Melanocytotoxic chemicals and their toxic mechanisms

Abstract

Melanocyte cell death can lead to various melanocyte-related skin diseases including vitiligo and leukoderma. Melanocytotoxic chemicals are one of the most well-known causes of nongenetic melanocyte-related diseases, which induce melanocyte cell death through apoptosis. Various chemicals used in cosmetics, medicine, industry and food additives are known to induce melanocyte cell death, which poses a significant risk to the health of consumers and industrial workers. This review summarizes recently reported melanocytotoxic chemicals and their mechanisms of toxicity in an effort to provide insight into the development of safer chemicals.

Introduction

Melanin is a biopolymer of phenolic substance synthesized by melanocytes that forms a complex of black pigments and protein [1]. Melanin is widely distributed in living organisms and determines the color of eye, skin and hair [2]. Additionally, melanin is effective in the absorption of UV radiation, and free radical scavenging in the skin. However, melanin can cause drug interaction that may lead to therapeutic or toxic effects [3]. In the skin, melanin is secreted by melanocytes and distributed as melanosome packages into adjacent keratinocytes in the epidermis, contributing to skin pigmentation and protecting the skin from UV radiation [4, 5].

Tyrosinase is an important enzyme in melanin production that catalyzes the hydroxylation of tyrosine to dihydroxyphenylalanine (DOPA) and the subsequent oxidation of DOPA to DOPA quinones. This is a common key step for the biosynthesis of the two types of melanin, eumelanin and pheomelanin [1, 6]. DOPA quinones are converted to DOPA chrome, which is converted to 5,6-dihydroxyindole (DHI) or via enzymatic tautomerization with the tyrosinase-related protein TRP-2 to 5,6-dihydroxyindole-2-carboxylic acid (DHICA). Both of these products are oxidized into quinones and polymerized to eumelanin. In the presence of sulfhydryl compounds (such as cysteine and glutathione), the end product of melanin production is pheomelanin [7, 8].

Tyrosinase and tyrosinase-related proteins TRP1 and TRP2 are the key enzymes for the multistep production of melanin, catalyzing the synthesis of brown-black eumelanin and /or red-yellow pheomelanin. The major transcription factor that directly regulates the transcription of genes associated with these melanogenic enzymes (TYR, TRP1, and TRP2) is the microphthalmia-associated transcription factor (MITF) [8]. MITF is widely involved in providing central links between transcription factors and signaling pathways in melanocytes such as cell survival, proliferation, and differentiation [9].

While an adequate amount of melanin is essential for the protection of the skin against UV radiation, excessive melanin accumulation in the skin can result in cosmetic defects such as melasma, freckles, solar lentigo and age spots [10]. Accordingly, to reduce the abnormal accumulation of melanin and correct skin hyperpigmentation, the use of cosmetics with anti-melanogenic agents such as hydroquinone, ascorbic acid, kojic acid, α-bisabolol and arbutin has been widespread in women around the world [11]. However, they are not without toxicity. Of note, many of these anti-melanogenic cosmetic ingredients are phenolic compounds.

Actually, phenolic derivatives are widely used as building blocks to make a variety of consumer chemical products including disinfectants, diaper creams, detergents, dyes, adhesives, pharmaceuticals, and food additives since they have ideal characteristics such as high water-solubility, low cost and weakly acidic pH. According to the Household Products Database (US Department of Health and Human Services 2013), 44 unique phenolic substances are being used in over 8400 consumer chemical products [12]. Interestingly, some of these phenolic substances are known to cause cytotoxicity against melanocytes. As a result, some phenolic substances and catechol derivatives induce the irreversible hypopigmentation of the skin or hair in humans and experimental animals [13], which is attributable to their toxicity inducing melanocyte cell death at least in part [14].

Some of these chemicals can covalently bind to tyrosinase resulting in the activation of UPR, reactive oxygen species (ROS) generation, and the induction of autophagy, and exosomes. Exosomes deliver new antigens to neighboring immune cells that trigger inflammation and activate autoreactive T cells, causing chemically induced stress to melanocytes and initiating an autoimmune response, resulting in the destruction of melanocytes [14, 15].

The antioxidant system, which consists of antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx), is the first line of defense against free radicals and ROS. Oxidative stress occurs when the rate of ROS formation exceeds the cell's capacity to remove it in melanocytes. Melanin has SOD activity and has been shown to be able to remove ROS produced in response to chemicals, UV radiation and environmental stress [16, 17].

Vitiligo is an acquired skin depigmentation disorder caused by the selective destruction of melanocytes that affects about 1% of the world's population [18]. The development of vitiligo is influenced by genetic, stochastic, and environmental factors. First degree relatives who are genetically predisposed to vitiligo have a 5- to sixfold increased risk and identical twins have a 23-fold increased risk. Probabilistically, it is caused by random recombination of T cell receptors and antibodies in the process of "building" autoreactive cells that attack melanocytes in leukoplakia. Depigmentation of the skin may occur as a result of or the destruction of melanocytes or the denaturation of chromoproteins. Environmentally, depigmentation of the skin by various chemicals occurs as one of the major occupational diseases, but common consumers who use cosmetics, household products, pharmaceuticals or food additives containing these chemicals (Table 1) are not free from it [19].

Table 1.

Classification of melanocytotoxic chemicals according to their usage

	Chemical Name
Cosmetic ingredients	Hydroquinone, rhododendrol, raspberry ketone, 4-hydroxyanisole, butylated hydroxytoluene, kojic acid, mercury
Pharmaceutical chemicals	Monobenzyl ether of hydroquinone, retinoic acid, cysteine, lomefloxacin, kanamycin, carvedilol, fluphenazine, prochlorperazine, chlorpromazine, perphenazine, thioridazine, imiquimod, nicotinic acid, picolinic acid
Industrial chemicals	Paraphenylenediamine, 4-tert-butylphenol, 4-tert-butylcatechol, acetophenone, 2-phenylphenol, sodium lauryl sulfate, arsenic
Food additives	Ethyl 4-hydroxybenzoate, cinnamic aldehyde
Miscellaneous	Flavokavain A, flavokavain B, tunicamycin

Depigmentation of the skin means denaturation of chromoproteins, and it is difficult to eliminate melanocyte toxicity in people exposed to high concentrations of chemicals. Phenol is widespread in many household items, and the subject of organizing the mechanisms of the chemicals that cause melanocyte toxicity is very important.

Chemicals used as cosmetic ingredients known to cause melanocyte toxicity

To improve skin tone, anti-melanogenic agents are widely used as skin-whitening cosmetics. These whitening cosmetic ingredients mostly target tyrosinase at the level of enzyme activity, protein expression or post-translational modification. Representative anti-melanogenic substances are hydroquinone, arbutin, α-bisabolol and kojic acid but new whitening agents are constantly being introduced into the market. However, these anti-melanogenic agents are not without toxicity and sometimes, they may cause adverse effects associated with melanocyte toxicity (Table 2) [4].

Table 2.

List of melanocytotoxic chemicals used for cosmetic ingredients

Chemical name	Structure	Mechanism of Toxicity	Effects
Hydroquinone *HQ, Idrochinone, Quinol		Inhibition of the tyrosinase activities, formation of quinones	Skin bleaching, exogenous ochronosis, discoloration and staining of the skin
Rhododendrol *RD, Rhododenol, 4-(4-hydroxyphenyl)-2-butanol		Tyrosinase-dependent apoptosis, activation of ER stress through the UPR, IL-8 production, and autophagy, ROS formation	Leukoderma, cytotoxicity
Raspberry ketone *RK, p-Hydroxybenzyl acetone		ROS formation	Impair the regular proliferation of cell
4-Hydroxyanisole *Mequinol, MeHQ, 4-methoxyphenol, 4-MP		Inhibition of the tyrosinase activities, formation of quinones	Skin bleaching, destruction of melanocytes, skin peeling, redness, hypopigmentation and discoloration
Butylated hydroxytoluene *BHT, dibutylhydroxytoluene, 2,6-Di-tert-butyl-4-methylphenol		Elevated levels of reactive oxygen species (ROS), formation of quinones	Cytotoxic of fibroblasts, keratinocytes, and melanocytes
Kojic Acid		Inhibition of the tyrosinase activities	Genetic toxicity, liver cancer and allergic dermatitis
Mercury	Hg	Interferes with transport of Cu2 + to apotyrosinase, inhibition of the tyrosinase activities	Skin whitening, dermal disorders, damage the nervous system, kidneys, liver and immune system

* Other names

Hydroquinone

Hydroquinone (HQ) was sold as an over-the-counter (OTC) drug for skin whitening in the United States. HQ, also known as benzene-1,4-diol or quinol, is an aromatic organic compound that is a type of phenol having the formula C₆H₄(OH)₂. HQ is a white solid with two hydroxyl groups attached to the benzene ring at the para position [19]. In the United States, HQ topical products usually contain HQ up to 2% and it could be used up to 4% as a prescription drug. HQ brightens the skin by suppressing melanin production through the inhibition of tyrosinase. However, melanocyte toxicity also contributes to the whitening effect of HQ. Indeed, HQ is a representative phenolic compound that is well known to cause toxicity against melanocytes [20]. HQ, is oxidized in melanocytes to produce highly toxic compounds such as quinone. These cytotoxic metabolites destroy melanocytes and cause skin bleaching. Using histochemistry and electron microscopy, Zinbow et al. found that HQ preferentially affects melanocyte cells by altering melanosomal formation, melanogenesis and degradation. Hydroquinone-induced leukoderma is most common after the exposure to HQ containing photographic developers [21].

The theory of "bypass effect" or "melanocyte recovery" suggests that after sustained exposure to HQ, melanocytes become resistant and can no longer absorb HQ and thereby triggering melanin overproduction. This phenomenon in turn, increases absorption of HQ by fibroblasts in the papillary dermis which causes elastic fiber deformation and abnormal fiber production [11, 22]. In addition, long-term use of HQ-containing creams or its oral intake can cause the development of exogenous ochronosis. Ochronosis is a syndrome caused by the accumulation of HQ and homogenic acid, and is a disease in which yellowish (ocher-like) pigment is deposited in tissues [23].

Rhododendrol (RD) and raspberry ketone (RK)

It has been reported that the phenolic compound rhododendrol (RD), which was used as a skin-whitening ingredient in cosmetics, induces leukoderma. Kim et al. demonstrated the relationship between the cytotoxicity of RD and reactive oxygen species (ROS) production [24]. In particular, RD showed higher cytotoxicity against B16 melanoma cells than the keratinocyte cell line, HaCaT. Tyrosinase, a metalloprotein containing copper, was speculated to be one of the mechanisms underlying the phenolic compound-induced cytotoxicity on melanocytes, which promotes ROS generation by functioning as a prooxidant. Indeed, hydroxyl radicals are generated by adding a mixture of tyrosinase and H₂O₂ to RD. Furthermore, radical generation was further increased by UVB irradiation, the representative ROS-generating stimulus in the skin supporting that the cytotoxicity of RD may be attributable to the generation of ROS [25].

Raspberry ketone (RK), a structurally related derivative of RD also increased intracellular ROS in B16F10 cells, and the increase in ROS was inhibited by N-acetylcysteine (NAC) [24]. Interestingly, the ROS generation induced by RK was increased even in HaCaT cells without melanocytes after the addition of tyrosinase [25].

Ultimately, RD treatment of human melanocytes induced tyrosinase-dependent apoptosis, UPR, IL-8 production, and activation of ER stress by autophagy [26]. RD toxicity was well correlated with the tyrosinase activity in human melanocyte cell lines tested from different donors of different racial backgrounds, which means that individual variability of tyrosinase activity at least in part explains the variable response to RD [27]. Histology of RD-induced skin lesions revealed a significant decrease in the number of melanocytes, and melanin content, while the infiltration of T cells increased [28, 29]. Affected patients had more melanocyte-specific CD8+ T cells in their blood than in the control group [30]. This suggests that these chemically induced changes in melanocyte by RD may also prime the autoimmune responses.

4-Hydroxyanisole

A skin-whitening phenolic chemical, 4-hydroxyanisole has been extensively studied for the inactivation of tyrosinase enzyme activity and reactive quinone formation by interacting with tyrosinase [31, 32]. Dermatologists usually prescribe medications containing 2% mequinol (4-hydroxyanisole)/0.01% tretinoin to treat solar lentigo and age spots. It is very effective against solar lentigines and associated hyperpigmented lesions [33]. When butylated hydroxyanisole (BHA) and its metabolites were applied on cultured rat and human embryo cells, the metabolites of BHA, tert-butylhydroquinone and tert-butylquinone were found to be more toxic than BHA [34]. These cytotoxic compounds can result in damage and destruction of melanocytes and cause skin bleaching. Application of topical products containing 4-hydroxyanisole and trans-retinoic acid (tretinoin, TRA) can cause side effects such as skin peeling, redness, hypopigmentation and discoloration (DrugBank. Mequinol). Specifically, 4-hydroxyanisole is a substrate for tyrosinase and it acts as a competitive inhibitor of melanin precursor formation. Nonetheless, 4-hydroxyanisole is considered a melanocyte cytotoxic chemical since when it is oxidized in melanocytes, it leads to the formation of toxic intermediates such as quinones.

Butylated hydroxytoluene

Butylated hydroxytoluene is an antioxidant with a concentration of 0.0002–0.5% and is used in various cosmetic preparations. BHT penetrates the skin, but is absorbed in relatively small amounts and remains primarily on the skin. A comparison of BHT and butylated hydroxyanisole (BHA) for the cytotoxicity against cultured human dermal fibroblasts, keratinocytes, and melanocytes [normal human epidermal melanocytes (NHEM) and melanoma cells (SK-MEL/27 melanoma cells)] revealed that BHT is more cytotoxic than BHA. BHT also promotes ROS generation by producing quinone methide metabolite, which contributes to its cytotoxicity [35]. Analysis of the cytotoxic IC₅₀ values (48 h) of BHT against mouse B16-F0 and human SK-MEL-28 cell lines revealed 1.9 mM and 2.0 mM, respectively. The cytotoxic IC₅₀ values of 3-hydroxytoluene and 2–4-hydroxytoluene (at 24 h and 48 h) was reported to be 2.1 mM, 2.5 mM and 3.2 mM, 3.2 mM, respectively [36].

Kojic acid

Kojic acid (KA) is a well-known anti-tyrosinase agent widely used in whitening cosmetics. KA is used in the treatment of hyperpigmentation, age spots and wrinkles [37] [38]. However, KA has various side effects such as genetic toxicity, liver cancer and allergic dermatitis [39].

B16F1 melanoma cells are a widely used model for assessing depigmentation activity [40]. Cell viability analysis using MTT in B16F1 melanoma cells showed that the number of surviving cells at KA concentrations of 125 μg/mL and 500 μg/mL was significantly reduced to less than 60%. Since the esters of KA have a lower cytotoxic effect than KA, it has been suggested that KA esters can be used as a safer skin-whitening agent [41]. KA and KA esters are known to inhibit melanin production by direct inhibition of tyrosinase. It has previously been proposed that inhibition of the upregulated tyrosinase enzyme in melanoma cells can suppress the cell proliferation of melanoma cells [36, 42]. This is due to the correlation between MITF and extracellular signal-regulated kinase (ERK) in melanocyte and melanoma pigmentation, proliferation and survival [43, 44].

Mercury

Inorganic mercury compounds have been used in the skin-whitening products since ancient times. Previous studies have demonstrated that mercury blocked the transfer of Cu²⁺ to apotyrosinase and investigated the mechanism of tyrosinase inactivation [3]. Kinetic analysis showed that HgCl₂ inhibits tyrosinase activity in an irreversible, and noncompatible manner. Intense intrinsic fluorescence quenching suggests that the formation of the HgCl₂-tyrosinase complex induces morphological changes in the enzyme, and that HgCl₂ has only a single binding site or a single class of binding sites in tyrosinase. Molecular docking and experiments have demonstrated that HgCl₂ binds to an histidine residue, the catalytic center of tyrosinase [45].

Chemicals used as pharmaceutics known to cause melanocyte toxicity

Melanocytes are special pigmented dendritic cells found in the skin, eyes, inner ears, brain, adipose tissue, heart and lungs. They play roles in photoprotection, ROS capture, metal ion chelating, and biomolecular binding with various drugs and xenobiotics. The interaction of the drug with the melanin biopolymer protects the organism from unwanted drug side effects, but on the other hand it can alter the pharmacodynamic properties of the drug and some drugs can increase the risk of cell damage by accumulating in melanin-containing tissues (Table 3) [46].

Table 3.

List of melanocytotoxic chemicals used for pharmaceuticals

Chemical name	Structure	Mechanism of toxicity	Effects
Monobenzyl ether of hydroquinone *MBEH, Monobenzone, 4-(Benzyloxy)phenol)		Release of high mobility group Box-1 protein, skin macrophages and natural killer (NK) cells activated by MBEH-stress	Bleaching of the skin, necrotic melanocyte cell death
Retinoic acid *RA		Inhibition of the tyrosinase activities and enzyme glutathione S-transferase, decreased SOD, CAT activity	Melanocyte toxicity, depigmentation
Cysteine		Oxygen radical	Hangover prophylaxis, toxic of melanoma cell line
Lomefloxacin *LFLX, Lomefloxacin hydrochloride, Maxaquin, Okacyn, Uniquin		Inhibition of the tyrosinase activities	Side effects on the central nervous system and phototoxicity, weak cytotoxicity
Kanamycin		Modulation of the activity of antioxidant enzymes, increased of free radicals, oxidative stress, imbalance of antioxidant defense	Tinnitus, hearing loss, kidney toxicity, and altered visual acuity, cytotoxicity
Carvedilol *Coreg		Downregulated MITF, tyrosinase, TRP-1, and TRP-2 downregulation of phosphor-cAMP response element-binding protein (CREB)	Skin eruption in the form of a macular rash with blisters, epidermal necrolysis, cytotoxicity
Fluphenazine *prolixin		Decrease of tyrosinase activity	Extrapyramidal symptoms ill effects on the eyes, skin disorders, reduce cell viability in human epidermal melanocytes
Prochlorperazine *Compazine		Decrease of tyrosinase activity	Reduce cell viability in human epidermal melanocytes
Chlorpromazine *Largactil, Thorazine, Sonazine		Increased the MITF content and tyrosinase activity, increased SOD activity, decreased CAT activity, oxidative stress	Hyperpigmentation, photosensitivity, dermatitis, retinopathy cataracts, cytotoxicity
Perphenazine *Trilafon		Oxidative stress	Extracorporeal symptoms, eye and skin disorders, cytotoxicity
Thioridazine *Mellaril, Melleril		Oxidative stress	Extracorporeal symptoms, eye and skin disorders, concentration-dependent decrease in cell viability
Imiquimod *Aldara		Expression of caspase-3, Bcl-2 and mitogen-activated protein kinase	Apoptosis, dry skin, itching, scabbing, skin breakdown, skin drainage, blisters, skin redness, scaling, skin ulceration, sores, swelling
Nicotinic acid *Niacin, Bionic, vitamin B3		Inhibition of the tyrosinase activities	Suppression of melanoma cell proliferation and cell viability, redness, warmth, itching, tingling of the skin
Picolinic acid		Inhibition of the tyrosinase activities	Suppression of melanoma cell proliferation and cell viability,

*Other names

Monobenzyl ether of hydroquinone

MBEH is an FDA-approved drug used to treat the depigmentation of advanced vitiligo. MBEH is also used to extract dyes for textile dyeing and is used in the rubber industry. It is the first chemical known to induce bleaching of the skin. Topical application of MBEH to animals increases melanosome excretion from melanocytes, which can explain the depigmenting effect of MBEH in humans. MBEH can cause melanocyte destruction and permanent bleaching [47]. MBEH induces a cytotoxic T cell response to melanocytes after inducing necrotic melanocyte cell death. Skin contact with phenolic or catechol compounds can cause local depigmentation, which can spread far away to unexposed parts of the body (occupational vitiligo). This bleaching is clinically or histologically indistinguishable from vitiligo [48]. Skin macrophages and natural killer (NK) cells activated by MBEH-stressed melanocytes can also be involved in melanocyte destruction [49]. MBEH induces non-apoptotic cell death without activating the caspase cascade or DNA fragmentation. The release of HMGB-1 (High Mobility Group Box-1) and ultrastructural features distinct of necrotic cell death was confirmed in MBEH-exposed melanocytes. Interestingly, MBEH exposure upregulated the levels of melanin-producing enzymes in cultured melanocytes and skin grafts while, a well-known melanocyte cytotoxic agent, 4-tert-butylphenol (4-TBP) reduced melanin-producing enzyme expression, suggesting that nonconventional cell death pathway was activated by MBEH [50].

Retinoic acid

Retinoids include retinoic acid (tretinoin), retinol, retinal, isotretinoin, and alitretinoin. Retinoic acid (RA) is a hormone-like substance that is mainly used in the treatment of cancer and acne and is involved in the regulation of cell differentiation [51]. Skin irritation is one of the most common side effects of RA. RA induces melanocyte toxicity and can cause depigmentation. In a study investigating the effects of total trans-retinoic acid (ATRA) on normal human melanocytes, 72 h treatment with 1.0 μM ATRA showed promelanogenic and proapoptotic effects, with decreased melanin content, tyrosinase, CAT and SOD compared to the control along with a time-dependent decrease in cell viability [52]. Inhibition of the enzyme glutathione S-transferase by RA may impair glutathione-dependent cytoprotection of melanocytes. Therefore, the combination of RA and melanocytotoxic compounds may increase the sensitivity of melanocytes to these compounds. Exemplifying this, MBEH's melanocyte toxicity and depigmentation are synergistically increased when combined with RA [53].

Cysteine

Cysteine has been proposed as hangover prophylaxis or antidote to some of the adverse effects of alcohol and neutralizes the toxic effects of acetaldehyde. Interestingly, cysteine was highly toxic to the MM96L melanoma cell line. The autoxidation of cysteine produces toxic radicals [54] and the toxicity of N-acetylcysteine to melanoma cells has been described in vitro [55]. MM96L and several other melanoma cell lines increase cysteine (cystine) uptake in endothelial cells [56] and are particularly sensitive to the cytotoxicity of cysteinyl radicals [57].

Lomefloxacin

Lomefloxacin is a powerful fluoroquinolone antibiotic used to treat bacterial transmission. It is used to prevent urinary tract infections before surgery. Lomefloxacin is associated with side effects on the central nervous system and phototoxicity. Of note, the use of lomefloxacin in the treatment of various infections is associated with serious side effects on the pigmented tissue. In normal human epidermal melanocytes, lomefloxacin showed weak cytotoxicity (ED50 0.75 mmol/l). Lomefloxacin has been shown to inhibit tyrosinase activity and reduce the melanin content of human skin melanocytes in a concentration-dependent manner [58]. It is suggested that lomefloxacin forms a stable complex with melanin after its accumulation to pigmented tissues.

Kanamycin

Kanamycin is recommended for the treatment of Gram-negative and some Gram-positive microbial infections, especially severe bacterial infections. Serious side effects of Kanamycin on humans include tinnitus or hearing loss, kidney toxicity, and altered visual acuity [59]. Kanamycin [60] has been shown to form a stable complex with a model synthetic melanin in vitro. The effects of various concentrations of kanamycin on the viability of normal human melanocytes were investigated which showed anti-proliferating activity at 6.0 mM along with decreased cell viability [61]. Kanamycin induced a concentration-dependent loss in normal human epidermal melanocyte viability. The IC₅₀ value was estimated to be 5.0 mM. A reduction in the antioxidant activity, an increase in free radical production and a decrease in melanin content were observed, confirming that kanamycin produces oxidative stress in melanocytes. Increased free radical levels by kanamycin can cause an imbalance in antioxidant defenses and a decrease in melanin content in melanocytes.

Carvedilol

Carvedilol is a drug used to treat hypertension, congestive heart failure (CHF), and left ventricular dysfunction [62]. In 2019, it received more than 20 million prescriptions, ranking as the 33rd most prescribed drug in the United States (Carvedilol—Drug Usage Statistics). Two days after the start of carvedilol treatment, a 70-year-old woman developed a skin eruption in the form of a macular rash with blisters that rapidly progressed to epidermal necrolysis. The suspicious drug was withdrawn, but the reaction was very rapid with fatal consequences [63]. The cytotoxicity of carvedilol to normal human melanocytes (NHM) and Mel-ab cells was investigated. Carvedilol showed cytotoxicity to both NHM and Mel-ab cells. Carvedilol downregulated MITF, tyrosinase, TRP-1 and TRP-2. Moreover, carvedilol treatment induced downregulation of the cAMP reactive element binding protein (CREB). Carvedilol, an adrenergic blocker, inhibits the cAMP/ CREB signaling pathway in human melanocytes and human skin explants and inhibits melanin synthesis [64].

Fluphenazine and prochlorperazine

Fluphenazine and prochlorperazine belong to the phenothiazine antipsychotics with piperazine-derived substituents, which are used specifically for the treatment of schizophrenia and bipolar disorder [65]. The most serious side effects of phenothiazine treatment are extrapyramidal symptoms (a Parkinsonism, and dystonias, akathisia) ill effects on the eyes (e.g., blurred vision and cataract), and skin disorders (e.g., rash, fixed drug reactions, skin photosensitivity and abnormal skin pigmentation) [66] [67]. Fluphenazine and prochlorperazine have been shown to reduce cell viability in weakly colored normal human epidermal melanocytes (HEMn-LP). Interestingly, the cytotoxicity of fluphenazine and prochlorperazine was weaker in heavily colored melanocytes (HEMn-DP) [68]. The binding ability and affinity of prochlorperazine to melanin is higher than that of fluphenazine [69]. Concentrations of unbound prochlorperazine in darkly colored cells can be much lower than in lightly colored melanocytes, which explains the stronger cytotoxicity of prochlorperazine to HEMn-LP cells. Nonetheless, fluphenazine and prochlorperazine reduced melanin content and tyrosinase activity [70].

Chlorpromazine

Chlorpromazine belongs to the phenothiazine family of neuroleptics and belongs to the first generation of antipsychotics. It is widely used in the treatment of mania, psychotic disorders, and bipolar disorders through its interaction with the nervous system [71, 72]. Hyperpigmentation, photosensitivity, dermatitis and eye effects (e.g. retinopathy and cataracts) have occurred as side effects of chlorpromazine [73]. Chlorpromazine binds to melanin granules and concentrates in the retinal pigmented epithelium and uveal tissues. Retinitis pigmentosa is a retinal lesion caused by a phototoxic process during the treatment with a phenothiazine derivative. Skin and eye reactions suggest a potential role for endogenous melanin in inducing these side effects in colored tissue [74, 75]. Chlorpromazine induced the concentration-dependent loss in the survival of normal human epidermal melanocytes (HEMn-DP) in vitro. At the sub-cytotoxic level, chlorpromazine increased melanin and MITF content and tyrosinase activity, but no changes in antioxidant status were observed. At higher concentrations, significantly increased SOD activity and decreased CAT activity were observed in normal melanocytes along with significant changes in antioxidant enzyme activity and oxidative stress, indicating that antioxidant defense systems do not function properly [76].

Perphenazine

Perphenazine is widely used in the treatment of psychosis and schizophrenia, but its use is associated with serious side effects such as extracorporeal symptoms as well as eye and skin disorders [77]. In human melanocytes, concentration-dependent losses in cell viability were induced by perphenazine. Perphenazine also inhibited melanin production and reduced MITF content. Perphenazine caused the depletion of melanocyte antioxidant status, which indicates the induction of oxidative stress [78].

Thioridazine

The antipsychotic drug thioridazine is widely used in the treatment of various psychotic disorders such as schizophrenia [79]. However, the treatment with this drug can cause extracorporeal symptoms as well as eye and skin disorders [80, 82]. The mechanism has not yet been fully established. Treatment of normal human melanocytes with thioridazine resulted in a concentration-dependent decrease in cell viability along with the imbalance of cellular antioxidant defense system and oxidative stress. In addition, thioridazine has been shown to significantly suppress melanin production [76].

Imiquimod

Imiquimod, a new imidazoquinoline immune response regulator, has been used for the topical treatment of genital warts and has been shown to be effective in superficial basal cell carcinoma and actinic keratosis [81, 82]. Imiquimod treatment induced melanocyte apoptosis as observed by TUNEL assay and Hoechst 33258 staining. Imiquimod-induced apoptosis was further demonstrated by disruption of mitochondrial membrane potential in melanocytes. The apoptotic activity of imiquimod was associated with the expression of caspase-3, Bcl-2 and mitogen-activated protein kinases in melanocytes. These results indicate that imiquimod induces apoptosis of melanocytes [83].

Nicotinic acid and picolinic acid

Nicotinic acid is one of the B vitamin complexes and is a component of nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP). It is also sold as a dietary supplement as an essential nutrient, and is sold as a prescription drug in the United States with niacin as the main ingredient [84]. Niacin and niacinamide are used for the prevention and treatment of pellagra [85]. Niacin also acts as a vasodilator and has the effect of lowering blood cholesterol levels, but niacinamide does not [86]. Picolinic acid is an isomer of nicotinic acid and has a carboxyl side chain at the 3-position. The dietary supplement of zinc dipicolinate has become widespread as it turns out to be an effective means of intaking zinc into the body [87]. Interestingly, treatment of melanoma cell lines with various concentrations of nicotinic acid and picolinic acid led to the suppression of melanoma cell proliferation and cell viability in a dose-dependent manner. The concentration that results in a 50% reduction in cell density (IC₅₀) is similar to the concentration that causes a 50% reduction in enzyme activity of tyrosinase, indicating that the observed cytotoxicity is likely due to tyrosinase inhibition [88].

Industrial chemicals that are known to be melanocytotoxic

Depigmentation of the skin by industrial chemicals has been recognized as a major occupational hazard for long (Table 4). Since chemical-induced skin depigmentation may be associated with allergic contact dermatitis, nonspecific post-inflammatory changes were considered to be accountable. However, chemical-induced skin depigmentation often occurs in the absence of overt dermatitis, and most people who experience contact dermatitis do not experience depigmentation. In fact, they usually cause excessive pigmentation after inflammation [89]. In addition, many subjects with chemical-induced skin depigmentation also develop lesions far away from the site of contact with the chemical [90]. Chemicals can simply accelerate the stress pathways already present in healthy melanocytes, but push them above the permissible threshold and induce autoimmune inflammation beyond the capacity that healthy cells can properly manage [14].

Table 4.

List of melanocytotoxic chemicals used for industrial chemicals

Chemical name	Structure	Mechanism of toxicity	Effects
Paraphenylenediamine *PPD, p-Phenylenediamine		Disruption of mitochondrial membrane potential, ROS formation, apoptosis through activation of caspase 8	Apoptosis, contact allergen, sensitization dermatitis, throat irritation, bronchial asthma
4-tert-butylphenol *4-TBP		Secretion of heat shock protein (HSP70), ROS formation, Activation of the unfolded protein response, production of the inflammatory cytokines IL-6 and IL-8, inhibition of the tyrosinase activities	Depigmentation, leukoderma, apoptosis
4-tert-butylcatechol *4-TBC		Quinone formation, inhibition of the tyrosinase activities	Inflammation, depigmentation, cytotoxicity
Acetophenone		Inhibition of the tyrosinase activities	Irritates the eyes, affects the central nervous system, unconsciousness, the liquid defats the skin
2-Phenylphenol *o-Phenylphenol, OPP		Formation of OPP metabolite, phenylhydroquinone (PHQ)	Leukomelanodermatosis, depigmentation, selective destruction of melanocytes
Sodium Lauryl Sulfate *SLS		Increased Lactate dehydrogenase (LDH) release Significant increase in S100B expression	Irritation, cytotoxicity
Arsenic	As	Increased cellular oxidative stress, oxidative DNA damage, ROS production, inhibits Poly(ADP-ribose)polymerase (PARP)-1 activity	Skin bleaching, keratoderma, hyperpigmentation, multiple cutaneous malignancies, intraepidermal carcinoma (Bowen disease), squamous cell carcinomas (SCC), basal cell carcinomas (BCC), Merkel cell carcinoma (MCC)

*Other names

Paraphenylenediamine

Paraphenylenediamine (PPD) has long been known as an essential ingredient in dyeing agents. The hair dye PPD has been reported to act purely as a topical dyeing agent [91]. For a long time, PPD has been used in the textile, leather and hair dye industries [92]. Sporadic side effects of PPD have been reported in human and mouse models [93]. PPD is converted to Brandrowski’s base by autoxidation and self-conjugation. In addition, keratinocytes have been shown to contain N-acetyltransferase 1 (NAT1), which modifies PPD by mono and di-N-acetylation to prevent toxicity [94]. The effects of PPD have been investigated in various cell types and have been found to induce apoptosis via ROS [95]. In human and mouse melanoma cell lines A375 and B16-F10, PPD induces apoptosis of these cells through disruption of mitochondrial membrane potential, ROS production and activation of caspase-8 [96].

4-tert-butylphenol

Typically, 4-tert-butylphenol (4-TBP) is used in the production of epoxy resins and as a hardener in polycarbonate resins. It was also used in the production of phenolic resins, and as a plasticizer. There was a case report about the depigmentation around the patient's mouth after using a lip liner containing 4-TBP. The chemical patch test was positive in patients [97]. Furthermore, there was a report about patients who had shown depigmentation after using adhesive resin in Bindi, a decorative item worn by many Indian women on their foreheads. Importantly, 4-TBP is a representative chemical that can cause leukoderma. Exposure to 4-TBP reduced the expression of melanin-producing enzymes in cultured melanocytes and skin explants [50]. The tyrosinase protein levels in pigmented melanoma cells were also reduced by 4-TBP [98]. The strong inhibitory effect of 4-TBP on melanin synthesis is likely to be from the reduced expression and activity of tyrosinase protein. The activation of the inflammatory cascade in dendritic cells cultured with melanocytes exposed to 4-TBP could indirectly induce the apoptosis of melanocytes. Melanocytes treated with 4-TBP exhibited a cellular stress response and secreted the inflammation-inducing heat shock protein HSP70. HSP70 activated dendritic cells cultured with melanocytes and subsequently resulted in the apoptosis of melanocytes. 4-TBP and MBEH induce ROS in melanocytes and activate unfolded protein response (UPR), which is a cellular stress response generating HSP70. The UPR's key mediator induced the production of the inflammatory cytokines interleukin (IL) -6 and IL-8. These data suggest that vitiligo-causing phenol stresses cells and indirectly induces melanocyte apoptosis by inducing the secretion of pro-inflammatory mediators [99].

4-tert-butylcatechol

Generally, 4-tert-butylcatechol (4-TBC) is used as a stabilizer in the manufacture of polyurethane foam and can be used as an antioxidant for synthetic rubbers, polymers and petroleum derivatives. In the 1970s, factory workers in the valve lifter assembly plant, who were exposed to 4-TBC present in the lubricating oil, developed acral depigmentation. All patients had severe inflammation prior to depigmentation of the contact area, and 75% of them also had depigmentation [100]. Dopa-stained split epidermal sheets showed that the number of melanocytes was reduced by less than half during the first month of 4-TBC exposure. Melanocytes often swelled or lost dendrites, and both premelanosomes and melanosomes showed ultrastructural changes [101]. After treatment with 4-TBC, some melanocytes showed reversible changes in cell shape and with higher concentrations of 4-TBC, melanocyte cell death was observed. The surviving cells contained ultrastructurally rare premelanosomes and melanosomes. In addition, the microfilaments were wavy [102]. Moreover, 4-TBC is oxidized by tyrosinase, and oxidation of 4-TBC produces more quinones in reaction with cysteine ​​or glutathione (GSH). It has been reported that 4-TBC and 4-TBP are converted to quinone derivatives by tyrosinase [103]. In studies in which tyrosinase in cultured human melanoma cells was extracted and treated with 4-TBC, enzyme activity was inhibited by both 4-TBC and hydroquinone. TBC and hydroquinone have been reported as tyrosinase inhibitors at concentrations higher than 1 mM [104].

Acetophenone

Acetophenone is primarily used for the manufacture of a commercially important resin by treating acetophenone with formaldehyde and bases. It is used in perfumes and gums as an ingredient in artificial scents such as jasmine, strawberries, almonds, cherries and sea bream [105]. NIOSH (NOES Survey 1981–1983) estimated that 78,624 workers in the United States were potentially exposed to acetophenone. Occupational exposure to acetophenone can occur by inhalation and skin contact with this compound in the workplace where acetophenone is manufactured or used. IC₅₀ value in the in vitro experiment with human melanoma cells was 2184.5 mg/l at 48 h (NIOSH 2017). Acetophenone irritates the eyes, affects the central nervous system, and can induce unconsciousness when exposed to high levels. After prolonged or repeated exposure, the liquid defats the skin (ICSC 2017). Acetophenone and its derivatives are known to be potent inhibitors of tyrosinase [106].

2-Phenylphenol

The main use of o-phenylphenol (OPP) is in agricultural fungicides, and it is also a common intermediate in chemical reactions. Phenylphenol compounds, including OPP, are known to cause leukomelanodermatosis in chemical factory workers. A weak depigmenting effect was found on the skin of hospital workers after using the phenolic detergent bactericide Ves-Phene® containing OPP [107] and a weak depigmenting effect was found on the skin of humans and guinea pigs after applying OPP [108]. After oral administration of OPP, depigmentation was observed in the hair of C57/BL black mice [110]. This phenomenon was not observed after local OPP application. Phenylhydroquinone (PHQ) is a major metabolite formed from OPP by the enzymatic action of the microsomal monooxygenase system [109]. In a study by Tayama, 2002 [110] OPP, applied topically to the skin on the back of black guinea pigs had little depigmenting activity, but its metabolite PHQ was irritating and induced depigmentation on the skin. When high concentrations of PHQ (1% or 5%) were treated, light microscopy showed few or no melanocytes compared to controls and damaged abnormal melanocytes. Under an electron microscope, decreased number of melanosomes was observed in melanocytes and keratinocytes along with the destruction of melanosomes, and membrane organelles in the 1% PHQ group, indicating that PHQ derived from OPP can induce selective destruction of melanocytes.

Sodium lauryl sulfate

Sodium lauryl sulfate (SLS) is a type of anionic surfactant used in the production of soaps, toothpaste, shampoos, etc. This utilizes the property of a surfactant that can mix a water-soluble substance and a fat-soluble substance, and SLS is widely used because of its low price [110]. After applying the doses of SLS inducing irritation, the effects of HQ and RA on co-cultures of keratinocytes and melanocytes or fibroblasts and melanocytes were investigated. The S100B protein was detected in melanocytes but not in keratinocytes or fibroblasts. In addition, the cell number of melanocytes decreased with significantly increased LDH (lactate dehydrogenase) release and S100B expression. The role of RAGE (the receptor for advanced glycation end-products) expression and CD166/ALCAM in melanocyte survival and cytotoxicity was suggested for the upregulation of S100B. As a result, S100B knockdown increased the apoptosis of melanocytes by inhibiting PI3K/AKT, NF-κB and ERK activation, which showed increased intracellular S100B expression by chemical stimulation may be a compensatory reaction to reduce cytotoxicity [111].

Arsenic

Arsenic is a naturally occurring environmentally toxic substance that is widely distributed in the Earth’s crust [112, 113]. The air, water and soil environment can be contaminated with arsenic from industrial smelting of metals, power generation from coal, pesticides and the application of herbicides [114]. Arsenic enters the human body through skin contact, inhalation, or food intake, but ingestion of contaminated drinking water is the most common route. The tissues most frequently affected by arsenic exposure are the skin, nasal passages, lungs, gastrointestinal tract, and liver [115]. Previous studies have reported that pyrotechnics had skin bleaching after contact with material contaminated with arsenic. Chronic human exposure to low-dose arsenic induces endothelial carcinomas such as intraepidermal carcinoma (Bowen disease), squamous cell carcinomas (SCC), basal cell carcinomas (BCC), and Merkel cell carcinoma (MCC). In the skin cancers resulting from arsenic exposure, arsenic-induced keratoderma, hyperpigmentation and multiple cutaneous malignancies were suggested to be involved (Maloney ME 1996). RAC1 regulates lamellipodia (cell marginal processes important for cell migration) and melanocyte dendritic formation [116, 117]. Increased localization or activity of RAC1 protein by arsenic results in increased melanocyte dendriticity and can cause hyperpigmentation [118]. In studies measuring cell viability after arsenic, UVR or combined exposure to compare the responses of normal human neonatal epidermal melanocytes (HEMn) to normal human neonatal epidermal keratinocytes (HEKn), both the cells showed a time-dependent decrease in cell viability by arsenite. Arsenic exposure also stimulates ROS production, which increases cellular oxidative stress [119] and oxidative DNA damage [120]. PARP-1 is a protein involved in strand destruction and oxidative DNA damage repair and has recently been shown to participate in direct DNA damage repair such as CPD [121, 122]. A dose-dependent decrease in PARP activity by arsenic was explained by the DNA damage occurred in both melanocytes and keratinocytes.

Food additives or other chemicals known to be melanocytotoxic (Table 5)

Table 5.

List of melanocytotoxic chemicals used for food additives or others

Chemical name	Structure	Mechanism of toxicity	Effects
Ethyl 4-hydroxybenzoate *4-HEB, Ethylparaben		o-Quinone formation, intracellular GSH depletion, ROS formation, mitochondrial toxicity	Cytotoxicity, antifungal preservative
Cinnamic aldehyde *cinnamaldehyde, trans-cinnamaldehyde		Inhibition of the tyrosinase activities	Skin irritant, cytotoxicity, allergic contact stomatitis, powerful fumigant against
Flavokavain A		Inhibition of the tyrosinase activities, suppression of Tyr, Trp-1, Trp-2 and Mitf genes	Cytotoxicity, hepatotoxicity
Flavokavain B		Inhibition of the tyrosinase activities, suppression of Tyr, Trp-1, Trp-2 and Mitf genes	Cytotoxicity, hepatotoxicity
Tunicamycin *TM		Endoplasmic reticulum (ER) stress, Up-regulate PERK, eif2α and CHOP	Apoptosis, reduced cell viability, cell cycle arrest

*Other names

Ethyl 4-hydroxybenzoate

Ethyl 4-hydroxybenzoate (4-HEB) is an ethyl ester of p-hydroxybenzoic acid. It is used as an antifungal preservative and food additive [123]. Specifically, 4-HEB induced cytotoxicity in human SK-MEL-28 melanoma cells with an IC₅₀ (day 2) of 75 μM. Dicoumarol, a diaphorase inhibitor, and 1-bromoheptane, a GSH-depleting agent, increased 4-HEB toxicity to SK-MEL-28 cells, suggesting that ο-quinone formation plays an important role in 4-HEB-induced cytotoxicity. In addition, 4-HEB caused a time-dependent decrease in intracellular GSH concentration prior to apoptosis. Moreover, 4-HEB also induced ROS formation in melanoma cells, exacerbated by dicoumarol and 1-bromoheptane [124]. There are studies comparing the effects of 4-HEB on four nonmelanoma cell lines (namely, SW-620, Saos-2, PC3, and BJ cells) and two melanoma cells (namely, SK-MEL-24 and C32 cells) that do not express functional tyrosinase. Selective toxicity was shown for the five melanogenic melanoma cell lines expressing functional tyrosinase, SK-MEL-28, SK-MEL-5, MeWo, B16-F0 and B16-F10 [36].

Cinnamic aldehyde

Cinnamic aldehyde is primarily used in the flavoring of gums, ice creams, candies, liquid phases and beverages. The usage level range is 9–4900 ppm (less than 0.5%). Cinnamaldehyde can be used as a food additive and is used in some perfumes with natural, sweet or fruity scents [125]. Trans-cinnamaldehyde is a powerful fumigant against adult mosquitoes and acts as a practical insect repellent [126]. B16 melanoma cells treated with trans-cinnamaldehyde showed potent inhibitory activity against melanin synthesis along with decreased cell viability [127]. Cinnamic aldehyde can suppress the oxidation of L-DOPA by mushroom tyrosinase. Recently, Cui et al., reported a series of alpha-substituted derivatives of cinnamaldehyde derivatives. SAR studies have shown that α-bromocinnamaldehyde, α-chlorocinnamaldehyde and α-methylcinnamaldehyde compounds reduce both monophenolase and diphenolase activity on tyrosinase. It has been suggested that the α-substituted cinnamaldehyde derivative is more potent than cinnamaldehyde [128].

Flavokavain

Flavokavain is an extract from kava root consumed on the Pacific islands and is associated with a reduced incidence of cancer [129]. Piper methysticum, commonly known as kava–kava, is a perennial shrub in the Pacific Islands. Kava extract contains chalcone and kavalactone. The three naturally occurring chalcones include flavocawain A, B and C (FLA, FLB and FLC) [130]. FLA and FLB significantly reduced the cell viability of B16/F10 melanoma cells. FLA and FLB also have anti-melanogenic activity. FLA and FLB reduced the melanin content in α-MSH-induced B16/F10 cells. At the same time, FLA and FLB reduced cell tyrosinase activity. Dose-dependent suppression of gene expression of the Tyr, Trp-1, Trp-2 and MITF genes was observed after treatment of FLA and FLB which may explain the mechanism of the anti-melanogenic effects of these compounds [131].

Tunicamycin

Tunicamycin (TM) is a mixture of nucleotide-based antibiotics produced by Streptomyces lysosuperificus [132]. Interestingly, TM suppresses cell viability in human primary epidermal melanocytes and promotes apoptosis. Western blot analysis showed that Bax and caspase-3 expression were upregulated and Bcl-2 expression was downregulated in TM-treated human melanocytes. The expression of ER stress-related proteins including PERK, eIF2α and CHOP was upregulated in a time-dependent manner. This means that TM activated ER stress in human melanocytes and reduced cell viability [133].

In this review, substances that induce toxicity on melanocytes are reviewed and their structures and mechanisms of toxicity are summarized. These melanocytotoxic substances are mostly aromatic compounds with phenol or catechol structures. Chemicals that cause depigmentation generally contain a phenolic group consisting of a benzene ring with a hydroxyl side chain. In particular, the depigmentation effect of 4-substituted phenol appears to be due to its toxicity to melanocytes, and treatment of these chemicals on melanocytes cultured in vitro causes toxicity at high concentrations indeed, suggesting that they are toxic to melanocytes. Phenols with nonpolar side chains at the para (or 4-) position appear to be the most potent depigmenting chemicals, especially if there is an ether group at that position [134]. This chemical structure is shared with the amino acid tyrosine, a building block of melanin in which tyrosinase and other melanin-producing enzymes catalyze modifications [89]. Therefore, the mechanism of phenol-induced melanocytotoxicity appears to be due to structural similarities with tyrosine. Open phenol appears to act as a tyrosine analog that interferes with melanin production. In addition, tyrosinase activity, which emerged as the main toxication mechanism of these chemicals, is the main characteristic of melanocytes, which is essential for producing melanin.

Tyrosinase is known to have copper in the active center [135] Proteins containing copper are known to promote oxidative reactions and induce apoptosis [136, 137]. Tyrosinase oxidizes large amounts of phenol and catechol to form ο-quinones. Vitiligo or leukoderma, which manifests as the main symptom of melanocyte toxicity, is a skin disorder characterized by pigment loss due to melanocyte death or dysfunction. Contact dermatitis can also occur after exposure to melanocytotoxic chemicals [21]. The effects of HQ on melanocyte-specific cytotoxicity and suppression of tyrosinase activity are known as depigmentation mechanisms. Arbutin, which is used as a skin-whitening agent, is known to have an inhibitory effect on tyrosinase activity. Previous studies used a mouse embryonic stem cell (ESC) culture system to examine the effects of HQ and arbutin at each stage of melanocyte differentiation. HQ has been shown to reduce the early stage of differentiation in which neural crest cells are produced and the late stage of differentiation in which melanin production is activated. On the other hand, arbutin did not affect the differentiation of melanocytes, specifically suppressed the increase in tyrosinase expression in the late stage of differentiation, and suppressed only melanin production [138]. Further research is needed as the discriminatory depigmentation mechanism of whitening and melanocytotoxic substances is not yet fully understood.

Recognizing chemicals that are toxic to the melanocytes is important for the prevention of vitiligo and other melanocyte-related diseases. We believe this review will help to develop safer chemicals used for cosmetics and pharmaceuticals.

Funding

This work was supported by the Grants from National Research Foundation of Korea (NRF-2020R1I1A1A01067636) and Ministry of Science and ICT (MSIT) (2018R1A5A2025286).

Declarations

Conflict of interest

All authors declare no conflict of interest for this work.