Altered gene expression in melanocytes exposed to 4-tertiary butyl phenol (4-TBP): Upregulation of the A2b adenosine receptor

Exposure to phenolic agents contributes to the development of occupational vitiligo. Proposed as a causative factor for leukoderma in vivo, the parasu bstituted phenol 4-tertiary butyl phenol was chosen to investigate early ce llular events responsible for selective disappearance of melanocytes from t he epidermis of individuals sensitive to such agents. To this end, differen tial display of melanocyte mRNA isolated from three separate cultures was p erformed following a 12 h exposure of cells to 250 mu M 4-tertiary butyl ph enol or to vehicle alone. Fragments of cDNA representing differentially exp ressed messages were cloned and subsequently confirmed by reverse dot blott ing. Alignment analysis revealed that the L30 ribosomal protein was upregul ated by the treatment, potentially reflecting altered levels of protein syn thesis in response to stress. In addition, a gene sequence upregulated foll owing exposure to 4-tertiary butyl phenol was identified as the A(2b) recep tor (a P1 receptor for adenosine). Differential expression of this gene was confirmed in an RNase protection assay. By reverse transcription-polymeras e chain reaction, the gene was shown to be expressed in keratinocytes and f ibroblasts as well. Flow cytometry confirmed differential expression in mel anocytes and fibroblasts, but not in keratinocytes. Interestingly, it has b een reported that pi purinoceptor stimulation can induce apoptosis, This is in concordance with results reported elsewhere demonstrating induction of apoptosis by 4-tertiary butyl phenol in human melanocytes, as well as with morphologic changes observed in this study in cells exposed to 250 mu M 4-t ertiary butyl phenol for 72 h. In conclusion, differential display is usefu l to establish melanocyte components involved in the cellular response to p henolic agents.