Effects of Oxygen Tension on the Growth and Pigmentation of Normal Human Melanocytes

The effects of oxygen tension on human melanocyte growth, tyrosinase activity, and melanin production were assessed. Melanocytes, seeded at 104 cells/cm2, were grown in modified Eagle's medium (MEM) with 5% fetal bovine serum (FBS) and 10 ng/ml 12-0-tetradecanoyl-phorbol-13-acetate (TPA). Flasks were equilibrated with gas mixtures containing 5% CO2 and various partial pressures of oxygen (PO2 7–620mm Hg) and kept in incubators, which were electronically maintained at the desired oxygen tensions. Melanocytes grew best at PO2 from 6–34mm Hg. Growth was reduced by 30% at PO2 142mm Hg, and even more at O2 tensions greater than 230mm Hg. A PO2 of 603 rum Hg was cytotoxic. Tyrosinase activity (assayed by the method of Pomerantz) was 300 μU/mg protein at PO2 7–34mm Hg. At PO2 235 and 355mm Hg tyrosinase activity decreased to about 100 μu/mg protein. The apparent Km for tyrosine was unchanged in melanocytes cultured at all experimental oxygen tensions. The Vmax, however, was decreased at the higher oxygen tensions (PO2 235mm Hg). At PO2 6–135mm Hg the melanin content was proportional to tyrosinase activity. At cytostatic oxygen tensions (PO2 235 and 355mm Hg) the intracellular melanin content increased somewhat, although tyrosinase activity was decreased. Low oxygen tension is favorable for both melanocyte proliferation and tyrosinase activity