The antioxidative activity and toxicity of DHEA: the interaction between DHEA and Vit. E in ratsand the effect of DHEA on HepG2 cells

第一部分 DHEA是男女性賀爾蒙的前驅物，其含量的下降或不足可能與人體許多慢性病和老化有關。本實驗主要的目的是探討補充DHEA後，其在大鼠各主要器官的分佈、累積、抗氧化或助氧化作用以及毒性。作法是將DHEA (0、50及100 mg/kg diet) 混合到維生素E (0及30 IU/kg diet) 的飼料中餵食大鼠6週。 結果顯示餵食DHEA 6週並未影響大鼠體重和器官之重量。缺乏Vit. E之大鼠，其血漿中DHEA含量有顯著增加 (p<0.001)。在腎上腺與腦中，不論有無餵食Vit. E，DHEA含量與其劑量成反比。缺乏Vit. E的大鼠，其血漿中GPT的活性隨著DHEA的含量增加而上升。肝臟TSH方面，有餵食Vit. E之大鼠無顯著差異；缺乏 Vit. E之大鼠，TSH含量與DHEA之餵食量成反比 (p<0.001)。 NPSH則是餵食Vit. E的組別明顯高於無餵食Vit. E者(p<0.001)，且缺乏Vit. E 之大鼠NPSH含量因DHEA的增加而下降。餵食Vit. E的組別，大鼠腦中G6PD活性隨著DHEA的劑量增加而下降。而餵食Vit. E之大鼠肝中G6PD活性亦顯著高於缺乏Vit. E之大鼠(p<0.01)。缺乏Vit. E的大鼠腦中，GST的活性隨DHEA的增加而上升。不論有無餵食Vit. E，大鼠溶血 (hemolysis) 的程度會隨著DHEA含量的增加而上升 (p<0.01)，缺乏Vit. E的組別尤其明顯。肝臟中之脂質過氧化傷害並不顯著，但經0.7 mM BHP誘發之後，缺乏Vit. E的大鼠體內，TBARS顯著提高(p<0.05)。 結論：餵食DHEA會提高血漿中DHEA的含量，但卻會減少腎上腺和腦中的分泌。缺乏Vit. E 時，DHEA會導致大鼠肝臟中TSH、NPSH含量下降以及BHP誘導之TBARS的增加，表示DHEA可能會造成氧化傷害。而溶血現象和GOT、GPT活性升高，還有肝臟中GST酵素活性的下降，表示DHEA會產生毒性；這說明了DHEA具有促氧化性和毒性，但在Vit.E 的保護下則可免於這些傷害。 第二部分 在第二部分中將以較簡單的細胞培養系統 HepG2 cells來做進一步探討，以解決並進一步釐清下列問題，包括：1.DHEA到底會不會促進脂質過氧化、2.DHEA是抗氧化劑或氧化劑、3.DHEA到底會使G6PD上昇或下降。此外，我們進一步想知道DHEA會不會產生脂質過氧化傷害，進而引起細胞壞死(necrosis)，還有DHEA會不會進入細胞中造成DNA傷害，甚至引發細胞程式性死亡(apoptosis)。 結果顯示：HepG2細胞經0~1000 M DHEA培養24小時後，細胞內最高濃度為0.089 ﹪最低為0.02 ﹪。而HepG2細胞數目明顯的隨著DHEA濃度成反比，當DHEA濃度到達1000 M時，細胞數目和蛋白質的含量都是隨著DHEA的濃度上升而下降，分別只有控制組(DMSO)的59 ﹪(p<0.01)和65 ﹪(p<0.05)。以300 M DHEA培養HepG2細胞24 hr後，TBARS比控制組增加26 ﹪(p<0.05)，但DHEA濃度再昇高時，TBARS則未顯著改變。而經Fe-NTA培養兩小時後，500 M DHEA的TBARS比控制組高了約6倍(p<0.01) 但DHEA濃度再昇高時，TBARS則未顯著改變，表示當DHEA濃度大於500 M時，加入Fe-NTA，DHEA不但沒有抗氧化的效果，反而是促氧化的角色。當DHEA濃度到達300 M以上時，G6PD活性明顯的下降了(p<0.001)，說明了DHEA確實會抑制G6PD的活性。GPT和LDH leakage都沒有隨著DHEA濃度的增加而有顯著性的差異，表示DHEA並不會造成細胞破裂或壞死(necrosis)。 電泳圖中看不出有DNA雙股斷裂的現象；UDS結果顯示：控制組與最高濃度DHEA (1000 M)之間並沒有顯著差異；而Comet assay結果也顯示：DNA的傷害並沒有與DHEA的濃度成比例，而且拖尾的DNA在各組中比例也在正常範圍（約2~5﹪），表示DHEA造成的DNA傷害並不嚴重。此外，DHEA也沒有改變5’-nucleotidase的活性，其結果並沒有顯著的差異。以DHEA濃度0~1000 M培養24 hr後，細胞內游離鈣離子濃度也沒有顯著的差異。 結論：雖然DHEA會促進脂質過氧化和細胞數目的減少，但其傷害的強度還不至於使細胞受傷甚至死亡；由LDH leakage、GPT與5’-nucleotidase的結果可知，細胞並沒有因DHEA的作用而使細胞膜破裂或受到傷害。DHEA確實會抑制G6PD的活性，使其抗氧化系統受損，甚至減少ribose的生化合成，使DNA複製減緩。UDS、DNA fragmentation與Comet assay的結果也顯示：HepG2細胞數目的減少，與DNA傷害應無直接關係。推測HepG2細胞數目的減少，並不是因為細胞壞死(necrosis)或細胞程式性死亡(apoptosis)，而是DHEA進入DNA中，影響DNA複製基因，而導致細胞數目的減少。Part 1 DHEA is the precursor of both male and female sex hormones and has been shown to have many functions including anti-obese, anti-cancer and antioxidant activities. Part 1 was to study the distribution and accumulation in rat tissues and the antioxidant or prooxidant effects as well as toxicity of supplemental DHEA. The approaches were to provide DHEA (0,50,and 100 mg/kg diet) to rats for 6 weeks in diets deficient or sufficient in vitamin E (30 IU/kg diet). The results show that feeding rats with DHEA did not affect rat body weights or organ weights. In Vit. E-deficient rats, dietary DHEA increased plasma DHEA concentration (p<0.001). In rats either deficient or sufficient in Vit. E, dietary DHEA decreased adrenal and brain DHEA contents. Plasma GPT activity was increased by DHEA supplementation in Vit. E deficient rats. Hepatic TSH contents were not significantly affected by dietary DHEA in Vit. E-sufficient rats but were decreased (p<0.001) by dietary DHEA in Vit. E deficient rats. Dietary Vit. E significantly increased hepatic NPSH content (p<0.001), and dietary DHEA significantly decreased hepatic NPSH contents (p<0.001). The brain G6PD activity in Vit. E-sufficient rats by supplemental DHEA; hepatic G6PD activity was higher in Vit. E-sufficient rats than in Vit. E- deficient rats (p<0.01). Brain GST activity was increased by supplemental DHEA contents in Vit. E-deficient rats. Regardless of dietary Vit. E, hemolysis was increased by supplemental DHEA but the extent of hemolysis was higher in Vit. E-deficient rats than in Vit. E-deficient (p<0.01). Hepatic ex-vivo TBARS were not significantly affected by DHEA or Vit. E. Hepatic in-vitro TBARS induced by 0.7 mM BHP was increased in Vit. E-deficient rats (p<0.05). Conclusion: supplemental DHEA increased plasma DHEA concentration but decreased adrenal and brain DHEA concentrations. DHEA decreased hepatic TSH and NPSH contents in both Vit. E-sufficient and Vit. E-deficient rats but increased hepatic TBARS in Vit. E deficient rats. These results suggest that DHEA may have induced oxidative damage. In addition, DHEA increased hemolysis and hepatic GOT and GPT activity and decreased GST activity. These results suggest that DHEA is pro-oxidative and possibly toxic when rats are deficient in Vit. E, but these effects can largely be avoided when rats are sufficient in Vit. E. Part 2 In part 2, we used a simple cell line, HepG2 cells, to strengthen the findings in part 1 and to clarify the following questions; they included: 1.whether DHEA induces lipid oxidation, 2.whether DHEA is an antioxidant or oxidant, and 3.whether DHEA decreased or increased G6PD activity. Furthermore, we investigated whether DHEA results in necrosis and apoptosis. The results show that after incubating with 0~1000 M DHEA for 24 hrs, cellular DHEA concentration was maximum at 0.089% and minimum at 0.02%. HepG2 cell numbers and protein contents were significantly decreased by DHEA. When DHEA concentration was 1000 M, cell numbers and protein contents were only 59% (p<0.01) and 65% (p<0.05) of control experiment. TBARS were increased 26% (p<0.05) by incubation with 300 M DHEA for 24 hrs. There was no significant difference in TBARS when DHEA was higher than 300 M. In HepG2 cells incubated with 500 M DHEA for 24hrs followed by incubation with Fe-NTA for 2 hrs, TBARS were 6-fold higher than the control (p