Abstracts of Posters EXTRACTIVE BIOCATALYSIS IN A TWO-LIQUID-PHASE SYSTEM WITH PLANT CELL SUSPENSIONS Lucilla Bassetti, Johannes Tramper Extractive fermentation is a technique used to reduce end product inhibition by removing the fermentation products in situ. The extraction can be per- formed by using an organic/aqueous two-phase system if the organic phase ex-hibits the following desirable properties: - biocompatibility; - non-biodegradability; - advantageous distribution coefficient for the product to be extracted; - thermal and chemical stability. Chemical permeabilization of plant cell membranes as well as metabolite extraction into an organic phase can be both affected by the potential toxicity of the compound(s) used. A good predictor of the cell viability retention is 'log P', i.e. the logarithm of the solvent partition coefficient in a standard two-phase system octanollwater. Solvents with log P > 5 were found to be not toxic for plant cell suspension cultures of Modnda citrifolia 1 and, among them, hexadecane (log P = 8.8) was selected as the main ex- tractive component. In this study, an organic/aqueous two-liquid-phase system was used to obtain the extraction of hydrophobic plant cell secondary metabolites. The selected compounds are the pigments anthraquinones, produced in high yields by cell suspension cultures of M. citrifolia (Rubiaceae). The organic phase consisted of a mixture of solvent plus co-solvent (hexadecane plus heptanol) 2. Cell viability was retained over the whole growth period. 1 L. Bassetti, J. Tramper, In J. Tramper et at. (eds), Biocatalysis in Non- Conventional Media, Elsevier, 1992, 617. 2 L.J. Bruce, A.J. Daugulis, Biotechnol. Prog., 7 (1991) 116. Food and Bioprocess Engineering Group, Department of Food Science, Wageningen Agricultural University, P.O. Box 8129, 6700 EV Wageningen, The Netherlands. THIOPHENES FORMED IN TAGETES HAIRY ROOT CULTURES AFTER ELICITATION WITH A CELL WALL EXTRACT OF FUSARIUM OXYSPORUM R.R.J. Arroo, A.P.M. de Brouwer, J.J.M.R. Jacobs, A.F. Croes, G.J. Wullems Thiophene derivatives are widely distributed in the Asteraceae (Compositae). They have been described as phytoalexins and thus play a role as natural protective agents. Several naturally occurring thiophenes show antibiotic, antifungal, and nematicidal activities when they are applied to living systems in the presence of long-wave UV light (UV-A). The biological activity of thio- phenes in th~liabsence of light is still a matter of discussion. Some activity is still expected, since roots are the main site of thiophene accumulation. The thiophenes that accumulate in roots of Tagetes species are structurally related. Transformed roots cultured in vitro accumulate the same thiophenas. These in vitro cultured roots were used as a model to investigate plant-micro- bial interactions. A cell wall extract of the fungus Fusarium oxysporum was added to a growing root culture, while a control culture was treated with demineralized water. After two days of incubation the thiophene contents of the cultiJres were compared. The overall thiophene content in cell-wall-treated roots was slightly higher than that of the control. More salient was that the thiophene spectrum of the cell- wall-treated roots differed from that of the control. Whereas the control mainly accumulated the hydrophobic thiophenes 5-(3-buten-l-ynyl)-2,2'-bithienyl (BBT) and 5-(4-acetoxy-l-butynyl)-2,2'-bithienyl (BBTOAc), the cell-wall-treated culture accumulated the more polar thiophenes 5-(4-hydroxy-l-butynyl)-2,2'- bithienyl (BBTOH) and 5-(3,4-dihydroxy-l-butynyl)-2,2'-bithienyl (BBT(OH)2). The polar thiophenes were partly excreted into the culture medium. Normally BBT is converted into BBTOH and BBT(OH)2, which in turn are converted into BBTOAc and BBT(OAc)2 by specific acetyl transferases. The acetyl esters are not converted further and are considered as end products in the biosynthetic pathway. It has been proposed that, after elicitation, specific esterasas would convert the acetoxy-bithienyls into alcohols ~. However, we found that [~S]-Iabeled BBTOAc and BBT(OAc)= were not converted into the corresponding alcohols after elicitation. Elicitation of Tagetes roots either activates the enzyme converting BBT into the thiophene alcohols or inhibits the acetyl transferases which use these alcohols as a substrate. 1 E. Kourany et al., Phys. Mol. Plant Path., 33 (1988) 287. NOVAPLANT Cell Biotechnology Group, Dept. of Experimental Botany, University of Nijmegen, Toernooiveld, 6525 ED Nijmegen, The Netherlands. CYTOTOXICITY OF ARTEMISININ AND DERIVATIVES AS EVALUATED BY THE CLONOGENIC AND MTT ASSAYS A.C. Beekman, H.J. Woerdenbaq, H.H. Kampinqa*, A.W.T. Koninqs* Artemisinin and its derivativss form a novel class of antimalarials. These drugs kill malada parasites efficiently at relatively low concentrations. Studies on the possible effect of these compounds to tumour cells may be indicative for the design of new anticancer agents. Therefore, we investi- gated their cytotoxicity to a rodent (EN 19) and a human cancer cell line (HeLa), using two methods: the clonogenic and MTT assays. In a number of published studies both methods gave comparable results. This may lead to the assumption that the MIF assay can be used as a substitute for the clonogenic assay to measure cell killing. Artemisinin showed considerable cytotoxicity in the M'IF assay, but was unable to reduce the clonogenic ability at concentrations up to 1 mM. The dimer of dihydroartemisinin was even more cytotoxic according to the M'IF test, but again no effect could be detected in the clonogenic test. The reason for this discrepancy is a critical difference in the endpoints detected by both tests. The M'I-F method cannot distinguish growth inhibition from cell killing, while the other detects actual clonogenic cell death. The cytotoxicity of artemisinin and the dimer of dihydroartemisinin as detected by the MFF teat must therefore solely be ascribed to cell growth delay and not to cell killing. For artemisitene and eupatoriopicnn, as well as for the reference drugs doxorubicin and cisplatin, identical IC~ values were found in both tests, demonstrating that for these compounds cell killing was measured in the MT-F test. Artemisitene and eupatoriopicrin both contain an execyclic methylene group which can alkyl- ate cellular macromoleculee apparently leading to drug-induced cell death. The endoperoxide bridge in artemisinin and its derivatives is sufficient to kill malarial parasites, but only inhibits turnout cell proliferation, even at high concentrations. Our experiments show that evaluation of cell killing potency of novel agents can not simply be performed with an M'FI" assay. Likewise, growth inhibition can not be assessed by the MTT test without checking the proportion of cell killing in a clonogenic assay. Department of Pharmacognosy, University Centre for Pharmacy, University of Groningen, A. Deusinglaan 2, 9713 AW Groningen, The Netherlands; *Department of Radiobiology, Faculty of Medicine, University of Groningen, Bloemsingel 1, 9713 8Z Groningen, The Netherlands. vo,um~ ,s N, 6,993 HS