Enhancement of Malignant Transformation of C3H Mouse Fibroblasts and Growth Stimulation of Primary Rat Hepatocytes1

Home , Alizarin, Rhein (molecule)

(CANCER RESEARCH 50. 6540-6544. October 15. 1990] Hydroxyanthraquinones as Tumor Promoters: Enhancement of Malignant Transformation of C3H Mouse Fibroblasts and Growth Stimulation of Primary Rat Hepatocytes1

Detlef NVÃ¶lfle,2ChristophSchmutte, Johannes Westendorf, and Hans Marquardt Department of Toxicology, L'nirersity of Hamburg Medical School, D-2001) Hamburg 13, Federal Republic of Germany

ABSTRACT discrepancy has been discussed (5). Since the mouse strain used in the in vivo experiments had a relatively high incidence of Because danthron, though carcinogenic, does not seem to be genotoxic, spontaneous hepatomas, the results do not support an initiating it and 8 other hydroxyanthraquinones were comparatively investigated activity of danthron in the liver. for activities associated with tumor promotion, such as stimulation of cell Nongenotoxic carcinogens often possess tumor-promoting proliferation and enhancement of malignant transformation. The in vivo treatment of primary rat hepatocytes with danthron, aloe-emodin, chry- activity (10-13). Fundamental characteristics of promoting sophanol, and rhein resulted in a 2-3-fold increase of D.NA synthesis, agents are the modulation of growth control (14-16) and dif lucidin and purpurin were less active, and emodin, purpuroxanthin, and ferentiation (16). In experimental hepatocarcinogenesis, tumor alizarin were essentially inactive. In addition, danthron, rhein, and chry- promotion can be achieved: (a) by cytotoxins that stimulate sophanol (preliminary data), but not alizarin, enhanced transformation of regenerate growth of toxin-resistant cells (17); or (b) by chronic C3H/M2 mouse fibroblasts initiated by /V-methyl-A''-nitro-A'-nitroso- administration of mitogens without toxic effects, i.e., inducers guanidine or 3-methylcholanthrene. The results of these in vitro studies of drug-metabolizing enzymes, peroxisome proliferators, or suggest that hydroxyanthraquinones, possessing 2 hydroxy groups in the hormones (14). Therefore, stimulation of growth/DNA synthe 1,8-positions, e.g., danthron, rhein, and chrysophanol, may have tumor- sis in vivo and in vitro is widely believed to be a reliable assay promoting activities. This conclusion is in accordance with the hypothesis for tumor-promoting activity of chemicals (15). Further evi that the in vivo carcinogenic activity of danthron may be associated with dence for a tumor-promoting capacity of chemicals can be tumor promotion. obtained in vitro by assaying the capacity to enhance malignant transformation of initiated fibroblasts (18). In this report, we INTRODUCTION investigated, therefore, the ability of nongenotoxic HA to en HA3 are synthetic (i.e., danthron) or naturally occurring hance malignant transformation in vitro and to stimulate the growth of primary rat hepatocytes. The significance of the structurally related compounds that are the active principle of results with regard to HA being tumor promoters is discussed. many phytotherapeutic drugs. HA are often used chronically as laxatives (aloe, senna, frÃ¡ngula,and rheum; Ref. 1) or as mild sedatives (Hypericum perforatum: Ref. 2). HA are also applied MATERIALS AND METHODS for the treatment of kidney and bladder stones (Rubia tincto- Animals. Male Wistar rats (Zentralinstitut fÃ¼rVersuchstierkunde, rum; Ref. 3). Previously, the mutagenic activity of various HA Hannover, Federal Republic of Germany) were fed a standard diet has been demonstrated in Salmonella typhimurium with a spe (Altromin; Lage, Federal Republic of Germany) and water ad libitum. cial sensitivity of the strain TA 1537 (4-6). Because of the They were housed under controlled conditions. unexplained particular susceptibility of this Salmonella strain, Chemicals. The following test compounds were purchased from the the significance of these findings in the bacterial assay with indicated companies: MNNG and TPA (Sigma. Munich, Federal Re respect to a human carcinogenic risk remains questionable. public of Germany): MCA (Eastman Organic Chemicals, Rochester, However, evidence for the mutagenicity of HA was also ob NY); danthron (1,8-dihydroxyanthraquinone; EGA-Chemie, Stein- tained in mammalian test systems, i.e., the V79 mutation assay heim. Federal Republic of Germany): purpurin and chrysophanic acid and the hepatocyte/DNA repair test (5-7). Recently, genotoxic (Aldrich-Chemie, Steinheim. Federal Republic of Germany); (aloe- HA were also found to transform mouse fibroblasts (5, 6). emodin. emodin, and rhein (Roth, Karlsruhe. Federal Republic of Germany); and purpuroxanthin and lucidin (synthesized in our labo Moreover, the chronic administration of danthron resulted in ratory; Ref. 6). The compounds were routinely checked for purity by the induction of intestinal tumors in rats and mice and hepa- high-performance liquid Chromatographie analysis and only accepted tocellular carcinomas in mice (8, 9). The mechanism of the for experiments if the purity was >99%. ['HjThymidine (specific activ danthron-induced tumor formation is poorly understood be ity, 40 mCi/mmol) and NTB-2 emulsion were obtained from Amersham cause this agent was found by us to be essentially negative in Buchler (Braunschweig, Federal Republic of Germany) and from East genotoxicity tests with mammalian cells (5). It should be noted man Kodak (Rochester, NY), respectively. that Mori et al. (7), in contrast to our results, reported an Malignant Transformation of C3H/M2 Mouse Fibroblasts in Vitro. induction of unscheduled DNA synthesis by danthron; this This assay was carried out as described previously (19) and adjusted to measure tumor promotion according to procedures described (20). Briefly, cells harvested from logarithmically growing stock cultures Received 4/10/90: accepted 7/13/90. (between passages 5 and 20) were plated on day 0 in basal Eagle's The costs of publication of this article were defrayed in part by the payment medium supplemented with 10% fetal calf serum into 60-mm dishes to of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. determine their plating efficiency (100 cells/dish) and the transforma 1Supported in part by the Deutsche Forschungsgemeinschaft (We 980/2-1) tion rate (1000 cells/dish). After 24 h, the cultures were treated for 24 and Roggenbuckstiftung zur Krebshilfe. h with initiating agents, i.e., MNNG (0.1 ^g/ml), MCA (1 ^g/ml), or J To whom requests for reprints should be addressed, at Department of solvent control, dimethyl sulfoxide (0.5%). Thereafter, the medium was Toxicology. University of Hamburg Medical School. Grindelallee 117. D-2000 renewed and the cells were allowed to grow in fresh medium. Beginning Hamburg 13. Federal Republic of Germany. ' The abbreviations used are: HA. hydroxyanthraquinones: MCA, 3-methyl on day 5 until the end of the experiment, with each of the (twice- cholanthrene; MNNG, A'-methyl-iV'-nitro-A'-nitrosoguanidine. weekly) medium renewals HA; the positive control, i.e., TPA (0.25 ug/ 6540

Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1990 American Association for Cancer Research. HVDROXYANTHRAQUINONES AS TUMOR PROMOTERS ml); or solvent were added. The cells were fixed and stained after 2 a slight viability loss, failed to promote the transformation of weeks (to determine their plating efficiency) or after 8 weeks (to initiated cells. determine the transformation yield). DNA Synthesis in Primary Rat Hepatocytes. The effect of DNA Synthesis in Primary Rat Hepatocytes. Rat hepatocytes were HA on DNA synthesis was studied in serum-free cultures of isolated by collagenase perfusion and plated at a final density of 10,000 cells/cm2 as described previously (21). After cell attachment, i.e., 2 h adult rat hepatocytes. As shown in Fig. 1, addition of danthron after plating, serum-free William's medium E, supplemented with in to the culture medium stimulated the incorporation of ['H] sulin (10"' M) and dexamethasone (10~7 M), was added containing HA thymidine into hepatocellular DNA by a factor of about 3 (dissolved in dimethyl sulfoxide at a final media concentration of (determined by liquid scintillation counting). Concentrations of 0.25%) or solvent. Epidermal growth factor (20 ng/ml) was added at 24 h after plating. [3H]Thymidine was added at 40 h after plating. To danthron higher than 10 ng/m\ resulted in cytotoxic effects measure DNA synthesis by liquid scintillation counting, cultures were leading to the detachment of cells. In order to distinguish allowed to grow in the presence of [3H]thymidine (0.5 ^Ci/dish) for 4 between replicative and repair synthesis, hepatocytes were cul h and were thereafter processed to determine the radioactivity of tured in the presence and absence of hydroxyurea (10 mivi). trichloroacetic acid-precipitable material as described previously (22). Hydroxyurea inhibited [3H]thymidine incorporation by 90% To determine DNA synthesis by autoradiography, cells were grown on and prevented the stimulating effect of danthron (Fig. 1). Thus, collagen-coated coverslips for 8 h in the presence of [3H]thymidine (1 the observed stimulation of DNA synthesis by danthron is not jiCi/dish), then washed and fixed with 4% buffered formalin and dipped in NTB-2 emulsion. After 12 days of exposure, the coverslips were due to the induction of DNA repair. developed (23). In further studies with hepatocyte cultures, we examined the effect of various HA on the DNA synthesis in primary rat hepatocytes (Fig. 2). Chrysophanol, aloe-emodin, danthron, and rhein were the most potent inducers of ['Hjthymidine RESULTS incorporation (2- to 3-fold increase). Lucidin and purpurin were Promotion of C3H/M2 Malignant Transformation. The effect less active and alizarin, purpuroxanthin, and emodin had no of danthron, rhein, and alizarin on the induction of transfor significant effect. The stimulatory effect upon DNA synthesis mation by the initiating agent MNNG or MCA was studied in observed with danthron and aloe-emodin could be confirmed C3H/M2 mouse fibroblasts (Table 1). The tumor-promoting by autoradiography (Table 2). phorbol ester, TPA, was included as a positive reference agent. To investigate the possibility that redox cycling leading to TPA alone did not induce cell transformation. However, in the generation of toxic oxygen species was involved in HA- MNNG or MCA-initiated cultures the numbers of transformed mediated effects on cultured hepatocytes, the medium was foci were significantly enhanced by subsequent exposure to supplemented with anti-oxidants. Ascorbic acid (0.5 mivi), to- TPA. In accordance with the previously reported findings (5), copherol (1 ITIM), and glutathione (1 HIM) reduced growth alizarin, danthron, and rhein were ineffective at initiating trans formation. However, exposure of MNNG- or MCA-pretreated stimulation by HA only to a small degree and even increased cultures to danthron or rhein resulted in an enhancement of the cytotoxic effects (data not shown). Furthermore, in cultures induction of transformed foci, similar to that observed with under a gas atmosphere with a reduced, more physiological TPA. In preliminary experiments (data not shown), chryso- oxygen concentration [6% oxygen (v/v) instead of air (24)], the phanol, at a concentration of 1 pg/m\, also was active at capacity of HA to stimulate DNA synthesis was essentially promoting MNNG- and MCA-induced transformation. In con unaffected. Thus, it appeared that reactive oxygen species were trast, alizarin, when tested up to a concentration that produced not causally involved in the effect on DNA synthesis observed.

Table 1 Enhancement ofC3H/M2 transformation by HA Results are a summary of 2 experiments for each compound. Cultures were pretreated with dimethyl sulfoxide or with the initiating agents, MNNG (0.1 jig/m') or

CompoundDMSOTPADanthronDMSOTPARheinDMSOTPAAlizarinConcentration(ng/ml)0.5%0.250.51.03.010.00.5%0.250.51.03.010.00.5%0.250.51.03.010.030.0PE24242126252424242321252324242626242618DMSO"Tx0/11 (0.00)"0/14(0.00)0/4

(0.00)0/13(0.00)0/14(0.00)0/11 (0.29)3/9 (0.33)13/15(0.87)9/4

(2.25)4/15(0.27)11/15(0.73)4/12(0.33)14/16(0.88)8/11

(0.00)0/1 1(0.00)0/15(0.00)0/15 1(0.64)2/10(0.20)11/15(0.73)3/6

(0.00)0/1 1(0.00)0/14(0.00)0/14(0.00)0/16(0.00)0/13(0.00)0/11(0.50)9/10(0.90)1/11 (0.73)11/13(0.85)2/16(0.13)10/15(0.67)2/9

(0.09)4/16(0.25)1/8(0.13)2/15(0.13)1/11

(0.22)1/16(0.06)2/11 (0.00)0/13(0.00)0/11 (0.09)0/14 (0.18)1/16(0.06)1/5(0.20) (0.00)0/4 (0.00)0/2 (0.00)PE25222125272220221618212123222223242315MNNGTx1/14(0.07)6/13(0.46)1/8(0.13)6/13(0.46)9/14(0.64)12/8(1.50)3/13(0.23)7/1(0.00)PE27252528292216182219212126292625252321MCATx3/13(0.23)14/14(1.00)2/7 " DMSO, dimethyl sulfoxide; PE, plating efficiency; Tx, number of transformed foci per dishes treated. 4 Numbers in parentheses, numerical value of the ratio. 6541

promoters in vitro, e.g., phÃ©nobarbital,hexachlorocyclohexane, estradiol, and nafenopin (25-27). Thus, hepatocellular carci nomas caused by danthron in rodents (9) may be the result of a promoting rather than an initiating activity. O W 400- The mechanisms of tumor promotion remain unknown. One hypothesis claims the involvement of oxygen radicals in this process (28-31). QuiÃ±onesare known to mediate the formation a u of oxygen radicals via redox cycling leading to lipid peroxida- tion (32), DNA damage (33), and oxidation of protein thiols I (34). HA have also been found to cause generation of Superoxide e by photo-mediated redox cycling (35). However, our present a O 200 H results with various HA do not correlate with the capacity of C these HA to generate Superoxides (35). e Alternatively, other authors have suggested that glutathione 3 depletion rather than redox cycling is involved in the tumor- E too B promoting activity of hydroquinone on initiated rat liver cells (36). However, in our studies with cultures of noninitiated rat hepatocytes, the addition of glutathione and antioxidants, i.e., tocopherol or ascorbic acid, known to suppress the toxic and O 001 01 1.0 10 100 tumor-promoting effects of phorbol esters (37, 38), only partly da nthron Ã|jg/ml) reduced the danthron-stimulated DNA replication and did not Fig. 1. Stimulation of DNA synthesis Â¡npriman cultures of rat hepatocytes as a function of danthron concentration. |'H|Thymidine incorporation was deter affect the cytotoxic effects of danthron (cell detachment) or the mined in presence and absence of hydroxyurea (HU, 10 mM). Data represent the genotoxic effects of emodin (DNA repair synthesis; Ref. 5). means Â±SD (triplicate cultures from 4 animals). Thus, the action of HA on initiated hepatocytes remains un known. Since cytotoxic interactions of quiÃ±ones and oxygen have 300"51Â¿ been demonstrated in different cell culture systems (39, 40), we also studied the effects of HA under a reduced oxygen partial pressure (6% v/v; Ref. 24). Under these conditions, the stimu lation of DNA synthesis by HA was unaffected and the cyto zooCSidine toxic effects of HA were even augmented. The latter observation â€¢â€¢â€¢i1i is in accordance with the suggestion that HA interact with incorpora components of the respiratory chain; such an effect has been reported in the case of rhein and alizarin (41). Our data suggest, in the case of HA, a structure-activity Â§Ein*--i1â€¢â€¢ relationship for at least the in vitro model systems for tumor- promoting activity, i.e., stimulation of DNA synthesis and enhancement of transformation (Table 3). Those HA possessing hydroxy groups in the 1,8-positions of the anthraquinone nu- CON CHR ALO DAN RHE LUC PUR ALI PUX EMO Fig. 2. Stimulation of DNA synthesis in primary cultures of rat hepatocytes by various HA. [JH]Thymidine incorporation at the effective doses (2-5 Mg/ml) Table 2 Effect of HA on hepatocyte DNA synthesis analyzed by autoradiography are indicated for control (COA'), chrysophanol (CHR). aloe-emodin (ALO), Hepatocytes were cultured as described in Fig. 1. danthron (DAN), rhein (RHE). lucidin (LUC), purpurin (PUR), alizarin (ALI). in purpuroxanthin (PUX). and emodin (EMO). Data represent the means Â±SD TreatmentControls dex(%)2.9 studied10 (triplicate cultures from at least 3 animals). Â±1.2Â° 83Â° Danthron (10 fig/ml) 9.7 Â±2.8* 12.4 Â±2.3*No. DISCUSSION Aloe-emodin (5 Â»jg/ml)Labeling Mean Â±SD. * P < 0.05. Previously, danthron was shown to induce tumors in rats and mice (8, 9). However, this agent was found by us to be inactive in mammalian cell genotoxicity and transformation assays. Table 3 In vitro effects of HA Thus, its carcinogenic action in vivo may possibly be due to Tumor promotion Genotoxicity and transformation" tumor promotion. This hypothesis is in accord with our in vitro data. First, danthron as well as rhein and chrysophanol (prelim formationassay+++++NDNDNDNDND0V79-HGPRTassay000+++++++0UDS*inPRH000+++++++++0TransformationofC3H/M2cells00ND+ inary data not shown) enhanced, similar to TPA and 2,3,7,8- CompoundDanthronRheinChrysophanolAloe-emodinLucidinPurpurinPurpuroxanthinEmodinAlizarinDNAsynthesis++c++++++++000Trans tetrachlorodibenzo-/>-dioxin ( 18, 20), the induction of cell trans formation in MNNG- or MCA-initiated C3H/M2 fibroblasts. Aloe-emodin, lucidin, purpurin, purpuroxanthin, and emodin +++++++++0 were active at transforming fibroblasts without prior initiation and, therefore, could not be studied for promoting activity in the transformation assay. Second, danthron as well as chryso phanol, aloe-emodin, and rhein stimulated the growth of pri " Data derived from Westendorf et al. (5). mary rat hepatocytes. The marked stimulation of DNA synthe * UDS. unscheduled DNA synthesis; PRH, primary rat hepatocytes. sis by these agents (2- to 3-fold) is in the range found with other c The results are presented semiquantitatively (from 0-++). 6542

Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1990 American Association for Cancer Research. HYDROXYANTHRAQUINONES AS TUMOR PROMOTERS cleus, i.e., danthron, rhein, and chrysophanol, exerted the most intestinal tumors in rats by chrysazin. Br. J. Cancer, 52: 781-783, 1985. Mori. H., Sugie, S., Niwa, K., Yoshimi, N.. Tanaka. T., and Hirono, I. pronounced effects. Aloe-emodin also was highly active at Carcinogenicity of chrysazin in large intestine and liver of mice. Jpn. J. stimulating hepatocellular DNA synthesis, though it could not Cancer Res.. 77:871-876, 1986. 10.Berenblum. I., and Shubik. P. The role of croton oil applications, associated be investigated in the transformation assay (see above). In with a single painting of a carcinogen, in tumor induction of the mouse's contrast, HA without these structural characteristics were either skin. Br. J. Cancer, /: 379-382, 1947. only weakly active (lucidin, and purpurin) or not active at all ' ' Pilot, H. C.. and Sirica, A. E. The stages of initiation and promotion in hepatocarcinogenesis. Biochim. Biophys. Acta. 605: 191-215. 1980. (alizarin) at stimulating DNA synthesis. In the transformation 12. Pilot. H. C. Progression: the terminal stage in carcinogenesis. Jpn. J. Cancer assay, these latter compounds were either directly transforming Res., SO: 599-607, 1989. 13. Trosko, J. E. Towards understanding carcinogenic hazards: a crisis in para (lucidin and purpurin) or inactive as initiator and as promoter digms. J. Am. Coll. Toxicol., 8: 1121-1132, 1989. (alizarin). Interestingly, the presence of hydroxy groups in the 14. Schuhe-Hermann. R., Ohde, G., Schuppler. J., and Timmermann-Trosiener, 1,8-positions is also associated with the laxative effect of HA. I. Enhanced proliferation of putative preneoplastic cells in rat liver following I niodiii represents an exception to the structure-activity rela treatment with the tumor promoters phÃ©nobarbital,hexachlorocyclohexane, steroid compounds and nafcnopin. Cancer Res., 41: 2556-2562, 1981. tionship suggested by us: the agent, though possessing hydroxy 15 Biisser. M. T.. and Lutz. W. K. Stimulation of DNA synthesis in rat and groups in the 1,8-positions, did not stimulate DNA synthesis mouse liver by various tumor promoters. Carcinogenesis (Lond.), 8: 1433- 1437. 1987. in primary rat hepatocytes. This inactivity may be due to the 16. Greenlee, W. F., Osborne, R., Dold, K. M., Hudson, L. G., Young, M. J., high cytotoxic potential of the compound or to different phys- and Toskano, W. A., Jr. Altered regulation of epidermal cell proliferation icochemical properties. It must be emphasized, however, that and differentiation by 2,3.7,8-tetrachlorodibenzo-/>-dioxin (TCDD). Rev. this structure-activity relationship observed by us in in vitro Biochem. Toxicol.. 8: 1-35. 1987. 17. Farber, E. Cellular biochemistry of the stepwise development of cancer with models for tumor promoters does not correlate to results ob chemicals. Cancer Res., 44: 5463-5474, 1984. tained on mouse skin: in skin-painting experiments, neither 18. Abernethy. D. J., and Boreiko. C. J. Promotion of C3H/10TI/2 morpholog ical transformation by polychlorinated dibenzo-yj-dioxin isomers. Carcino danthron nor chrysophanol was active as a tumor promoter genesis (Lond.), 8: 1485-1490. 1987. (42, 43). In general, the reasons for the well-known species- 19. Marquardt. H., Philips, F. S., and Sternberg, S. S. Tumorigenicity in vivo and tissue-specificity of tumor promoters are unknown. and induction of malignant transformation and mutagenicity in cell cultures by adriamycin and daunomycin. Cancer Res., 36: 2065-2069, 1976. Previously, we reported (5, 44) that HA with 2 hydroxy 20. Sanchez, J. H., Abernethy. D. L.. and Boreiko. C. J. Reversible expression groups in the 1,3-positions of the molecule (i.e., emodin, pur- of morphological transformation in C3H 10T1/2 mouse embryo cultures exposed to 12-0-tetradecanoylphorbol-13-acetate. Carcinogenesis (Lond.), puroxanthin, and purpurin) or HA with a hydroxymethyl side 7: 1793-1796. 1986. chain (i.e., lucidin and aloe-emodin) are active as tumor initia- 21. WÃ¶lfle,D., MÃ¼nzel,P.,Fischer, G., and Bock, K. W. Altered growth control tors. Now we suggest that HA with hydroxy groups in the 1,8- of rat hepatocytes after treatment with 3.4.3'.4'-tetra-chlorobiphenyl in viro and in vitro. Carcinogenesis (Lond.), 9: 919-924, 1988. positions may have tumor-promoting activity. Agents with both 22 Michalopoulos. G.. Sattler. G. L.. O'Connor. L.. and Pilot, H. C. Unsched structural characteristics, i.e., aloe-emodin, may be regarded as uled DNA synthesis induced by procarcinogens in suspensions and primary complete carcinogens. In contrast, alizarin, a compound devoid cultures of hepatocytes on collagen membranes. Cancer Res., 38:1866-1871, 1978. of these structures, was found to be inactive in test systems 23. Koch, K., and Leffert, H. L. Growth control of differentiated fetal rat determining initiating as well as promoting activity. hepatocytes in primary monolayer cultures. J. Cell Biol., 62: 780-791. 1974. Obviously, these conclusions were derived from in vitro in- 24 WÃ¶lfle,D., and Jungermann. K. Long-term effects of physiological oxygen concentrations on glycolysis and gluconeogenesis in hepatocyte cultures. Eur. vestigations only. Therefore, the long-term effect of HA in J. Biochem.. 151: 299-303. 1985. animals must urgently be studied to prevent a potential human 25 Edwards, A. M., and Lucas, C. M. PhÃ©nobarbitaland some other liver tumor promoters stimulate DNA synthesis in cultured rat hepatocytes. Biochem. risk by these agents, which are often chronically administered Biophys. Res. Commun., 131: 103-108, 1985. as phytotherapeutic drugs. 26 Shi, Y. E., and Yager, J. D. Effects of the liver tumor promoter ethinyl estradici on epidermal growth factor-induced DNA synthesis and epidermal growth factor receptor levels in cultured rat hepatocytes. Cancer Res.. 49: ACKNOWLEDGMENTS 3574-3580, 1989. 27. Bieri. F.. Bentley. P.. Waechter, F.. and StÃ¤ubli.W. Use of primary cultures The authors are grateful to E. Becker, A. Piasecki, and A. Ruge for of adult rat hepatocytes to investigate mechanisms of action of nafenopin. a hepatocarcinogenic peroxisome proliferator. Carcinogenesis (Lond.), 5: technical assistance, and C. Mink for editorial assistance. 1033-1039, 1984. 28. Kappus. H. Oxidative stress in chemical toxicity. Arch. Toxicol.. 60: 144- 149. 1987. REFERENCES 29. Schwarz, M., Peres, G., Kunz, W., FÃ¼rstenberger,G., Kittstein, W., and Marks, F. On the role of Superoxide aniÃ³nradicals in skin tumor promotion. 1. Barnhart, E. R. Physician's Desk Reference. 41st Edition. Oradell. NJ: Carcinogenesis (Lond.), 5: 1663-1670. 1984. Medical Economics Co.. 1987. 30. Cerutti, P. Prooxidant states and tumor promotion. Science (Washington 2. Gessner, O., and Orzechowski, G. Gift- und Arzneipflanzen von Mitteleu DC), 227:375-381. 1985. ropa, pp. 316-317. Heidelberg: Carl Winter-UniversitÃ¤tsverlag, 1974. 31. Nakamura, Y., Colburn, N. H., and Gindhard, T. D. Role of reactive oxygen 3. Gebhardt, M. Erste vorlÃ¤ufigeErgebnisse einer Studie Ã¼berdielitholytische in tumor promotion: implication of Superoxide aniÃ³nin promotion of neo- Wirksamkeit von Urol auf Calcium-Oxalatsteine. Fortschr. Urol. Nephrol.. plastic transformation in JB-6 cells by TPA. Carcinogenesis (Lond.), 6: 229- 14 (Suppl.); 34-40, 1979. 235. 1985. 4. Brown, J. P.. and Dietrich, P. S. Mutagenicity of anthraquinone and benzan- 32. Mimnaugh, E. G., Trush, M. A., Bhatnagar. M.. and Gram, T. E. Enhance throne derivatives in the SaimoneHa/microsome test: activation of anthra ment of reactive oxygen-dependent mitochondria) membrane lipid peroxi- quinone glycosides by enzymic extracts of rat cecal bacteria. MutÃ¢t.Res.. 66: dation by the anticancer drug adriamycin. Biochem. Pharmacol.. 34: 847- 9-24, 1979. 856. 1985. 5. Westendorf, J.. Marquardt, H., Poginsky, B., Dominiak, M., Schmidt. J.. 33. Lewis, J. G., Stewart, W.. and Adams. D. O. Role of oxygen radicals in and Marquardt. H. Genotoxicity of naturally occurring hydroxyanthraqui- induction of DNA damage by metabolites of benzene. Cancer Res., 48:4762- nones. MutÃ¢t.Res., 240: 1-12, 1990. 4765, 1988. 6. Westendorf, J., Poginsky. B., Marquardt, H.. Groth, G., and Marquardt, H. 34. DiMonte. D.. Bellomo, G.. Thor, H., Nicotera, P.. and Orrenius. S. Mena- The genotoxicity of lucidin. a natural component oÃRubia tinctorum L., and dione-induced cytotoxicity is associated with protein-thiol oxidation and lucidinethylether. a component of ethanolic Rubia extracts. Cell Biol. Toxi- alteration in intracellular Ca!* homeostasis. Arch. Biochem. Biophys., 235: col., 4: 225-239, 1988. 343-350, 1984. 7. Mori, H., Kawai. K.. Ohbayashi, F., Kuniyasu, T.. Yamazaki, M., Hamasaki, 35. Hartman, P. E., and Goldstein, M. A. Superoxide generation by photome- T., and Williams, G. M. The genotoxicity of a variety of mycotoxins in the diated redox cycling of anthraquinones. Environ. Mol. Mutagen., 14:42-47, hepatocyte primary culture/DNA repair test using rat and mouse hepatocytes. 1989. Cancer Res., 44: 2918-2923. 1984. 36. Stenius, U., Warholm, M., Rannug. A., Walles. S., Lundberg, !.. and HÃ³g- 8. Mori, H., Sugie, S.. Niwa, K., Takahashi, M., and Kawai. K. Induction of berg, J. The role of GSH depletion and toxicity in hydroquinone-induced 6543

development of enzyme-altered foci. Carcinogenesis (Lond.), 10: 593-599, 41 Kawai. K., Mori, H., Sugie, S., Yoshimi, N., Inoue, T., Nakamaru, T., 1989. Nozawa, Y., and Matsushima, T. Genotoxicity in the hepatocyte/DNA repair 37. Armato, U.. Andreis. P. G., and Romano. F. Exogenous Cu, Zn-superoxide test and toxicity to liver mitochondria of 1-hydroxyanthraquinone and several dismutase suppresses the stimulation of neonatal rat hepatocytes' growth by dihydroxyanthraquinones. Cell Biol. Toxicol., 2: 457-467, 1986. tumor promoters. Carcinogenesis (Lond.). 5: 1547-1555, 1984. 42. Van Duuren, B. L., Segal, A., Tseng, S. S., Rusch, G. M.. Loewengart, G.. 38. Perchellet, J. P., Owen. M. D., Posey, T. D., Orten, D. K., and Schneider, Mate, U., Roth, D., Smith, A., Melchionne, S., and Seidman, 1. Structure B. A. Inhibitory effects of glutathione level-raising agents and D-n-toeopherol and tumor-promoting activity of analogues of anthralin (1.8-dihydroxy-9- on omnium decarboxylase induction and mouse skin tumor promotion by anthrone). J. Med. Chem., 21: 26-31, 1978. 12-0-tetradecanoylphorbol-13-acetate. Carcinogenesis (Lond.), 6: 567-573, 43. DiGiovanni, J., Kruszewski, F. H.. Coombs, M. M. Bhatt, T. S., and 1985. Pezenshk, A. Structure-activity relationships for epidermal ornithine decar 39. Lorentzen, R. J., Lesko. S. A., McDonald, K.. and Ts'o, P. O. P. Toxicity of boxylase induction and skin tumor promotion by anthrones. Carcinogenesis metabolic benzo(a)pyrenediones to cultured cells and the dependence upon (Lond.), 9: 1437-1443, 1988. molecular oxygen. Cancer Res., 39: 3194-3198. 1979. 44 WÃ¶lfle,D., Westendorf. J., Schmutte, C., Dominiak, M., Poginsky, B., and 40. Mimnaugh, E. G., Druse, L.. Atwell. J., and Myers, C. E. Differential oxygen Marquardt, H. Genotoxicity. cell transformation, and growth stimulation radical susceptibility of adriamycin-sensitive and -resistant MCF-7 human (promotion) in vitro induced by hydroxyanthraquinones. Proc. Am. Assoc. breast tumor cells. Cancer Res.. 49: 8-15, 1989. Cancer Res., 30: 562, 1989.

6544

Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1990 American Association for Cancer Research. Hydroxyanthraquinones as Tumor Promoters: Enhancement of Malignant Transformation of C3H Mouse Fibroblasts and Growth Stimulation of Primary Rat Hepatocytes

Detlef Wölfle, Christoph Schmutte, Johannes Westendorf, et al.

Cancer Res 1990;50:6540-6544.

Updated version Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/50/20/6540

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Subscriptions Department at [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://cancerres.aacrjournals.org/content/50/20/6540. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.