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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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Related in: MedlinePlus

The Olive Tail Moment values for fresh lymphocytes treated with oestrogenic compounds or H2O2 for 120 min and after 24 h repair in fresh medium. The mean of the values obtained from 7 independent experiments are shown (n = 7 different donors). Average age is 27.57 ± 2.41. 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, no repair versus repair for each of the oestrogenic compounds was compared. The levels of significance are included: n.s. not significant; *p ≤ 0.05; **p ≤ 0.01 and ***p ≤ 0.001.
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f2-ijms-12-02783: The Olive Tail Moment values for fresh lymphocytes treated with oestrogenic compounds or H2O2 for 120 min and after 24 h repair in fresh medium. The mean of the values obtained from 7 independent experiments are shown (n = 7 different donors). Average age is 27.57 ± 2.41. 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, no repair versus repair for each of the oestrogenic compounds was compared. The levels of significance are included: n.s. not significant; *p ≤ 0.05; **p ≤ 0.01 and ***p ≤ 0.001.

Mentions: There was consistency amongst all oestrogenic compounds in the detection of oxidized bases after 24 h repair in lymphocytes but they were not statistically significant. It is known that a critical step in the detection of oxidized bases after exposure to genotoxicants is the need for a recovery period in order to repair the strand breaks generated leaving the oxidized bases exposed which will be cleaved by FPG and EndoIII. The selection of a repair time which is too short results in confusion between strand breaks produced by the investigated genotoxicant and the ones originated by the enzymes (FPG ad EndoIII). The fact that the DNA repair period reduced the Olive tail moment to nearly negative control levels (Figure 2) for all the treatments confirms that 24 h repair is an adequate recovery period. However, the possibility cannot be ruled out that a slightly longer repair, bringing Olive tail moment values down to negative control levels, might allow a more optimized detection of the oxidized bases.


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 fresh lymphocytes treated with oestrogenic compounds or H2O2 for 120 min and after 24 h repair in fresh medium. The mean of the values obtained from 7 independent experiments are shown (n = 7 different donors). Average age is 27.57 ± 2.41. 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, no repair versus repair for each of the oestrogenic compounds was compared. 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

f2-ijms-12-02783: The Olive Tail Moment values for fresh lymphocytes treated with oestrogenic compounds or H2O2 for 120 min and after 24 h repair in fresh medium. The mean of the values obtained from 7 independent experiments are shown (n = 7 different donors). Average age is 27.57 ± 2.41. 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, no repair versus repair for each of the oestrogenic compounds was compared. The levels of significance are included: n.s. not significant; *p ≤ 0.05; **p ≤ 0.01 and ***p ≤ 0.001.
Mentions: There was consistency amongst all oestrogenic compounds in the detection of oxidized bases after 24 h repair in lymphocytes but they were not statistically significant. It is known that a critical step in the detection of oxidized bases after exposure to genotoxicants is the need for a recovery period in order to repair the strand breaks generated leaving the oxidized bases exposed which will be cleaved by FPG and EndoIII. The selection of a repair time which is too short results in confusion between strand breaks produced by the investigated genotoxicant and the ones originated by the enzymes (FPG ad EndoIII). The fact that the DNA repair period reduced the Olive tail moment to nearly negative control levels (Figure 2) for all the treatments confirms that 24 h repair is an adequate recovery period. However, the possibility cannot be ruled out that a slightly longer repair, bringing Olive tail moment values down to negative control levels, might allow a more optimized detection of the oxidized bases.

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