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Toxicology and health risks of hydroquinone in skin lightening formulations

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Abstract

Background Hydroquinone has been used for decades as a skin lightening agent. As a result of concerns about mid-term effects like leukomelanoderma en confetti and exogenous ochronosis, its use in cosmetics has been banned since January 2001. Objective Until recently no attention was paid to potential long-term side-effects, despite the fact that there are indications that these may exist. It was decided that a clearer picture of these potential long term effects was needed. Methods A literature search was conducted with an emphasis on the biochemistry and toxicology of hydroquinone, benzene and related molecules. Results It appeared that since 1996 an enormous amount of articles have been published on the carcinogenicity of hydroquinone, benzene and related molecules. The literature search on hydroquinone as a skin lightening agent suggests that possible long-term effects like carcinogenesis may be expected. Conclusion The risks of long-term effects (cancer) of topically applied hydroquinone may no longer be ignored. Based on recent evidence of the potential risks, which are higher than has been assumed up until now, we plead that the use of hydroquinone as a skin lightening agent will be stopped completely.
... There are many industrial uses for hydroquinone: it is used in photographic developers as a polymer inhibitor, as an antioxidant for fats and oils, and as an intermediate for rubber processing chemicals. It also serves as a depigmenting agent in concentrations ranging from 2% to 10% [40]. ...
... Hydroquinone's use for depigmenting purposes became widespread in the following decades and is currently utilised in managing various hyperpigmentation disorders such as melasma, chloasma, freckles, age spots and post-inflammatory hyperpigmentation caused by acne or trauma [34]. Hydroquinone is present in numerous prescription-only preparations; for example, in the Netherlands, pharmaceutical wholesalers supplied pharmacies with 90 kg of hydroquinone and it is estimated that 5000-10,000 formulations of 2% hydroquinone and 10,000-15,000 formulations of 5% hydroquinone were prescribed in a single year [40]. Although hydroquinone has been the dermatological gold standard for skin lightening for over 50 years, in recent times regulatory agencies in Japan, Europe and the United States have questioned its safety [41]. ...
... Although hydroquinone has been the dermatological gold standard for skin lightening for over 50 years, in recent times regulatory agencies in Japan, Europe and the United States have questioned its safety [41]. Considering the risks to human health associated with its application, hydroquinone has been banned from cosmetics in Europe since 2001 [40] and its use in skin-lightening cosmetic formulations is currently illegal (see the "Regulations" section for more details). Cosmetics containing hydroquinone have also been banned in several other countries, such as the United Kingdom, Australia and Japan. ...
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Abstract: The use of bleaching products can have a medical or cosmetic purpose; in the latter case, skin whitening is most widespread in countries where darker skin tones prevail and can be driven by psychosocial, cultural and economic reasons. Skin-whitening products containing highly toxic active ingredients (in particular mercury derivatives, hydroquinone and corticosteroids) are easily found on the market; the use of these depigmenting agents can be followed by a variety of adverse effects, with very serious and sometimes fatal complications, and is currently an emerging health concern in many countries. This article concisely discusses the reasons for the current prevalence of skin lightening products and provides an overview of the skin lightening agents that pose a threat to human health. The review also reports market surveillance data on the circulation of banned skin lighteners in Europe, obtained through the Safety Gate system.
... Long term chronic exposure to arsenic can lead to multisystem failure and various cancers [13] while the chronic exposure to lead is reported to have effects on brain, leading to convulsions and sometimes coma [14]. An enormous number of articles have since 1996, been published on the effects including carcinogenicity and leukomelanoderma of hydroquinone, benzene and related molecules [10,11,15,16]. These underlying effects of hydroquinone necessitated that its use in cosmetics be banned in the Netherlands since January 2001 [16] even though it had for decades been used as a skin lightening agent. ...
... An enormous number of articles have since 1996, been published on the effects including carcinogenicity and leukomelanoderma of hydroquinone, benzene and related molecules [10,11,15,16]. These underlying effects of hydroquinone necessitated that its use in cosmetics be banned in the Netherlands since January 2001 [16] even though it had for decades been used as a skin lightening agent. ...
... Notably, despite the various reported health side effects of these heavy metals and hydroquinone, the data regarding their levels in skin lightening creams sold on the Ugandan market is lacking, even though it is obvious that several of such products are seemingly ISSN 2520-4638 (Online) Vol.4, Issue 1, pp [15][16][17][18][19][20][21][22][23][24][25][26][27][28][29][30][31][32][33][34]2021 www.ajpojournals.org circulating in the market. ...
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In this study, Skin-lightening creams commonly sold in Mbarara municipality were analyzed for chemical parameters (pH, thermal stability and fatty substance content), total hydroquinone, Lead, Mercury, and Arsenic contents. Total heavy metal content was determined by atomic absorption spectrophotometry. The levels of hydroquinone were determined using High Performance liquid chromatography (HPLC). All the creams showed detectable mean levels of mercury, ranging from 0.07±0.01ppm to 0.33±0.01ppm. Only 26.31% of the creams showed detectable levels of lead and 15.79% creams recorded detectable levels of arsenic. The mean levels of hydroquinone ranged from 0.54±0.02% to 4.47±0.02%. All the creams passed the thermal stability and fatty substance content tests. However, all the creams had very low pH values below the recommended 4.5- 8.5 pH ranges by Uganda National Bureau of Standards (UNBS). The levels of mercury, arsenic and lead in the samples were less than the UNBS, European Union and US Food and Drug Administration’s acceptable limits. Only 84.2% of the cream samples analyzed contained hydroquinone levels higher than the recommended WHO limit of 2%. The use of such creams may lead to serious health hazards. While the low concentrations of heavy metals detected in the cream samples analyzed do not pose any potential risk to consumers, repeated application of these creams may cause a cumulative effect over prolonged exposure. The low pH values may cause skin irritations. Therefore, the community needs to be sensitized on the implications of using skin lightening creams and UNBS should conduct periodic analysis to ascertain the levels of hydroquinone, heavy metals, and chemical requirements of skin lightening creams sold in Uganda as well as encourage manufacturers to state the exact bleaching agents in their creams.
... Melanin provides UV photoprotection and scavenges free radicals; however, an excessive production of melanin in the skin can lead to hyperpigmentation and is associated with medical skin disorders such as melasma, postinflammatory hyperpigmentation (PIH), lentigo senilis (LS) and seborrheic keratosis (SK), which can cause significant psychosocial burdens. Melanocytes connect to neighboring keratinocytes in the epidermis by dendrites; one melanocyte contacts up to [30][31][32][33][34][35][36][37][38][39][40] keratinocytes to transfer melanin [3]. The process involves the synthesis, packaging, transfer and uptake of melanin by keratinocytes [4], which is ultimately responsible for perceived skin coloration. ...
... Commercial skin whiteners, such as kojic acid (KA), hydroquinone (HQ) and arbutin (glycosylated HQ) are known to exhibit serious side effects [31,32], prompting a need for safer and nontoxic alternatives for the treatment of hyperpigmentation disorders of the skin [33]. Curcumin, a polyphenolic bioactive compound, isolated from the rhizomes of turmeric, Curcuma longa, is a popular nutraceutical compound with a broad spectrum of pleiotropic activities that is used as an oral supplement in the management of various cutaneous conditions [34] and is reported to inhibit cellular melanogenesis [35][36][37][38][39]. ...
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Hyperpigmentation is a dermatological condition characterized by the overaccumulation and/or oversecretion of melanin pigment. The efficacy of curcumin as an anti-melanogenic therapeutic has been recognized, but the poor stability and solubility that have limited its use have inspired the synthesis of novel curcumin analogs. We have previously reported on comparisons of the anti-melanogenic activity of four novel chemically modified curcumin (CMC) analogs, CMC2.14, CMC2.5, CMC2.23 and CMC2.24, with that of parent curcumin (PC), using a B16F10 mouse melanoma cell model, and we have investigated mechanisms of inhibition. In the current study, we have extended our findings using normal human melanocytes from a darkly pigmented donor (HEMn-DP) and we have begun to study aspects of melanosome export to human keratinocytes. Our results showed that all the CMCs downregulated the protein levels of melanogenic paracrine mediators, endothelin-1 (ET-1) and adrenomedullin (ADM) in HaCaT cells and suppressed the phagocytosis of FluoSphere beads that are considered to be melanosome mimics. All the three CMCs were similarly potent (except CMC2.14, which was highly cytotoxic) in inhibiting melanin production; furthermore, they suppressed dendricity in HEMn-DP cells. CMC2.24 and CMC2.23 robustly suppressed cellular tyrosinase activity but did not alter tyrosinase protein levels, while CMC2.5 did not suppress tyrosinase activity but significantly downregulated tyrosinase protein levels, indicative of a distinctive mode of action for the two structurally related CMCs. Moreover, HEMn-DP cells treated with CMC2.24 or CMC2.23 partially recovered their suppressed tyrosinase activity after cessation of the treatment. All the three CMCs were nontoxic to human dermal fibroblasts while PC was highly cytotoxic. Our results provide a proof-of-principle for the novel use of the CMCs for skin depigmentation, since at low concentrations, ranging from 5 to 25 µM, the CMCs (CMC2.24, CMC2.23 and CMC2.5) were more potent anti-melanogenic agents than PC and tetrahydrocurcumin (THC), both of which were ineffective at melanogenesis at similar doses, as tested in HEMn-DP cells (with PC being highly toxic in dermal fibroblasts and keratinocytes). Further studies to evaluate the efficacy of CMCs in human skin tissue and in vivo studies are warranted.
... Phenolic chemicals, such as hydroquinone, are among the many substances that have excellent whitening properties [8,9]. However, various side effects of hydroquinone have been reported, including toxicity to normal skin fibroblasts and a dialectic effect on tissue after prolonged usage [10]. Therefore, its use as a primary chemical in whitening treatments is restricted and used exclusively in medicine. ...
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Whitening research is of particular interest in the cosmetics market. The main focus of whitening research is on melanogenesis inhibition through tyrosinase activity. The mechanism of melanogenesis is involved with tyrosinase activity and p-PKC signaling. In this study, we used Momordica cochinchinensis (Lour.) spreng, a tropical fruit found throughout Southeast Asia, to investigate the inhibitory effect of melanogenesis. M. cochinchinensis contains a high concentration of polyphenols, flavonoids, and unsaturated fatty acids, which might be related to antioxidant activity. This study aimed to determine whether M. cochinchinensis extracts inhibit melanin synthesis in melan-A cells by inhibiting tyrosinase activity and p-PKC signaling. M. cochinchinensis was divided into pulp and aril and extracted under various conditions, and it was confirmed that all pulp and aril extracts have high contents of both phenols and flavonoids. Melan-A cells were treated with PMA for three days to induce melanin synthesis. After PMA treatment, M. cochinchinensis extracts were added to cultured media in a dose-dependent manner. Melanin contents and MTS were used to determine the amount of melanin in live cells. M. cochinchinensis extracts were evaluated for their effects on tyrosinase activity and p-PKC signaling pathways by Western blotting. It was found that M. cochinchinensis extract treatment decreased the amount of melanin and suppressed p-PKC expression. Additionally, tyrosinase activity was reduced after M. cochinchinensis extract treatment in a dose-dependent manner. Therefore, it was concluded that M. cochinchinensis could be used in antimelanogenesis and functional cosmetic materials to improve whitening.
... As a result of the key role, played by tyrosinase in melanin biosynthesis, most marketed skin lightening products use a tyrosinase inhibitor as the active ingredient (e.g., hydroquinone, kojic acid and arbutin). However, the use of these products is marred by safety and/or effectiveness concerns [3][4][5] . ...
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In Jordanian folkloric medicine, several medicinal plants–based recipes are used for skin lightening. Local recipes for skin lightening were collected and the tyrosinase inhibitory activity of the plants reported in these recipes, as a potential depigmentation mechanism was evaluated in vitro on both, mushroom and murine melanoma tyrosinase. The surveyed recipes included a total of 25 traditional medicinal plants belonging to 19 families. Kojic acid and licorice (Glycyrrhiza glabra) extract were used as positive controls. Thirteen extracts exhibited good mushroom tyrosinase inhibitory potential (>70%), and 7 extracts showed moderate tyrosinase inhibition activity (30-70%) while 5 extracts showed poor mushroom tyrosinase inhibitory activity (<30%). Four of the tested extracts; Juniperus communis L. (Juniper), Rosa indica L. (Rose), Amygdalus communis var. amara L. (Bitter almond), and Carthamus tinctorius L. (Safflower) showed good inhibitory activity (>70%) against both, mushroom and melanoma tyrosinase enzymes that was similar or better than that of kojic acid. While, 6 tested extracts, obtained from Raphanus sativus L. (radish), Juniperus communis L. (juniper), Petroselinum sativum Hoffm. (parsely), Salvia triloba L. (sage), Viola odorata L. (garden violet), and Mentha piperita L. (mint), showed almost similar mushroom tyrosinase inhibitory activity as licorice extract (73.4%). Tyrosinase inhibitory activities observed in many of the tested plant extracts validate their traditional use.
... Among these compounds, pure chemicals have become commonly used skin-lightening products because of their excellent lightening effects. However, pure chemicals may cause side effects such as irritation (2), allergy (3), and risk of cancer when used over a long period (4). Pure natural plant extracts have been proposed as a feasible alternative to pure chemicals, but their lightening effect is unreliable due to the limitation of the components that make up the pure natural plant extracts. ...
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Background: With the emergence of various new skin-lightening products, there is an urgent need to scientifically evaluate the efficacy and toxicology of these products, and provide scientific guidance for their use based on physiological differences between individuals. Visualized imaging methods and quantitative evaluation criteria play key roles in evaluating the efficacy of skin-lightening products. In order to quantify the changes in the multilayered morphology and endogenous components of human skin before and after the use of lightening products, high-resolution three-dimensional (3D) imaging of human skin is required. Methods: In this study, photoacoustic microscopy (PAM; SSPM-532, Guangdong Photoacoustic Medical Technology Co., Ltd.) was used to capture the morphological structures of human skin and reveal skin components quantitatively. The efficacy and safety of skin-lightening products were evaluated by measuring skin melanin concentration and observing skin morphology. The melanin concentration in the epidermis was obtained by examining the linear relationship between photoacoustic (PA) signals. Further, the epidermal thickness and the melanin distribution were obtained in the cross-sectional (x-z) and lateral (x-y) images. Finally, the efficacy of skin-lightening products was evaluated according to the concentration and distribution of melanin in the epidermis, and the safety of cosmetics was assessed by observing the vascular morphology in the dermis. Results: PAM noninvasively could assess the multilayered morphological structures of human skin, which allowed for quantification of epidermal thickness and melanin concentration of different skin sites. Based on this, the efficacy and safety of skin-lightening products in multilayer structures were quantitatively evaluated. Conclusions: As a quantitative imaging method, PAM, has the potential to accurately evaluate the use of skin-lightening products. The method can also be extended to assessments within the larger field of aesthetic medicine.
... This is the potential mechanism of action of hydroquinone as a skin lightening agent as well as its toxicity mechanism. The use of this ingredient in cosmetics has been banned since 2001 because of the high risk of carcinogenesis in case of prolonged exposure to hydroquinone [6,7]. Arbutin (Chemical structure 2, Figure 1) is a compound with a structure in which one molecule of D-glucose is bound to hydroquinone. ...
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Arbutin is a compound of hydroquinone and D-glucose, and it has been over 30 years since there have been serious studies on the skin lightening action of this substance. In the meantime, there have been debates and validation studies about the mechanism of action of this substance as well as its skin lightening efficacy and safety. Several analogs or derivatives of arbutin have been developed and studied for their melanin synthesis inhibitory action. Formulations have been developed to improve the stability, transdermal delivery, and release of arbutin, and device usage to promote skin absorption has been developed. Substances that inhibit melanin synthesis synergistically with arbutin have been explored. The skin lightening efficacy of arbutin alone or in combination with other active ingredients has been clinically evaluated. Combined therapy with arbutin and laser could give enhanced depigmenting efficacy. The use of arbutin causes dermatitis rarely, and caution is recommended for the use of arbutin-containing products, especially from the viewpoint that hydroquinone may be generated during product use. Studies on the antioxidant properties of arbutin are emerging, and these antioxidant properties are proposed to contribute to the skin depigmenting action of arbutin. It is hoped that this review will help to understand the pros and cons of arbutin as a cosmetic ingredient, and will lead to future research directions for developing advanced skin lightening and protecting cosmetic products.
... Tyrosinase inhibitors are used for skin-whitening agents and the inhibition of undesired browning in the food industry. Kojic acid, hydroquinone and arbutin are known tyrosinase inhibitors, but they exhibit side effects (Ishikawa, Sasaki, Kawaguchi, Mochizuki, & Nagao, 2006;Kooyers & Westerhof, 2006;Ota et al., 2009). ...
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Rice bran protein hydrolysates (RBPHs) obtained by Alcalase® have attracted attention because of their bioactivity properties (antioxidant, anti-acetylcholinesterase and anti-butyrylcholinesterase). However, there has been no report on their improved inhibition of melanogenesis. This study aimed to investigate the inhibitory tyrosinase activity of RBPH. The defatted Khao Hom Mali RD15 rice bran protein was extracted into water (RBP1), 2% NaCl (RBP2) and 0.1 N NaOH (RBP3) fractions. All protein fractions were hydrolyzed with Alcalase® to produced RBPH1, RBPH2 and RBPH3. The protein content, % yield, degree of hydrolysis (DH) and molecular weight patterns of each fraction were investigated. The RBPH2 and RBPH3 showed high potential inhibition on mushroom tyrosinase activity, the IC50 values were determined to be 1.92 mg/ml and 0.46 mg/ml, respectively. The inhibition kinetics showed that RBPH2 was an uncompetitive mechanism, with inhibition constants (Ki and Kis) were 6.1 mg/ml and 4.5 mg/ml, respectively. Moreover, the RBPH3 displayed a non-competitive mechanism and Ki and Kis were 2.8 mg/ml and 2.5 mg/ml, respectively. Therefore, the RBPHs could be potential candidates for use in the cosmetics and food industries.
... Tyrosinase contains two copper atoms in its active site [11] and thus compounds which can chelate copper can inhibit tyrosinase activity. The most popular commercial tyrosinase inhibitors, such as kojic acid (KA), hydroquinone and arbutin (glycosylated hydroquinone) exhibit serious sideeffects: KA causes pigmented contact dermatitis [12], hydroquinone is carcinogenic [13] and arbutin has potent genotoxicity [14]. These limitations have prompted a surge in identification of novel and natural plant-derived compounds that are associated with fewer adverse effects for reduction in food browning and for treatment of hyperpigmentation both in cosmetic and clinical settings. ...
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Skin hyperpigmentation disorders arise due to excessive production of the macromolecular pigment melanin catalyzed by the enzyme tyrosinase. Recently, the therapeutic use of curcumin for inhibiting tyrosinase activity and production of melanin have been recognized, but poor stability and solubility have limited its use, which has inspired synthesis of curcumin analogs. Here, we investigated four novel chemically modified curcumin (CMC) derivatives (CMC2.14, CMC2.5, CMC2.23 and CMC2.24) and compared them to the parent compound curcumin (PC) for inhibition of in vitro tyrosinase activity using two substrates for monophenolase and diphenolase activities of the enzyme and for diminution of cellular melanogenesis. Enzyme kinetics were analyzed using Lineweaver-Burk and Dixon plots and nonlinear curve-fitting to determine the mechanism for tyrosinase inhibition. Copper chelating activity, using pyrocatechol violet dye indicator assay, and antioxidant activity, using a DPPH radical scavenging assay, were also conducted. Next, the capacity of these derivatives to inhibit tyrosinase-catalyzed melanogenesis was studied in B16F10 mouse melanoma cells and the mechanisms of inhibition were elucidated. Inhibition mechanisms were studied by measuring intracellular tyrosinase activity, cell-free and intracellular α-glucosidase enzyme activity, and effects on MITF protein level and cAMP maturation factor. Our results showed that CMC2.24 showed the greatest efficacy as a tyrosinase inhibitor of all the CMCs and was better than PC as well as a popular tyrosinase inhibitor-kojic acid. Both CMC2.24 and CMC2.23 inhibited tyrosinase enzyme activity by a mixed mode of inhibition with a predominant competitive mode. In addition, CMC2.24 as well as CMC2.23 showed a comparable robust efficacy in inhibiting melanogenesis in cultured melanocytes. Furthermore, after removal of CMC2.24 or CMC2.23 from the medium, we could demonstrate a partial recovery of the suppressed intracellular tyrosinase activity in the melanocytes. Our results provide a proof-of-principle for the novel use of the CMCs that shows them to be far superior to the parent compound, curcumin, for skin depigmentation.
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Sepiwhite is a novel anti‐pigmenting agent that is derived from fatty acid and phenylalanine and used for hyperpigmentation induced by light exposure or inflammation. In this study, a simple and validated HPLC method for quantitation of sepiwhite was developed. Optimized forced degradation of sepiwhite at thermal, acid/base, photolysis, oxidative and heavy metal ions condition were evaluated and the effect of each of them to production of specific 10–30% degradants were studied by approach of design of experiment. Sepiwhite accelerated study was conducted and toxicity of sepiwhite at each condition were tested. An optimized HPLC method was validated by a face‐centered central composition design. Ten different degradants were identified from sepiwhite and degradation behavior under different conditions were studied. Sepiwhite and its degradant products show no cytotoxicity. This optimized HPLC method can be apply for quality control assay and sepiwhite degradation behavior may be considered in manufacturing of sepiwhite products. This article is protected by copyright. All rights reserved
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Exogenous ochronosis is clinically and histologically similar to its endogenous counterpart; however, it exhibits no systemic effects and is not an inherited disorder. It is characterized by an asymptomatic hyperpigmentation of the face, sides and back of the neck, back, and extensor surfaces of the extremities. The associated ochronotic discoloration most commonly results from use of products containing hydroquinone. It also occurs following use of antimalarials and products containing resorcinol, phenol, mercury or picric acid. The etiology of hydroquinone-induced hyperpigmentation in exogenous ochronosis remains speculative. The majority of patients with this condition are Black, but it has been reported to occur in Hispanics and Caucasians. Exogenous ochronosis is prevalent among South African Blacks, but is believed relatively uncommon within the US. The reasons for this phenomenon are not clear, but it could be a result of the use of skin care products containing resorcinol in combination with hydroquinone or the use of hydroquinone in a hydroalcoholic lotion. Treatment of this condition is difficult. The offending agent must be avoided, but improvement occurs only slowly. A number of topical agents have been studied as have dermabrasion and the use of lasers. Controlled studies in larger numbers of patients are require to determine the true efficacy of newer treatments.
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The triphenolic metabolite of benzene, 1,2,4-benzenetriol (BT), is readily oxidized to its corresponding quinone via a semiquinone radical. During this process, active oxygen species are formed that may damage DNA and other cellular macromolecules. The ability of BT to induce micronuclei (MN) and oxidative DNA damage has been investigated in both human lymphocytes and HL60 cells. An antikinetochore antibody based micronucleus assay was used to distinguish MN containing kinetochores and potentially entire chromosomes (kinetochore-positive, K+) from those containing acentric chromosome fragments (kinetochore-negative, K−). BT increased the frequency of MN formation twofold in lymphocytes and eightfold in HL60 cells with the MN being 62% and 82% K+, respectively. A linear dose-related increase in total MN, mainly in K+-MN, was observed in both HL60 cells and lymphocytes. Addition of copper ions (Cu2+) potentiated the effect of BT on MN induction threefold in HL60 cells and altered the pattern of MN formation from predominantly K+ to K−. BT also increased the level of 8-hydroxy-2′-deoxyguanosine (8-OH-dG), a marker of active oxygen-induced DNA damage. Cu2+ again enhanced this effect. Thus, BT has the potential to cause both numerical and structural chromosomal changes in human cells. Further, it may cause point mutations indirectly by generating oxygen radicals. BT may therefore play an important role in benzene-induced leukemia. © 1993 Wiley-Liss, Inc.
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Hydroquinone (HQ) was administered to F344 rats and B6C3F1 mice of both sexes at a level of 0.8% in the diet for two years. This treatment induced renal tubular hyperplasia as well as adenomas, predominantly in males of both species, and was associated with chronic nephropathy in rats. In addition, the occurrence of epithelial hyperplasia of the renal papilla was increased in male rats. Foci of cellular alteration of the liver were significantly reduced in number by HQ in rats, but in contrast, were increased in mice, where development of hepatocellular adenoma was also enhanced in males. The incidence of squamous cell hyperplasia of the forestomach epithelium was significantly higher in mice of both sexes given HQ than in the controls, but no corresponding increase in tumor development was observed. The present study strongly indicates potential renal carcinogenicity of HQ in male rats and hepatocarcinogenicity in male mice. Thus, it is possible that HQ, which is present in the human environment, may play a role in cancer development in man.
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Previously, we identified p-benzoquinone (BQ) and 1,2,4-benzenetriol (BT) as toxic metabolites of benzene on the basis of their inhibitory effect on DNA synthesis. In the present study, the capability of benzene and the two metabolites to induce DNA strand breaks was investigated in either the in vivo or the in vitro system by comparing the DNA elution rate on a fine membrane filter at alkaline pH. In the in vitro system were bone marrow cells were reacted with test chemicals for 60 min, both BQ and BT induced a dose-related increase in alkali-labile DNA single-strand breaks (SSBs) of bone marrow cells. However, when glutathione (350 micrograms/ml) was added to the same reaction system, the DNA damaging effect of BQ (24 microM) and BT (24 microM) was blocked by 100 and 53%, respectively. Catalase (130 units/ml) completely blocked the DNA damaging effect of BT, while no protection was afforded with BQ. Consistent with these observations, no induction of alkali-labile DNA SSBs was observed in the in vivo system by an anesthetic dose of benzene (1760 mg/kg, ip or po) at 1, 24, and 36 hr postadministration in both male and female ICR mice. These results suggest that benzene exposure would not induce direct DNA strand breaks in vivo under realistic work-related or accidental exposure conditions and also indicate that caution should be exercised in the interpretation of in vitro data for whole-body toxicity evaluation.
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The triphenolic metabolite of benzene, 1,2,4-benzenetriol (BT), is readily oxidized to its corresponding quinone via a semiquinone radical. During this process, active oxygen species are formed that may damage DNA and other cellular macromolecules. The ability of BT to induce micronuclei (MN) and oxidative DNA damage has been investigated in both human lymphocytes and HL60 cells. An antikinetochore antibody based micronucleus assay was used to distinguish MN containing kinetochores and potentially entire chromosomes (kinetochore-positive, K+) from those containing acentric chromosome fragments (kinetochore-negative, K-). BT increased the frequency of MN formation twofold in lymphocytes and eightfold in HL60 cells with the MN being 62% and 82% K+, respectively. A linear dose-related increase in total MN, mainly in K(+)-MN, was observed in both HL60 cells and lymphocytes. Addition of copper ions (Cu2+) potentiated the effect of BT on MN induction threefold in HL60 cells and altered the pattern of MN formation from predominantly K+ to K-. BT also increased the level of 8-hydroxy-2'-deoxyguanosine (8-OH-dG), a marker of active oxygen-induced DNA damage. Cu2+ again enhanced this effect. Thus, BT has the potential to cause both numerical and structural chromosomal changes in human cells. Further, it may cause point mutations indirectly by generating oxygen radicals. BT may therefore play an important role in benzene-induced leukemia.
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Iron catalysed bleomycin (an antitumor antibiotic)-dependent degradation of DNA was investigated in the presence of glutathionyl hydroquinone (GHQ). DNA degradation was enhanced twelve-fold in the presence of iron and GHQ and three-fold in the presence of iron and glutathione (GSH) as compared to iron alone. The degradation of DNA was linear with the increase in concentration of GHQ or GSH keeping the iron, bleomycin and other factors constant. The presence of oxyradical scavengers, viz., thiourea, mannitol, albumin, superoxide dismutase, catalase and dimethyl sulfoxide caused significant inhibition of degradation of DNA by GHQ and iron. All the externally added GHQ to bone marrow cell lysate was completely demonstrable by the assay of iron-catalyzed bleomycin-dependent degradation of DNA. Superoxide radical generation was demonstrable during the incubation of GHQ. Thus, the present study revealed that GHQ is a potent pro-oxidant and this observation is significant in understanding the mechanism of benzene toxicity with the possibility of GHQ as one of the toxic metabolites of benzene.
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The cytotoxicity of benzene (BZ) and its major metabolites phenol (PHE), hydroquinone (HQ), catechol (CAT), 1,4-benzoquinone (BQ), 1,2,4-benzenetriol (BT), trans,trans-muconic acid (ttMA) and S-phenylmercapturic acid (S-PMA) was assessed by exposing Chinese Hamster Ovary (CHO) cells to these compounds. Benzene was the least toxic (LD50 = 20 mM), while BQ showed the highest potency (LD50 = 10 microM), followed by HQ (LD50 = 40 microM). It was found that the trend of cytotoxicity was: BQ > HQ > > CAT > ttMA > BT > S-PMA > > PHE > BZ. 1,4-Benzoquinone and HQ also demonstrated considerable ability to induce DNA strand breaks in CHO cells, which was assayed using the fluorimetric analysis of DNA unwinding. The other metabolites were unable to cause DNA strand breaks. When HQ was administered in combination with other metabolites, no synergism was observed in the induction of DNA strand breaks. From these results, it can be seen that BQ and HQ are the most bioreactive species among the benzene metabolites when tested on CHO cells. Differences between the results obtained in our study and other studies were discussed.
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Benzene toxicity involves both bone marrow depression and leukemogenesis caused by damage to multiple classes of hematopoietic cells and a variety of hematopoietic cell functions. Study of the relationship between the metabolism and toxicity of benzene indicates that several metabolites of benzene play significant roles in generating benzene toxicity. Benzene is metabolized, primarily in the liver, to a variety of hydroxylated and ring-opened products that are transported to the bone marrow where subsequent secondary metabolism occurs. Two potential mechanisms by which benzene metabolites may damage cellular macromolecules to induce toxicity include the covalent binding of reactive metabolites of benzene and the capacity of benzene metabolites to induce oxidative damage. Although the relative contributions of each of these mechanisms to toxicity remains unestablished, it is clear that different mechanisms contribute to the toxicities associated with different metabolites. As a corollary, it is unlikely that benzene toxicity can be described as the result of the interaction of a single metabolite with a single biological target. Continued investigation of the metabolism of benzene and its metabolites will allow us to determine the specific combination of metabolites as well as the biological target(s) involved in toxicity and will ultimately lead to our understanding of the relationship between the production of benzene metabolites and bone marrow toxicity.
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Hydroquinone is a ubiquitous chemical readily available as monographed in cosmetic and nonprescription forms for skin lightening, and is an important industrial chemical. The in vivo bioavailability for 24-h application in humans was 45.3+/-11.2% of dose from a 2% cream formulation containing [14C]hydroquinone, with the majority of radioactivity excreted in the first 24 h. Timed skin wash and skin tape-stripping sequences showed a rapid and continuous movement of hydroquinone into the stratum corneum of human volunteers. Plasma levels taken both ipsilateral and contralateral to the topical dosing site contained radioactivity at the first 0.5-h sampling time. Peak plasma radioactivity was at 4 h in the 8-h blood sampling period. In vitro percutaneous absorption with fresh viable human skin gave a bioavailability of 43.3% of dose, and flux was calculated at 2.85 microg/cm2/h. In vitro, some of the skin samples were pretreated with the metabolic inhibitor sodium azide, which had no effect on percutaneous absorption. Receptor fluid accumulations and 24-h skin samples were extracted and the extracts subjected to thin-layer chromatography (TLC). Control [14C]hydroquinone extraction and TLC had one radioactivity peak, hydroquinone. Receptor fluid and skin extraction had a second peak with the same Rf as benzoquinone, which was decreased with azide treatment. No other peaks were found. Ethyl acetate extraction of urine from the in vivo study showed all radioactivity to be only water-soluble, free hydroquinone released following glucuronidase treatment. Risk assessment should not only involve the bioavailability of intact topical hydroquinone, but also consider phase I and phase II metabolism in both humans and any animal for which toxicity potential was assessed.
Article
Benzo(a)pyrene and benzene are human carcinogens. The metabolic activation of these compounds into ultimate mutagenic and carcinogenic metabolites is prerequisite for their carcinogenic effects. In this report, the mutagenicity and carcinogenicity of hydroquinones of benzo(a)pyrene and benzene was investigated to address two important questions: (1) do hydroquinones contribute to benzo(a)pyrene and benzene carcinogenicity; and (2) how safe is it to increase the levels of NAD(P)H:quinone oxidoreductase 1 (NQO1), a key enzyme in the generation of hydroquinone. The supF tRNA of the plasmid pSP189 was used as the mutational target in a cell-free and Chinese hamster ovary (CHO) cell system to study hydroquinone mutagenicity. RNA and protein-free pSP189 DNA was incubated in a cell-free system with benzo(a)pyrene-3,6-quinone and purified NQO1 or with benzoquinone hydroquinone to generate adducted pSP189 DNA. The adducted pSP189 DNA was transfected in human embryonic kidney cells Ad293. In the CHO cell system, monolayer cultures of CHO cells and CHO cells overexpressing NQO1 or P450 reductase were transfected with pSP189 vector DNA, treated with benzo(a)pyrene-3,6-quinone. The adducted and replicated pSP189 DNA was rescued from transfected Ad293 (cell-free system) and CHO cells (CHO cell system), digested with the restriction enzyme Dpn1 to remove unreplicated DNA followed by transformation in Escherichia coli MBM7070. The mutant colonies [white/pale blue on 5-bromo-4-chloro-3-indolyl beta-D-galactoside/isopropyl beta-D-thiogalactoside (X-gal/IPTG) plates] were selected, regrown and analysed by DNA sequencing. Mutagenesis experiments demonstrated that hydroquinones cause sequence-specific frameshift mutations involving deletion of a single cytosine from the DNA sequence 5'-172-CCCCC176-3' or a single guanosine from the complementary strand sequence 5'-GGGGG-3' in the supF tRNA gene. This mutation was specific to the hydroquinones, as it was not observed with quinones and other components of the redox cycling (semiquinones and reactive oxygen species). Exposure of BALBc/3T3 cells to hydroquinones resulted in cellular transformation leading to the loss of contact inhibition and regulation of cell growth. The transformation efficiency of BALBc/3T3 cells exposed to hydroquinones was significantly increased by the tumour promoter 12-O-tetradecanoylphorbol-13-acetate (TPA), indicating that hydroquinones are excellent initiators that require additional co-carcinogens or promoters to exert an effect. The hydroquinone + TPA as well as hydroquinone-transformed BALBc/3T3 cells, when injected s.c. in severe combined immunodeficient (SCID) mice, produced tumours at 100% frequency. These results establish that hydroquinones lead to mutagenicity and carcinogenicity. Images Figure 1 Figure 2 Figure 3