Formation of a glucuronide conjugate of 12-O-tetradecanoylphorbol-13-acetate by LLC-PK1 renal epithelial cells in culture

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Abstract
The metabolism of [ 3 H]tetradecanoylphorbol-13-acetate [ 3 H]TPA) was studied in LLC-PK 1 cells, a differentiated renal epithelial cell line. In contrast to earlier studies in both fibroblastic and epithelial cells, a major metabolite of [ 3 H]-TPA by these cells was a new, previously unobserved species which was much more hydrophilic than TPA or its major metabolite in other cells, phorbol-13-acetate. Incubation of culture medium containing this metabolite with β-glucuronidase greatly reduced the amount of this polar metabolite, accompanied by a nearly equal increase in the amount of [ 3 H]-TPA extractable by organic solvents from this medium. In several experiments this TPA-glucuronic acid conjugate accounted for 20–50% of the total metabolites present after several days of incubation of LLC-PK1 cells with [ 3 H]TPA. Two other polar renal epithelial cell lines, derived from canine (MDCK) and bovine (MDBK) kidney, were incapable of converting [ 3 H]TPA to a β-glucuronidase sensitive product. A time course study indicated that once formed, the TPA-glucuronic acid conjugate was stable in LLC-PK 1 cells. Incubation of LLC-PK 1 conditioned medium containing the conjugate with other cell types such as primary hamster embryo cells or primary newborn hamster epidermal cells indicated that this material was stable in these cultures also. However, primary cultures of rat hepatocytes, and to a lesser extent a human hepatoma cell line, were capable of further metabolism of this conjugate to a form insensitive to β-glucuronidase treatment. This is the first evidence for the formation of a TPA-glucuronic acid conjugate either in vivo or in vitro , and while it is stable in the presence of the cells which produce it, this compound can also be further metabolized, presumably via β-glucuronidase in at least one cell type (i.e., hepatocytes). The biological significance of the formation and cleavage of this TPA-glucuronic acid conjugate remains to be determined.

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