Figure 3. NNT inhibitors are non-toxic and induce pigmentation of primary melanocytes in vitro and in human skin explants.

(A) Murine melanocytes (Melan-A) showed increased melanin content after incubation with 2 mM 2,3BD or DCC, but not after incubation with palmitoyl-CoA; n =3, analyzed by ordinary one-way ANOVA with Dunnett’s post-test. (B-C) Treatment of primary human melanocytes with different doses of DCC (B, n = 4) or 2,3BD (C, n = 6) for 24 hours yielded decreased GSH/GSSG ratios; analyzed by ordinary one-way ANOVA with Tukey’s (B) or Dunnett’s (C) post-test. (D) A single, one-time topical treatment with 2,3BD (1M or 11M) induces human skin pigmentation after 5 days. Left Panel: Representative images of at least three individual experiments are displayed. Right panel: Reflective colorimetry measurements of skin treated with 2,3BD (higher L* values represent lighter skin tones); n = 3, analyzed by ordinary one-way ANOVA with Dunnett’s post-test. (E) Fontana-Masson staining of melanin in human skin after 2,3BD (50 mM) (i) and hematoxylin & eosin staining (ii) compared with vehicle control (DMSO). (iii) Supranuclear capping in human keratinocytes of 2,3BD- and vehicle control-treated skin displayed by Fontana-Masson staining. (F) NNT inhibitors, 2,3BD or DCC, applied daily at a 50 mM dose resulted in skin darkening after 5 days. Left Pane: Representative images of three individual experiments are displayed. Right panel: Reflective colorimetry measurements of human skin treated with 2,3BD, DCC, or DMSO vehicle (higher L* values represent lighter skin tones;) n = 3, analyzed by ordinary one-way ANOVA with Dunnett’s post-test. (G) Immunofluorescence staining for CPD formation (Red) in human skin treated with 50 mM 2,3BD for 5 consecutive days. On the last day, skin was irradiated with 1000 mJ/cm² UVB. The results show a protective role for 2,3BD from UVB-induced CPD damage. Representative images of three individual experiments are displayed. Scale bar 50μM. Quantified results were normalized to the total number of cells; n = 3, analyzed by ordinary two-way ANOVA with Šídák’s post-test. (H) Measurement of γ-H2AX (Red) in human skin revealed no significant toxicity of 2,3BD, while 2,3-BD-induced pigmentation protected from UVB-induced γ-H2AX formation. Representative images of three individual experiments are displayed Scale bar 50μM. Quantified results were normalized to the total number of cells; n = 3, analyzed by ordinary two-way ANOVA with Šídák’s post-test.

All data are expressed as mean ± SEM. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001