Vitamin B12 Deficiency Induces Imbalance in Melanocytes

International Journal of Molecular Sciences Article Vitamin B12 Deficiency Induces Imbalance in Melanocytes Homeostasis—A Cellular Basis of Hypocobalaminemia Pigmentary Manifestations Zuzanna Rzepka 1, Michalina Respondek 1, Jakub Rok 1, Artur Beberok 1,* , Keith ó Proinsias 2, Dorota Gryko 2 and Dorota Wrze´sniok 1,* 1 Department of Pharmaceutical Chemistry, School of Pharmacy with the Division of Laboratory Medicine in Sosnowiec, Medical University of Silesia in Katowice, Jagiellońska4, 41-200 Sosnowiec, Poland; [email protected] (Z.R.); [email protected] (M.R.); [email protected] (J.R.) 2 Institute of Organic Chemistry, Polish Academy of Science, Kasprzaka 44/52, 01-224 Warsaw, Poland; [email protected] (K.ó.P.); [email protected] (D.G.) * Correspondence: [email protected] (A.B.); [email protected] (D.W.); Tel.: +48-32-364-16-11 (A.B. & D.W.) Received: 19 August 2018; Accepted: 18 September 2018; Published: 19 September 2018 Abstract: Vitamin B12 deficiency causes significant changes in cellular metabolism leading to various clinical symptoms, such as hematological, psychiatric, and neurological disorders. We hypothesize that skin pigmentation disorders may be a diagnostically important manifestation of vitamin B12 deficiency, however the cellular and molecular mechanisms underlying these effects remain unknown. The aim of this study was to examine the effect of vitamin B12 deficiency on melanocytes homeostasis. Hypocobalaminemia in vitro model was developed by treating epidermal melanocytes with synthesized vitamin B12 antagonist—hydroxycobalamin(c-lactam). The cells were examined using immunoenzymatic, spectrophotometric, and fluorimetric assays as well as image cytometry. Significant melanogenesis stimulation—the increase of relative melanin content and tyrosinase activity up to 131% and 135%, respectively—has been indicated. Cobalamin-deficient cells displayed the elevation (by 120%) in reactive oxygen species level. Moreover, the redox status imbalance was stated. The study provided a scientific evidence for melanocytes homeostasis disturbance under hypocobalaminemia, thus indicating a significant element of the hyperpigmentation mechanism due to vitamin B12 deficiency. Furthermore, the implication between pigmentary and hematological and/or neuropsychiatric symptoms in cobalamin-deficient patients may be an important issue. Keywords: vitamin B12; cobalamin deficiency; hyperpigmentations; melanogenesis; oxidative stress 1. Introduction Vitamin B12 (cobalamin) deficiency is an important health issue, particularly among the elderly. The main causes of hypocobalaminemia are insufficient dietary intake (e.g., in vegans and vegetarians) and malabsorption of the vitamin [1]. The nearly ubiquitous use of drugs that can interrupt cobalamin absorbtion, i.e., gastric acid–blocking agents and metformin, may also contribute to a growing prevalence of cobalamin deficiency [2]. Vitamin B12 plays a key role in cellular metabolism. It is a cofactor of two enzymes: Methionine synthase, which catalyzes a methylation of homocysteine to methionine with simultaneous reconstitution of tetrahydrofolate and methylmalonyl-CoA mutase, which catalyzes the conversion of methylmalonyl-CoA to succinyl-CoA. At the cellular level, cobalamin deficiency results in the Int. J. Mol. Sci. 2018, 19, 2845; doi:10.3390/ijms19092845 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2018, 19, 2845 2 of 13 inhibition of purine synthesis and in the accumulation of methylmalonic acid and homocysteine (Hcy) [1]. Hcy has been previously demonstrated to act as a prooxidant in various types of cells [3,4]. Melanin is a natural pigment and the major determinant of skin and hair color. Biosynthesis of melaninInt. takes J. Mol. placeSci. 2018 in, 19 melanocytes—dendritic, x cells located mainly in the basal layer of the2 of epidermis 13 and hair follicles [5,6]. The ability to synthesize melanin plays an important role in skin physiology and inhibition of purine synthesis and in the accumulation of methylmalonic acid and homocysteine pathology, and determines skin responsiveness to ultraviolet radiation. Melanogenesis is a complex (Hcy) [1]. Hcy has been previously demonstrated to act as a prooxidant in various types of cells [3,4]. process controlledMelanin byis a differentnatural pigment modes and including the major receptor-mediated determinant of skin and pathways hair color. activated Biosynthesis by hormones, of cytokines,melanin neurotransmitters, takes place in melanocytes—dendritic and eicosanoids, as cells well located as receptor-independent mainly in the basal layer mechanisms of the epidermis modulated by nutrients,and hair microelements, follicles [5,6]. The pH, ability ions, to andsynthesize redox melanin homeostasis plays an [5 important,7]. role in skin physiology Theand major pathology, symptoms and determines of cobalamin skin responsiveness deficiency are to hematological, ultraviolet radiation. psychiatric, Melanogenesis and neurological is a disorderscomplex [8]. Less process known controlled manifestations by different modes include including reversible receptor-mediated cutaneous hyperpigmentation,pathways activated by mostly hormones, cytokines, neurotransmitters, and eicosanoids, as well as receptor-independent localized in the dorsum of hands and feet, fingers, knees, lateral surfaces of the legs, skin folds, and oral mechanisms modulated by nutrients, microelements, pH, ions, and redox homeostasis [5,7]. mucosa [9–The12]. major Interestingly, symptoms severalof cobalamin clinical deficiency cases are of vitiligohematological, and depigmentation psychiatric, and neurological of scalp hair due to cobalamindisorders depletion [8]. Less haveknown also manifestations been reported include [13 reversible,14]. Thus, cutaneous the impact hyperpigmentation, of cobalamin mostly deficiency on melanogenesislocalized andin the melanocytes dorsum of hands homeostasis and feet, fingers, is not knees, obvious. lateral surfaces of the legs, skin folds, and Currentoral mucosa scientific [9–12]. findings Interestingly, about several the molecular clinical cases patomechanism of vitiligo and depigmentation of hypocobalaminemia-induced of scalp hair due to cobalamin depletion have also been reported [13,14]. Thus, the impact of cobalamin deficiency disorders are limited by the lack of a specific experimental system. The in vivo model of vitamin on melanogenesis and melanocytes homeostasis is not obvious. B12 deficiencyCurrent is difficult scientific to findings prepare about because the molecula of pre-existingr patomechanism tissue of cobalamin hypocobalaminemia-induced stores in experimental animalsdisorders [15,16]. are Therefore, limited by cell the culture lack of a systems specific experimental might be useful system. for The elucidating in vivo model mechanisms of vitamin B of12 various disordersdeficiency induced is bydifficult hypocobalaminemia. to prepare because However, of pre-existing under tissue standard cobalamin conditions, stores inin experimental which the growth media areanimals supplemented [15,16]. Therefore, with cell serum, culture the systems deficiency might be cannot useful for be elucidating achieved mechanisms by simple meansof various as serum disorders induced by hypocobalaminemia. However, under standard conditions, in which the contains protein-bound vitamin B12. This problem may be overcome by cell culturing in the presence growth media are supplemented with serum, the deficiency cannot be achieved by simple means as of vitamin B12 antagonists, e.g., analogue of hydroxycobalamin: (OH)Cbl(c-lactam). According to the serum contains protein-bound vitamin B12. This problem may be overcome by cell culturing in the c nomenclaturepresence of of cobalamin vitamin B derivatives,12 antagonists, (OH)Cbl( e.g., analogue-lactam) of hydroxycobalamin: is the analogue with (OH)Cbl( a modificationc-lactam). of the amide groupAccording present to the at nomenclature the c-position of cobalamin of B pyrrolic derivatives, ring [17 (OH)Cbl(]. c-lactam) is the analogue with a It hasmodification been shown of the amide that melanogenesisgroup present at the process c-position and of homeostasisB pyrrolic ring [17]. in normal melanocytes may be modulatedIt has by been various shown agents, that melanogenesis e.g., fluoroquinolones process and homeostasis [18,19], aminoglycoside in normal melanocytes antibiotics may be [20–22], tetracyclinesmodulated [23,24 by], various and nicotine agents, [ 25e.g].., Recently,fluoroquinolones it hasbeen [18,19], demonstrated aminoglycoside that antibiotics melatoninergic [20–22], system, tetracyclines [23,24], and nicotine [25]. Recently, it has been demonstrated that melatoninergic with itssystem, final product, with its final can product, both have can anti-oxidativeboth have anti-oxidative and anti-melanogenic and anti-melanogenic properties properties [26 [26].]. The aimThe of aim this of study this study was to was develop to develop an in an vitro in vitromodel model for for specific specific investigation investigation of of the the effecteffect of vitamin B12 deficiencyvitamin onB12 melanin-producing deficiency on melanin-producing cells, thus providing cells, thus a scientificprovidingexplanation a scientific explanation of the hitherto of the unknown cellularhitherto and molecular unknown mechanisms cellular and of hypocobalaminemiamolecular mechanisms pigmentary of hypocoba manifestations.laminemia pigmentary manifestations. 2. Results 2. Results 2.1. Synthesis and Identification of

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