Willets J M, Lambert D G, Lunec J, Griffiths H R, Phillipson O
Division of Chemical Pathology, C.M.H.T., Hodgkin Building, University of Leicester, Leicester LE1 9HN, UK.
Environ Toxicol Pharmacol. 1996 Aug 15;2(1):59-68. doi: 10.1016/1382-6689(96)00030-0.
We have investigated the potential neurotoxicity of the catecholamine depleting agent 1,2,3,4-tetrahydro-2-methyl-4,6,7-isoquinolinetriol (TMIQ) in SH-SY5Y neuroblastoma cells. TMIQ induced a time and dose related inhibition of 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide; thiazoyl blue (MTT) reduction and an increase in lactate dehydrogenase release. After 72 h TMIQ (30 μM) significantly (P < 0.05) inhibited MTT reduction, and significantly increased LDH release. TMIQ cytotoxicity was not prevented by the inclusion of monoamine oxidase inhibitors (clorgyline or deprenyl), antioxidants (α-tocopherol or Trolox C) or the uptake(1) inhibitor imipramine. TMIQ also induced a dose dependent stimulation of [(3)H]noradrenaline (NA) uptake, with maximum at 100 μM and EC(50) of 8 μM. This stimulation of [(3)H]NA uptake was not prevented by the inhibition of protein kinase C, or activation of adenylate or guanylate cyclases. In addition, TMIQ significantly (P < 0.05) displaced [(3)H]nisoxetine binding from the uptake(1) recognition site with a K(i) of 71 ± 8 μM. However, as this interaction occurs at concentrations of TMIQ well above the EC(50) for [(3)H]NA uptake, it is unlikely to explain TMIQ stimulated NA uptake. Furthermore, TMIQ inhibited potassium evoked [(3)H]NA release from SH-SY5Y cells, with an IC(50) of 490 μM. Thus, TMIQ is cytotoxic to SH-SY5Y cells. However, the exact mechanism of toxicity requires further investigation, since it appears not to involve monoamine oxidase bioactivation, and is not mediated through membrane based free radical damage. Furthermore, although TMIQ inhibits mitochondrial Complex I (IC(50) = 1.5 mM) with potency apparently greater than MPTP (2.7 mM), mitochondrial respiration was unaffected. The present studies suggest that the mechanism of toxicity differs from that causing depletion of catecholamines and inhibition of tyrosine hydroxylase by TMIQ described in previous studies.
我们研究了儿茶酚胺耗竭剂1,2,3,4-四氢-2-甲基-4,6,7-异喹啉三醇（TMIQ）对SH-SY5Y神经母细胞瘤细胞的潜在神经毒性。TMIQ诱导了与时间和剂量相关的3-[4,5-二甲基噻唑-2-基]-2,5-二苯基四氮唑溴盐；噻唑蓝（MTT）还原抑制以及乳酸脱氢酶释放增加。72小时后，TMIQ（30μM）显著（P<0.05）抑制MTT还原，并显著增加LDH释放。加入单胺氧化酶抑制剂（氯吉兰或司来吉兰）、抗氧化剂（α-生育酚或Trolox C）或摄取（1）抑制剂丙咪嗪并不能防止TMIQ的细胞毒性。TMIQ还诱导了[(3)H]去甲肾上腺素（NA）摄取的剂量依赖性刺激，在100μM时达到最大值，EC(50)为8μM。这种[(3)H]NA摄取的刺激不受蛋白激酶C抑制或腺苷酸或鸟苷酸环化酶激活的影响。此外，TMIQ显著（P<0.05）从摄取（1）识别位点取代[(3)H]尼索西汀结合，K(i)为71±8μM。然而，由于这种相互作用发生在TMIQ浓度远高于[(3)H]NA摄取的EC(50)时，因此不太可能解释TMIQ刺激的NA摄取。此外，TMIQ抑制钾诱导的[(3)H]NA从SH-SY5Y细胞释放，IC(50)为490μM。因此，TMIQ对SH-SY5Y细胞具有细胞毒性。然而，确切的毒性机制需要进一步研究，因为它似乎不涉及单胺氧化酶生物活化，也不是通过基于膜的自由基损伤介导的。此外，尽管TMIQ抑制线粒体复合物I（IC(50)=1.5mM），其效力明显大于MPTP（2.7mM），但线粒体呼吸未受影响。目前的研究表明，毒性机制与先前研究中描述的TMIQ导致儿茶酚胺耗竭和酪氨酸羟化酶抑制的机制不同。
