In this work, the cytotoxic effects of some spirostane derivatives were examined in cultured hepatocytes and V79 fibroblasts using different viability assays. The derivatives were obtained by modifying the A and B rings of diosgenin. Diosgenin and its derivatives were more toxic in V79 fibroblasts (IC50 40-300 µM) than in hepatocytes (IC50 280-1000 µM). Inhibition of cytochrome P450IIIA in cultured hepatocytes by incubation with 1 mM cimetidine did not alter the toxicity of these compounds in these cells. These observations suggest that other pathways of detoxification may be involved in hepatocytes. In conclusion, the compounds studied merit investigation for their potential pharmacological and industrial applicability.
1 Piironen V , Lindsay DV , Miettinen TA , Toivo J , Lampi AM . Plant sterols: biosynthesis, biological function and their importance to human nutrition . J Sci Food Agr2000; 80: 939-966 .
2.
2 Matsuda H , Pongpiriyadacha Y , Morikawa T , Kishi A , Ktaoka S , Yoshikawa T . Protective effects of steroid saponins from Paris polyphylla var. yunnanensis on ethanol- or indomethacin-induced gastric mucosal lesions in rats: structural requirement for activity and mode of action . Biorganic & Med Chem Lett2003; 13: 1101-1106 .
3.
3 Lacaille Dubois MA , Wagner H . A review of the biological and pharmacological activities of saponins . Phytomedicine1996; 2: 363-386 .
4.
4 Hayashi K , Hayashi H , Hiraoka N , Ikeshiro Y . Inhibitory activity of soyasaponin II on virus replication in vitro . Planta Med1997; 63: 102-105 .
5.
5 Kamisako T , Ogawa H . Regulation of biliary cholesterol secretion is associated with abcg5 and abcg8 expressions in the rats: effects of diosgenin and ethinyl estradiol . Hepatol Res2003; 26: 348-352 .
6.
6 Moghadasian MH . Pharmacological properties of plant sterols in vivo and in vitro observations . Life Sci2000; 67: 605-615 .
7.
7 Wester I . Cholesterol-lowering effect of plant sterols . Eur. I Lipid Sci Tech2000; 102: 37-44 .
8.
8 Awad AT , Fink C . Phytosterols as anticancer dietary components: evidence and mechanism of action . Am Soc Nutr Sci2000; 2127-2130 .
9.
9 Moalic S , Liagre B , Corbière C , Bianchi A , Dauça M , Bordji K , Beneytout JL . A plant steroid, diosgenin, induces apoptosis, cell cycle arrest and COX activity in osteosarcoma cells . FEBS Lett2001; 506: 225-230 .
10.
10 Hepburn PA , Horner SA , Smith M . Safety evaluation of phytosterol esters. Part 2. Subchronic 90-day oral toxicity study on phytosterol esters–a novel functional food . Food Chem Toxicol1999; 37: 521-532 .
11.
11 Moreau RA , Whitaker BD , Hicks KV . Phytosterols, phytostanols, and their conjugates in foods: structural diversity, quantitative analysis, and health-promoting uses . Prog Lipid Res2002; 41: 457-500 .
12.
12 Dvořák Z , Kosina P , Walterová D , Šimánek V , Bachleda V , Ulrichová J . Primary cultures of human hepatocytes as a tool in cytotoxicity studies: cell protection against model toxins by flavolignans obtained from Silybum marianum . Toxicol Lett2003; 137: 201-212 .
13.
13 Ulrichová J , Dvořák Z , Vičar J , Lata J , Smržová J , Šedo A , Šimánek V . Cytotoxicity of natural compounds in hepatocyte cell culture models. The case of quaternary benzo[c]phenanthridine alkaloids . Toxicol Lett2001; 125: 125-132 .
14.
14 Melo PS , Cavalcante HMM , Barbosa-Filho JM , Diniz MFFM , Medeiros IA , Haun M . Warifteine and milonine, alkaloids isolated from Cissampelos sympodialis Eichl: cytotoxicity on rat hepatocyte culture and in V79 cells . Toxicol Lett2003; 142: 143-151 .
15.
15 Hammond AH , Fry JR . Effects of culture duration, cytochrome P450 inhibitor and glutathione depletion on toxicity of diverse xenobiotics . Toxicol In Vitro1996; 10: 315-321 .
16.
16 Seibert H , Balls M , Fentem JH , Bianchi V , Clothier RH , Dierickx PJ , Ekwall B , Garle MJ , Gómez-Lechón MJ , Gribaldo L , Gülden M , Liebsch M , Rasmussen E , Roguet R , Shrivastava R , Walum E . Acute toxicity testing in vitro and the classification and labelling of chemicals . Altern Labor Anim1996; 24: 499-510 .
17.
17 Paillard F , Finot F , Mouche I , Prenez A , Vericat JA . Use of primary cultures of rat hepatocytes to predict toxicity in the early development of new chemical entities . Toxicol In Vitro1999; 13: 693-700 .
18.
18 Martelli A , Allavena A , Campart GB , Canonero R , Ghia M , Mattioli F , Mereto E , Robbiano L , Brambilla G . In vitro and in vivo testing of hydralazine genotoxicity . J Pharmacol Exp Ther1995; 273: 113-120 .
19.
19 Rodriguez JA , Haun M . Cytotoxicity of trans-dehydrocrotonin from Croton cajucara on V79 cells and rat hepatocytes . Planta Med1999; 65: 1-5 .
20.
20 De Conti R , De Oliveira DA , Fernandes AMP , Melo PS , Rodriguez JA , Haun M , De Castro SL , Souza-Brito ARM , Durán N . Application of a multi-endpoint cytotoxicity assay to the trypanocidal compounds 2-propen-1-amine derivatives and determination of their acute toxicity . In Vitro & Mol Toxicol1998; 11: 153-160 .
21.
21 Melo PS , Durán N , Haun M . Cytotoxicity of derivatives from dehydrocrotonin on V79 cells and Escherichia coli . Toxicol2001; 159: 135-141 .
22.
22 Melo PS , Durán N , Haun M . Cytotoxicity of prodigiosin and benznidazole on V79 cells . Toxicol Lett2001; 16: 237-242 .
23.
23 Zullo MAT , Ramos MTB , Monteiro DA , Godoy Jr. G . Extraction and isolation of diosgenin from yams . Bragantia1987; 46: 9-15 .
24.
24 Adam G , Marquardt V , Vorbrodt HM , Hörhold C , Andreas W , Gartz J . Aspects of synthesis and bioactivity of brassinosteroids. In Cutler HG , Yokota T , Adam G eds. Brassinosteroids–Chemistry, Bioactivity and Applications (ACS Symposium Series 474). Washington: American Chemical Society , 1991: 74-85.
25.
25 Anastasia M , Alllevi P , Ciuffreda P , Oleotti A . A convenient isomerization of 6-oxo-3α,5α-cyclo-5α-steroids to 6-oxo-δ2-5α-steroids . Steroids1985; 45: 561-564 .
26.
26 Shing TKM , Tai VWF , Tam EKW . Practical and rapid vicinal dihydroxylation of alkenes by catalytic ruthenium tetraoxide, AngewChem Int Edit1994; 33: 2312-2313 .
27.
27 Melo PS , Durán N , Haun M . Derivatives of dehydrocrotonin, a diterpene lactone isolated from Croton cajucara: cytotoxicity in rat cultured hepatocytes and in V79 cells . Human & Exp Toxicol2002; 21: 281-288 .
28.
28 Guguen-Guillouzo C , Guillouzo A . Isolated and cultured hepatocytes. In Guillouzo A , Gugen-Guillouzo C eds. Les EditionsINSERM and Libbey Eurotext, Paris , 1986.
29.
29 Omura T , Sato R . The carbon monoxide-binding pigment of liver microsomes . J Biol Chem1964; 239: 2379-2385 .
30.
30 Zurlo J , Arterbun LM . Characterization of a primary hepatocyte culture system for toxicological sudies . In Vitro Cell Dev Anim1996; 32: 211-220 .
31.
31 Bianchi V , Fortunati E . Cellular effects of an anionic surfactant detected in V79 fibroblasts by different cytotoxicity tests . Toxicol In Vitro1990; 4: 9-16 .
32.
32 Denizot F , Lang R . Rapid colorimetric assay for cell growth and survival. Modifications to the tetrazolium dye procedure giving improved sensitivity and reliability . J Immunol Methods1986; 89: 271-277 .
33.
33 Borefreund E , Puerner JA . A simple quantitative procedure using monolayer cultures for cytotoxicity assays (Htd/NR 90) . J Tissue Cult Methods1984; 9: 7-9 .
34.
34 Runnegar MT , Shou-Ming K , Ya-Zhen Z , Lu SC . Inhibition of reduced glutathione synthesis by cyanobacterial alkaloid cylindrospermopsin in cultured rat hepatocytes . Biochem Pharmacol1995; 49: 219-225 .
35.
35 Jurima-Romet M , Huang HS , Paul CJ , Thomas BH . Enalapril cytotoxicity in primary cultures of rat hepatocytes. 1. Effects of cytochrome P450 inducers and inhibitors . Toxicol Lett1991; 58: 257-267 .
36.
36 Walum E , Forsby A , Clemedson C , Ekwall B . Dynamic qualities of validation and the evolution of new in vitro toxicological tests . Altern Labor Anim1996; 24: 333-338 .
37.
37 Andrews MJ , Garle MJ , Clothier RH . Reduction of the new tetrazolium dye, alamar blue,™ in cultured rat hepatocytes and liver fractions . Alt Lab Anim1997; 25: 641-653 .
38.
38 Rakba N , Melhaoui A , Rissel M , Morel I , Loyer P , Lescoat A . Irniine, a pyrrolidine alkaloid, isolated from Arisarum vulgare can induce apoptosis and/or necrosis in rat hepatocyte cultures . Toxicon2000; 38: 1389-1402 .
39.
39 Walker TM , Rhodes PC , Westmoreland C . The differential cytotoxicity of methotrexate in rat hepatocyte monolayer and spheroid cultures . Toxicol In Vitro2000; 14: 475-485 .
40.
40 Jover R , Ponsoda X , Castell JV , Gómez-Lechón MJ . Evaluation of the cytotoxicity of ten chemicals on human culture hepatocytes: predictability of human toxicity and comparison with rodent cell culture systems . Toxicol In Vitro1992; 6: 47-52 .
41.
41 Hammond AH , Garle MJ , Sooriakumaran P , Fry JR . Modulation of hepatocyte thiol content by medium composition: implications for toxicity studies . Toxicol In Vitro2002; 16: 259-265 .
42.
42 Fry JR , Hammond AH , Atmaca M , Dhanjal P , Wilkinson DJ . Toxicity testing with hepatocytes: some methodological aspects . Alt Labor Anim1995; 23: 91-96 .
43.
43 Plant NJ , Gibson GG . Evaluation of the toxicological relevance of CYP3Anduction . Curr Opin Drug Discov Devel6: 50-56 .
44.
44 Jansing RL , Chao ES , Kaminsky LS . Phase II metabolism of warfarin in primary culture of adult rat hepatocytes . Mol Pharmacol1991; 41: 209-215 .
45.
45 Amigo L , Mendoza H , Zanlungo S , Miquel JF , Rigotti A , González S , Nervi F . Enrichment of canalicular membrane with cholesterol and sphingomyelin prevents bile salt-induced hepatic damage . J Lipid Res1999; 40: 533-542 .
46.
46 Ikeda T , Tsumagari H , Honbu T , Nohara T . Cytotoxic activity of steroidal glycosides from Solanum plants . Biol Pharm Bull2003; 26: 1198-1201 .
47.
47 Ling WH , Jones PJH . Dietary phytosterols: a review of metabolism, benefits and side effects . Life Sci1995; 57: 195-206 .
