- Email: [email protected]
Circulating microRNA biomarkers of paracetamol hepatotoxicity in zebrafish
Abstracts / Toxicology Letters 229S (2014) S40–S252
ethanol and DPBS supplemented with BSA (5%), DMSO (5%) or SDS (5%). The results confirmed, that the receptor fluid used for dermal absorption studies in vitro has an influence on the penetration properties. The total absorption of caffeine varied depending on the receptor fluid composition between 45% (DMSO and BSA) and 15% (50% ethanol and SDS). http://dx.doi.org/10.1016/j.toxlet.2014.06.209
P-1.31 Toxicity of selected bioactivated compounds in primary rat hepatocytes cultured in micropatterned co-cultures Okechukwu Ukairo 1,∗ , Julianne Shi 1 , Justin Jackson 1 , Kelly Rose 2 , Melvin Andersen 2 , Edward LeCluyse 2 1
Hepregen Corporation, Medford, MA, USA, 2 The Hamner Institutes for Health Sciences, Research Triangle Park, NC, USA Drug-induced liver injury is often caused by cytochrome P450dependent activation of drugs into reactive metabolites. In vitro models, which can mimic in vivo responses and allow the evaluation of initial and adaptive responses to bioactivated compounds over prolonged periods, offer potentially valuable tools for toxicological assessment. We have previously developed a model in which primary hepatocytes are seeded onto ECM-coated domains of optimized dimensions and subsequently co-cultivated with murine embryonic fibroblasts [i.e. micropatterned cocultures (MPCC)]. This model retains key biochemical functions of in vivo liver with long term stability. Here, we assess the bioactivation and cytotoxicity of acetaminophen (APAP) and other compounds in the 96-well rat MPCC. APAP exerts its toxic effects through bioactivation associated, in part, with cytochrome P450 3A (CYP3A). Rat MPCCs were exposed to increasing concentrations of APAP (over 5 days) and assessed for changes in hepatic ATP content, glutathione (GSH) levels and urea synthesis. Similar concentration-dependent cytotoxicity profiles were obtained over the course of the 4-week study. Addition of 200 M l-buthionine (S, R)-sulfoximine, an inhibitor of GSH synthesis, or 10 M dexamethasone, an inducer of rat CYP3A1/2, to rat MPCCs potentiated APAP-induced hepatotoxicity in these cultures irrespective of culture age (over 4 weeks). These findings are consistent with the known in vivo mechanisms of APAP toxicity in rats. Rat MPCCs provided reproducible APAPinduced cell cytotoxicity profiles over a 4 week period and can be used to assess the effects of chronic exposure to bioactivated compounds. The toxicity profiles of other bioactivated compounds are also reported here. http://dx.doi.org/10.1016/j.toxlet.2014.06.210
P-1.32 Description of the MoA/AOP linked with PPARgamma receptor dysregulation leading to liver fibrosis Vessela Vitcheva 1,2,∗ , Merilin Al Sharif 1 , Ivanka Tsakovska 1 , Petko Alov 1 , Aleksandra Mostrag-Szlichtyng 3 , Mark T.D. Cronin 4 , Chihae Yang 3 , Ilza Pajeva 1 1
Institute of Biophysics and Biomedical Engineering, Sofia, Bulgaria, Department of Pharmacology, Pharmacotherapy and Toxicology, Faculty of Pharmacy, Medical University, Sofia, Bulgaria, 3 Altamira, 2
S49
LLC, Columbus, OH, USA, 4 School of Pharmacy and Chemistry, Liverpool John Moores University, Liverpool, UK Modes of Action and Adverse Outcome Pathways (MoAs/AOPs) are key elements in the toxicological knowledge framework that are being built to support chemical risk assessment based on mechanistic reasoning. Peroxisome Proliferator-Activated Receptor gamma (PPARgamma) is a nuclear receptor that regulates adipocyte differentiation, insulin sensitivity and lipid synthesis and storage in adipocytes. PPARgamma activation in hepatocytes is regarded as one of the molecular initiating events (MIE) involved in liver steatosis/stetatohepatitis. However its inhibition in the hepatic stellate cells (HSCs) results in their activation that is essential for the pathogenesis of liver fibrosis. In the current study a systematic literature search has been performed and a MoA scheme based on the PPARgamma dysregulation in stellate cells and resulting in hepatic fibrosis is proposed. Literature data revealing the role of PPARgamma in HSCs are consistent and associate its depletion with HSC activation and fibrosis, whereas increasing PPARgamma expression results in HSC quiescence. A large body of literature confirms that PPARgamma agonists have anti-proliferative and anti-fibrotic effects on activated HSCs. Two applications have been defined from the AOP for fibrosis from PPARgamma dysfunction in stellate cells: (i) the description of possible MIEs triggering PPARgamma inhibition/downregulation that result in fibrosis and allowing for the development of structural alerts; (ii) the identification of key events downstream from PPARgamma dysregulation leading to fibrosis that would be suitable for assay development. Acknowledgement: The funding from the European Community’s 7th Framework Program (FP7/2007-2013) COSMOS Project under grant agreement no. 266835 and from Cosmetics Europe is gratefully acknowledged. http://dx.doi.org/10.1016/j.toxlet.2014.06.211
P-1.33 Circulating microRNA biomarkers of paracetamol hepatotoxicity in zebrafish Bastiaan Vliegenthart 1,∗ , Philip Starkey Lewis 2 , Carl Tucker 1 , Jorge Del Pozo 1 , Seb Rider 1 , Dan Antoine 2 , Valerie Dubost 3 , Magdalena Westphal 3 , Pierre Moulin 3 , Matthew Bailey 1 , Jonathan Moggs 3 , Chris Goldring 2 , Kevin Park 2 , James Dear 1 Edinburgh University, Edinburgh, UK, 2 Liverpool University, Liverpool, UK, 3 Novartis, Basel, Switzerland
1
Paracetamol (acetaminophen) is the commonest cause of acute liver failure in the Western world and biomarkers are needed that report early hepatotoxicity. The liver enriched microRNA, miR-122, is a promising biomarker currently being qualified in humans. For biomarker development and drug toxicity screening the zebrafish has advantages over rodents, however, blood acquisition in this model remains technically challenging. We developed a method for collecting blood from the adult zebrafish by retro-orbital (RO) bleeding and compared it to the commonly used ‘lateral incision’ (LI) method. The RO technique was more reliable in terms of the blood yield and minimum amount per fish. This new RO technique was used in a zebrafish model of paracetamol toxicity. Paracetamol induced dose-dependent increases in liver cell necrosis, serum ALT activity and mortality. In situ hybridization localised expression of miR-122 to the cytoplasm of zebrafish hepatocytes. After collection by RO bleeding, serum miR-122 could be measured and this
S50
Abstracts / Toxicology Letters 229S (2014) S40–S252
microRNA was substantially increased by paracetamol 24 h after exposure, an increase that was prevented by delayed (3 h post start of paracetamol exposure) treatment with acetylcysteine. In summary, collection of blood by RO bleeding facilitated measurement of miR-122 in a zebrafish model of paracetamol hepatotoxicity. The zebrafish represents a new species for measurement of circulating microRNA biomarkers that are translational and can bridge between fish and humans. http://dx.doi.org/10.1016/j.toxlet.2014.06.212
P-1.34 DNA damage and antioxidant capacity produced by beauvericin, zearalenone and its metabolites in CHO-K1 cells Beatriz Mallebrera, Elena Tatay, Guillermina Font, Maria-Jose Ruiz ∗ Faculty of Pharmacy, University of Valencia, Burjassot, Valencia, Spain Beauvericin (BEA) and zearalenone (ZEA) are secondary metabolites of Fusarium fungus. These mycotoxins are often as contaminants of cereals and vegetables, thus can reach man through diet. BEA produces lipid peroxidation (LPO), cytotoxicity and reactive oxygen species (ROS) in mammalian cells. ZEA and its metabolites (␣-Zearalenol, ␣-ZOL and -Zearalenol, ZOL) demonstrated cytotoxicity, genotoxicity, modified steroid metabolism and functional problems in reproductive organs. The aims of this study were to determine the genotoxiciy of BEA, ZEA and its metabolites causing DNA damage in Chinese Hamster Ovary (CHO-K1) cells and to investigate if these mycotoxins produce an adaptive response increasing the expression of glutathione (GSH) and antioxidant enzymes (glutathione peroxidase, GPx; catalase, CAT; and superoxide dismutase, SOD) in CHO-K1 cells. The results obtained demonstrated that al mycotoxins significantly decreased GSH levels (from 18% to 36%). The GPx activity increased from 34% to 66%. The CAT activity increased 70% for BEA and from 35% to 53% for ZEA and its metabolites. The SOD activity increased after BEA (from 37% to 134%) and ZEA (from 48% to 69%) exposure. The comet assay was used to determinate the effect of these mycotoxins on DNA damage. All of them showed DNA damage on CHO-K1 cells at the lowest concentrations assayed. Results suggested that BEA, ZEA and its metabolites cause DNA damage in CHO-K1; furthermore, GSH and related enzymes play an important role in antioxidant defense in CHO-K1 cells exposed to BEA, ZEA and its metabolites. http://dx.doi.org/10.1016/j.toxlet.2014.06.213
P-1.34A Alternative animal model of urethane-induce lung cancer: A pilot study Sameh Mohamed A. 1,∗ , Amira M. Abou-Yousef 1 , Heba F. Salem 2 , Mohamed A. Hamzawy 3 1
Pharmacology & Toxicology Department, Faculty of Pharmacy, Beni Suef University, Beni Suef, Egypt, 2 Pharmaceutics Department, Faculty of Pharmacy, Beni Suef University, Beni Suef, Egypt, 3 Pharmacology & Toxicology Department, College of Pharmacy, Misr University for Science & Technology, 6th October City, Egypt Background: Lung cancer is a leading cause of death and accompanied with poor five years survival rate. Mouse models for lung
cancer can serve as a valuable tool not only for understanding lung tumor biology, but also for the development of new tumor medication. Aim: The current study aimed to investigate the promotive effect of multi-injection of high dose of urethane as alternative animal model for lung cancer in BALB/c mice. Method: Three groups of male BALB/c mice were treated for 120 days included negative control; the group treated with single i.p injection of urethane (1.5 g/Kg b.w) at the first day of treatment period; the group treated with first i.p injection of urethane (1.5 g/kg b.w) at the first day and re-injected at day 60 of treatment period. Lung samples from each group were collected for histological examinations. Results: The results revealed that no incidence for tumor nodules in the control group. However, mice treated in the first day with single urethane exhibited lung tumor nodules accompanied with severe changes like adenomas and papilloma. Animals treated with 2 doses of urethane (at day 1 and 60) exhibited an increase of cancerous lesion and significant advancement of tumor incidence, hyperplasia and lung adenocarcinoma. Conclusion: It can be concluded that multiple injection of high dose of urethane is a comparable model for lung carcinogenesis and mimics the human adenocarcinoma with significant increase of representative cancerous animals in a short period. This effect may be due to its effect on mitochondrial dysfunction. http://dx.doi.org/10.1016/j.toxlet.2014.06.903
P-1.34B The influence of betulinic acid formulated as nanoemulsion on UVB activity as a tumor promoter in a mouse model of skin carcinoma Dorina Coricovac 1,∗ , Corina Danciu 1 , Daniela Ionescu 1 , Cristina Trandafirescu 1 , Georgeta Simu 1 , Codruta Soica 1 , Lajos Kemeny 3 , Simona Ardelean 2 , Cristina Dehelean 1 1 University of Medicine and Pharmacy Victor Babes Timisoara, Timisoara, Romania, 2 University Vasile Goldis Arad, Arad, Romania, 3 University of Szeged, Szeged, Hungary
Betulinic acid is a pentacyclic triterpene, known to possess multiple pharmacological effects and currently studied for its potential use in skin cancer therapy. The aim of the present study was to evaluate the effects of a betulinic acid nanoemulsion topically applied in a mouse model of skin carcinoma induced by chronic exposure to UVB. SKH-1 mice (n = 6/group) were randomly divided into 3 groups: Sham group: no intervention was applied, Not-treated group: in the first week of experiment a single application of tumor initiator, 7,12-dimethylbenz[a]anthracene (DMBA) solution was followed by exposure to UVB (3 times/week for 24 weeks) and by topical application of the nanoemulsion blank 30 min before exposure, and Treated group: same interventions as not-treated group and betulinic acid nanoemulsion was topically applied 30 min prior exposure to UVB. Skin and tumor samples were histological analyzed. Specific skin parameters, including transepidermal water loss (TEWL), melanin and erythema were measured by noninvasive methods. Our results indicated that the treated group presented a smaller number of skin lesions and tumors as compared to the not-treated group. Moreover, the development and occurrence of skin tumors were delayed by betulinic acid treatment. An improvement of skin parameters was, also, observed in the treated group.
ethanol and DPBS supplemented with BSA (5%), DMSO (5%) or SDS (5%). The results confirmed, that the receptor fluid used for dermal absorption studies in vitro has an influence on the penetration properties. The total absorption of caffeine varied depending on the receptor fluid composition between 45% (DMSO and BSA) and 15% (50% ethanol and SDS). http://dx.doi.org/10.1016/j.toxlet.2014.06.209
P-1.31 Toxicity of selected bioactivated compounds in primary rat hepatocytes cultured in micropatterned co-cultures Okechukwu Ukairo 1,∗ , Julianne Shi 1 , Justin Jackson 1 , Kelly Rose 2 , Melvin Andersen 2 , Edward LeCluyse 2 1
Hepregen Corporation, Medford, MA, USA, 2 The Hamner Institutes for Health Sciences, Research Triangle Park, NC, USA Drug-induced liver injury is often caused by cytochrome P450dependent activation of drugs into reactive metabolites. In vitro models, which can mimic in vivo responses and allow the evaluation of initial and adaptive responses to bioactivated compounds over prolonged periods, offer potentially valuable tools for toxicological assessment. We have previously developed a model in which primary hepatocytes are seeded onto ECM-coated domains of optimized dimensions and subsequently co-cultivated with murine embryonic fibroblasts [i.e. micropatterned cocultures (MPCC)]. This model retains key biochemical functions of in vivo liver with long term stability. Here, we assess the bioactivation and cytotoxicity of acetaminophen (APAP) and other compounds in the 96-well rat MPCC. APAP exerts its toxic effects through bioactivation associated, in part, with cytochrome P450 3A (CYP3A). Rat MPCCs were exposed to increasing concentrations of APAP (over 5 days) and assessed for changes in hepatic ATP content, glutathione (GSH) levels and urea synthesis. Similar concentration-dependent cytotoxicity profiles were obtained over the course of the 4-week study. Addition of 200 M l-buthionine (S, R)-sulfoximine, an inhibitor of GSH synthesis, or 10 M dexamethasone, an inducer of rat CYP3A1/2, to rat MPCCs potentiated APAP-induced hepatotoxicity in these cultures irrespective of culture age (over 4 weeks). These findings are consistent with the known in vivo mechanisms of APAP toxicity in rats. Rat MPCCs provided reproducible APAPinduced cell cytotoxicity profiles over a 4 week period and can be used to assess the effects of chronic exposure to bioactivated compounds. The toxicity profiles of other bioactivated compounds are also reported here. http://dx.doi.org/10.1016/j.toxlet.2014.06.210
P-1.32 Description of the MoA/AOP linked with PPARgamma receptor dysregulation leading to liver fibrosis Vessela Vitcheva 1,2,∗ , Merilin Al Sharif 1 , Ivanka Tsakovska 1 , Petko Alov 1 , Aleksandra Mostrag-Szlichtyng 3 , Mark T.D. Cronin 4 , Chihae Yang 3 , Ilza Pajeva 1 1
Institute of Biophysics and Biomedical Engineering, Sofia, Bulgaria, Department of Pharmacology, Pharmacotherapy and Toxicology, Faculty of Pharmacy, Medical University, Sofia, Bulgaria, 3 Altamira, 2
S49
LLC, Columbus, OH, USA, 4 School of Pharmacy and Chemistry, Liverpool John Moores University, Liverpool, UK Modes of Action and Adverse Outcome Pathways (MoAs/AOPs) are key elements in the toxicological knowledge framework that are being built to support chemical risk assessment based on mechanistic reasoning. Peroxisome Proliferator-Activated Receptor gamma (PPARgamma) is a nuclear receptor that regulates adipocyte differentiation, insulin sensitivity and lipid synthesis and storage in adipocytes. PPARgamma activation in hepatocytes is regarded as one of the molecular initiating events (MIE) involved in liver steatosis/stetatohepatitis. However its inhibition in the hepatic stellate cells (HSCs) results in their activation that is essential for the pathogenesis of liver fibrosis. In the current study a systematic literature search has been performed and a MoA scheme based on the PPARgamma dysregulation in stellate cells and resulting in hepatic fibrosis is proposed. Literature data revealing the role of PPARgamma in HSCs are consistent and associate its depletion with HSC activation and fibrosis, whereas increasing PPARgamma expression results in HSC quiescence. A large body of literature confirms that PPARgamma agonists have anti-proliferative and anti-fibrotic effects on activated HSCs. Two applications have been defined from the AOP for fibrosis from PPARgamma dysfunction in stellate cells: (i) the description of possible MIEs triggering PPARgamma inhibition/downregulation that result in fibrosis and allowing for the development of structural alerts; (ii) the identification of key events downstream from PPARgamma dysregulation leading to fibrosis that would be suitable for assay development. Acknowledgement: The funding from the European Community’s 7th Framework Program (FP7/2007-2013) COSMOS Project under grant agreement no. 266835 and from Cosmetics Europe is gratefully acknowledged. http://dx.doi.org/10.1016/j.toxlet.2014.06.211
P-1.33 Circulating microRNA biomarkers of paracetamol hepatotoxicity in zebrafish Bastiaan Vliegenthart 1,∗ , Philip Starkey Lewis 2 , Carl Tucker 1 , Jorge Del Pozo 1 , Seb Rider 1 , Dan Antoine 2 , Valerie Dubost 3 , Magdalena Westphal 3 , Pierre Moulin 3 , Matthew Bailey 1 , Jonathan Moggs 3 , Chris Goldring 2 , Kevin Park 2 , James Dear 1 Edinburgh University, Edinburgh, UK, 2 Liverpool University, Liverpool, UK, 3 Novartis, Basel, Switzerland
1
Paracetamol (acetaminophen) is the commonest cause of acute liver failure in the Western world and biomarkers are needed that report early hepatotoxicity. The liver enriched microRNA, miR-122, is a promising biomarker currently being qualified in humans. For biomarker development and drug toxicity screening the zebrafish has advantages over rodents, however, blood acquisition in this model remains technically challenging. We developed a method for collecting blood from the adult zebrafish by retro-orbital (RO) bleeding and compared it to the commonly used ‘lateral incision’ (LI) method. The RO technique was more reliable in terms of the blood yield and minimum amount per fish. This new RO technique was used in a zebrafish model of paracetamol toxicity. Paracetamol induced dose-dependent increases in liver cell necrosis, serum ALT activity and mortality. In situ hybridization localised expression of miR-122 to the cytoplasm of zebrafish hepatocytes. After collection by RO bleeding, serum miR-122 could be measured and this
S50
Abstracts / Toxicology Letters 229S (2014) S40–S252
microRNA was substantially increased by paracetamol 24 h after exposure, an increase that was prevented by delayed (3 h post start of paracetamol exposure) treatment with acetylcysteine. In summary, collection of blood by RO bleeding facilitated measurement of miR-122 in a zebrafish model of paracetamol hepatotoxicity. The zebrafish represents a new species for measurement of circulating microRNA biomarkers that are translational and can bridge between fish and humans. http://dx.doi.org/10.1016/j.toxlet.2014.06.212
P-1.34 DNA damage and antioxidant capacity produced by beauvericin, zearalenone and its metabolites in CHO-K1 cells Beatriz Mallebrera, Elena Tatay, Guillermina Font, Maria-Jose Ruiz ∗ Faculty of Pharmacy, University of Valencia, Burjassot, Valencia, Spain Beauvericin (BEA) and zearalenone (ZEA) are secondary metabolites of Fusarium fungus. These mycotoxins are often as contaminants of cereals and vegetables, thus can reach man through diet. BEA produces lipid peroxidation (LPO), cytotoxicity and reactive oxygen species (ROS) in mammalian cells. ZEA and its metabolites (␣-Zearalenol, ␣-ZOL and -Zearalenol, ZOL) demonstrated cytotoxicity, genotoxicity, modified steroid metabolism and functional problems in reproductive organs. The aims of this study were to determine the genotoxiciy of BEA, ZEA and its metabolites causing DNA damage in Chinese Hamster Ovary (CHO-K1) cells and to investigate if these mycotoxins produce an adaptive response increasing the expression of glutathione (GSH) and antioxidant enzymes (glutathione peroxidase, GPx; catalase, CAT; and superoxide dismutase, SOD) in CHO-K1 cells. The results obtained demonstrated that al mycotoxins significantly decreased GSH levels (from 18% to 36%). The GPx activity increased from 34% to 66%. The CAT activity increased 70% for BEA and from 35% to 53% for ZEA and its metabolites. The SOD activity increased after BEA (from 37% to 134%) and ZEA (from 48% to 69%) exposure. The comet assay was used to determinate the effect of these mycotoxins on DNA damage. All of them showed DNA damage on CHO-K1 cells at the lowest concentrations assayed. Results suggested that BEA, ZEA and its metabolites cause DNA damage in CHO-K1; furthermore, GSH and related enzymes play an important role in antioxidant defense in CHO-K1 cells exposed to BEA, ZEA and its metabolites. http://dx.doi.org/10.1016/j.toxlet.2014.06.213
P-1.34A Alternative animal model of urethane-induce lung cancer: A pilot study Sameh Mohamed A. 1,∗ , Amira M. Abou-Yousef 1 , Heba F. Salem 2 , Mohamed A. Hamzawy 3 1
Pharmacology & Toxicology Department, Faculty of Pharmacy, Beni Suef University, Beni Suef, Egypt, 2 Pharmaceutics Department, Faculty of Pharmacy, Beni Suef University, Beni Suef, Egypt, 3 Pharmacology & Toxicology Department, College of Pharmacy, Misr University for Science & Technology, 6th October City, Egypt Background: Lung cancer is a leading cause of death and accompanied with poor five years survival rate. Mouse models for lung
cancer can serve as a valuable tool not only for understanding lung tumor biology, but also for the development of new tumor medication. Aim: The current study aimed to investigate the promotive effect of multi-injection of high dose of urethane as alternative animal model for lung cancer in BALB/c mice. Method: Three groups of male BALB/c mice were treated for 120 days included negative control; the group treated with single i.p injection of urethane (1.5 g/Kg b.w) at the first day of treatment period; the group treated with first i.p injection of urethane (1.5 g/kg b.w) at the first day and re-injected at day 60 of treatment period. Lung samples from each group were collected for histological examinations. Results: The results revealed that no incidence for tumor nodules in the control group. However, mice treated in the first day with single urethane exhibited lung tumor nodules accompanied with severe changes like adenomas and papilloma. Animals treated with 2 doses of urethane (at day 1 and 60) exhibited an increase of cancerous lesion and significant advancement of tumor incidence, hyperplasia and lung adenocarcinoma. Conclusion: It can be concluded that multiple injection of high dose of urethane is a comparable model for lung carcinogenesis and mimics the human adenocarcinoma with significant increase of representative cancerous animals in a short period. This effect may be due to its effect on mitochondrial dysfunction. http://dx.doi.org/10.1016/j.toxlet.2014.06.903
P-1.34B The influence of betulinic acid formulated as nanoemulsion on UVB activity as a tumor promoter in a mouse model of skin carcinoma Dorina Coricovac 1,∗ , Corina Danciu 1 , Daniela Ionescu 1 , Cristina Trandafirescu 1 , Georgeta Simu 1 , Codruta Soica 1 , Lajos Kemeny 3 , Simona Ardelean 2 , Cristina Dehelean 1 1 University of Medicine and Pharmacy Victor Babes Timisoara, Timisoara, Romania, 2 University Vasile Goldis Arad, Arad, Romania, 3 University of Szeged, Szeged, Hungary
Betulinic acid is a pentacyclic triterpene, known to possess multiple pharmacological effects and currently studied for its potential use in skin cancer therapy. The aim of the present study was to evaluate the effects of a betulinic acid nanoemulsion topically applied in a mouse model of skin carcinoma induced by chronic exposure to UVB. SKH-1 mice (n = 6/group) were randomly divided into 3 groups: Sham group: no intervention was applied, Not-treated group: in the first week of experiment a single application of tumor initiator, 7,12-dimethylbenz[a]anthracene (DMBA) solution was followed by exposure to UVB (3 times/week for 24 weeks) and by topical application of the nanoemulsion blank 30 min before exposure, and Treated group: same interventions as not-treated group and betulinic acid nanoemulsion was topically applied 30 min prior exposure to UVB. Skin and tumor samples were histological analyzed. Specific skin parameters, including transepidermal water loss (TEWL), melanin and erythema were measured by noninvasive methods. Our results indicated that the treated group presented a smaller number of skin lesions and tumors as compared to the not-treated group. Moreover, the development and occurrence of skin tumors were delayed by betulinic acid treatment. An improvement of skin parameters was, also, observed in the treated group.