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[CANCER RESEARCH 38, 4527-4533, December 1978] 0008-5472/78/0038-0000$02.00 Effects of Harman and Norharman on Spontaneous and Ultraviolet Light induced Mutagenesis in Cultured Chinese Hamster Cells Chia-cheng Chang,' Marc Castsblazzl,2Thomas W. Glover, and James E. Trosko3 Department of Human Development, College of Human Medicine, Michigan State University, East Lansing. Michigan 48824 were identified. These compounds are known to exist in cigarette tar (18), charred meat, and naturally occurring Nontoxic concentrations of harman and norharman foods (21). It was found that, when these 2 compounds are were tested in cultured Chinese hamster cells for their added to a reaction mixture containing rat liver enzymes effects on DNA repair and mutagenesis. The following and other chemical mutagens, a significant increase in effects of harman were observed: (a) the survival of mutagenicity was observed with the Ames assay (11, 15). uitravloletbight-or X-ray-damagedcells was reduced;(b) Moreover,aniline ando-tobuidine,which are not mutagenic, the ultravioletlight-inducedunscheduledDNA synthesis were capable of inducing mutations in the presence of was slightly inhibited; and (C) the frequency of sponta harmanor norharman (14). Nagaoet a!. (15) introduced the neousor ultravioletbight-induced ouabain-resIstant(oua―)term, “comutagenesis,― to describe this phenomenon, or 6-thioguanine-resistant(6-TG')mutationswas reduced. which in principle seems to parallel similar observations Furthermore, the effect of harman on survival and muta made by Van Duuren and Goldschmidt (27) related to the genesis was greater than that of norharman and was phenomenon of “cocarcinogenesis.― detected primarily In treatments in which cells were cx Experimental evidence exists that links the role of muta posed to harman immediately following uftraviolet light genesis to carcinogenesis (9, 24). From a wide variety of in irradiation. Our data clearly indicate that harman de vitro studies with either tumor promoters (23) or antitumor creases the capacity to repair DNA damage and fix muta promoters (2), it now appears that carcinogenic initiation tions in Chinese hamstercells, possiblybecause of the might be due to a mutagenic process, whereas carcino intercalation properties of this compound. genic promotion might be due to an epigenetic effect (22). Accordingly, the experiments reported here were designed to demonstrate whether such a comutagenic phenomenon INTRODUCTION exists in eukaryotic cells and whether this would be related A wide variety of studies has implicated tryptophan (and to a cocarcinogenic effect in vivo. As a first step in a some of its precursors and metabolites) in the carcinogenic comparative series of molecular and biological in vitro and process in mice, rats, hamsters, and dogs. A cocarcino in vivo experiments, we report here the effect of harman genic effect of DL-tryptophanhasbeendemonstrated in rats and norharman on the recovery of spontaneous and UV (3) and dogs (19). Indole, a precursor of tryptophan, has induced mutations in cultured Chinese hamster V79 cells. been shown to enhance the carcinogenicity of 2-acetylami Using2 genetic markers, resistanceto 6-thioguanine and to nofluorene in the bladder of rats (17) and hamsters(16) and ouabain, we have shown that harman and norharman in yet suppress the carcinogenicity of 2-acetylaminofluorene hibit rather than enhance mutagenesis in our assay sys in the liver of rats (17) and hamsters (16). Matsumoto et a!. tems. ABSTRACT (10) have shown that indole appears to act as an antitumor promoter of dibutylnitrosammne-mnduced bladder cancers in hamsters. In search of possible chemical factors in the typical Japanese diet that might be correlated with a high fre quency of stomach cancer in the Japanese population, charred material of fish and meat has been tested for its mutagenic potential with the use of the Ames assay(13). It was found that mutagenic principles were formed by pyrob ysis of protein but not of other compounds (12, 13). Subse quently, some mutagenic principles in pyrolytic products of MATERIALSAND METHODS Cell Culture. A Chinese hamster cell line (V79), derived originally from lung tissue (6), was used for the experi ments. Cells were grown in modified Eagle's medium (5) (Earle's balanced salt solution with a 50% increase of essential amino acids and vitamins) supplemented with “nonessential― amino acids (100%increase), 1 mM sodium pyruvate, and 5% fetal calf serum. Under the incubation condition with 5% CO2 in humidified air at 37°,the cells DL-tryptophan were identified (20). In addition, 2 nonmuta havea generation time of about 12 hr. genic f3-carboline derivatives, harman and norharman, UnscheduledDNASynthesis.To determinewhetherhar which are the main products of DL-tryptOphanpyrolysate, man affects DNA repair synthesis (“unscheduled― DNA synthesis), we used the method described by Trosko and Yager(25)in a slightly modified form. Cells were inoculated I Recipient of National Institute of Environmental Health Sciences Young Environmental Scientist Award E501809-01. To whom requests for reprInts should be addressed. a On postdoctoral leave from Unite do Genétique Cellulaire, Institut do Rochorches en Biologie Moléculaire,2. p1. Jussieu, F-75221 Paris, Cedex 05. France. 3 Recipient of National Cancer Instftute Grant CA 21104-01. Received May 15, 1978; accepted September 7. 1978. into6-cm Falcon plastic @tri plates(3 x 10°/plate) and grown overnight to confluency. The medium was then replaced with an arginine-deficient medium for 3 days. One hr prior to UV treatment, hydroxyurea (Sigma Chemical Co., St. Louis, Mo.) was added (5 mM final concentration). DECEMBER 1978 Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1978 American Association for Cancer Research. 4527 C-c. Chang et a!. The medium was decanted, and the cells were then exposed to various UV doses. The decanted medium, now contain ing [3H]thymidine (5 MCi/mI; 50 Ci/mmol; New England Nuclear, Boston, Mass.), was added, and the cells were incubated for various times before they were collected and frozen. Cells were later analyzed for DNA content and rad ioactivity incorporated into DNA. Table 1 Effect of harman inChinese and norharman on colony-forming cellsCells periodof were exposed to harman or norharman colony development.No.of Harman Platingml) (lLg/ Mutagenesisand Cell Survival.Quantitativemutagene sis, using ouabain resistance as a genetic marker, has been systematically characterized (1). Cells were trypsinized with 0.01% crystalline trypsin in phosphate-buffered saline with out calcium and magnesium ions and plated for attachment for 3.5 hr before UV irradiation. With the medium removed from the plates, the attached cells were exposed to UV from a (General Electric G25T8-25W) germicidal lamp positioned to deliver a dose rate of 10 ergs/sq mm/sec (1 J/sq m/sec). Growth medium with or without harman (Aldrich Chemical Company, Milwaukee, Wis.) was added at various times for various durations described in the protocols accompanying the tables. After sufficient expression times (when colonies contain 8 to 16 cells, usually 2 to 4 days depending on the severity of UV dose), the cells were exposed to selective medium with 1 mM ouabain. The resistant colonies that developed in selective medium were scored 1 week later. The number of cells seeded and plates (9 cm) used for each experiment are indicated in the tables. The cells plated for survival studies were treated the same way as the cells for mutation. experiments, except they contained fewer cells and were not exposed to the selective medium. The per centage of survivors was determined by dividing the total number of colonies developed in 3 to 4 plates by the total number of cells plated, multiplied by 100. For mutation experiments in which 6-thioguanine resist ance was used as a genetic marker, the replating technique was used.The cellswere replated at2 x 10°/plate (9cm) after sufficient expression time and exposed to 6-thiogua nine (10 @tg/mI)after they had attached (3.5 hr after cell plating). The 6-thioguanine-resistant mutations require 6 to 8 days for maximum mutation expression. The mutation frequencies are, however, very stable after the maximum expression time (1, 28). Norharman (@.tg/ml) efficiency— cells plated 1001 1025(5.5)° (28) 515b20(110) 10 (55) — — — — 01 985 (5.9) (30) 9320(120) 10 (60) a Numbers in parentheses, b Colony size slightly C size Colony ability hamster(V79) greatly for the entire No.ofcol onies formed 600 603 600 600 600 600 613 605 600 600 600 593 583 563k 97 600 391@' 65 100 0 @LM. reduced. reduced. reduced to 10 @M. The sensitization effect of harman was found in a narrow range (10 to 50 SM). Higher concentra tions of harman were toxic to cells. Results presented in Table 2 indicate that the reducing effect of harman on survival was found only when the chemical is present immediately following UV irradiation. Itwas alsofoundthat, at same molar concentration, norharman was not as effec tive as harman for this sensitization. The effect of harman on reduction of the survival of radiation-damaged cells was not limited to UV. A similar effect was also found in X-ray-irradiated cells (Chart 1b). Effect of Harmanon UnscheduledDNA Synthesis.The unscheduled DNA synthesis induced by UV was slightly, but consistently, inhibited by the presence of nontoxic concen trations of harman (Chart 2). The inhibition appears to be specific for unscheduled DNA synthesis, since normal DNA synthesis was found not to be decreased by this treatment for 3 hr in exponentially growing cells (data not shown). Effect of Harman on UV Mutagenesis.In a preliminary experiment with the use of only 1 UV dose (18 J/sq m) (Table 2), harman and norharman were found to reduce the RESULTS frequency of UV-induced ouabain-resistant mutations. The Cytotoxicity of Harman and Norharman. The effects of reducing effect was greater when the cells were exposed to different concentrations of harman or norharman on the harman or norharman immediately following UV irradiation. At a similar concentration, harman showed a greatereffect colony-forming ability of Chinese hamster cells were tested. As shown in Table 1 the colony-forming ability of Chinese than did norharman. For this reason harman was chosen hamster cells was slightly reduced by 10 @g (55 @M) harman for a more detailed study. Two additional experiments testing the effect of harman per ml and completely eliminated by 20 @g (110 @M) harman on mutation induction by different doses of UV at the treatment per ml. Furthermore, at the same molar concen trations, harman was more toxic than was norharman to ouabain-resistant locus were carried out. The results of one experiment showing that harman reduces both the survival Chinese hamster cells. of UV-damaged cells and the frequencies of UV-induced Effects of Harman on Survival of UV and X-ray-irradiated Cells. The survivalof UV-damagedChinesehamstercells mutations are presented in Chart 3. The results were essen when exposed to nontoxic concentratrons of harman after tially repeated in another experiment (Table 3) in which irradiation was significantly reduced compared to cells not more UV doses were used. To confirm the observation that harman treatment re treated with harman. The effect was concentration depend duces the frequency of UV-induced mutations, we repeated ent (Chart la). The cells were more sensitive to UV killing when exposed to higher concentrations of harman. The experiments in a different mutation system, namely, for effect was not detectable when the concentration was ward mutation from 6-thioguanine sensitivity to 6-thiogua 4528 CANCERRESEARCHVOL. 38 Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1978 American Association for Cancer Research. Inhibition of Mutagenesis by Harman 100 ‘@‘ lb Ia \‘ 10 l( ‘V \ — - Harmon ——— + Harmon (40MM) \ “\ ‘\ \ . 01 > \ ‘ U) 0 ‘ \ S. > > ‘ \ ‘ ‘ •@ \ \ ‘ \ \ \ “ V \ \ C,) .‘ @Contro@ ,,‘ \ ‘ ‘¼@ \ \ \ \ \@ \ ‘@Horman \ \ .‘ .1 \ (l0@M) \ \ \ \ \ @Hormon (30@J4 ‘.Korma@ (50MM) 0 5 10 15 20 2@ uv Dose(J/sqm) 30 X-Ray Dose(R) Chart 1 a, survival of UV-irradiated Chinese hamster cells posttreated with various nontoxic concentrations of harman. Cells were exposed to harman immediately after UV irradiation for the entire period. b, survival of X-ray-irradiated Chinese hamster cells posttreated with harman. Cells were exposed to harman immediately after X-ray-irradiation for the entire period. X-rays were generated by a General Electric Maxitron 300 X-ray machine. The exposure rate was180R/min (250kV,20 ma,with 3 mm aluminumfiltration). nine resistance. Since the ouabain mutation system appar ouar mutants does not increase as the cell number in ently can detect only “ point―mutations (excluding deletion creases; therefore the frequencies are bower than the con type mutations) (1), the 6-thioguanine system was chosen trol not treated with harman. Once the cells were exposed to pick up any possible effect of harman on the recovery of to the selective agent, the presence of harman did not mutations,includingdeletion-type mutations.The results affect the mutant frequency. from 2 experiments involving a replating technique after an The harman effect on frequencies of spontaneous muta 8-day expression time (Table 4) consistently showed that tions was similarly observed in the two 6-TG―mutation harman treatment reduces UV-induced 6@TGrmutations. To experiments (Table 4). Therefore the observation was re test whether the reduction is due to a delay in the mutation peatable in 2 different mutation systems. expression because of the harman treatment, a 12-day Effectof Harmanon Survivalof Preexistingouar and 6expression time was included in one of the experiments. TGr Mutants. Treatment by nontoxic concentrations of The results clearly indicate that the mutation reduction by harman has been shown todecreasebothspontaneousand harman treatment is not due to a delay effect on mutation UV-induced oua'@mutants. The results could be due to its expression. effect on mutagenesis or to its specific inhibitory effect on Effect of Harman on the Spontaneous Mutation Fre the survival ofouar mutants in growth or selective medium. quency. Experimentspresentedin Table 3 and Chart 3 As a test of the latter possibility, 2 reconstruction experi show that harman reduces both the spontaneous and UV ments involving wild-type and ouar mutant cells were done, induced oua'@mutations. Since spontaneous frequencies although results presented in Table 5 (Protocol A) have are very low, the difference could be due to a random shown that the frequencies of spontaneous ouar mutants variation. Three more experiments were carried out to verify were not reduced by harman treatment in selective medium. whether the difference is real. The results from these 5 The results from these reconstruction experiments clearly experiments (Table 5) consistently show that, when cells show that harman treatment, comparable to that used in replicate in the presence of harman before exposure to the mutation experiments, did not reduce the recovery of selective agent (ouabain), the spontaneous frequency of preexisting ouar mutants derived from a single clone or DECEMBER1978 Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1978 American Association for Cancer Research. 4529 C-c. Chang et a!. Table2 Effect of harman and norharman treatment on the frequency of UV-induced ouar mutations For Experiment1, the number of cells per plate (9 cm) usedwas reduced; the UV-induced unscheduled DNA synthesis was slightly inhibited; and the frequencies of spontaneous or UV-induced mutations were reduced. Furthermore the ef 2.1 x 10°/21 . For Experiments 2 to 6, the number of cells per plate (9 cm) used was 4.2 x 10°/21. Protocol: 3.5hr 1.5days Cell plating 2days UV Score for mutants Ouabain I//I/I/I, Control [@JTdR Incorporation —lweek ______________ f//f//I', za o—o———oI.5hr Harmon (50MM) 0—a——a 3 hr Horm@ (25MM) 0' E 0. ±Harman, norharman >@ UV (18J/ (28 survivors1———9912 sq m)Harman@M)Norharman(30 j.tM)Sur fre quency/10° vival (%)Mutation (25)°2 (160) + 3 + 4 (73)6+—+ 5+ +— a Numbers + ([email protected])b + (1 .5—3.5) — —— 300― (39) 521d + (0-1 .5)b6.1 47625 37CC (1.5-3.5)5.9609 in parentheses, b Numbers 3.1 5.8 in parentheses, number of mutants. time of treatment (days) d Mutation frequency of harman- or U Control Harmon(5O@M) Hormon([email protected]) U U 4, 0. U) (127) (151) after UV irradiation. C' > norharman-treated cells compared to control cells at equivalent dose of UV was significantly reduced (Footnote c , p < 1%; Footnote d, p < 5%). 5 UVDose (J/sq m) Chart 2. Effect of harman on uv-induced unscheduled DNA synthesis in Chinese hamster cells. Various concentrations of harman were added immediately after uv irradiation for 1.5 or 3.0 hr. Statistical tests (t test on paired observations) indicate that harman treatments significantly reduced the unscheduled DNA synthesis with the exception of 25 @M harman (1.5 hr measurement). from a mixture of many clones (Table 6). A reconstruction experiment with 6@TGrmutants and a protocol similar to that of the UV mutagenesis experiments also indicates that harman did not reduce the recovery of preexisting sponta neous or UV-induced 6-TGT mutants (Table 7). 0 S DISCUSSION Harman and norharman have been shown to exhibit a variety of effects on biological systems. Both these /3carboline derivatives have been shown to intercalate into DNA in vitro, causing the unwinding of the double helix (7). The binding was more efficient with harman than norhar man. Both compounds also inhibit the binding of benzo(a)pyrene metabobites to DNA in vitro (8). In bacteria, both compounds have been shown either to enhance (11, 15, 26) or to inhibit (8) the mutagenicity of mutagens, as well as to develop the mutagenicity of nonmutagens (14) in the presence or absence of microsomal enzymes. In the mouse both harman and norharman inhibited benzo(a) pyrene metabolism as measured by aryl hydrocarbon hy droxylase activity (8). On the basis of these observations, 3 possible comutagenic mechanisms have been considered (26): alteration of the extracellular metabolic activation .@ C/) C of mutagens; alteration of the bacterial metabolism involved in mutagenesis; and a more direct action such as interac tion of comutagens with DNA. The first mechanism affects the initiation of DNA damage. The latter 2 mechanisms could be combined as one mechanism, i.e., alteration in the process of DNA repair and mutagenesis. From present studies in Chinese hamster cells, the fob lowing effects of nontoxic concentrations of harman were observed: the survival of UV or X-ray damaged cells was 4530 I U) UVDose (J/sqm) Chart 3. The effect of a nontoxic concentration of harman on the survival (left ordinate)and ouar mutations(right ordinate)of UV-irradlatedChinese hamster cells. The results were obtained from the same experiment with the same population of cells. The protocol used was identical with that presented in Table 3. The mutation frequencies of harman-treated cells compared to control cells at an equivalent dose of UV were significantly reduced (p < 1%). CANCER RESEARCH VOL. 38 Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1978 American Association for Cancer Research. Inhibition Table 4 Effect of harman 6@TGrmutants treatment on frequenciesof UV-induced Table 3 Effect of harman treatment on frequencies of UV-induced ouar mutants in Chinesehamster(V79)cells For Experiments 1 to 3 and 6 to 8, the number cellsProtocol:3.5hr in Chinese hamster (V79) of cells per plate 3days (9 cm) used was 2.1 x 10'/21 ; for Experiments 4 and 9, the number of cells per plate (9 cm) used was 4.2 x 10°/21; for Experiments 5 and 10, the number of cells per plate (9 cm) used was 21.0 x 10°/ Cell plating 4days Replating for mu tation analysis ,,,,,,,,,,, 3.5hr 48hr Cell plat ing 8days UV 21. Protocol: @ of Mutagenesis by Harman l6hr 2Ohr —lweek Ouabain UV ± Harman Scorefor mutants Mutation frequency―Il 0' ‘‘‘‘I''''' survivors8-day ±Harman 12-dayUV (40 expression (J/sq time01 m)ExperimentHarman timeb @M)expression Expres UV (JI time quencyIl0° (40@tM)sion vors10—4810317 sqm)Harman (hr)Survival (%)Mutation fre suM + (37)a25—489553 22― — 2— 88 +44 52―51 79 56― (106)310—6455109 (126)420—844.4351 + (65)530—840.61525 (67)6 2— 284@'101 159― — 318 +287 207― + 322― 316 (9) 70 (66)8 5+ +48 48104 954― 8444 2.492 38b (85) +64 910 20+ (20)1030+840.28404 2— 199@' (24) a Numbers in parentheses, b Mutation frequency number of mutants. a Number of cells used — 569 567 +447 349― 394C for mutation assay: 4.2 x 10' (cells not trol cells at equivalent dose of UV was significantly reduced (p < irradiated with UV) and 1 .5 x 10' (5 or 10 J/sq m UV-irradiated). b Time interval between removal of harman and exposure of cells 1%). to selectivemedium. of harman-treated cells compared to con C Mutation frequency trol cells at equivalent of harman-treated cells compared dose of UV was significantly to reduced con (p < 1%). fect of harman on survival and mutation frequencies was greater than that of norharman and was detected primarily in treatments in which cells were exposed to harman immediately following UV irradiation. By using UV light as a mutagen, the possible effect of harman on metabolic activation and initiation of DNA dam age in our system can be eliminated.The timing and the specific effect of harman on survival of radiation-damaged cells appear to indicate that harman inhibits the DNA repair of radiation-damaged cells. This notion is supported by the observed inhibiting effect of harman on unscheduled DNA synthesis. However, since unscheduled DNA synthesis pri manly measures the error-free excision repair process [as suming that excision repair in Chinese hamsters cells is error free as it seems to be in human cells (9)] and if harman specifically inhibits this repair process, an increase in mutation frequency would be expected. The results ob tamed apparently contradict this prediction. An alternative hypothesis that can explain all these results is that unsched uled DNA synthesis could include an error-prone postrepli cation repair component and that harman specifically in hibits this process. Experiments to test this hypothesis are now inprogress. Harman was also found to reduce the survival of X-ray damaged cells. There is a report that X-ray-induced DNA single strand breaks seem to be repaired except those that are sufficiently close to each other on complementary strands, which would constitute double strand breaks, which are not repairable (4). The harman treatment could possibly affect the final yield of these double strand breaks as a consequence of its intercalation into DNA. Alterna tiveby, there might be a common step in the repair of UV and X-ray-induced DNA damage that is inhibitable by har man. Reduction of mutagenesis by harman has been demon strated in 2 different mutation systems in Chinese hamster cells. Experiments have shown that it is not due to its effect on mutation expression or its specific effect on survival of oua'@or 6-TG'@mutants. The effect is more likely due to a harman effect on DNA repair and mutagenesis similar to that observed for benzo(a)pyrene in bacteria (8). From the resultspresented here and resultsreportedinthe literature, an interesting correlation is observed. Norharman is more effective than harman for inducing a comutagenic effect with carcinogens tested in the Ames assay (6, 25). On the contrary, harman is more effective than norharman in its ability to intercalate into the DNA (7), to reduce the surival of radiation-damaged cells (our data), and to reduce the mutagenic effect of the damage caused by another mutagen (Ref. 8; present results). This correlation not only seems to separate the 2 major effects of /3-carboline derivatives, i.e., the effect on mutagen activation and mutation fixation, but also points to the possible causal relationship between DNA DECEMBER 1978 Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1978 American Association for Cancer Research. 4531 C-c. Chang et a!. Table 6 Effect of harman on the recovery of wild-type and ouabain resistant Chinese hamster cells Protocol: 3.5 hr 1 day —1 week Table 5 Effect of harman on recovery of spontaneous ouar mutants in Chinese hamster (V79) cells Protocol: A. 3.5 hr —1week Score for Ouabain Cell plating Score for Cell plat mutants ing ± Ouabain colonies ‘‘‘I'''''' HarmanNo. ± Harman B. 3.5hr Cell plat ing 1_1.5daysa —1week ± ofcells/plate (9cm)Har Score for Ouabain ‘‘“‘‘‘/‘“/ mutants %ouar@Wild Oua No. of col (1 @M)bain mM)oniesformed―Recov (ouar@141)mixture@man ery°200——167841200 (40 typeouar@135 ± Harman @ (200) (40 @.tM)survivorsProtocol B1— A — + — 200(200) — — + + —— +165 200(200)+ 74(85)1xlO'++0.51x Protocol 165 (168)d (84)d 170(168) 85(84) 153(173) 77(87) — 200(200) mutants/lO'Experiment'@Harman Frequency ofouar xlO'200 147(169) 083 17.1― 4.1c2— +— — 3.6e3— +— — 2.2°4— +0.13@ ozi@ <O.9@5— +025h 0.49― 10'200(200)—+148(170)74(85)1x 10'200 (200)++146 (84)200——18894200200 8.8' <0.69i 94 — — 188 — + 200 176 — + 192 200 +— +182 18291 911xlO'—+01xlO'++01xlO'200—+192961xlO°200++18894 200+ 6.7' +<0.69' (167)73 9.0° 0.7' 88 96 a One day for Experiment 1 , 1 .5 days for Experiments 2 to 5. b Experiments 1 and 3 are experiments presented in Table 3 and Chart 3, respectively. C Numberofcellstested:2.1 d Number x of cells tested: 10g. a Determined by dividing the total number of colonies developed 1 .65 x 10'. by the total number of cells plated that were expected to grow in the medium provided, multiplied by 100. e Number of cells tested: 1 .8 x 10g. I Numberofcellstested: 4.2 U Numberofcellstested: 1.05 h Numberofcellstested: 8.4 x 10'. 1 1.5 x Numberofcellstested: x 10'. b Mixture x 10'. of an equal C 10g. Average of (I Numbers intercalation, cell survival, and the process of mutagenesis. Harman was also found to reduce the frequency of spontaneous mutations. If the reduction is due to the intercalation of harman into DNA, we suspect that sponta neous mutagenesis may share a common step with an error-prone postreplication repair process. If the harman effecton spontaneousmutagenesiscan be found to be a general phenomenon, harman can be an important chemi cal to study the mechanism of spontaneous mutations. If mutagenesis does play a role in carcinogenesis, and because of the potential role of harman and norharman in nutritionally related human carcinogenesis, additional from 17 indepe―-' it 4 plates. in parentheses, results for ouar@141. Table 7 Effect of harman on the recovery of preexisting 6@TGrmutants in Chinese hamster cells Protocol: 3.5hr 9days@' 3days Replating for recoveryof /////‘‘‘‘‘‘‘‘‘///// Cell plating 6-TGT mu tants ± Harman Proportion of initial cell mixtureHarman of 6@TGrmutant type(xlO')6@TGr recoveredWild (12O)'@1 (44) 1 1 125― (spontaneous) l25@(spontaneous) 1 125d (UV-induced) — 10 125d +0.9 a Cells were b Numbers The authors gratefully acknowledgethe technical assistanceof Judy (40Frequency (origin)@M)(x 10')10—3.3 ACKNOWLEDGMENTS 4532 of cells stud ies must be made to examine the implications of our results. Comparative studies, with the use of mutagenesis assays in both bacteria and cultured human fibroblasts as well as the initiation-promotion mouse skin assay, are now in progress. Funston and the typing assistance of Sharon Austin. number spontaneous ouar clones. C A mixture d A mixture + (UV-induced)+ subcultured every in parentheses, of — 18 63 (368) 67 (368) 123 (940) 139 (1062) 2 days. number of mutants. independent6@TGr clones. of 116 independent6@TGr clones. CANCER RESEARCH VOL. 38 Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1978 American Association for Cancer Research. Inhibition of Mutagenesis by Harman of Norharman and Harman. Proc. Japan Acad., 53: 95-98, 1977. 16. Oyasu, A.. Kitajima, T., Hopp. M. L., and Sumie, H. Enhancement of Chang.C. C., Trosko,J. E., andAkera,T. Characterizationof Ultraviolet Urinary Bladder Tumorigenesis in Hamsters by Coadministration of 2Light-Induced Ouabain-Resistant Mutations in Chinese Hamster Cells. 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