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The in vitro effects on melanogenesis of γ-oryzanol (1), a rice bran-derived phytosterol, were investigated. The melanin content in B16F1 cells was significantly and dose-dependently reduced (-13% and -28% at 3 and 30 μM, respectively). Tyrosinase enzyme activity was inhibited by 1 both in a cell-free assay and when analyzed based on the measurement of cellular tyrosinase activity. Transcriptome analysis was performed to investigate the biological pathways altered by 1, and it was found that gene expression involving protein kinase A (PKA) signaling was markedly altered. Subsequent analyses revealed that 1 stimulation in B16 cells reduced cytosolic cAMP concentrations, PKA activity (-13% for cAMP levels and -40% for PKA activity), and phosphorylation of the cAMP-response element binding protein (-57%), which, in turn, downregulated the expression of microphthalmia-associated transcription factor (MITF; -59% for mRNA and -64% for protein), a key melanogenic gene transcription factor. Accordingly, tyrosinase-related protein 1 (TRP-1; -69% for mRNA and -82% for protein) and dopachrome tautomerase (-51% for mRNA and -92% for protein) in 1-stimulated B16F1 cells were also downregulated. These results suggest that 1 has dual inhibitory activities for cellular melanogenesis by inhibiting tyrosinase enzyme activity and reducing MITF and target genes in the PKA-dependent pathway.

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Rationale: Drug use during adolescence results in a life-long risk to develop substance-use disorders. Adolescent rats are less reactive to cocaine-associated cues compared to adults; however, the contribution of adolescent-formed context-drug-associations to elicit relapse-like behavior is underexplored. Although it is known that social isolation can impact drug-seeking behavior, the effects of housing conditions on context-induced cocaine-seeking during adolescence vs adulthood is unknown.Objectives: The present study compared the effect of adolescent vs adult-formed context-drug associations under differential housing conditions (pair vs single) on cocaine-seeking behavior during adolescence or adulthood. This objective was accomplished using operant cocaine self-administration (Coc-SA) under a standard, non-abbreviated (Non-ABRV) or modified, abbreviated (ABRV) paradigm. Methods:In experiment 1, adolescent and adult rats received Non-ABRV Coc-SA in a distinct context (2 hr, 1x/day, 10 days), extinction training (EXT) in a second context (1 hr, 1x/day, 8 days) with reinstatement test (TEST) during adulthood in the cocaine-paired context. In experiments 2-3, rats received all behavioral phases during adolescence or adulthood: ABRV Coc-SA (2 hr, 2x/day, 5 days), EXT (1 hr, 4x/day, 2 days) with TEST in a cocaine-paired or novel, unpaired context. All experiments included pair and single-housing conditions.Results & Conclusions: Age at cocaine exposure did not influence behavior in Non-ABRV or ABRV paradigms. Under Non-ABRV conditions, adolescent and adult single-housed rats had higher seeking behavior than pair housed. These data suggest that social isolation influences context-induced cocaine-seeking regardless of age at drug exposure and provides a condensed, ABRV paradigm to investigate context-induced, cocaine-seeking behavior during adolescence.

Long-term topical skin care by traditional anti-melanogenic agents can raise several safety concerns. An understanding of the molecular mechanisms of active compounds on melanogenesis is, therefore, necessary to address pigmentation issues. Here we revealed that stimulation with 1 mM betaine, an abundant component in rice bran, significantly reduced 21% of intracellular melanin content by suppressing tyrosinase activity and microphthalmia-associated transcription factor (MITF) expression in B16-F1 murine melanocytes. The expression of MITF was suppressed at both mRNA and protein levels by 43 and 44%, respectively. Subsequently, the betaine-stimulated melanocytes showed inhibition of PKA-CREB signaling axis but activation of extracellular-signal-regulated kinase and AKT-GSK3β signaling pathways. This inhibition and activation led to downregulation of MITF expression at both the transcriptional and post-translational levels to suppress melanin synthesis. These findings collectively suggested that betaine is a potential anti-melanogenic compound for functional foods and cosmetics.