DNA Methylation and Detoxification in the Earthworm Lumbricus terrestris Exposed to Cadmium and the DNA Demethylation Agent 5-aza-2′-deoxycytidine
<p>(<b>a</b>) mRNA copy numbers of Metallothionein 2 (MT2) in <span class="html-italic">L. terrestris</span> coelomocytes and (<b>b</b>) tissue of control (C) and exposed individuals (5-aza-2′-deoxycytidine (A); 200 mg/kg CdCl<sub>2</sub> (Cd); 5-aza-2′-deoxycytidine and 200 mg/kg CdCl<sub>2</sub> (CdA)). Stars indicate significant differences between treatments and the control (<span class="html-italic">p</span> < 0.05 (*); <span class="html-italic">p</span> < 0.01 (**)). Replicates per treatment analyzed in coelomocytes: C = 5, A = 6, Cd = 5, CdA = 3. Replicates per treatment analyzed in tissue: C = 7, A = 5, Cd = 8, CdA = 12.</p> "> Figure 2
<p>mRNA copy numbers of DNA-methyltransferase 1 (DNMT1) and ten–eleven translocation (TET) in <span class="html-italic">L. terrestris</span> coelomocytes and tissue of control (C) and exposed individuals (5-aza-2′-deoxycytidine (A); 200 mg/kg CdCl<sub>2</sub> (Cd); 5-aza-2′-deoxycytidine and 200 mg/kg CdCl<sub>2</sub> (CdA)). Stars indicate significant differences between treatments and control (<span class="html-italic">p</span> < 0.05 (*)). Replicates per treatment analyzed in coelomocytes: C = 5, A = 6, Cd = 5, CdA = 3. Replicates per treatment analyzed in tissue: C = 6, A = 4, Cd = 6, CdA = 6.</p> "> Figure 3
<p>(<b>a</b>) Global DNA methylation levels in coelomocytes extruded from control (C, <span class="html-italic">n</span> = 4) and exposed <span class="html-italic">L. terrestris</span> specimens (5-aza-2′-deoxycytidine (A), <span class="html-italic">n</span> = 4; 200 mg/kg Cd (Cd) <span class="html-italic">n</span> = 5; 5-aza-2′-deoxycytidine and 200 mg/kg CdCl<sub>2</sub> (CdA), <span class="html-italic">n</span> = 4) after 48 h of exposure. (<b>b</b>) Total DNMT protein activity (DNMT1 and DNMT3) of coelomocytes extruded from control (C, <span class="html-italic">n</span> = 3) and Cd-exposed (200 mg/kg CdCl<sub>2</sub> (Cd, <span class="html-italic">n</span> = 6)) <span class="html-italic">L. terrestris</span> individuals after 48 h of exposure.</p> "> Figure 4
<p>(<b>a</b>) MDS analysis using tissue samples and variables DNMT1, TET, MT2 and 5mC. Significance in group separation is based on the MDS2 dimension (shown by letters). The ellipse around the treatment groups indicates a confidence of 90%. (<b>b</b>) MDS analysis using coelomocyte samples and variables DNMT1, TET, MT2 and 5mC. Significance in group separation is based on the MDS1 dimension (shown by letters). The ellipse around treatment groups indicates a confidence of 90%.</p> ">
Abstract
:1. Introduction
2. Materials and Methods
2.1. Experimental Setup and Sampling
2.2. RNA Extraction and cDNA Synthesis
2.3. Quantitative Real-Time PCR
2.4. Protein Extraction and DNMT Activity
2.5. Quantification of 5-Methylcytosine (5mC) and 5-Hydroxymethylcytosine (5hmC)
2.6. Statistical Analysis
3. Results
3.1. Quantification of Gene Expression
3.2. Global Methylation Level and DNMT Activity
3.3. Global 5hmc
3.4. Multidimensional Scaling
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Gene | Treatment | Statistical Test | p Value |
---|---|---|---|
MT2 | C | Student’s t-test | 0.0117 |
A | Mann–Whitney U test | 0.0606 | |
Cd | Welch’s test | 0.0324 | |
CdA | Student’s t-test | <0.0001 | |
DNMT1 | C | Welch’s test | 0.0216 |
A | Welch’s test | 0.019 | |
Cd | Mann–Whitney U test | 0.0455 | |
CdA | Welch’s test | 0.2101 | |
TET | C | Welch’s test | 0.0025 |
A | Student’s t-test | 0.0208 | |
Cd | Welch’s test | 0.0025 | |
CdA | Student’s t-test | 0.0008 |
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Aigner, G.P.; Nenning, P.; Fiechtner, B.; Šrut, M.; Höckner, M. DNA Methylation and Detoxification in the Earthworm Lumbricus terrestris Exposed to Cadmium and the DNA Demethylation Agent 5-aza-2′-deoxycytidine. Toxics 2022, 10, 100. https://doi.org/10.3390/toxics10020100
Aigner GP, Nenning P, Fiechtner B, Šrut M, Höckner M. DNA Methylation and Detoxification in the Earthworm Lumbricus terrestris Exposed to Cadmium and the DNA Demethylation Agent 5-aza-2′-deoxycytidine. Toxics. 2022; 10(2):100. https://doi.org/10.3390/toxics10020100
Chicago/Turabian StyleAigner, Gerhard P., Pamela Nenning, Birgit Fiechtner, Maja Šrut, and Martina Höckner. 2022. "DNA Methylation and Detoxification in the Earthworm Lumbricus terrestris Exposed to Cadmium and the DNA Demethylation Agent 5-aza-2′-deoxycytidine" Toxics 10, no. 2: 100. https://doi.org/10.3390/toxics10020100