Mouse Hepa 1c1c7 hepatoma cells produce complement component C3; 2,3,7,8-tetrachlorodibenzo-p-dioxin fails to modulate this capacity.

Previous studies from this laboratory have shown that 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) decreased complement component C3 levels in female B6C3F1 mouse serum following in vivo acute or subchronic exposure (White et al., 1986). Since TCDD is a hepatotoxic compound and more than 90% of serum C3 is produced by the liver, studies were undertaken using mouse Hepa 1c1c7 (Hepa 1) hepatoma cell line to determine if TCDD acts directly on hepatocytes to inhibit C3 production. The C3-producing capacity of Hepa 1 cells was first examined. When confluent Hepa 1 cell monolayers were cultured in 24-well plates with serum-free medium, a detectable amount of C3 (14.1 +/- 0.8 ng/ml) was secreted as early as 1 h after culture and reached a plateau at 12 h (68.3 +/- 4.9 ng/ml). Furthermore, the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis demonstrated that the molecular weight of C3 in culture supernatant corresponded to that present in mouse serum. Human recombinant IL-1 beta (hrIL-1 beta), a known inducer of complement C3, at doses as low as 1 unit/ml increased the C3 production to 158% of control after 24 h of incubation. The effect of hrIL-1 beta was dose dependent, and the maximum tested dose of 10 units/ml increased C3 production to 256% of control. When cells were directly exposed to TCDD at concentrations from 10(-10) to 10(-6) M, there was no inhibitory effect on production of C3. TCDD also failed to block the stimulatory effect of 10 units/ml hrIL-1 beta added to the culture 1 h later. To verify that cultured Hepa 1 cells were able to respond to TCDD, 7-ethoxyresorufin O-deethylase (EROD) activity was measured under the same conditions. TCDD dose-dependently increased EROD activity of Hepa 1 cells at 24 h following exposure. The activity reached 56.7 +/- 3.0 pmol/min/mg protein with 10(-9) M TCDD, compared with 10.7 +/- 1.7 pmol/min/mg protein of vehicle-exposed cells. Our results indicate that the direct interaction of TCDD with Hepa 1 cells does not affect their C3-producing capacity, although EROD activity, a characteristic response mediated by the cellular TCDD/Ah receptor, was induced. The lack of effect of TCDD in vitro suggests that the decrease of serum C3 levels observed in vivo may result from an indirect effect of TCDD on hepatocytes.