Ultrastructural Observations on the Effect of 4-Hydroxyanisole on Normal Human Melanocytes in Tissue Culture

Abstract

Riley's classic 1970 experiment showing a specific cytotoxic effect of 4-hydroxyanisole (4-OHA) on tissue-cultured melanocytes of black guinea pig ear skin was repeated on normal human melanocytes, and the results were examined by electron microscopy. In dispersed tissue culture, no specific toxic effect on human melanocytes was observed following equally timed exposures to similar (10(-3) M) or even higher (10(-2) M) concentrations of the drug; plasma membrane, nucleus, and cytoplasmic organelles, including melanosomes were unaffected. The same applied to melanocytes of whole epidermis exposed for 5 hr to the same concentrations of 4-OHA in culture medium. Melanocytes of PUVA treated skin similarly exposed for up to 24 hr to 10(-2) and 10(-3) M 4-OHA, likewise exhibited no evident morphological damage at the ultrastructural level. The discrepancy of results between guinea pig and man could have a variety of explanations, one of which could be due to a possible relatively low level of active tyrosinase in the human melanocytes (Riley believes the cytotoxic effect of 4-OHA to be due to the fact that it acts as a substrate for tyrosinase, toxic intermediates being liberated as a result). However, the lack of effect on the PUVA-activated melanocytes indicates that this cannot be the entire explanation.

Ultrastructural and biochemical observations on the effect of 4-hydroxyanisole plus tyrosinase on normal human melanocytes and keratocytes in tissue culture

“…The observations presented here extend those of a previous ultrastructural study (Breathnach et al, 1981) which failed to demonstrate a specific tyrosinase-dependent cytotoxic effect of 4-OHA on The other peaks in the figure represent the intermediate metabolic products leading to the melanoid polymer. They were not identified and peak areas were not measured.…”

“…Although the present results clearly show that normal human melanocytes can be damaged by external exposure to products of the oxidation of 4-OHA by tyrosinase, they still do not account for the discrepancy between results on human and guinea-pig melanocytes exposed to 4-OHA alone (Riley, 1970). The possibilities discussed previously (Breathnach et al, 1981) still remain. One further point of interest emerges from the present investigations.…”

“…Failure of a previous attempt (Breathnach et al, 1981) to produce damage to cultured melanocytes by exposure to 4-OHA alone can be partly explained by the present results. The damage observed in present cultures washed free of medium and exposed to 10-3M 4-OHA plus tyrosinase for 1 h, confirms the biochemical findings of production of a free toxic 4-OHA quinone during this period.…”

“…According to Riley, the toxic effect of 4-OHA is tyrosinase dependent and, in line with this, addition of tyrosinase to cultures of Harding-Passey melanoma cells has been said to have a marked effect on their sensitivity to 4-OHA (Dewey et al, 1977). However, in a previous study (Breathnach et al, 1981), we failed to demonstrate any specific damage to normal melanocytes in primary dispersed and organ cultures following exposure to 4-OHA at concentrations equal to, and even higher than, those reported to be markedly toxic to guinea-pig melanocytes (Riley, 1970). The discrepancy between results in the two instances could be attributed to a number of factors, one of which might have been a low level of tyrosinase activity within the human melanocytes.…”

2 Final Report on the Safety Assessment of p-Hydroxyanisole

“…In the previously cited reports of Riley(46) and Breathnach et al, (53) p-Hydroxyanisole was cytotoxic to guinea pig melanocytes at a concentration of 1 0-3 M but had no toxic effect at M on human melanocytes. The apparent discrepancy of results may be due t~: (~~,~~) (1) species differences between man and the guinea pig (although the basic structure of the melanocyte and general process of melanogenesis are, as far as is known, identical in the two species), (2) an inability of p-Hydroxyanisole to traverse the plasma membrane and enter the human melanocyte, (3) differences i n the reaction between human tyrosinase and p-Hydroxyanisole and in the reaction between the guinea pig enzyme and p-Hydroxyanisole, and (4) the need for human tyrosinase to be present in particularly high concentrations (in Riley's view, the presence of active tyrosinase within the cell is essential for p-Hydroxyanisole to exert its toxic effect).…”