Multiple roles of polyhalogenated biphenyls in causing increases in cytochrome P450 and uroporphyrin accumulation in cultured hepatocytes

Abstract

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.