds and H2O2 in all cases with the exception of DMSO. The reduction was statistically significant in some instances. By contrast, no reduction by BHA was observed at any treatment in sperm.


Mechanistic investigation of ROS-induced DNA damage by oestrogenic compounds in lymphocytes and sperm using the comet assay.

Cemeli E, Anderson D - Int J Mol Sci (2011)

The Olive Tail Moment values for lymphocytes (a) and sperm (b) treated with oestrogenic compounds or H2O2 for 30 min at 37 °C in the presence and absence of BHA (500 μM). The figures express the mean of the values obtained from 7 independent experiments (n = 7 different donors) in (a) and 5 independent experiments (n = 5 different donors) in (b). For (a) the average age was 27.57 ± 2.41 and for (b) the average age was 30.80 ± 2.35. Normality of the data was evaluated with the Shapiro-Wilks test. Data were normally distributed. The Student’s T-test was applied. For statistical interpretation of the data, treatment in the absence of BHA was compared to treatment with the same compound with its presence. The levels of significance are included: n.s. not significant; *p ≤ 0.05; **p ≤ 0.01 and ***p ≤ 0.001.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC3116156&req=5

f1-ijms-12-02783: The Olive Tail Moment values for lymphocytes (a) and sperm (b) treated with oestrogenic compounds or H2O2 for 30 min at 37 °C in the presence and absence of BHA (500 μM). The figures express the mean of the values obtained from 7 independent experiments (n = 7 different donors) in (a) and 5 independent experiments (n = 5 different donors) in (b). For (a) the average age was 27.57 ± 2.41 and for (b) the average age was 30.80 ± 2.35. Normality of the data was evaluated with the Shapiro-Wilks test. Data were normally distributed. The Student’s T-test was applied. For statistical interpretation of the data, treatment in the absence of BHA was compared to treatment with the same compound with its presence. The levels of significance are included: n.s. not significant; *p ≤ 0.05; **p ≤ 0.01 and ***p ≤ 0.001.
Mentions: In Figure 1a and b, the effect of BHA on the genotoxicity of oestrogenic compounds on lymphocytes and sperm is displayed. The negative control for lymphocytes and sperm in the absence of BHA provided an Olive tail moment value which was within the historical records in our laboratory as observed in publications by this group. BHA at a concentration of 500 μM exhibited a pro-oxidant effect which generated DNA damage in a statistically significant manner in both cell types. All oestrogenic compounds, the solvent (DMSO) and the positive control (H2O2) induced an increase in the values of Olive tail moment when compared to the negative control in both cell types. In lymphocytes (Figure 1a), BHA reduced the DNA damage generated by the oestrogenic compounds and H2O2 in all cases with the exception of DMSO. The reduction was statistically significant in some instances. By contrast, no reduction by BHA was observed at any treatment in sperm.

Bottom Line: No effects were observed for sperm.The successful detection of oxidized bases in the negative control (untreated) of sperm provides an opportunity for its application in biomonitoring studies.Rapid rejoining of DNA, in a matter of hours, is a characteristic of DNA damaged by ROS.

View Article: PubMed Central - PubMed

Affiliation: Genetic and Reproductive Toxicology Group, Division of Biomedical Sciences, University of Bradford, Bradford, West Yorkshire, BD7 1DP, UK; E-Mail: Eduardo_Cemeli@bat.com.

ABSTRACT
Past research has demonstrated that oestrogenic compounds produce strand breaks in the DNA of sperm and lymphocytes via reactive oxygen species (ROS). In the current investigation, sperm and lymphocytes were treated in vitro with oestrogenic compounds (diethylstilboestrol, progesterone, 17β-oestradiol, noradrenaline and triiodotyronine) and several aspects of DNA damage were investigated. Firstly, mediation of DNA damage by lipid peroxidation was investigated in the presence of BHA (a lipid peroxidation blocker). BHA reduced the DNA damage generated by 17β-oestradiol and diethylstilboestrol in a statistically significant manner. No effects were observed for sperm. Secondly, the presence of oxidized bases employing FPG and EndoIII were detected for lymphocytes and sperm in the negative control and after 24 h recovery in lymphocytes but not immediately after treatment for both cell types. The successful detection of oxidized bases in the negative control (untreated) of sperm provides an opportunity for its application in biomonitoring studies. DNA repair at 24 h after exposure was also studied. A nearly complete recovery to negative control levels was shown in lymphocytes 24 h recovery after oestrogenic exposure and this was statistically significant in all cases. Rapid rejoining of DNA, in a matter of hours, is a characteristic of DNA damaged by ROS.

Show MeSH
Related in: MedlinePlus