Hydroxyl radicals (OH center dot) were previously hypothesized as the principal ROS triggering lip-H2 expression in liquid cultures of Phanerochaete chrysosporium, through signal transduction. We therefore focused on determining the relationship between protein kinase C (PKC), ROS and LIP-H2. Significantly lower PKC activity was measured in high-LIP-producing (oxygenated) vs. low-LIP-producing (aerated grown with free exchange of atmospheric air) cultures. In oxygenated cultures, inactivation of PKC activity by calphostin C, staurosporin or H7 caused significant elevation in lip-H2 and also in MnSOD1 expression. Stimulation of PKC by phorbol 12-myristate 13-acetate (PMA) caused the reverse effect. Significantly low levels of lip-H2 expression were detected when O-2(center dot-), H2O2 or OH center dot were scavenged by 4,5-dihydroxy-1,3-benzene disulfonic acid (Tiron), pyruvate or dimethylsulfoxide (DMSO), whether the level of PKC was normal, stimulated or inactivated. In situ generation of OH center dot, via addition of Fenton reagents to aerated cultures, reduced pkc expression. In contrast, OH center dot scavenging stimulated pkc expression. This work suggests that due to high OH center dot levels, fungal cells activate a complex defensive system which regulates the levels of Fenton components by repressing PKC and stimulating LIP, MnSOD1 and catalase. (C) 2010 Elsevier Inc. All rights reserved.