A Dual and Opposite Effect of Calendula officinalis Flower Extract: Chemoprotector and Promoter in a Rat Hepatocarcinogenesis Model

Abstract

Calendula officinalis extracts have protective and cytotoxic effects. We previously reported the dual activity of C. officinalis in primary rat hepatocyte cultures treated with N-nitrosodiethylamine. At nM concentrations it was anti-genotoxic while at μM concentrations it exhibited genotoxic effects. Here we tested the activity of Calendula officinalis in vivo in male Fischer 344 rats initiated with N-nitrosodiethylamine, promoted with 2-acetylaminofluorene, and 70 % partially hepatectomized. Liver γ-glutamyltranspeptidase positively altered hepatocyte foci 25 days after initiation were our end point. The protective effect of C. officinalis started at 0.1 mg/kg concentration, increased at 0.5 mg/kg and reached its maximum at 2.5 mg/kg, when it decreased the area and number of altered foci by 55 % and 49 %, respectively, in comparison with rats treated only with carcinogen. At 5 mg/kg the number and area of altered hepatocyte foci were still lower, but almost reached the figures of carcinogen-treated rats. Ten and 20 mg/kg doses produced a notorious increment in the area and number of altered hepatic foci, and at 40 mg/kg of extract the increment was 40 % and 53 %, respectively. Additionally, when 2-acetylaminofluorene was substituted by a 40 mg/kg C. officinalis extract, a promoting effect was observed with increments of 175 % and 266 % in area and number of altered hepatocyte foci with respect to controls. When N-nitrosodiethylamine was substituted by 40 mg/kg of extract, the latter did not show initiator activity. In summary, we showed a protecting activity of C. officinalis at low doses, but doses above 10 mg/kg increased altered hepatocyte foci. This dual effect is an example of the phenomenon of hormesis. Furthermore, 40 mg/kg of dry weight extract administered instead of 2-acetylaminofluorene induced a clear promoting activity. These in vivo results are similar and consistent with those reported by us in primary rat liver cell cultures.

Abbreviations

DDT:1,1-bis(p-chlorophenyl)-2,2,2-trichloroethane

DEN:N-diethylnitrosamine

AHF:altered hepatocytes foci

RH:resistant hepatocyte model

AEE:Aqueous-ethanol extract

GGT:γ-glutamyltranspeptidase

2-AAF:2-acetylaminofluorene

PH:partial hepatectomy

Key words

Antigenotoxicity - genotoxicity - promoter - chemopreventive effect - hepatocarcinogenesis - Calendula officinalis - hormesis

References

• 1 Owen R W, Giacosa A, Hull W E, Haubner R, Spiegelhalder B, Bartsch H. The antioxidant/anticancer potential of phenolic compounds isolated from olive oil.  Eur J Cancer. 2000;  36 1235-47
• 2 García-Gasca T, Fattel S, Villa-Treviño S, González de Mejía E. Effect of carotenoids against genotoxicity of diethylnitrosamine on rat hepatocytes.  Toxicol In Vitro. 1998;  12 691-8
• 3 Lu Y P, Lou Y R, Lin Y, Shih W J, Huang M T, Yang C S. et al . Inhibitory effects of orally administered green tea, black tea, and caffeine on skin carcinogenesis in mice previously treated with ultraviolet B light (high-risk mice): relationship to decreased tissue fat.  Cancer Res. 2001;  61 5002-9
• 4 Puatanachokchai R, Kishida H, Denda A, Murata N, Konishi Y, Vinitketkumnuen U. et al . Inhibitory effects of lemon grass (Cymbopogon citratus, Stapf) extract on the early phase of hepatocarcinogenesis after initiation with diethylnitrosamine in male Fischer 344 rats.  Cancer Lett. 2002;  183 9-15
• 5 Boucaud-Maitre Y, Algernon O, Raynaud J. Cytotoxic and antitumoral activity of Calendula officinalis extracts.  Pharmazie. 1988;  43 220-1
• 6 Popoca J, Aguilar A, Alonso D, Villarreal M L. Cytotoxic activity of selected plants used as antitumorals in Mexican traditional medicine.  J Ethnopharmacol. 1998;  59 173-7
• 7 Lastra H, Piquet R. Calendula officinalis. Rev.  Cubana Farm. 1999;  33 188-94
• 8 Della Loggia R, Tubaro A, Sosa S, Becker H, Saar S, Isaac O. The role of triterpenoids in the topical anti-inflammatory activity of Calendula officinalis flowers.  Planta Med. 1994;  60 516-20
• 9 Zitterl-Eglseer K, Sosa S, Jurenitsch J, Schubert-Zsilavecz M, Della Loggia R, Tubaro A. et al . Anti-oedematous activities of the main triterpendiol esters of marigold (Calendula officinalis L.)  J Ethnopharmacol. 1997;  57 139-44
• 10 Ramos A, Edreira A, Vizoso A, Betancourt J, Lopez M, Decalo M. Genotoxicity of an extract of Calendula officinalis L .  J Ethnopharmacol. 1998;  61 49-55
• 11 Pietta P, Bruno A, Mauri P, Rava A. Separation of flavonol 2-O-glycosides from Calendula officinalis and Sambucus nigra by high-performance liquid and micellar electrokinetic capillary chromatography.  J Chromatogr. 1992;  593 165-70
• 12 Asad S, Singh A, Ahmad A, Hadi S M. Flavonoids: antioxidant in diet and potential anticancer agent.  Med Sci Res. 1998;  26 723-8
• 13 Laughton M J, Halliwell B, Evans P J, Hoult J R. Antioxidant and pro-oxidant actions of the plant phenolics quercetin, gossypol and myricetin. Effects on lipid peroxidation, hydroxyl radical generation and bleomycin-dependent damage to DNA.  Biochem Pharmacol. 1989;  38 2859-65
• 14 Calabrese E J. Hormesis: a revolution in toxicology, risk assessment and medicine.  EMBO Rep. 2004;  5 37-40
• 15 Sukata T, Uwagawa S, Ozaki K, Ogawa M, Nishikawa T, Iwai S. et al . Detailed low-dose study of 1,1-bis(p-chlorophenyl)-2,2,2-trichloroethane carcinogenesis suggests the possibility of a hormetic effect.  Int J Cancer. 2002;  99 112-8
• 16 Kinoshita A, Wanibuchi H, Morimura K, Wei M, Shen J, Imaoka S. et al . Phenobarbital at low dose exerts hormesis in rat hepatocarcinogenesis by reducing oxidative DNA damage, altering cell proliferation, apoptosis and gene expression.  Carcinogenesis. 2003;  24 1389-99
• 17 Lalanne M C, Doutremepuich C, de Seze O, Belon P. What is the effect of acetylsalicylic acid at ultra low dose on the interaction platelets/vessel wall?.  Thromb Res. 1990;  60 231-6
• 18 Murray R W, Melchior E P, Hagadorn J C, Marotti K R. Staphylococcus aureus cell extract transcription-translation assay: firefly luciferase reporter system for evaluating protein translation inhibitors.  Antimicrob Agents Chemother. 2001;  45 1900-4
• 19 Perez-Carreon J I, Cruz-Jimenez G, Licea-Vega J A, Arce Popoca E, Fattel Fazenda S, Villa-Trevino S. Genotoxic and anti-genotoxic properties of Calendula officinalis extracts in rat liver cell cultures treated with diethylnitrosamine.  Toxicol In Vitro. 2002;  16 253-8
• 20 Carrasco-Legleu C E, Marquez-Rosado L, Fattel-Fazenda S, Arce-Popoca E, Perez-Carreon J I, Villa-Trevino S. Chemoprotective effect of caffeic acid phenethyl ester on promotion in a medium-term rat hepatocarcinogenesis assay.  Int J Cancer. 2004;  108 488-92
• 21 Rutenburg A M, Kim H, Fischbein J W, Hanker J S, Wasserkrug H L, Seligman A M. Histochemical and ultrastructural demonstration of gamma-glutamyl transpeptidase activity.  J Histochem Cytochem. 1969;  17 517-26
• 22 Dumenil G, Chemli R, Balansard C, Guiraud H, Lallemand M. Evaluation of antibacterial properties of marigold flowers (Calendula officinalis L.) and mother homeopathic tinctures of C. officinalis L. and C. arvensis L .  Ann Pharm Fr. 1980;  38 493-9
• 23 Lin L T, Liu L T, Chiang L C, Lin C C. In vitro anti-hepatoma activity of fifteen natural medicines from Canada.  Phytother Res. 2002;  16 440-4
• 24 Calabrese E J, Baldwin L A. Inorganics and hormesis.  Crit Rev Toxicol. 2003;  33 215-304
• 25 Linde K, Jonas W B, Melchart D, Worku F, Wagner H, Eitel F. Critical review and meta-analysis of serial agitated dilutions in experimental toxicology.  Hum Exp Toxicol. 1994;  13 481-92
• 26 Jonas W B. The future of hormesis: what is the clinical relevance to hormesis?.  Crit Rev Toxicol. 2001;  31 655-8
• 27 Cordova C A, Siqueira I R, Netto C A, Yunes R A, Volpato A M, Cechinel Filho V. et al . Protective properties of butanolic extract of the Calendula officinalis L. (marigold) against lipid peroxidation of rat liver microsomes and action as free radical scavenger.  Redox Rep. 2002;  7 95-102