RESUMEN
Objective:To learn about the levels of 5-methylcytosine (5-mC) and 5-hydroxymethylcytosine (5-hmC) in bone tissue of rats with different types of skeletal fluorosis and analyze their correlation.Methods:Thirty 4-week-old SPF grade healthy SD rats were selected. After adaptive feeding for 1 week, the rats were divided into control group (4 ml·kg -1·bw deionized water + standard maintenance diet), osteosclerosis group [20 mg·kg -1·bw sodium fluoride (NaF) + standard maintenance diet], and osteoporosis/osteomalacia group (20 mg·kg -1·bw NaF + low-calcium and low-protein partial diet) according to their body weight (100 - 120 g) by random number table method, with 10 rats in each group, half male and half female; gavaged 6 days each week and the experimental period was 5 months. At the end of the experiment, samples of rat heart blood and lower limb femur were collected. The contents of serum methyl donor S-adenosylmethionine (SAM) and its metabolite S-adenosylhomocysteine (SAH) in serum, and the levels of 5-mC and 5-hmC in bone tissue were measured by enzyme-linked immunosorbent assay (ELISA). Western blot was used to determine the expression of DNA methyltransferase (DNMTs) and DNA hydroxymethylase (TETs) in bone tissue of rats. The correlation between serum SAM content, SAM/SAH ratio and bone tissue 5-mC level, and between the bone tissue 5-mC level and 5-hmC level was analyzed. Results:Serum SAM [11.03 (7.06, 18.63), 3.96 (2.32, 9.09), 3.91 (2.35, 4.46) nmol/L], SAH content [(4.69 ± 0.55), (5.41 ± 1.13), (13.90 ± 1.09) ng/L], SAM/SAH ratio [2.58 (1.54, 4.12), 0.62 (0.52, 1.69), 0.14 (0.13, 0.15)] and bone tissue 5-mC [103.39 (97.37, 109.35), 52.50 (50.19, 68.13), 55.03 (49.97, 59.57) ng/L], 5-hmC levels [(32.61 ± 8.84), (56.96 ± 8.48), (20.34 ± 6.22) ng/L] in the control group, osteosclerosis group and osteoporosis/osteomalacia group were compared, and the differences were statistically significant beween three groups ( H/ F = 12.81, 284.24, 21.85, 19.37, 55.23, P < 0.01). Compared with the control group, the content of SAM, the ratio of SAM/SAH, the level of 5-mC in the osteosclerosis group and osteoporosis/osteomalacia group, and the level of 5-hmC in the osteoporosis/osteomalacia group were lower ( P < 0.05), while the content of SAH in the osteoporosis/osteomalacia group and the level of 5-hmC in the osteosclerosis group were higher ( P < 0.05). Compared with the osteosclerosis group, the content of SAH in the osteoporosis/osteomalacia group was higher, while the ratio of SAM/SAH and the level of 5-hmC were lower ( P < 0.05). Western blot showed that there were statistically significant differences in the expression levels of DNMT1, DNMT3A, DNMT3B, TET1 and TET2 protein in bone tissue of rats in the control group, osteosclerosis group, and osteoporosis/osteomalacia group ( F = 285.45, 345.58, 239.83, 311.52, 318.24, P < 0.001). Among them, the expression levels of DNMT1, DNMT3A and DNMT3B protein in the osteosclerosis group and osteoporosis/osteomalacia group were lower than those in the control group, and the expression levels of DNMT1, DNMT3A and DNMT3B protein in the osteosclerosis/osteomalacia group were lower than those in the osteosclerosis group ( P < 0.05); the expression levels of TET1 and TET2 protein in osteosclerosis group were higher than those in the control group and osteoporosis/osteomalacia group, and the expression levels of TET1 and TET2 protein in the osteoporosis/osteomalacia group were lower than those in the control group ( P < 0.05). The results of Spearman rank correlation analysis showed that the content of SAM and the ratio of SAM/SAH in the control group, osteosclerosis group and osteoporosis/osteomalacia group were positively correlated with the level of 5-mC in bone tissue ( rs = 0.89, 0.92, 0.81, 0.73, 0.87, 0.73, P < 0.05). The levels of 5-mC and 5-hmC in bone tissue of rats in each group were negatively correlated ( rs = - 0.69, - 0.68, - 0.72, P < 0.05). Conclusions:The level of 5-mC in bone tissue of osteosclerotic fluorosis rats is low, and the level of 5-hmC is high, while those of osteoporosis/osteomalacia fluorosis rats are lower. The difference of 5-mC level in bone tissue of rats with different types of skeletal fluorosis is not significant, which may be related to the difference of 5-hmC level in bone tissue.
RESUMEN
BACKGROUND: Alcohol is known to affect two epigenetic phenomena, DNA methylation and DNA hydroxymethylation, and iron is a cofactor of ten-eleven translocation (TET) enzymes that catalyze the conversion from methylcytosine to hydroxymethylcytosine. In the present study we aimed to determine the effects of alcohol on DNA hydroxymethylation and further effects of iron on alcohol associated epigenetic changes. METHODS: Twenty-four male Sprague-Dawley rats were fed either Lieber-DeCarli alcohol diet (36% calories from ethanol) or Lieber-DeCarli control diet along with or without iron supplementation (0.6% carbonyl iron) for 8 weeks. Hepatic non-heme iron concentrations were measured by colorimetric assays. Protein levels of hepatic ferritin and transferrin receptor were determined by Western blotting. Methylcytosine, hydroxymethylcytosine and unmodified cytosine in DNA were simultaneously measured by liquid chromatography/mass spectrometry method. RESULTS: Iron supplementation significantly increased hepatic non-heme iron contents (P < 0.05) but alcohol alone did not. However, both alcohol and iron significantly increased hepatic ferritin levels and decreased hepatic transferrin receptor levels (P < 0.05). Alcohol reduced hepatic DNA hydroxymethylation (0.21% ± 0.04% vs. 0.33% ± 0.04%, P = 0.01) compared to control, while iron supplementation to alcohol diet did not change DNA hydroxymethylation. There was no significant difference in methylcytosine levels, while unmodified cytosine levels were significantly increased in alcohol-fed groups compared to control (95.61% ± 0.08% vs. 95.26% ± 0.12%, P = 0.03), suggesting that alcohol further increases the conversion from hydroxymethylcytosine to unmodified cytosine. CONCLUSIONS: Chronic alcohol consumption alters global DNA hydroxymethylation in the liver but iron supplementation reverses the epigenetic effect of alcohol.