Impact of hyperpigmentation on superoxide flux and melanoma cell metabolism at mitochondrial complex II.

Melanogenesis is a highly conserved process of cytophotoprotection from UV radiation present in many species. Although both mitochondrial function and UV radiation insults are well-documented promoters of increased cellular stress, their individual molecular relationships with skin pigmentation have not been clearly resolved. This study provides evidence for a direct relationship between cellular melanin content, superoxide flux, and mitochondrial function at complex II. Direct and significant correlation between increased pigmentation and complex II turnover was observed in genetically different melanoma cell lines of varied basal pigmentation states (P < 0.01). The same trend was also observed when comparing genetically identical cell cultures with increasing levels of induced pigmentation (P < 0.005). The observation of increased steady-state levels of the catalytic complex II succinate dehydrogenase subunit A alongside hyperpigmentation suggested coregulation of activity and pigment production (P < 0.01). The study also presents novel evidence for a relationship between hyperpigmentation and increased superoxide-generating capacity at complex II. By amperometrically monitoring superoxide flux from differently pigmented FM55 melanocytes and their isolated mitochondria, a dynamic and responsive relationship between pigmentation, complex II function, and intracellular superoxide generation was observed (P < 0.005). The data support hyperpigmentation as a protective antioxidant mechanism in response to complex II-mediated reactive oxygen species generation.

A role for human mitochondrial complex II in the production of reactive oxygen species in human skin.

The mitochondrial respiratory chain is a major generator of cellular oxidative stress, thought to be an underlying cause of the carcinogenic and ageing process in many tissues including skin. Previous studies of the relative contributions of the respiratory chain (RC) complexes I, II and III towards production of reactive oxygen species (ROS) have focussed on rat tissues and certainly not on human skin which is surprising as this tissue is regularly exposed to UVA in sunlight, a potent generator of cellular oxidative stress. In a novel approach we have used an array of established specific metabolic inhibitors and DHR123 fluorescence to study the relative roles of the mitochondrial RC complexes in cellular ROS production in 2 types of human skin cells. These include additional enhancement of ROS production by exposure to physiological levels of UVA. The effects within epidermal and dermal derived skin cells are compared to other tissue cell types as well as those harbouring a compromised mitochondrial status (Rho-zero A549). The results show that the complex II inhibitor, TTFA, was the only RC inhibitor to significantly increase UVA-induced ROS production in both skin cell types (P<0.05) suggesting that the role of human skin complex II in terms of influencing ROS production is more important than previously thought particularly in comparison to liver cells. Interestingly, two-fold greater maximal activity of complex II enzyme was observed in both skin cell types compared to liver (P<0.001). The activities of RC enzymes appear to decrease with increasing age and telomere length is correlated with ageing. Our study showed that the level of maximal complex II activity was higher in the MRC5/hTERT (human lung fibroblasts transfected with telomerase) cells than the corresponding wild type cells (P=0.0012) which can be considered (in terms of telomerase activity) as models of younger and older cells respectively.

Real-time monitoring of superoxide generation and cytotoxicity in neuroblastoma mitochondria induced by 1-trichloromethyl-1,2,3,4-tetrahydro-beta-carboline.

The potential neurotoxin 1-trichloromethyl-1,2,3,4-tetrahydro-beta-carboline (TaClo) has recently been suggested to be a causative factor in the clinical development of parkinsonian symptoms after long-term exposure to precursor compounds such as the hypnotic chloral hydrate. TaClo is known to cause cell death in dopaminergic neuronal cells, however, the pathway and mechanisms remain undefined. This study reports for the first time that TaClo promotes cytotoxicity in SH-SY5Y neuroblastoma cells within 2 hours of initial exposure. TaClo also caused superoxide production from isolated mitochondria, which was comparable in response time and magnitude to production elicited by more established respiratory inhibitors such as rotenone and antimycin A. These findings present new evidence in support of TaClo-induced neuronal death via superoxide signalling and oxidative stress.