MULTIPLE ROLES OF POLYHALOGENATED BIPHENYLS IN CAUSING INCREASES IN CYTOCHROME-P450 AND UROPORPHYRIN ACCUMULATION IN CULTURED-HEPATOCYTES

Uroporphyrin (URO) accumulation occurs in chick embryo hepatocytes tre ated with a number of polyhalogenated aromatic hydrocarbons (PHAHs) th at are known inducers of cytochrome P4501As (CYP1A). Previous dose res ponse studies had shown that URO accumulation does not begin until CYP 1A, as indicated by ethoxyresorufin O-deethylase (EROD) activity, is m aximally induced. The reason why the concentrations of PHAHs required for URO accumulation were higher than those required to induce EROD ha d not been explained. PHAHs, such as 3, 3', 4, 4'-tetrachlorobiphenyl (PCB77, IUPAC nomenclature, TCB) stimulate uroporphyrinogen (UROGEN) o xidation by microsomes from 3-methylcholanthrene (MC)-treated chick em bryos. Here we used a new protocol to investigate whether the requirem ent for more TCB to stimulate in vitro microsomal UROGEN oxidation ext ended to TCB-induced URO accumulation in intact cultured hepatocytes. Cultures were treated with increasing concentrations of TCB or other P HAHs to induce CYP1As, then with cycloheximide (CX) to prevent further P450 synthesis. The CX treatment was shown to block any further incre ases 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 pos tinduction TCB, the inducing concentrations of TCB which caused URO to begin to accumulate were now much lower than in the absence of postin duction TCB. Increases in CYP1A proteins, measured immunochemically, w ere 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 sensi tivity of the culture system for detection of PHAHs with EC50s (nM) fo r 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 c ulture dish caused detectible increases in URO accumulation. We conclu de that URO accumulation in chick hepatocyte cultures is Limited not o nly by the induction of CYP1A, but also by the stimulation of intracel lular UROGEN oxidation. (C) 1997 Academic Press.