Sinclair P R, Walton H S, Gorman N, Jacobs J M, Sinclair J F
VA Medical Center, White River Junction, Vermont 05009, USA.
Toxicol Appl Pharmacol. 1997 Dec;147(2):171-9. doi: 10.1006/taap.1997.8273.
Uroporphyrin (URO) accumulation occurs in chick embryo hepatocytes treated with a number of polyhalogenated aromatic hydrocarbons (PHAHs) that are known inducers of cytochrome P4501As (CYP1A). Previous dose response studies had shown that URO accumulation does not begin until CYP1A, as indicated by ethoxyresorufin O-deethylase (EROD) activity, is maximally induced. The reason why the concentrations of PHAHs required for URO accumulation were higher than those required to induce EROD had not been explained. PHAHs, such as 3,3',4,4'-tetrachlorobiphenyl (PCB77, IUPAC nomenclature, TCB) stimulate uroporphyrinogen (UROGEN) oxidation by microsomes from 3-methylcholanthrene (MC)-treated chick embryos. Here we used a new protocol to investigate whether the requirement for more TCB to stimulate in vitro microsomal UROGEN oxidation extended to TCB-induced URO accumulation in intact cultured hepatocytes. Cultures were treated with increasing concentrations of TCB or other PHAHs to induce CYP1As, then with cycloheximide (CX) to prevent further P450 synthesis. The CX treatment was shown to block any further increases in CYP1A as determined by immunoblots. 5-Aminolevulinic acid and a high concentration of TCB ("postinduction TCB") were then added to stimulate intracellular UROGEN oxidation. Using the protocol with postinduction TCB, the inducing concentrations of TCB which caused URO to begin to accumulate were now much lower than in the absence of postinduction TCB. Increases in CYP1A proteins, measured immunochemically, were detected at about the same inducing TCB concentrations that began to increase URO accumulation. The new protocol, with postinduction TCB, using URO accumulation as the end point, greatly increased the sensitivity of the culture system for detection of PHAHs with EC50s (nM) for 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), TCB, 3,3',4,4',5,5'-hexachlorobiphenyl, MC, and hexachlorobenzene being about 0.003, 0.11, 0.75, 3.5, and 30, respectively. As little as 2-4 fmol TCDD per culture dish caused detectible increases in URO accumulation. We conclude that URO accumulation in chick hepatocyte cultures is limited not only by the induction of CYP1A, but also by the stimulation of intracellular UROGEN oxidation.
尿卟啉（URO）蓄积发生在用多种多卤代芳烃（PHAHs）处理的鸡胚肝细胞中，这些多卤代芳烃是已知的细胞色素P4501As（CYP1A）诱导剂。先前的剂量反应研究表明，直到细胞色素P4501A被最大程度诱导（以乙氧基异吩恶唑酮O - 脱乙基酶（EROD）活性表示）时，URO蓄积才开始。URO蓄积所需的PHAHs浓度高于诱导EROD所需浓度的原因尚未得到解释。多卤代芳烃，如3,3',4,4'-四氯联苯（PCB77，国际纯粹与应用化学联合会命名法，TCB）可刺激来自经3 - 甲基胆蒽（MC）处理的鸡胚的微粒体氧化尿卟啉原（UROGEN）。在此，我们采用一种新方案来研究，对于在体外刺激微粒体UROGEN氧化需要更多TCB这一情况，是否也适用于完整培养的肝细胞中TCB诱导的URO蓄积。用浓度递增的TCB或其他PHAHs处理培养物以诱导CYP1As，然后用环己酰亚胺（CX）来阻止进一步的P450合成。免疫印迹显示，CX处理可阻止CYP1A的任何进一步增加。然后加入5 - 氨基乙酰丙酸和高浓度的TCB（“诱导后TCB”）以刺激细胞内UROGEN氧化。使用含诱导后TCB的方案，导致URO开始蓄积的TCB诱导浓度现在比无诱导后TCB时低得多。通过免疫化学方法测量，在开始增加URO蓄积的大约相同的TCB诱导浓度下检测到CYP1A蛋白增加。以URO蓄积为终点的含诱导后TCB的新方案极大地提高了培养系统检测PHAHs的灵敏度，对于2,3,7,8 - 四氯二苯并 - p - 二恶英（TCDD）、TCB、3,3',4,4',5,5'-六氯联苯、MC和六氯苯，其半数有效浓度（EC50s，单位为纳摩尔）分别约为0.003、0.11、0.75、3.5和30。每个培养皿低至2 - 4飞摩尔的TCDD就能引起可检测到的URO蓄积增加。我们得出结论，鸡肝细胞培养物中的URO蓄积不仅受CYP1A诱导的限制，还受细胞内UROGEN氧化刺激的限制。
