Arachidonic acid cascade inhibitors modulate phorbol ester-induced oxidative stress in female ICR mouse skin: differential roles of 5-lipoxygenase and cyclooxygenase-2 in leukocyte infiltration and activation

We recently developed a short-term assay for skin H2O2 generation induced by double 12-O-tetradecanoylphorbol-13-acetate (TPA) applications for mechanistic study on skin epidermal carcinogenesis. In the present study, we investigated the individual roles of arachidonic acid metabolism in H2O2 generation in mouse skin inflammation. The experiments using inhibitors of arachidonic acid (AA) metabolism showed that corticosteroid and a lipoxygenase (LO) inhibitor expectedly suppressed double TPA application-induced H2O2 generation through the interference of chemotactic action but not by direct decomposition or scavenging. We also demonstrated that the treatment of AA (1 mumol) and 5-LO metabolites including leukotriene B4 (LTB4) partly mimicked, though soybean LO-derived lipid hydroperoxide and prostaglandins did not, the priming effect evaluated by edema formation and leukocyte infiltration. We also confirmed that inflammatory leukocytes accumulated by LTB4 generated a significant amount of H2O2 by TPA stimulation. These results suggested that 5-LO metabolites of AA are the potential key molecules in the TPA-induced priming event. Interestingly, the cyclooxygenase (COX-) 2-selective inhibitor nimesulide (NS) and celecoxib (CXB) showed different responses than those of other inhibitors. These agents showed no specific potential to inhibit the priming event but significantly suppressed H2O2 generation, lipid peroxidation, and hyperplasia in mouse skin. From the results based on an in vitro leukocyte differentiation model, we speculated that the antioxidant effect of the COX-2 inhibitors might be partly associated with both counteraction of proinflammatory cytokine-enhanced ROS generation and inhibition of CD11b, an important molecule for cell adhesion, expression. Indeed, the topical application of NS attenuated the number of infiltrated leukocytes induced by TPA in mouse skin. Thus, these gathered data indicated the differential roles of 5-LO and COX-2 in leukocyte adhesion, infiltration, and H2O2 generation.     

Topically applied vitamin E prevents massive cutaneous inflammatory and oxidative stress responses induced by double application of 12-O-tetradecanoylphorbol-13-acetate (TPA) in mice

Vitamin E (alpha-tocopherol) is a promising chemopreventive and pharmacologically safe agent, which can be exploited or tested against skin cancer. It is an established antioxidant with an ability to ameliorate the UV-induced skin damage and chemically induced inflammation in lungs. However, there are some conflicting reports about its role as a modulator of chemically induced promotion. We evaluated its efficacy in preventing the inflammatory and oxidative stress responses in a double 12-O-tetradecanoylphorbol-13-acetate (TPA) application tumor skin promotion protocol. Double application of TPA was undertaken to produce massive inflammatory and oxidative stress responses. Topical TPA treatment adversely altered many of the marker responses of stage I skin tumor promotion. Vitamin E application 30 min prior to TPA treatment (10 nmol) inhibited induction of hydrogen peroxide, myeloperoxidase (MPO) activity, xanthine oxidase (XO) activity and lipid peroxidation (LPO). Vitamin E also positively modulated altered antioxidants of mouse skin. Histological examination also revealed marked improvement. These results confirm the efficacy of vitamin E against early inflammatory and oxidative stress responses, which are hallmark of tumor promotion and provide rational basis for chemopreventive action of vitamin E in skin cancer.     

Benzyl isothiocyanate inhibits oxidative stress in mouse skin: Involvement of attenuation of leukocyte infiltration

The exposure of benzyl isothiocyanate (BITC) to mouse skin resulted in the attenuation of 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced oxidative damage through not only inhibition of the NADPH oxidase system but also leukocyte clearance at inflamed region. In spite of little ability to affect TPA-induced edema formation, pretreatments of mouse skin with BITC before the first or second TPA treatment significantly decrease the H2O2 level. A histological study also demonstrated that BITC enhanced the terminal deoxynucleotidyl transferase-dUTP nick end labeling (TUNEL)-positive index in mouse skin, suggesting that BITC might accelerate the disappearance of infiltrated leukocytes. Thus, these gathered data further supported that BITC has a potential as an anti-inflammatory agent.     

慢性应激通过减少毛囊黑色素细胞和酪氨酸酶活性导致雌性C57小鼠毛发颜色变浅

越来越多的证据表明压力可能会导致头发颜色发生变化,但其潜在机制尚不完全清楚。本研究采用雌性C57BL/6小鼠脚底电刺激结合束缚来建立慢性应激小鼠模型,并用比色法检测小鼠皮肤和B16F10黑色素瘤细胞中黑色素含量和酪氨酸酶活性;通过酶联免疫吸附实验(ELISA)测定小鼠皮肤中肿瘤坏死因子α(tumor necrosis factorα, TNF-α)、白细胞介素1β(interleukin-1β, IL-1β)和白细胞介素6 (interleukin-6, IL-6)含量;通过免疫荧光染色评估小鼠皮肤中核因子κB (nuclear factorκB, NFκB)/p65亚基的含量。结果显示:C57BL/6小鼠在慢性应激下由于皮肤中的毛囊黑色素细胞和酪氨酸酶活性降低,其毛皮颜色从暗色变为棕色。同时,慢性应激小鼠皮肤炎症反应增加,表现为皮肤中NFκB活性和TNF-α表达增加。在体外,TNF-α以剂量依赖性方式降低B16F10黑色素瘤细胞中黑色素生成和酪氨酸酶活性。以上结果表明,慢性应激通过降低雌性C57BL/6小鼠的毛囊黑色素细胞和酪氨酸酶活性来诱导皮毛颜色改变,而TNF-α可能在应激诱导的毛色改变中起重要作用。     

Rescue of the albino phenotype by introduction of a functional tyrosinase gene into mice.

The c-locus of the mouse is thought to encode tyrosinase, the key enzyme for melanin synthesis in melanocytes of the skin and the eye. Recently, a mouse cDNA was isolated and shown to confer tyrosine activity on a cell line which expressed no specialized functions for melanin synthesis. To verify that the isolated tyrosinase gene is encoded at the genetically well characterized c-locus, a minigene was assembled from tyrosinase cDNA and tyrosinase genomic DNA and used for generation of transgenic mice. Following microinjection of this construct into fertilized eggs of an albino mouse strain, transgenic mice were obtained which showed pigmentation in skin and eyes. By in situ hybridization, we show expression of the transgene in melanocytes of the hairbulb and in the pigmented cell layers of the eye. We conclude that we have rescued the albino mutation (c/c) by introduction and expression of a functional tyrosinase gene.     

Glycyrrhizin exhibits potential chemopreventive activity on 12-O-tetradecanoyl phorbol-13-acetate-induced cutaneous oxidative stress and tumor promotion in Swiss albino mice

Glycyrrhizin and its aglycone, glycyrrhetic acid has been found useful for various therapeutic purposes. Glycyrrhizin has been shown to possess many physiological functions like anti-inflammatory activity, detoxification and inhibition of carcinogenic promoters. 12-O-Tetradecanoyl phorbol-13-acetate (TPA), a well-known phorbal ester is known for its tumor promotion activity. The induction of inflammation in skin mediated by TPA is believed to be governed by cyclooxygenase (COX), lipoxygenase and ornithine decarboxylase (ODC). These markers of inflammatory responses are important for skin tumor promotion. In our present study, we studied the chemopreventive effect of glycyrrhizin on TPA (20 nmol/0.2 mL acetone/animal, topically)-induced oxidative stress and hyperproliferation markers in skin. TPA enhanced lipid peroxidation with reduction in the level of catalase, glutathione, glutathione peroxidase, glutathione reductase and glutathione-s-transferase. TPA treatment also enhanced ODC activity and [3H] thymidine incorporation into cutaneous DNA. Prophylactic treatment of mice with glycyrrhizin (2.0 & 4.0 mg/0.2 mL acetone/animal, topically) resulted in a significant decrease in cutaneous microsomal lipid peroxidation (P < 0.001) and recovery of cutaneous glutathione content (P < 0.001) and its dependent enzymes. A significant inhibition in ODC activity and DNA synthesis (P < 0.001) was also observed. Thus, the results demonstrate that pretreatment with glycyrrhizin is protective against TPA-induced oxidative stress and tumor promotion in Swiss albino mice.     

Glycyrrhizin exhibits potential chemopreventive activity on 12-O-tetradecanoyl phorbol-13-acetate-induced cutaneous oxidative stress and tumor promotion in swiss albino mice

Glycyrrhizin and its aglycone, glycyrrhetic acid has been found useful for various therapeutic purposes. Glycyrrhizin has been shown to possess many physiological functions like anti-inflammatory activity, detoxification and inhibition of carcinogenic promoters. 12-O-Tetradecanoyl phorbol-13-acetate (TPA), a well-known phorbal ester is known for its tumor promotion activity. The induction of inflammation in skin mediated by TPA is believed to be governed by cyclooxygenase (COX), lipoxygenase and ornithine decarboxylase (ODC). These markers of inflammatory responses are important for skin tumor promotion. In our present study, we studied the chemopreventive effect of glycyrrhizin on TPA (20 nmol/0.2 mL acetone/animal, topically)-induced oxidative stress and hyperproliferation markers in skin. TPA enhanced lipid peroxidation with reduction in the level of catalase, glutathione, glutathione peroxidase, glutathione reductase and glutathione-s-transferase. TPA treatment also enhanced ODC activity and [³H] thymidine incorporation into cutaneous DNA. Prophylactic treatment of mice with glycyrrhizin (2.0 & 4.0 mg/0.2 mL acetone/animal, topically) resulted in a significant decrease in cutaneous microsomal lipid peroxidation (P < 0.001) and recovery of cutaneous glutathione content (P < 0.001) and its dependent enzymes. A significant inhibition in ODC activity and DNA synthesis (P < 0.001) was also observed. Thus, the results demonstrate that pretreatment with glycyrrhizin is protective against TPA-induced oxidative stress and tumor promotion in Swiss albino mice.     

TPA (12-O-tetradecanoyl-phorbol-13-acetate) as a carcinogen for mouse skin. A positive dose-response relationship

In order to study a possible dose/response relationship in the tumorigenic effect of 12-O-tetradecanoyl-phorbol-13-acetate (TPA), groups of hairless mice were treated topically on the back skin with different doses and treatment schedules of TPA in acetone. A group of 104 mice (56 M/48 F) received a single application of 20 nmol TPA; 80 mice (32 M/48 F) received 2 X 20 nmol TPA at 3 day intervals; 95 mice (48 M/47 F) received five applications; and 32 mice (16 M/16 F) received 50 applications of 20 nmol TPA twice weekly. Two groups of 32 mice (16 M/16 F) were painted topically on the back skin twice weekly with doses of 17 and 10 nmol TPA, respectively, in each application for 60 weeks or until the animals died. All applications were given in 0.1 ml reagent grade acetone. A control group was treated twice weekly with acetone alone for 60 weeks. Long-term application of 17 nmol TPA was toxic to the animals, and only 20% survived 10 months. Doses of 10 nmol TPA twice weekly were less toxic, and 80% of the animals survived 14 months. The other doses were fairly non-toxic. The results were assessed statistically by accepted methods for tumor rates and yields, respectively. There was a significant tumor incidence after the higher doses of TPA and a clear dose-response relationship. This further strengthens the evidence that TPA is a complete carcinogen, and not merely a promoter.     

Inhibition of phorbol ester-caused synthesis of mouse epidermal ornithine decarboxylase by retinoic acid

The mechanisms by which topically applied retinoic acid to mouse skin inhibits tumor promoter 12-O-tetradecanoylphorbol 13-acetate (TPA)-induced epidermal ornithine decarboxylase activity were analyzed. Retinoic acid inhibition of the induction of epidermal ornithine decarboxylic activity was not the result of nonspecific cytotoxicity, production of a soluble inhibitor of ornithine decarboxylase, or direct effect on its activity. In addition, inhibition of TPA-caused increased ornithine decarboxylase activity does not appear to be due to enhanced degradation and/or post-translational modification of ornithine decarboxylase by transglutaminase-mediated putrescine incorporation. We found that retinoic acid inhibits the synthesis of ornithine decarboxylase caused by TPA. Application of 10 nmol TPA to mouse skin led to a dramatic induction of epidermal ornithine decarboxylase activity which was paralled by increased [3H]difluoromethylornithine binding and an increased incorporation of [35S]methionine into the enzyme. Application of 17 nmol retinoic acid 1 h prior to application of 10 nmol TPA to skin resulted in inhibition of the induction of activity which accompanied inhibition of [3H]difluoromethylornithine binding and [35S]methionine incorporation into ornithine decarboxylase protein as determined by the tube-gel electrophoresis of the enzyme immunoprecipitated with monoclonal antibodies to it. Inhibition of ornithine decarboxylase synthesis was not the result of the inhibitory effect of retinoic acid on general protein synthesis. The results indicate that retinoic acid possibly inhibits TPA-caused synthesis of ornithine decarboxylase protein selectively.     

Generation of albino Xenopus tropicalis using zinc‐finger nucleases

To generate albino lines of Xenopus tropicalis, we injected fertilized eggs with mRNAs encoding zinc‐finger nucleases (ZFNs) targeting the tyrosinase coding region. Surprisingly, vitiligo was observed on the skin of F0 frogs that had been injected with ZFN mRNAs, indicating that both tyrosinase genes in the genome were disrupted in all melanocytes within the vitiligo patches. Mutation analysis using genomic DNA from the skin revealed that two mosaic F0 frogs underwent spatially complex tyrosinase gene mutations. The data implies that the ZFN‐induced tyrosinase gene ablations occurred randomly over space and time throughout the entire body, possibly until the young tadpole stage, and that melanocyte precursors lacking functional tyrosinase proliferated and formed vitiligo patches. Several albino X. tropicalis, which are compound heterozygotes for biallelic tyrosinase mutations, were obtained by mating the mosaic F0 frogs. To our knowledge, this is the first report of the albino vertebrates generated by the targeted gene knockout.     

Avarol inhibits TNF-alpha generation and NF-kappaB activation in human cells and in animal models

Avarol is a marine sesquiterpenoid hydroquinone with interesting pharmacological properties including anti-inflammatory and antipsoriatic effects. In the present study we evaluated the pharmacological effect of avarol on some inflammatory parameters related to the pathogenesis of psoriasis. Avarol inhibited tumor necrosis factor-alpha (TNF-alpha) generation in stimulated human monocytes (IC(50) 1 microM) and TNF-alpha-induced activation of nuclear factor-kappaB (NF-kappaB)-DNA binding in keratinocytes. In the mouse air pouch model, administration of avarol produced a dose-dependent reduction of TNF-alpha generation (ED(50) 9.2 nmol/pouch) as well as of interleukin (IL)-1beta, prostaglandin E(2) (PGE(2)) and leukotriene B(4) (LTB(4)) levels in pouch exudates. In the psoriasis-like model of 12-O-tetradecanoylphorbol-acetate-induced mouse epidermal hyperplasia, topical administration of avarol (0.6-1.2 micromol/site) reduced edema, myeloperoxidase activity, IL-1beta, IL-2 and eicosanoid levels in skin. Histopathological study confirmed the inhibition of epidermal hyperplasia as well as leukocyte infiltration. The reduction of cutaneous TNF-alpha by avarol was also detected by immunohistochemical analysis. Avarol was also capable of suppressing in vivo NF-kappaB nuclear translocation, determined in mouse skin. Our results suggested that antipsoriatic properties of avarol previously described could be mediated in part by the downregulation of several inflammatory biomarkers, such as TNF-alpha and NF-kappaB in psoriatic skin.     

Molecular Characterization of the Mouse Tyrosinase Gene:Pigment Cell-Specific Expression in Transgenic Mice

Tyrosinase is the key enzyme in melanin synthesis, and is expressed in the pigment epithelium of the retina, a cell layer derived from the optic cup; and in neural crest-derived melanocytes of skin, hair follicle, choroid, and iris. The tyrosinase gene has been cloned and shown to map to the well-characterized c-locus (albino locus) of the mouse. Subsequent studies demonstrated that a functional tyrosinase minigene was able to rescue the albino phenotype in transgenic mice. The transgene was expressed in a cell type-specific manner in skin and eye. During development of the mouse, the tyrosinase gene is expressed in the pigment epithelium of the retina as early as day 10.5 of gestation. In the hair follicle, tyrosinase gene expression is detected from day 16.5 onwards. This cell-type–specific expression is largely reproduced in transgenic mice. Our results suggest that sequences in the immediate vicinity of the mouse tyrosinase gene are sufficient to provide cell type-specificity and developmental regulation in melanocytes and the pigment epithelium.     

Ebselen, a glutathione peroxidase mimetic seleno-organic compound, as a multifunctional antioxidant. Implication for inflammation-associated carcinogenesis.

Ebselen, a seleno-organic compound showing glutathione peroxidase-like activity, is one of the promising synthetic antioxidants. In the present study, we investigated the antioxidant activities of ebselen using a 12-O-tetradecanoylphorbol-13-acetate (TPA)-treated mouse skin model. Double pretreatments of mouse skin with ebselen significantly inhibited TPA-induced formation of thiobarbituric acid-reacting substance, known as an overall oxidative damage biomarker, in mouse epidermis, suggesting that ebselen indeed acts as an antioxidant in mouse skin. The antioxidative effect of ebselen is attributed to its selective blockade of leukocyte infiltration and activation leading to attenuation of the H(2)O(2) level. In in vitro studies, ebselen inhibited TPA-induced superoxide generation in differentiated HL-60 cells and lipopolysaccharide-induced cyclooxygenase-2 protein expression in RAW 264.7 cells. In addition, we demonstrated for the first time that ebselen potentiated phase II enzyme activities, including NAD(P)H:(quinone-acceptor) oxidoreductase1 and glutathione S-transferase in cultured hepatocytes and in mouse skin. These results strongly suggest that ebselen, a multifunctional antioxidant, is a potential chemopreventive agent in inflammation-associated carcinogenesis.     

The P2X7 receptor is not essential for development of imiquimod-induced psoriasis-like inflammation in mice

Psoriasis is a chronic inflammatory skin disorder, characterised by epidermal hyperplasia (acanthosis) and leukocyte infiltration of the skin. Current therapies are inadequate, highlighting the need for new therapeutic targets. The P2X7 receptor is implicated in the pathogenesis of psoriasis. This study investigated the role of P2X7 in imiquimod (IMQ)-induced psoriasis-like inflammation. Topically applied IMQ caused twofold greater ear swelling in BALB/c mice compared to C57BL/6 mice, which encode a partial loss-of-function missense mutation in the P2RX7 gene. However, there was no difference in histological skin pathology (acanthosis and leukocyte infiltration) between the two strains. IMQ treatment up-regulated P2X7 expression in skin from both mouse strains. Additionally, IMQ induced ATP release from cultured human keratinocytes, a process independent of cell death. Injection of the P2X7 antagonist Brilliant Blue G (BBG) but not A-804598 partly reduced ear swelling compared to vehicle-injected control mice. Neither antagonist altered skin pathology. Moreover, no difference in ear swelling or skin pathology was observed between C57BL/6 and P2X7 knock-out (KO) mice. Flow cytometric analysis of IMQ-treated skin from C57BL/6 and P2X7 KO mice demonstrated similar leukocyte infiltration, including neutrophils, macrophages and T cells. In conclusion, this study demonstrates that P2X7 is not essential for development of IMQ-induced psoriasis-like inflammation but does not exclude a role for this receptor in psoriasis development in humans or other mouse models of this disease.     

Pro/antioxidant Status in Murine Skin Following Topical Exposure to Cumene Hydroperoxide Throughout the Ontogeny of Skin Cancer

Organic peroxides used in the chemical and pharmaceutical industries have a reputation for being potent skin tumor promoters and inducers of epidermal hyperplasia. Their ability to trigger free radical generation is critical for their carcinogenic properties. Short-term in vivo exposure of mouse skin to cumene hydroperoxide (Cum-OOH) causes severe oxidative stress and formation of spin-trapped radical adducts. The present study was designed to determine the effectiveness of Cum-OOH compared to 12-O-tetradecanoylphorbol-13-acetate (TPA) in the induction of tumor promotion in the mouse skin, to identify the involvement of cyclooxygenase-2 (COX-2) in oxidative metabolism of Cum-OOH in keratinocytes, and to evaluate morphological changes and outcomes of oxidative stress in skin of SENCAR mice throughout a two-stage carcinogenesis protocol. Dimethyl-benz[a]anthracene (DMBA)-initiated mice were treated with Cum-OOH (32.8 micro mol) or TPA (8.5 nmol) twice weekly for 20 weeks to promote papilloma formation. Skin carcinoma formed only in DMBA/Cum-OOH-exposed mice. Higher levels of oxidative stress and inflammation (as indicated by the accumulation of peroxidative products, antioxidant depletion, and edema formation) were evident in the DMBA/Cum-OOH group compared to DMBA/TPA treated mice. Exposure of keratinocytes (HaCaT) to Cum-OOH for 18 h resulted in expression of COX-2 and increased levels of PGE(2). Inhibitors of COX-2 efficiently suppressed oxidative stress and enzyme expression in the cells treated with Cum-OOH. These results suggest that COX-2-dependent oxidative metabolism is at least partially involved in Cum-OOH-induced inflammatory responses and thus tumor promotion.     

Deletion of the mouse meprin beta metalloprotease gene diminishes the ability of leukocytes to disseminate through extracellular matrix

Meprins are metalloendopeptidases expressed by leukocytes in the lamina propria of the human inflamed bowel, that degrade extracellular matrix proteins in vitro implicating them in leukocyte transmigration events. The aims of these studies were to 1) examine the expression of meprins in the mouse mesenteric lymph node, 2) determine whether macrophages express meprins, and 3) determine whether deletion of the meprin beta gene (Mep-1beta) mitigated the ability of leukocytes to disseminate through extracellular matrix in vitro. These studies show that meprin alpha and beta are expressed in leukocytes of the mouse mesenteric lymph node, and meprin alpha, but not beta, decreased during intestinal inflammation. Deletion of Mep-1beta gene decreased the ability of leukocytes to migrate through matrigel compared with wild-type leukocytes. Meprin beta, but not alpha, was detected in cortical and medullary macrophages of the lymph node. Thus overall, meprin beta is expressed by leukocytes in the draining lymph node of the intestine, regardless of the inflammatory status of the animal, and is likely to contribute to leukocyte transmigration events important to intestinal immune responses. Thus, the expression of meprins by leukocytes of the intestinal immune system may have important implications for diseases such as inflammatory bowel diseases, which are aggravated by leukocyte infiltration.     

Extracellular cyclophilins contribute to the regulation of inflammatory responses

The main regulators of leukocyte trafficking during inflammatory responses are chemokines. However, another class of recently identified chemotactic agents is extracellular cyclophilins, the proteins mostly known as receptors for the immunosuppressive drug, cyclosporine A. Cyclophilins can induce leukocyte chemotaxis in vitro and have been detected at elevated levels in inflamed tissues, suggesting that they might contribute to inflammatory responses. We recently identified CD147 as the main signaling receptor for cyclophilin A. In the current study we examined the contribution of cyclophilin-CD147 interactions to inflammatory responses in vivo using a mouse model of acute lung injury. Blocking cyclophilin-CD147 interactions by targeting CD147 (using anti-CD147 Ab) or cyclophilin (using nonimmunosuppressive cyclosporine A analog) reduced tissue neutrophilia by up to 50%, with a concurrent decrease in tissue pathology. These findings are the first to demonstrate the significant contribution of cyclophilins to inflammatory responses and provide a potentially novel approach for reducing inflammation-mediated diseases.     

An avocado constituent, persenone A, suppresses expression of inducible forms of nitric oxide synthase and cyclooxygenase in macrophages, and hydrogen peroxide generation in mouse skin

We investigated the suppressive effects of an avocado constituent, persenone A, on lipopolysaccharide- and interferon-y-induced inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX-2) in a mouse macrophage cell line RAW 264.7. Persenone A at concentration of 20 microM almost completely suppressed both iNOS and COX-2 protein expression. In mouse skin, double treatments with persenone A (810 nmol) significantly suppressed double 12-O-tetradecanoylphorbol-13-acetate (TPA, 8.1 nmol) application-induced hydrogen peroxide (H2O2) generation. Treatment with persenone A before the second TPA treatment was sufficient to inhibit H2O2 generation, while the first treatment was not. This study thus suggests that persenone A is a possible agent to prevent inflammation-associated diseases including cancer.     

Localized in vivo genotypic and phenotypic correction of the albino mutation in skin by RNA-DNA oligonucleotide

We recently demonstrated that an RNA-DNA oligonucleotide corrected a point mutation in the mouse tyrosinase gene, resulting in permanent and inheritable restoration of tyrosinase enzymatic activity, melanin synthesis, and pigmentation changes in cultured melanocytes. In this study, we extended gene correction of melanocytes from tissue culture to live animals, using a chimeric oligonucleotide designed to correct a point mutation in the tyrosinase gene. Both topical application and intradermal injection of this oligonucleotide to albino BALB/c mouse skin resulted in dark pigmentation of several hairs in a localized area. The restored tyrosinase enzymatic activity was detected by dihydroxyphenylacetic acid (DOPA) staining of hair follicles in the treated skin. Tyrosinase gene correction was also confirmed by restriction fragment length polymorphism analysis and DNA sequencing from skin that was positive for DOPA staining and melanin synthesis. Localized gene correction was maintained three months after the last application of the chimeric oligonucleotides. These results demonstrated correction of the tyrosinase gene point mutation by chimeric oligonucleotides in vivo.