MOUSE HEPA 1C1C(7) HEPATOMA-CELLS PRODUCE COMPLEMENT COMPONENT C3 - 2,3,7,8-TETRACHLORODIBENZO-P-DIOXIN FAILS TO MODULATE THIS CAPACITY

Previous studies from this labortory have shown that 2,3,7,8-tetrachlo rodibenzo-p-dioxin (TCDD) decreased complement component C3 levels in female B6C3F1 mouse serum following in vivo acute or subchronic exposu re (White et al., 1986). Since TCDD is a hepatotoxic compound and more than 90% of serum C3 is produced by the liver, studies were undertake n using mouse Hepa 1c1c7 (Hepa 1) hepatoma cell line to determine if T CDD acts directly on hepatocytes to inhibit C3 production. The C3-prod ucing capacity of Hepa 1 cells was first examined. When confluent Hepa 1 cell monolayers were cultured in 24-well plates with serum-free med ium, a detectable amount of C3 (14.1 +/- 0.8 ng/ml) was secreted as ea rly as 1 h after culture and reached a plateau at 12 h (68.3 +/- 4.9 n g/ml). Furthermore, the sodium dodecyl sulfate-polyacrylamide gel elec trophoresis (SDS-PAGE) analysis demonstrated that the molecular weight of C3 in culture supernatant corresponded to that present in mouse se rum. Human recombinant IL-1beta (hrIL-1beta), a known inducer of compl ement 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-1beta 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 inhibito ry effect on production of C3. TCDD also failed to block the stimulato ry effect of 10 units/ml hrIL-1beta added to the culture 1 h later To verify that cultured Hepa 1 cells were able to respond to TCDD, 7-etho xyresorufin O-deethylase (EROD) activity was measured under the same c onditions. TCDD dose-dependently increased EROD activity of Hepa 1 cel ls 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 di rect interaction of TCDD with Hepa 1 cells does not affect their C3-pr oducing capacity, although EROD activity, a characteristic response me diated by the cellular TCDD/Ah receptor, was induced. The lack of effe ct of TCDD in vitro suggests that the decrease of serum C3 levels obse rved in vivo may result from an indirect effect of TCDD on hepatocytes .