Revised: May 28, 2014
Accepted: June 27, 2014
Published online: August 2, 2014
AIM: To establish whether d-limonene can protect against induction of cyclobutane pyrimidine dimers (CPDs) and sunburn in ultraviolet irradiation (UVR) irradiated mouse skin.
METHODS: The d-limonene was given in 4 daily oral 20 μL aliquots at different concentrations as follows: 100%, 10% or 1% in liponate and 100% liponate as control. One day after the final d-limonene treatment, the mice were anesthetized with i.p. sodium pentobarbital and placed in boxes to allow a rectangular (2 cm × 4 cm) region of dorsal skin to be irradiated with a single, ultraviolet radiation dose of 1.5 kJ/m2. Skin samples from UVR irradiated area were obtained at 5 min after UVR exposure for CPD detection, at 6 d after UVR exposure, skin samples were obtained for in situ analysis for N-myc downstream regulating gene 1 (NDRG1) (a stress response gene), proliferating cell nuclear antigen (PCNA) (an S-phase marker) and filaggrin (a barrier integrity gene). Based on immunohistochemistry staining, the number of CPD, NDRG1 and PCNA positive cells, as well as unstained cells was counted in 3 different individually selected areas and percentage of positive cells was established.
RESULTS: CPD reduction occurred as follows: liponate only-none; 1% d-limonene-54.3% reduction of CPDs; 10% d-limonene-73.4% reduction of CPDs; 100% d-limonene-86.1% reduction of CPDs, the latter equivalent to a UV dose of only 0.21 kJ/m2. Sunburn was also dose-dependently reduced by d-limonene. The NDRG1 protein was strongly induced by UVR (70.0% ± 10.4% positive cells), but 1% d-limonene reduced the response to 64.6% ± 9.2%, 10% d-limonene reduced the response to 16.2% ± 3.4% and 100% d-limonene reduced the response to 6.3% ± 1.7%. Similarly, PCNA was 52.4% ± 9.9% positive in UVR exposed skin, and 1% d-limonene reduced it to 42.9% ± 8.1%, 10% d-limonene reduced it to 36.2% ± 6.7% and 100% d-limonene reduce it to 13.8% ± 3.4%. NDRG1 and PCNA were increased by d-limonene or UVR separately, but combined they produced less than either agent separately owing to the protective effect of pre-exposure to d-limonene.
CONCLUSION: Overall d-limonene acted to protect against ultraviolet B-induced DNA photodamage and sunburn in UVR exposed skin.
Core tip: Skh-1 hairless mice were given 4 daily 20 μL aliquots of different concentrations of d-limonene, and then irradiated to a single ultraviolet irradiation. Skin samples from the ultraviolet-exposed area of mice showed that ultraviolet irradiation induced cyclobutane pyrimidine dimers formation, N-myc downstream regulating gene 1 and proliferating cell nuclear antigen expression, pretreatment of d-limonene significantly reduced these responses. Pure d-limonene also induced the expression of epidermal barrier protein filaggrin. In conclusion, d-limonene protected the mice skin from UV-induced DNA photodamage and sunburn in mice skin.