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Short-term toxicity and genotoxicity studies of BIX003, a natural preparation from porcine cartilage
Abstracts / Toxicology Letters 238S (2015) S56–S383
P02-014 Effects induced by polyphenol-rich natural extracts against lipoperoxidation – Protection against toxicant induced oxidative stress D. Margina 1,∗ , O. Olaru 1 , M. Ilie 1 , C. Gutu 1 , S. Petrache 2 , L. Florea 1 , D. Gradinaru 1 , A. Dinischiotu 2 1
UMF Carol Davila Bucharest, Biochemistry, Bucharest, Romania Bucharest University, Biochemistry and Molecular Biology, Bucharest, Romania 2
Background: During the past decades, there has been a substantial body of research on the pharmacological properties of the phytochemicals from Curcuma longa L. (turmeric) as well as Vitis vinifera leaf. Aim: The aim of the present study is to assess the effects of aqueos and alcoholic extracts from C. longa root and V. vinifera leaf available on Romanian market. Methods: The extracts were assessed regarding their antioxidant potential using a cell model (Jurkat lymphocytes); cells cultivated in RPMI (11.11 mM glucose) as well as in oxidative stress conditions (35 mM glucose) were exposed for 1 h and 24 h respectively to extracts containing 10 M polyphenols. DPPP (diphenyl-1pyrenylphosphine) was used as a fluorescent marker to evaluate the response to lipid peroxidation induced with cumene hydroperoxyde. Results: Exposure for 1 h to high glucose levels induces a prooxidant effect in cell membranes. The potency of the antioxidant effect is higher for extracts prepared in 96◦ ethanol, compared to the aqueos ones. The Curcuma extracts have high anti-oxidant effects both in normal glucose media and in high glucose ones. The antioxidant effects are highly impaired under high glucose media, after 24 h of exposure. Conclusions: The antioxidant effects of the Vitis extracts are impaired by the oxidative stress conditions, which could be induced by pathological conditions as well as by some toxic substances. Still, the Curcuma extracts maintain these effects both after short time and 24 h exposure. This suggests that Curcuma extracts have both preventive and reparatory effects for redox associated pathology. Acknowledgement: The study was performed in the framework of the Global Research Initiative 2014 sponsored by Bionorica. http://dx.doi.org/10.1016/j.toxlet.2015.08.241
P02-015 Short-term toxicity and genotoxicity studies of BIX003, a natural preparation from porcine cartilage A. Torrent 1,∗ , E. Montell 1 , J. Vergés 1 , A. Sokolowski 2 , L. Canut 3 , S. García 3 , A. Casadesús 3 1
Bioiberica S.A., Pre-Clinical R&D, Palafolls, Spain Harlan Laboratories, Germany 3 Harlan Laboratories, Barcelona, Spain 2
Purpose: Preclinical toxicology estudies were performed to evaluate the safety of BIX003, a preparation of porcine cartilage rich in glycosaminoglycans, collagen type II and other bioactive substances due to the mild conditions used in the manufacturing process (patent pending). Previous research in rats has shown that this extract is a potential therapeutic product for treatment of osteoarthritis and osteoporosis.
S69
Methods: The toxicity of BIX003 was assessed in a short-term study in rats and in a bacterial reverse mutation assay. In the repeated dose study, Wistar Hannover rats (12 males, 12 females) were administered BIX003 by oral gavage at dose levels of 0, 500, 1000 and 2000 mg/kg body weight/day for 14 consecutive days. The Ames test was performed according to the OECD Test Guideline 471. Two independent experiments were performed, both without and with metabolic activation, using triplicate plates for each treatment condition. Results: All animals survived after oral administration for 14 days and exhibited normal body weight gain through the study. No significant adverse effects on food consumption, hematology and urinalysis were recorded and no noteworthy differences were observed in clinical biochemistry. Macroscopic lesions recorded were within the range of normal background lesions in rats of this strain and age. Furthermore, no target organ was identified. Therefore, under the conditions of the study, BIX003 administered by oral gavage to Wistar Hannover rats daily for 14 consecutive days at 2000 mg/kg/day caused no signs of toxicity. BIX003 did not induce mutagenicity in the bacterial reverse mutation test in Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100, and the Escherichia coli strain WP2 uvrA, with or metabolic activation. Conclusion: Overall, the results from the toxicity studies support the safety of the porcine cartilage extract in food. Further studies will be performed to evaluate the long-term safety. http://dx.doi.org/10.1016/j.toxlet.2015.08.242
P02-017 In vivo alkaline and Endo III and FPG-modified comet assays of Carvacrol, an Oregano essential oil compound M. Llana-Ruiz-Cabello, M. Puerto, A.I. Prieto, P. Mellado-Garcia, D. Gutierrez-Praena, S. Pichardo, A.M. Cameán ∗ Area of Toxicology, University of Sevilla, Sevilla, Spain Oregano essential oil is one of the oils selected by food industry as additive to develop new active packaging systems with the aim to improve shelf life of perishable products. Carvacrol is the main component of this essential oil; therefore its safety should be confirmed by different toxicity assays. The comet assay was performed because it provides information about mechanisms of genotoxicity and is a rapid and sensitive method. In the present study, the in vivo genotoxicity of carvacrol, the main component of oregano essential oil, was evaluated in the liver and stomach of wistar rats. Animals of both sexes were exposed to carvacrol (81, 270 and 810 mg/kg BW) orally by gavage, according to recommendations of OECD 489. The standard comet assay and detection of oxidative DNA damage with enzyme-comet assay were used. The nucleoids of cells were incubated with endonuclease III (End III) and formamido pyrimidine glycosylase (Fpg) proteins to detect levels of oxidized pyrimidines and purines in DNA, respectively. The results for the standard comet assay revealed no significant increase in DNA strand breakage at any concentrations assayed. Similarly, post-treatment with End III- or FPG showed no difference between treated and untreated groups. These results, coupled with previous studies developed by our laboratory, suggest that low concentrations of carvacrol seem to be safe for use in food packaging. Acknowledgements: The authors wish to thank the Spanish Ministry of Science and Innovation (AGL2012-38357-C02-01) cofinanced by FEDER Funds, and Junta de Andalucía (AGR-7252) for the financial support for this study. María Llana Ruiz-Cabello also
P02-014 Effects induced by polyphenol-rich natural extracts against lipoperoxidation – Protection against toxicant induced oxidative stress D. Margina 1,∗ , O. Olaru 1 , M. Ilie 1 , C. Gutu 1 , S. Petrache 2 , L. Florea 1 , D. Gradinaru 1 , A. Dinischiotu 2 1
UMF Carol Davila Bucharest, Biochemistry, Bucharest, Romania Bucharest University, Biochemistry and Molecular Biology, Bucharest, Romania 2
Background: During the past decades, there has been a substantial body of research on the pharmacological properties of the phytochemicals from Curcuma longa L. (turmeric) as well as Vitis vinifera leaf. Aim: The aim of the present study is to assess the effects of aqueos and alcoholic extracts from C. longa root and V. vinifera leaf available on Romanian market. Methods: The extracts were assessed regarding their antioxidant potential using a cell model (Jurkat lymphocytes); cells cultivated in RPMI (11.11 mM glucose) as well as in oxidative stress conditions (35 mM glucose) were exposed for 1 h and 24 h respectively to extracts containing 10 M polyphenols. DPPP (diphenyl-1pyrenylphosphine) was used as a fluorescent marker to evaluate the response to lipid peroxidation induced with cumene hydroperoxyde. Results: Exposure for 1 h to high glucose levels induces a prooxidant effect in cell membranes. The potency of the antioxidant effect is higher for extracts prepared in 96◦ ethanol, compared to the aqueos ones. The Curcuma extracts have high anti-oxidant effects both in normal glucose media and in high glucose ones. The antioxidant effects are highly impaired under high glucose media, after 24 h of exposure. Conclusions: The antioxidant effects of the Vitis extracts are impaired by the oxidative stress conditions, which could be induced by pathological conditions as well as by some toxic substances. Still, the Curcuma extracts maintain these effects both after short time and 24 h exposure. This suggests that Curcuma extracts have both preventive and reparatory effects for redox associated pathology. Acknowledgement: The study was performed in the framework of the Global Research Initiative 2014 sponsored by Bionorica. http://dx.doi.org/10.1016/j.toxlet.2015.08.241
P02-015 Short-term toxicity and genotoxicity studies of BIX003, a natural preparation from porcine cartilage A. Torrent 1,∗ , E. Montell 1 , J. Vergés 1 , A. Sokolowski 2 , L. Canut 3 , S. García 3 , A. Casadesús 3 1
Bioiberica S.A., Pre-Clinical R&D, Palafolls, Spain Harlan Laboratories, Germany 3 Harlan Laboratories, Barcelona, Spain 2
Purpose: Preclinical toxicology estudies were performed to evaluate the safety of BIX003, a preparation of porcine cartilage rich in glycosaminoglycans, collagen type II and other bioactive substances due to the mild conditions used in the manufacturing process (patent pending). Previous research in rats has shown that this extract is a potential therapeutic product for treatment of osteoarthritis and osteoporosis.
S69
Methods: The toxicity of BIX003 was assessed in a short-term study in rats and in a bacterial reverse mutation assay. In the repeated dose study, Wistar Hannover rats (12 males, 12 females) were administered BIX003 by oral gavage at dose levels of 0, 500, 1000 and 2000 mg/kg body weight/day for 14 consecutive days. The Ames test was performed according to the OECD Test Guideline 471. Two independent experiments were performed, both without and with metabolic activation, using triplicate plates for each treatment condition. Results: All animals survived after oral administration for 14 days and exhibited normal body weight gain through the study. No significant adverse effects on food consumption, hematology and urinalysis were recorded and no noteworthy differences were observed in clinical biochemistry. Macroscopic lesions recorded were within the range of normal background lesions in rats of this strain and age. Furthermore, no target organ was identified. Therefore, under the conditions of the study, BIX003 administered by oral gavage to Wistar Hannover rats daily for 14 consecutive days at 2000 mg/kg/day caused no signs of toxicity. BIX003 did not induce mutagenicity in the bacterial reverse mutation test in Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100, and the Escherichia coli strain WP2 uvrA, with or metabolic activation. Conclusion: Overall, the results from the toxicity studies support the safety of the porcine cartilage extract in food. Further studies will be performed to evaluate the long-term safety. http://dx.doi.org/10.1016/j.toxlet.2015.08.242
P02-017 In vivo alkaline and Endo III and FPG-modified comet assays of Carvacrol, an Oregano essential oil compound M. Llana-Ruiz-Cabello, M. Puerto, A.I. Prieto, P. Mellado-Garcia, D. Gutierrez-Praena, S. Pichardo, A.M. Cameán ∗ Area of Toxicology, University of Sevilla, Sevilla, Spain Oregano essential oil is one of the oils selected by food industry as additive to develop new active packaging systems with the aim to improve shelf life of perishable products. Carvacrol is the main component of this essential oil; therefore its safety should be confirmed by different toxicity assays. The comet assay was performed because it provides information about mechanisms of genotoxicity and is a rapid and sensitive method. In the present study, the in vivo genotoxicity of carvacrol, the main component of oregano essential oil, was evaluated in the liver and stomach of wistar rats. Animals of both sexes were exposed to carvacrol (81, 270 and 810 mg/kg BW) orally by gavage, according to recommendations of OECD 489. The standard comet assay and detection of oxidative DNA damage with enzyme-comet assay were used. The nucleoids of cells were incubated with endonuclease III (End III) and formamido pyrimidine glycosylase (Fpg) proteins to detect levels of oxidized pyrimidines and purines in DNA, respectively. The results for the standard comet assay revealed no significant increase in DNA strand breakage at any concentrations assayed. Similarly, post-treatment with End III- or FPG showed no difference between treated and untreated groups. These results, coupled with previous studies developed by our laboratory, suggest that low concentrations of carvacrol seem to be safe for use in food packaging. Acknowledgements: The authors wish to thank the Spanish Ministry of Science and Innovation (AGL2012-38357-C02-01) cofinanced by FEDER Funds, and Junta de Andalucía (AGR-7252) for the financial support for this study. María Llana Ruiz-Cabello also