Mutagenicity Experiments on Agroclavines, New Natural Antineoplastic Compounds1

[CANCER RESEARCH 47, 1811-1814, April l, 1987] Mutagenicity Experiments on Agroclavines, New Natural Antineoplastic Compounds1

Hansruedi Glatt,2 Eckart Eich, Heinz Pert/, Christoph Becker, and Franz Oesch

Institut fir Toxikologie der UniversitÃ¤tMainz, Obere Zahlbacher Strosse 67, Â¡H.G., F. O.], Fachbereich Pharmazie der UniversitÃ¤tMainz, Saarstrasse 2l, Â¡E.E., H. P., C. BJ 0-6500 Mainz, Federal Republic of Germany

ABSTRACT sumption that this inhibition is mediated by interaction with one of the receptors mentioned above could be refuted (9). The Agroclavine, an alkaloid produced by some species of fungi and dico tyledon plants, and its 1-alkylated derivatives are potentially useful as mechanism of action seems to be a fundamentally new one for antineoplastic drugs, since they exert potent and selective cytostatic ergoline compounds. effects. In the present study, we have investigated agroclavine and its 1- We were able to show that the inhibition of DNA synthesis propyl and 1-pentyl derivatives for mutagenicity. The genetic end point is not due to a direct effect on DNA polymerase o or ÃŸ(9). studied was the reversion of strains of Salmonella typhimurium (TA 100, Knowledge about possible mutagenic properties should be of TA 98, TA 1537) and Escherichia coli (WP2 uvrA), auxotrophic for interest in this connection. Therefore mutagenicity experiments histidine and tryptophan, respectively. The compounds were tested di with agroclavine and its 1-propyl and 1-pentyl derivatives were rectly and in the presence of a mammalian xenobiotic-metabolizing carried out and are reported here. system. In the direct test, agroclavine and the two alkylated derivatives examined exhibited substantial bacteriotoxicity but no mutagenicity. Addition of NADPH-fortified postmitochondrial supernatant fraction of MATERIALS AND METHODS rat liver homogenate led to a clear-cut decrease in bacteriotoxicity' and Compounds. Agroclavine was isolated from surface cultures of Clav to the formation of mutagenic products. Each compound was effective in iceps strain 47a by a phase separation procedure. The 1-alkyl derivatives all three strains of S. typhimurium used. In E. coli only spurious effects were synthesized from the natural alkaloid by alkylation of the indole were seen. 1-Pentylagroclavine, the most hydrophobic compound in the nitrogen via a reaction with potassium and the corresponding alkyl series, was the strongest mutagen. Agroclavine, the least hydrophobic iodide in ammonia (9, 10). The purity of the compounds was higher compound, was the weakest. The mutagenic potencies and efficacies of than 98%, as determined by Chromatographie methods as well as by all these test compounds were much weaker than those of the positive elementary analysis. Agroclavine was present as a free base. Due to the controls, which were known mutagens and carcinogens. Moreover, the instability of the free bases of the alkylated derivatives, 1-propylagro- differential effect of metabolism by liver enzymes demonstrates that the clavine was stored and normally used as hydrogen (2/J,3/?)-tartrate, toxicity and mutagenicity of agroclavine and its derivatives are caused by and 1-pentylagroclavine as (-)-di-O,O'-p-toluoyl hydrogen (2R,3R)- different chemical species. Hence, it may be possible to develop deriva tartrate. In selected mutagenicity experiments, the salts were converted tives that are cytotoxic but not mutagenic. to the free bases immediately prior to their use. Tissue Preparations. Male Sprague-Dawley rats (200-300 g) were INTRODUCTION treated with Aroclor 1254 in order to induce xenobiotic-metabolizing enzymes (11). Aroclor 1254 was diluted with sunflower oil (1:5, v/v) Agroclavine (Fig. 1), an 8-methyl-ergoline type clavine alka and i.p. injected at a dose of 500 mg/kg body weight on the sixth day loid, is produced by fungi of the genera Claviceps, PÃ©nicillium, before the rats were killed. The livers were homogenized in 3 volumes and Aspergillus, and by certain species of the dicotyledon family of sterile, cold solution of 150 mM K("l in 10 mM sodium phosphate Convoivulaceae (1). It was discovered at the beginning of the buffer, pH 7.4. The homogenate was centrifuged at 10,000 x g for 10 fifties by Abe (2, 3). In contrast to the therapeutically used min. One volume of the resulting supernatant was mixed with two lysergic acid amide type ergot alkaloids (e.g., ergotamine) agro volumes of a solution which contained 12 mM MgCl2, 50 HIMKC1, 6 clavine is characterized by a methyl substituent at C-8 and a mM NADP, and 7.5 mM glucose 6-phosphate in 75 mM sodium phosphate buffer, pH 7.4. This preparation is termed S9 mix. Liver double bond in position 8,9. Nevertheless, it interacts, like the homogenate was always prepared on the day of the experiment and lysergic acid amides, with dopamine and serotonin receptors used immediately. and even with a-adrenoceptors, although with a lower affinity Bacteria. The histidine-auxotrophic Salmonella typhimurium strains (4). TA 98 and TA 1537 are derived from prototrophic bacteria by frame- Recently we reported that agroclavine possesses antibiotic shift mutations (12). The histidine auxotrophy of S. typhimurium TA activity against some pathogenic and apathogenic bacterial 100 (12) and the tryptophan auxotrophy of Escherichia coli WP2 uvrA species (5, 6) and remarkable cytostatic activity in the LSI78Y (13) are due to substitution mutations. The bacteria were grown over mouse lymphoma cell system (7). This in vitro activity was 10- night in nutrient broth (25 g Oxoid nutrient broth No. 2/liter). For fold higher than that for non-tumor cells (murine spleen lym inoculation, stock cultures which were stored at â€”70Â°Cwereused. phocytes). Agroclavine was also active against L5178Y in vivo. Before the assay, bacteria were centrifuged, resuspended in medium B (1.6 Bacto nutrient broth plus 5 g NaCl), and adjusted nephelometri- Substitution at N-\ by an alkyl group (C3-C7) led to highly cally to a titer of 1.5-2 x 10' bacteria (colony-forming units)/ml. active compounds; for 1-pentylagroclavine a 50% cytostatic Mutagenicity Experiments. The mutagenicity experiments with his' concentration in vitro was observed which was as low as 0.87 S. typhimurium strains were performed as described by Ames et ai. (12) fiM (8, 9). It was also shown that 1-propylagroclavine inhibited with minor modifications. The test compound (in 10-30 n\ dimethyl the incorporation of thymidine into DNA. The obvious pre- sulfoxide), 500 n\ S9 mix (or 500 /tl 150 mM KCI), 100 Â¡i\of the bacterial suspension, and 2 ml of 45' C warm top agar (0.55% agar- Received 3/3/86; revised 12/18/86; accepted 1/7/87. 0.55% NaCl-50 n\i histidine-50 /JM tryptophan-50 UM biotin-25 IHM The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in sodium phosphate buffer, pH 7.4) were mixed in a test tube and poured accordance with 18 U.S.C. Section 1734 solely to indicate this fact. onto a Petri dish with minimal agar (1.5% agar-Vogel-Bonner E me 1This study was supported by the Deutsche Forschungsgemeinschaft (SFB dium with 2% glucose). Experiments with the trp~ E. coli strain (13) 302) and the Deutsche Krebshilfe e.V.-Dr. Mildred-Scheel-Stiftung/Deutsche Stiftung fÃ¼rKrebsforschung. No. 4 of the series "Clavines as Antitumor Agents"; were conducted the same way. Where indicated, the epoxide hydrolase for No. 3 see Ref. 9. inhibitor (14) and glutathione depletor (15), 1,1,1-trichloropropene 2,3- 2To whom requests for reprints should be addressed. oxide, was added (0.3 ^I/plate) in 10 M'dimethyl sulfoxide. Correspond- 1811 Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1987 American Association for Cancer Research. MUTAGENICITY OF AGROCLAVINES

colony-forming units) were added as an internal standard to otherwise normal mutagenicity plates. The his" bacteria used were spontaneous revenants from TA 1537. They were added to plates together with the strain that gives the lowest numbers of revenant colonies, TA 1537. The difference in the number of colonies on plates with and without added his* bacteria, in the presence of test compound, is compared to the value obtained with solvent controls. The ratio of these two values gives the surviving fraction. Râ€”N

RESULTS R = H agroclavme Bacterial Cytotoxicity. Agroclavine and the two 1-alky laied = (CH^CHg 1-propyl-agroclavine derivatives tested showed bacteriotoxic effects (Fig. 2). The bacteriotoxic potency paralleled the hydrophobicity of the three = (CH^CH-j 1-pentyl-agroclavine compounds in that 1-pentylagroclavine was most toxic and Fig. 1. Structures of the investigated compounds. agroclavine was least toxic. Addition of a mammalian xenobiotic-metabolizing system, liver S9 mix, drastically reduced the bacteriotoxicity of all three test compounds. Mutagenicity Experiments. The number of mutants on sol g 1.0 tâ€” vent control plates and on plates containing known mutagens (Table 1) were within the range previously observed in our CE u_ laboratory (16-18). Detailed mutagenicity results with agro clavine and its derivatives are presented in Figs. 3 and 4. The S 0.5 results are summarized in Table 2. None of the compounds was > directly mutagenic with any of the four bacterial strains used. o However, when liver S9 mix was added as a metabolizing 00 system, all three compounds exhibited mutagenicity in each 0.0 0.02 0.2 20 strain. The effects in E. coli WP2 uvrA were extremely weak (data not shown) and quantification was therefore not possible. TEST COMPOUND [ umol per plate l At high concentrations, the compounds were toxic in /;'. coli, Fig. 2. Cytotoxicity of agroclavine (O, â€¢),1-propylagroclavine (A, A), and 1- as was seen from a decrease in the trp~ background lawn. With pentylagroclavine (D, â€¢)toS. typhimurium. Toxicity was determined directly (O, the other substitution-mutated strain used, 5. typhimurium TA A, D) and with addition of rat liver S9 mix as a mammalian metabolic system (â€¢,A,â€¢).Toxicity was determined by adding his* mutants as an internal standard 100, each test compound showed about 3-fold increases in the to mutagenicity plates and measuring the surviving fraction of these mutants, as compared to solvent control plates. l-Propylagroclavine and 1-pentylagroclavine number of revenants per plate above solvent control (Fig. 3). were tested as hydrogen (2A,3/?)-tartrate and (-)-di-0,0'-p-toluoyl hydrogen The dose required for eliciting this effect was lowest when 1- (2Ã„,3Ã„)-tartrate, respectively. (2Ã„,3Ã„)-Tartaric acid used up to a dose of 100 numi piati- and (â€”)-di-O,O'-/Koluoyl (2/?,3/?)-tartaric acid used up to a dose of pentylagroclavine was used and was highest when agroclavine 4 ,/inD| plaif were not toxic (surviving fraction, Â»0.9).At a dose of 12 /imol, (-)- was used. This was true independently of whether the alky lateci di-O.O'-p-loluoyt (2A,3A)-tartaric acid reduced the surviving fraction to 0.46 in agroclavines were tested as the salts (Fig. 3, left), the form in the presence of S9 mix. which they can be stored as stable compounds, or as the free bases (Fig. 3, right). The differences (about 2-fold) in the ing plates without 1,1,1-trichloropropene 2,3-oxide received 10 n\ di methyl sulfoxide. After incubation for 3 days in the dark, colonies (his'' concentrations of the salts and the bases leading to equal effects or trp* revenants) were counted. are within the variation of the assay, but could also have their reason in a partial decomposition of the free bases during their Incubations were made in duplicate (TA 98 and TA 100) or triplicate (TA 1537), except for the solvent controls, for which four and six handling prior to the mutagenicity experiments. The effects caused in the frameshift-mutated strains TA 1537 and TA 98 replicate incubations, respectively, were made. In addition, each exper iment was repeated, at least in the relevant dose range. usually were weak (Fig. 4). However, potent effects (up to 13- Toxicity Experiments. To estimate toxicity under the incubation fold increases in the number of revenants) were seen in strain conditions of the mutagenicity experiments, his' bacteria (about 600 TA 1537 with 1-pentylagroclavine as the test compound.

Table 1 Solvent and positive controls of mutagenicity experiments Revenant colonies/plate TA 100Â° TA 98 TA 1537 WP2 uvrA Experi- Experi- Experi- Experi- Experiment Experi- Experi- Experi- Experi- Compound, dose/plate S9 mix ment 1 ment 2 ment 1 ment 2 2; TOPO* ment 1 ment 2 ment 1 ment 2 SolventKM)Benzo(a)pyrene blank (dimethylsulfoxide, /ig)\ 4,5-oxide (1 /ig)W-Ethyl-W-nitro-JV-nitrosoguanidineMftli\i V nilru .V nitrosonuaimlini- ( 10 /ig)Solvent (15 //I)Benzo(a)pyreneblank (dimethylsulfoxide, 10 130+ /ig)Benzo(e)pyrene(10 1,500+ /ig)2-Aminoanthracene(50 240+ /ig)3-Methylcholamhrene(10 4,200+ (90 ng)120e2,70027,00019,000+ 3,5001002,40027,00018,0001101,5003004,2003,500192,8003932052540862,5001,400122,3003536028460612,7001,700NTNTNTNT344402902,2001,60074201102321210NT3101501246096421018024260130722504306,0007918086440*87511602806,3004818055410*72 " Data of a third experiment (Fig. 3, right) are not shown. Values deviated by factors of less than 1.5 from those shown here. * TCPO, 1,1,1-trichloropropene 2,3-oxide (0.3 ^I/plate) was added in order to inhibit microsomal epoxide hydrolase and to deplete glutathione; NT, not tested. 1 Mean of two to six replicate incubations. '' The dose in the experiments with /;'. coli WP2 uvrA was 50 /ig-

300 Â¡S 300

a. Å“ 200 Â£ 200 LU o. Ã»_ tâ€” iâ€”on l 1 100 S TOO g Â£ -ih 0.002 0.02 0.2 20 0 0.2 20 TEST COMPOUND umol per plate ] TEST COMPOUND [ nmol per piate ] Fig. 3. Mutagenicity of agroclavine (O, â€¢),1-propylagroclavine (A, A), and 1-pentylagroclavine (D, â€¢)in S. typhimurium TA 100 (V, T; solvent controls). Mutagenicity was determined directly (O, A, V, G) and with addition of rat liver S9 mix as a mammalian metabolic system (â€¢,A, V, â€¢).Values are means from duplicate determinations. The individual values deviated by less than 10% from the means, a, experiments in which 1-propyl- and 1-pentylagroclavine were used as hydrogen (2Ã„,3Ã„)-tartrate and (-)-di-O,O'-p-toluoyl hydrogen (2/?,3/?)-tartrate, respectively. (2Ã„,3Ã„)-Tartaric acid (30 nmol-100 ^mol/plate) and (-)-di-O.O'-p- toluoyl (2A,3/?)-tartaric acid (10 nmol-10 ^mol/plate) were not mutagenic in either the absence or presence of S9 mix. (The increases in the number of colonies per plate above the solvent control values were less than 30.) Ã¨,a repeat experiment, in which 1-propyl- and 1-pentylagroclavine were converted from their salts to the free bases and immediately tested for mutagenicity.

Table 2 Mutagenic potency of agroclavine and derivatives The specific mutagenicity in this study is defined as the number of revenants which can be maximally induced per nmol test compound (at optimal selection of the dose). Usually this coincided with the slope of the initial part of the dose-response curve. In experiments in which the (-)-di-O,O'-p-toIuoyl tartrate of 1- pentylagroclavine was used, the effect was increased more than linearly with increasing doses of test compound, and the maximum ratio of effect and dose is shown. The values were calculated from the data shown in Figs. 3, left and 4. The results of repeat experiments were similar. compound)TA Specific mutagenicity (revertants/pmol 100Test 98With 1537Direct<30Â° uvrADirect<30" compoundAgroclavine S9mix100 S9mix60 S9mix10 S9mix<10Â»

1-Propylagroclavine <400Â° 300 <200Â° SO <200Â° 30850WP2 <200Â° <20* 1-PentylagroclavineDirect<60"<1000Â°With 780Direct<30Â°<500Â°TA 140TA <500Â°With <500Â°With <20Â» Â°There was no mutagenicity, but there was, however, strong toxicity with all three test compounds when they were tested in the absence of liver S9 mix. The values are the estimated detection limits of the mutagenicity experiments. * In the liver S9 mix-mediated experiments with strain WP2 uvrA, statistically significant increases in the number of revenants above solvent control were seen, but the effects were too weak to allow quantification.

di-O,O'-p-toluoyl tartrate was used, were noteworthy in that no effect was seen at doses of up to 72 nmol/plate while at moderate subsequent increases of the dose (to 227 nmol), a strong mutagenic activity was observable. Comparable devia 300 tions from linearity resulting in a threshold dose for mutagen icity were not seen with the other two test compounds and in the (single) experiment in which 1-pentylagroclavine was used in the free base form (Fig. 3, left). At high doses of the test 200 compounds, the observed effects were usually decreased, the most probable reason being bacteriotoxicity (cf. Fig. 2). In one experiment (using TA 98 as the target strain), 1,1,1- Â»-ff- trichloropropene 2,3-oxide was added to the S9 mix in order 0.002 002 0.2 20 to inhibit microsomal epoxide hydrolase and to deplete gluta- TESTCOMPOUND[jimol per piafe 1 thione. This treatment resulted in a strong potentiation of the Fig. 4. Liver S9 mix-mediated mutagenicity of agroclavine (O, â€¢),1-propyl mutagenicity of benzo(e)pyrene(Table 1)but had no measurable agroclavine (A, A), 1-pentylagroclavine (D, â€¢)and solvent controls (V, V) in S. typhimurium TA 1537 (O, A, V, G) and TA 98 (â€¢,A, T, â€¢).Values are means effect on the mutagenicity of agroclavine, 1-propylagroclavine, from duplicate (TA 98) or triplicate determinations (TA 1537). The individual and 1-pentylagroclavine(data not shown). values deviated by less than 10% and/or less than 5 colonies from the means. I- Propyl- and 1-pentylagroclavine were used as hydrogen (2/Ã®,3/f)-tartrateand (â€”)- di-O,O'-p-toluoyl hydrogen (2Ã„,3Ã„)-tartrate, respectively. (2Ã„,3Ã„)-Tartaricacid DISCUSSION (30 nmol-100 pmol/plaic) and (-)-di-O,O'-/>-toluoyl (2Ã„,3Ã„)-tartaric acid (10 nmol-10 fimol/plate) were not mutagenic in either bacterial strain. (The increases Agroclavine and the investigated alkylated derivatives by in the number of colonies per plate above the solvent control value were less than themselves were cytotoxic but not mutagenic to bacteria. Ad 10.) dition of a subcellular rat liver preparation resulted in a sub stantial decrease of cytotoxicity and in the formation of muta- The dose-mutagenicity curves of 1-pentylagroclavine (in all gens. The differential effect of the mammalian metabolic system Salmonella strains used), in the experiments in which the (-)- demonstrates that cytotoxicity and mutagenicity of the agro- 1813 Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1987 American Association for Cancer Research. MUTAGENICITY OF AGROCLAVINES clavines are caused by different chemical species. Hence, it may ACKNOWLEDGMENTS be possible to develop derivatives that are cytotoxic but not (proximately nor ultimately) mutagenic. We thank K. Pauly for her excellent technical assistance. Our discovery that cytostatically active agroclavines also show antibiotic properties (5, 6) has been demonstrated once more by the bacteriotoxicity results of this study. Cytostatic and antibiotic activities seem to be mediated by the same REFERENCES mechanism of action because we could observe a strongly 1. Floss, H. G. Biosynthesis of ergot alkaloids and related compounds. Tetra marked parallelism of activity against LSI78Y mouse lym- hedron, 32:873-912, 1976. 2. Abe, M. Research on ergot fungus, 9: isolation and properties of an alkaloid phoma cells and bacteria species (5-9). The fact that on the one "agrotlavine" produced by ergot fungus in saprophytic cultures. Rep. Takeda hand agroclavines are not directly mutagenic whereas on the Res. Lab., 10: 145-152, 1951. 3. Abe, M. Research on ergot fungus, 11: isolation of agroclavine from natural other hand their mutagenic metabolites show only drastically ergot. Rep. Takeda Res. Lab., JO: 162-166, 1951. reduced bacteriotoxicity could be considered as evidence for the 4. Berde, B., and Schild, H. O. (eds.). Ergot alkaloids and related compounds. assumption that the remarkable cytostatic/antibiotic activities Handbook of Experimental Pharmacology, Vol. 49. Berlin: Springer-Verlag, 1978. of agroclavines are not mediated by mutations. 5. Schwarz, G., and Eich, E. Influence of ergot alkaloids on growth ofStrepto- The mutagens formed in the presence of the liver preparation myces purpurascens and production of its secondary metabolites. Planta Mod.. were able to cause both frameshift and substitution mutations Â¿7:212-214,1983. 6. Eich, E., Eichberg, D., Schwarz, G., CÃas,F., and Loos, M. Antimicrobial in 5. typhimurium, whereas in E. coli only spurious effects were activity of clavines. Arzneim.-Forsch., 35: 1760-1762, 1985. noted. Whether this result reflects a true species difference or 7. Eich, E., Eichberg, D., and MÃ¼ller,W.E. G. Clavinesâ€”new antibiotics with cytostatic activity. Biochem. Pharmacol., 33: 523-526, 1984. not is unknown. It is possible that the particular mutations 8. Eich, E., Becker, 0'., and MÃ¼ller,W. E. G. Zur zytostatischen Wirkung required for reversion of the E. coli strain used are less effi partialsynthetischer Clavine. Pharmaz. Ztg., Â¡29:2209-2210, 1984. ciently induced by the metabolites of the agroclavines than 9. Eich, E., Becker, C, Sieben, R., Maidhof, A., and MÃ¼ller,W.E. G. Clavines as antitumor agents, 3: cytostatic activity and structure/activity relationships those mutations which lead to reversion of the Salmonella of 1-alkyl-agroclavines and 6-alkyl-6-nor-agroclavines. J. Antibiot. (Tokyo), strains used. In Salmonella, among the three compounds tested, 39: 804-812, 1986. agroclavine was the weakest and 1-pentylagroclavinethe strong 10. Eich, E., Sieben, R., and Becker, C. Partialsynthese neuer Ergolinderivate aus Clavinalkaloiden, 4: N-1-, C-2- und /V-6-monosubstituierte Agroclavine. est mutagen. Compared to the positive controls used (Table 1) Arch. Pharm. (Weinheim), 318: 214-218, 1985. and cytostatic drugs for which mutagenicity data have been 11. Alvares, A. P., Bickers, D. R., and Kappas, A. Polychlorinated biphenyls: a published (19), metabolized agroclavine and its metabolized 1- new type of inducer of cytochrome P-448 in the liver. Proc. Nati. Acad. Sci. USA, 70:1321-1325, 1973. alkyl derivatives were weak mutagens in the Ames test. Fur 12. Ames, B. N.. McCann, J., and Yamasaki, E. Methods for detecting carcino thermore, in some experiments with 1-pentylagroclavine, ef gens and mutagens with the 5a/monÂ«//a/mammalian-microsome mutagenic ity test. MutÃ¢t.Res., 31: 347-364, 1975. fects did not occur below a threshold dose (or, at least, were 13. Green, M.H.I... and Muriel, W. J. Mutagens testing using trp' reversion in much less than expected by linear interpolation from the effects Escherichia coli. MutÃ¢t.Res., 38: 3-32, 1976. at higher doses). Saturable metabolic detoxification or DNA 14. Oesch, F., Kaubisch, N., Jerina, D. M., and Daly, J. W. Hepatic epoxide hydrolase, structure-activity relationships for substrates and inhibitors. Bio repair processes are potential explanations for this threshold chemistry, 70:4858-4866, 1971. effect. 15. Oesch, F., and Daly, J. Conversion of naphthalene to frunv-naphthalene The nature of the mutagenic metabolites is unknown. The dihydrodiol: evidence for the presence of a coupled aryl monooxygenase- epoxide hydrase system in hepatic microsomes. Biochem. Biophys. Res. molecules of agroclavine and its derivatives offer a number of Commun., 46: 1713-1720, 1972. possible sites for oxidative transformations by liver prepara 16. Glatt, H. R., Ohlsson, A., Asure 11.S.,and Oesch, F. A'-Tetrahydrocannabinol and la,2a-epoxyhexahydrocannabinol: mutagenicity investigation in the tions. Wilson et al. (20) showed that rat liver microsomes Ames test. MutÃ¢t.Res., 66: 329-335, 1979. demethylated agroclavine at N-6 to 6-nor-agroclavine (main 17. Glatt, H. R., Metzler, M., and Oesch, F. Diethylstilbestrol and 11 derivatives: intact ergoline metabolite, 5.6%) and oxidized the C-8 methyl a mutagenicity study with Salmonella typhimurium. MutÃ¢t.Res., 67: 113- 121, 1979. to a hydroxymethyl group (elymoclavine, 1.8%). Furthermore, 18. Glatt, H. R., Jung, R., and Oesch, F. Bacterial mutagenicity investigation of agroclavine was transformed to setoclavine, isosetoclavine, and epoxides: drugs, drug metabolites, steroids, and pesticides. MutÃ¢t.Res., ///. 99-118,1983. penniclavine. Finally, the paper also mentions that phenolic 19. McCann, J., Choi, E., Yamasaki, E., and Ames, B. N. Detection of carcino ergoline derivatives and unidentified non-ergolines were gens as mutagens in the Salmonella/microsome test: assay of 300 chemicals. formed. Further metabolic studies should show which metabo Proc. Nati. Acad. Sci. USA, 72: 5135-5139, 1975. 20. Wilson, B. J., Ramstad, E., Janssen, I., and Orrenius, S. Conversion of lites are responsible for the liver S9 mix-mediated mutagenicity agroclavine by mammalian cytochrome I' 450. Biochim. Biophys. Acta, 252: of the agroclavines. 348-356, 1971.

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Hansruedi Glatt, Eckart Eich, Heinz Pertz, et al.

Cancer Res 1987;47:1811-1814.

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