Use of oral N-acetylcysteine for protection of melanocytic nevi against UV-induced oxidative stress: towards a novel paradigm for melanoma chemoprevention

Abstract

Purpose: Induction of oxidative stress has been implicated in UV-induced melanoma. We sought to determine whether the antioxidant N-acetylcysteine (NAC) could be safely administered to protect melanocytic nevi from the oxidative stress resulting from acute UV exposure.

Experimental design: Patients at increased risk for melanoma were recruited from a screening clinic. Induction and detection of oxidative stress (reactive oxygen species and glutathione depletion) was optimized in nevi following ex vivo UV irradiation. Nevi were removed from patients before, and following, oral ingestion of a single (1,200 mg) dose of NAC, and then these nevi were UV irradiated (4,000 J/m(2)).

Results: Oxidative stress was induced in nevi 24 to 48 hours following ex vivo UV irradiation. A single oral dose of NAC was well tolerated in all patients (n = 72). Basal levels of reduced glutathione and the NAC metabolite cysteine were well correlated between similar-appearing nevi from the same patient and were significantly increased in nevi removed 3 hours after NAC ingestion compared with nevi removed before drug ingestion. In approximately half (9 of 19) of patients tested, UV-induced glutathione depletion was attenuated in the postdrug (compared with predrug) nevus.

Conclusions: NAC can be safely administered to patients for the purpose of modulating UV-induced oxidative stress in nevi. This study suggests the feasibility of patients taking NAC prophylactically before acute UV exposure, to prevent pro-oncogenic oxidative stress in nevi and ultimately reduce long-term melanoma risk.

Fig. 1

Inflammatory and oxidative responses in human nevi following ex-vivo UV irradiation. A, Fragments from an individual nevus were untreated or UV-irradiated (4000 J/m²), then at the indicated time points were formalin-fixed and paraffin-embedded sections were stained with hematoxylin and eosin (original magnification ×400). Inflammatory cells are recognized by their small size and dark nuclei. B, Fragments from individual nevi were untreated or UV-irradiated (4000 J/m²), then ROS levels in protein equivalent lysates were measured by DCFDA fluorescence either 24 h (n=5 nevi) or 48 h (n=7 nevi) later. For each nevus, fluorescence value for the UV-treated fragment was normalized to the untreated fragment which was set at 1. *P<0.001 (two-tailed one sample t test). C, Fragments from individual nevi were untreated or UV-irradiated (4000 J/m²), then GSH content (normalized to nevus weight) was measured by chromatography coupled with fluorescence detection either 24 h (n=6 nevi) or 48 h (n=4 nevi) later. For each nevus, GSH content for the UV-treated fragment was normalized to the untreated fragment which was set at 100. *P<0.001, **P=0.008 (two-tailed one sample t tests).

Fig. 2

Inter-nevus correlation of oxidative biomarkers, and NAC-mediated oxidative modulation in nevi. A, Two similar-appearing nevi were removed from individual patients (n=8), and Cys and GSH content (normalized to nevus weight) was determined (left panel). For each patient, values for Cys (left plot, open circles) and GSH (right plot, filled circles) are expressed as a single data point reflecting each pair of nevi (nevus A, abscissa; nevus B, ordinate). Dotted lines represent theoretical correlation where data points should fall for pairs of nevi with identical values. For Cys measurements, correlation coefficient (r)=0.77 (95% confidence interval 0.14-0.95, P=0.03). For GSH measurements, correlation coefficient (r)=0.69 (95% confidence interval -0.03 – 0.94, P=0.06). B, Two similar-appearing nevi were removed from individual patients immediately prior to, and either 1.5 h, 3 h, 6 h, or 24 h (n=5-6 at each time point) following ingestion of 1200 mg NAC (left panel). Cys and GSH content (normalized to nevus weight) was determined for each nevus, and data expressed as percent increase in post-drug vs. pre-drug nevus (right panels). *P=0.047; **P=0.016 (Wilcoxon Signed Rank tests).

Fig. 3

NAC-mediated protection against UV-induced oxidative stress suggests potential utility of novel paradigm for melanoma chemoprevention. A, Two similar-appearing nevi were removed from 19 patients immediately prior to, and 3 h following ingestion of 1200 mg NAC. Fragments of each nevus were either untreated or UV-irradiated (4000 J/m²), then cultured for either 24 h (8 patients) or 48 h (11 patients). GSH content (normalized to nevus weight) was determined for each nevus fragment. B, Data expressed as percent UV-induced GSH depletion (UV-treated vs. untreated) in fragments of pre-drug nevi (open bars) and post-drug nevi (filled bars) from each patient. For nevi cultured 24 h, there was protection (ie. less GSH depletion) in 3/8 patients (left panel); for nevi cultured 48 h, there was protection in 6/11 patients. C, Schematic illustrating proposed novel paradigm for melanoma chemoprevention based on these pilot studies. Patients could take NAC in anticipation of sun exposure, to protect their nevi and other skin melanocytes from UV-induced oxidative stress (lower panel), and reduce their long-term risk of melanoma if applied over many years and sun exposures. This approach would circumvent the major pitfalls of conventional chemoprevention (upper panel) by timing drug administration with activation of a relevant oncogenic pathway, and episodic drug ingestion would avoid potential deleterious effects that may be associated with chronic use of any drug.