Environmental and Molecular Mutagenesis 20:289-296 (1992)
Certain Tryptophan Photoproducts Are Inhibitors of Cytochrome P450-Dependent Mutagenicity U. Rannug, E. Agurell, A. Rannug, and H. Cederberg Department of Genetics (U.R.) and Department of Genetic and Cellular Toxicology (E.A., H.C.), Wallenberg Laboratory, Stockholm University, Stockholm, and Department of Toxicology, National Institute of Occupational Health, Solna (A.R.),Sweden Two photoproducts, derived from UV-irradiation of the amino acid L-tryptophan and with high Ah (TCDD) receptor binding affinity, were tested for genotoxic and antimutagenic effects. The two indolo[3,2-b]carbazole derivatives, with the molecular weights of 284 and 312, respectively, were tested in Saccharomycescerevisiae strain D7 for mitotic gene conversion and reverse mutation and in strain RS112 for sister chromatid conversion and gene conversion. No significant (P > 0.05) genotoxic effects were found in strain D7, while strain RS112 showed a small but significant increase in the frequency of sister chromatid conversions. In Chinese hamster ovary (CHO) cells the two compounds induced a statistically significant but less than twofold increase in the frequency of sister chromatid exchanges (SCE). No mutations were detected when the compounds were tested in Sal-
monella typhimurium strains TA98 and TA100. However, both 284 and 312 acted as antimutagens on strain TA100+S9 in the presence of benzo(a)pyrene. The decrease in mutagenicity by the most potent compound 284 was 20 revertants/nmol. This effect could be explained by an inhibitory effect on the cytochrome P450dependent ethoxyresorufin O-deethylase (EROD) activity as seen in rat hepatocytes. The two compounds were also tested with hamster cells expressing rat cytochrome P-4501A1. The results support the conclusion that this cytochrome P-450 isozyme is inhibited by the tryptophan photoproducts. Similar results were also seen with two other high affinity Ah receptor ligands the quinazolinocarboline alkaloids rutaecarpine and dehydrorutaecarpine. 0 1992 Wiley-Liss, Inc.
Key words: Saccharomyces, Salmonella, HPRT-mutation, SCE, antimutagen, nitrogen heterocycles, EROD activity
INTRODUCTION Ultraviolet or near ultraviolet irradiation of the amino acid L-tryptophan gives rise to a considerable number of photoproducts. Although several of these compounds have been identified [Creed 1984; Borkman et al., 19861 they only constitute a minor fraction of all derivatives formed. As in the case of pyrolysis of tryptophan, mutagenic derivatives are also formed after UV photolysis [Sjogren et al., 19921. After UV irradiation of tryptophan solutions, however, at least two compounds which may be unique and of special interest in this context are formed. The compounds, which are indolo[3,2-b]carbazole derivatives, will be described elsewhere [Rannug, manuscript in preparation]. One of these compounds has a molecular weight of 284 and the molecular weight of the other is 3 12. Both compounds have a high affinity for the Ah or TCDD (2,3,7,8-tetrachlorodibenzo-para-dioxin) receptor. The affinity of compound 312 is in the same range as that of TCDD, while 284 shows an affinity at least one order of magitude higher [Rannug et al., 19871. 284 has also been shown to induce AHH activity in rat hepatoma cells in vitro [Rannug and Rannug, 19891. 0 1992 Wiley-Liss, Inc.
Both compound 284 and 3 I2 were tested for genotoxic effects and for modulating effects on cytochrome P450dependent metabolism and mutagenicity. The genotoxic effects were evaluated using different systems, both prokaryotic and eukaryotic, based on several genetic endpoints. The initial mutagenicity tests were carried out with Salmonella typhimurium strains TA98 and TA100. In addition, the compounds were studied in two different systems in yeast, Saccharomyces cerevisiae. One of the strains used, D7 has been developed by Zimmermann et al. [1975]. This strain is a multipurpose system for detection of various biological endpoints such as gene mutations, mitotic crossing-over and mitotic gene conversion. The other yeast strain, RSI 12, recently developed by Schiestl et al. [ 1988, 19891 is used to score intrachromosomal recombinational events. In RS 1 12 a plasmid containing an internal fragment of the HIS3 gene
Received March 5, 1992; revised and accepted July 20, 1992. Address reprint requests to U. Rannug, Dept. of Genetics, Wallenberg Lab., Stockholm University, S-106 91, Stockholm. Sweden.
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has been integrated at the genomic HIS3 site. This resulted in two copies of the HIS3 gene, one with a terminal deletion at the 3' end and the other with a terminal deletion at the 5' end. Reversion of this gene disruption can be obtained by mitotic sister chromatid conversion [Schiestl et al., 19881. Mitotic gene conversion at the adenine locus can also be detected in RS 112. The compounds 284 and 3 12 were tested for mitotic gene conversion and reverse mutation in Saccharomyces cerevisiae strain D7 and for mitotic sister chromatic conversion and mitotic gene conversion in strain RSI 12. The compounds were also tested for sister chromatid exchanges (SCE) and HPRT mutations in mammalian cells in vitro. The antimutagenic effects initially noted with Salmonella typhimurium in combination with benzo(a)pyrene, were studied further in mammalian cells. For this purpose the V79 Chinese hamster cells expressing specific rat cytochrome P450 isozymes, developed by Doehmer and coworkers [Doemer et al., 1988; Dogra et al., 19901, were chosen. The modifying effect of our test compounds on the induction of HPRT mutations by benzo(a)pyrene-frans-7,8diol (BPD) was studied in XEM2 cells and XEMd-Mz cells expressing cytochrome P4501A 1 and IA2, respectively. The effect on cytochrome P450IA-dependent metabolism, i.e., 0-deethylation of 7-ethoxyresorufin or EROD activity, was tested in the presence of compound 284 using primary hepatocyte suspensions from male Sprague-Dawley rats. The antimutagenic effects in Salmonella of the tryptophan photoproducts were compared with ellipticine and 9-hydroxyellipticine, known to be antimutagenic against benzo(a)pyrene in this system [Lesca et al,. 19791. In the mammalian systems the effects were compared with the structurally related plant alkaloid rutaecarpine and its derivative 7,8-dehydrorutaecarpine. Both these substances are, like compounds 284 and 312, high affinity Ah receptor ligands [Gillner et al., 1989; Rannug unpublished]. MATERIALS AND METHODS Chemicals
Compounds 284 and 312 were produced by UV-irradiation of tryptophan solutions as described by Rannug et al. [ 19871 and Sjogren et al. [ 19921. The purification procedure and chemical characterization of these two compounds will be described elsewhere [ Rannug, manuscript in preparation]. Ellipticine was purchased from Aldrich-Chemie GmbH & Co. K.G., Germany. 9-Hydroxyellipticine, rutaecarpine, and 7,8-dehydrorutaecarpine were synthesized and kindly provided by J. Bergman. Benzo(a)pyrene (BaP), N6,2'-0-dibutyryladenosine 3'5'-cyclic mono-phosphate (CAMP),3-isobutyl-1 -methyl-xanthine (IBMX), and camptothecin were purchased from Sigma Chemical Company (St Louis, MO). Benzo(a)pyrene-rrans-7.8-dihydrodiol (BPD) were purchased from NCI, Midwest Research Institute (Kansas City, MO).
Genotoxicity in Saccharomyces cerevisiae: Yeast strains.
The diploid S. cerevisiae D7 (MAT a/a ilvl-92/ilvl-92 trp5- 12ltrp5-27 cyhR2/CYHS2 ade2-40lade2- 1 19), kindly provided by F.K. Zimmermann and RS I 12 (MAT a/a ura3521ura3-52 leu2-3,I 12/leu2-A98 trp5-27lTRP5 arg4-31 ARG4 ade2-40lade2- 101 ilvl-91lILVl HtS3:: pRS61his3A200 lys2-801), kindly provided by R.H. Schiestl, were used. Genotoxicity tests.
The assays for gene conversion and reverse mutation were performed according to Zimmermann et al. [I9751 with minor modifications [Agurell et al., 1991). Yeast strain RS 1 12 was used in the assay for sisterchromatid conversion and gene conversion, which was performed according to Schiestl et al. [ 19891. The two compounds were dissolved in dimethylsulfoxide (DMSO). The test tubes were sealed and incubated for 17 h at 30°C with vigorous shaking. Mutagenicity Test in Salmonella typhimurium
The Salmonella typhimurium strains TA98 and TAlOO were kindly provided by B.N. Ames. The mutagenicity tests were performed both as plate incorporation tests and as preincubation tests [Maron and Ames, 19831. The tests for antimutagenic effects were carried out only as preincubation tests. In the latter case the following additions to the test tubes were made 30 min prior to incubation at 37°C: 0.1 ml of an overnight culture of the tester strain (TA98 or TAIOO); 0.5 ml of a S9-mix prepared from livers of Arochlor 1254 pre-treated male Sprague-Dawley rats; 30 ~1 DMSO or benzo(a)pyrene (BaP, 20 nmol/plate) dissolved in DMSO; and 40 1.p of a solution of the test compounds or the corresponding volume of solvent (DMSO). As positive controls, i.e., compounds with known antimutagenic effects, ellipticine and 9-hydroxyellipticine were used [Lesca et al., 19791. These compounds and 284 were tested at concentrations of 5, 10, and 20 nmol per plate and 312 at 4.5, 9, and 18 nmol per plate. After the preincubation, 2.5 ml soft agar was added to each tube and the contents thoroughly mixed and poured onto minimal agar medium [Maron and Ames, 19831. Histidine and biotin were added to the minimal medium instead of to the top agar. The plates were incubated for 48 hr and then the revertant colonies were counted on a Biotran I1 colony counter. Metabolism Studies
Primary hepatocytes were prepared from male SpragueDawley rats (ALAB, Sollentuna, Sweden) by the two step collagenase perfusion technique (Hogberg and Kristofersson, 19771. The rats, weighing 200 g, were maintained on ordinary lab chow (R3, ASTRA-Ewos, Sweden) and water
Tryptophan Photoproducts Are Antimutagenic 2000
1500
rev./plate
1000
500
0 0
2
4
6
B
10
12
14
16
18
20
Wl
Fig. 1. Antimutagenic effects of photoproduct 284 (m), ellipticine ( + ) and 9-hydroxyellipticine (A) in Sulmonellu typhimurium TA IOO+S9 in the presence of 20 nmol BaP.
ad lib. The enzymatic activity was measured as 7-ethoxyresorufin 0-deethylation, which is primarily catalyzed by cytochrome P4501Al [ h r o m and DePierre, 19861, essentially as described by Lake and Paine (1983). The 2 ml reaction mixture contained 50 mM Tris-HC1, 50 mM MgS04, 5 mM NADP+, 75 mM DL-isocitric acid, 5 U/ml isocitric dehydrogenase, and 5 pM 7-ethoxyresorufin. After preliminary dose-finding experiments, 0.02, 0.2, and 2.0 pM concentrations of 284 (in 5 pI DMSO) were chosen for the incubation. To one of the series a mixture of CAMPand IBMX was added. The reaction was started by the addition of 5 pM 7-ethoxyresorufin. The dealkylation was monitored spectrofluorometrically on a Perkin-Elmer fluorometer with excitation at 536 nm and emission at 582 nm. Genotoxicity Tests With Mammalian Cells
Sister chromatid exchanges (SCE) were studied in a UV sensitive strain of Chinese hamster ovary (CHO) cells. The experiments were basically carried out as described by Perry and Wolf [1974]. The cells were cultured in Ham’s F12 medium supplemented with glutamine, 10% newborn-calf serum and penicillin (10 IU/ml), fungizone (1.5 pg/ml), and streptomycin (10 pg/ml). The cells were reseeded 24 hr before treatment and the treatment time was 2 or 4 hr in 20 ml Hanks’ balanced salt solution (HBSS). The test com-
291
pounds were dissolved in DMSO and added in a volume of 100 or 200 pl. After the treatment period the cells were washed with HBSS and fresh F12 medium containing BrdUrd (0.01 mM) was added. The cells were then incubated in the dark for 25 hr. Two hours before harvest colcemid (0.1 pg/ml) was added. The cells were then washed with HBSS, trypsinized, treated hypotonically with 1% trisodium citrate for 15 min, fixed in ethano1:acetic acid (3:l), dropped onto ice-cold wet slides, and air dried. Sister chromatid differentiation was obtained with the fluorescence+Giemsa technique [Perry and Wolf, 19741. The SCE frequency was determined by counting 50 complete metaphase plates from each treatment concentration. In addition to 284 and 312 three other plant derived, nitrogen heterocycles, rutaecarpine, 7,8-dehydrorutaecarpine, and camptothecin were also tested for SCE induction. The latter compound is known to induce SCE both in vitro and in vivo [Lim et al., 1986; Backer et al., 19901. The two V79 derived cell lines, XEM2 expressing rat cytochrome P450IA 1 and XEMd-Mz expressing P450IA2, used in the HPRT mutagenicity tests, were kindly provided by Dr. J. Doehmer. The cells were maintained in DulbecCO’Smodified Eagle’s medium (DMEM) supplemented with 10% fetal calf serum according to Doehmer et al. [1988]. The medium was supplemented with Hepes (10 mM), penicillin (10 IU/ml), streptomycin (10 pg/ml), and fungizone (1.5 pg/ml). The day before treatment 25 cm2 flasks were inoculated with 750,000 cells in complete medium (DMEM). Before treatment the medium was changed to HBSS with Ca2+ and Mg2+ and the test substance, dissolved in DMSO, was added. The treatment time was 3 hr. After treatment the cells were washed with HBSS, trypsinized and 300,000 cells were added to 9 cm2 petri dishes in DMEM and reseeded every third day for a total expression time of nine days. After the expression time cells were plated in 6-thioguanine containing medium (6-TG, 7 pg/ml), 200,000 cells/plate, for determining the number of 6-TG resistant cells and in 6-TG free medium for calculating cloning efficiency (CEF) and mutation frequency. The cells were treated with 0.1 pM BPD in the absence or presence of the compounds 284 or 312. For comparison, rutaecarpine, and dehydrorutaecarpine were also tested for antimutagenic effects. Statistical Analyses
The genotoxicity tests, the antimutagenic tests and the EROD activities were analyzed by linear regression analyses with the following significance levels: n.s., P > 0.05; *0.01 < P < 0.05; **0.001 < P < 0.01; ***P < 0.001. The standard deviation is indicated in each figure.
RESULTS The results from the yeast assays are shown in Tables I and 11. From the tables it can be seen that neither 284 nor
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Rannug et al.
TABLE 1. Genotoxicity Tests of Compounds 284, 312 and Camptothecin in Saccaromyces cerevisiae D7t Compound 284 Exp. 1
Slope 284 Exp. 2
Slope 312
Slope Camptothecin Exp. I
Slope Camptothecin Exp. 2
SloDe
Conc . (pM)
CFU t S.D.
61 + I I 59 + 7 65 + 7 103 + 20 . 78 2 8 117 2 10 0.36 2 0.08*** 29 2 4 0 17 +. 2 7.46 23 ? 4 18.66 24 2 8 37.32 27 c 6 74.65 149.30 31 + 6 0.09 + 0.03** 0 192 f 16 158 + 2 0.68 1.71 152 + 16 3.42 146 + 8 198 + 5 6.84 13.68 I84 + 7 1.63 f 1.10 0 157 + 14 176 + 18 0.12 0.34 169 + 12 I .06 155 + 6 3.19 146? 10 9.56 133 + 5 28.71 102 + 9 -2.21 ? 0.29*** 0 89 ? 3 0.12 80 + 8 0.34 YO + 2 103 t 2 1.06 3.19 80 + 9 9.56 84 + 9 35 c 5 28.71 - I .86 + 0.23***
0 7.46 18.66 37.32 74.65 149.30
%
100
97 I07 I69 I28 I92 100
59 79 83 93 128
Gene-conversion (colonies t S.D.1 38 2 36 + 43 t 792 79f 69t
3 3 7 II 13 15
12.5 c 0.9 12.4 + 1.0 13.4 + 2.0 15.4 f 2.0 20.3 + 3.4 9.9 + 0.9 -0.006 + 0.017 21 .0 2.5 10.0 + 4.6 21.3 + 6.1 4.5 + 2.4 21.9 2 3.0 6.8 f 0.7 -0.051 c 0.036 9.52 2 1.16 10.63 + 2.81 8.52 ? 0.74 8.07 f 1.07 12.10 f 1.53 9.12 + I .84 0.019 0.102 10.30 + 2.08 8.90 1.32 10.75 ? 1.48 11.56 + 0.97 14.52 + 3.14 29. I4 f 5.65 55.72 3. I6 1.623 + 0.067*** 19.66 + 0.00 14.23 f 1.84 13.28 2 2.92 16.61 + 2.80 24.17 + 1.00 43.35 + 2.11 98.57 + 1.43 2.916 + 0.064***
*
24 + 3 7 2 3 19 t 6 4 t 2 23 t 3 102 1
I00 83 79 76 103 96
73 + 9 67 f 18 52 + 5 47 2 6 9 6 f 12 6 7 + 14
I00 I12 I08 99 93 85 65
6 5 2 13 63 + 9 73 2 10 72 t 7 85 f 18 120 2 44 2 2 7 + 13
100
70 f 0 45 f 6 48t II 5 8 2 15 65 2 15 146 2 7 138 + 2
90 101 I I6 99 94 39
T R P + / I Ocells ~ f S.E.
* *
*
Reverse mutation (colonies t S.D.)
ILV+/lOh cells t S.E.
14 + 5 9 f 3 II 2 3 9 + 2 14 + 2 5+1
0.47 f 0.17 0.30 ? 0. I 1 0.34 ? 0.09 0. I8 t 0.04 0.35 f 0.06 0.08 ? 0.02 -0.002 + 0.001** 4.6 t 1.0 3.0 2 2.0 3.7 f 0.3 7.2 t 1.5 4.2 2 0.4 3.2 t 0.7 -0.007 f 0.007 I .46 f 0.20 2.68 t 1.00 1.13 f 0.17 1.46 t 0.09 1.73 + 1.03 2.13 t 0.37 0.035 t 0.033 1.01 t 0.28 1.87 2 0.78 2.81 t 0.58 0.70 + 0. I7 1.05 t 0.55 2.60 f 0.44 2.33 2 0.12 5.09 2 1.30** 2.01 f 0.81 1.99 f 0.33 1.34 f 0.21 1.73 2 0.62 2.13 f 0.59 1.41 f 0.32 5.60 2 1.15 0.129 f 0.018***
II r 2 4 2 3 7+1 14 3
*
9+1 10 + 2
22 + 3 34 + 13 14 + 2 17+ I 28 f 16 31 + 6 13t4 262 II 38 f 8 9 f 2 12 t 6 28 + 5 19t I 14 f 6 13 f 2 10+2 14 2 5 14 + 4 10 f 2 16 + 3
?The number of colony forming units (CFU) as well as the number of gene conversion colonies and reverse mutation colonies + S.D. are shown. The frequencies (fS.E.) of gene conversion and reverse mutation are calculated from the dose response curves by means of linear regression analysis. *0.01 < P < 0.05; **0.001 < P < 0.01; ***P < 0.001.
312 were found to be genotoxic in strain D7, but that 284 gave a decrease in the reverse mutation frequency which was statistically significant ( P < 0.01) in one of the two experiments (Table I). In strain RSI 12, however, both compounds gave a significant ( P < 0.05) increase in the frequency of sister chromatid conversion and 284 also gave a significant increase in the gene conversion frequency at the adenine locus. A 15-fold difference in the spontaneous background levels for the adenine locus in RS 1 12 was seen. This instability of the gene conversion rate at this locus has also been demonstrated by Schiestl et al. [ 19891, who noted a 10-fold difference in the spontaneous frequency between experiments. A variability in the results from the reverse mutation experiments was also seen and was most likely due to difficulties in counting the number of revertant colonies, since the size of the colonies in some cases was extremely
small. A small but significant increase in the number of colony forming units (CFU) was seen in three of the four experiments performed with 284 (Tables I, 11). Camptothecin gave much more pronounced effects. It gave a clear reduction in CFU ( P < 0.001) in both yeast strains. Camptothecin induced gene conversions, I .6-2.9 TRP+/106 cells per p M ( P < O.OOl), and reverse mutations 0.1-5.1 ILV'I lo6 cells per pM in strain D7. The frequency of sister chromatid conversions in strain RS 1 12 was increased significantly ( P < O.OOl), by camptothecin, corresponding to between 53 and 73 conversions/106 cells per p M . No reproducible statistically significant effects were noted in the two Salmone[lu tester strains TA98 and TA 100 with 284 or 312 (data not shown). In the combined mutagenicity tests on Salmonella with BaP a significant ( P < 0.001) reduction in the BaP mutagenicity was seen
293
Tryptophan Photoproducts Are Antimvtagenic
TABLE II. Genotoxicity Tests of Compounds 284, 312 and Camptothecin in Succuromyces cerevisiue RS112t
Conc. Compound
(uM)
284 Exp. I
0 7.46 18.66 37.32 74.65 149.30
Slope 284 Exp. 2.
Slope 312
Slope Camptothecin Exp. 1
Slope Camptothecin Exp. 2
SloDe
0 7.46 18.66 37.32 74.65 93.31
0 0.68 1.71 3.42 6.84 13.68 0 0.12 0.34 I .06 3.19 9.56 28.71 0 0.12 0.34 1.06 3.19 9.56 28.71
Survival (CFU t S.D.) ll0t II 121 f 17 224 f 20 179 t 16 150 t 70 1 4 4 t 10 0.00 t 0.22 120 f 10 9 9 t 15 119 f 19 105 f I 133 6 133 t 15 0.26 f 0.09* 192 f 26 1 6 6 t 13 202 f 14 173 2 14 163 t 15 171 f 7 - 1.34 t 0.97 105 f 12 101 f 13 l l 0 t 12 88 f 5 106 f 2 902 13 38 f I -2.24 t 0.25*** 83 t 4 108 f 12 113t 5 89 t 6 106 t 14 91 f 18 45 t 7 - 1.88 t 0.33***
*
%
100
I10 204 I63 I36 131 100
121 99 88 Ill Ill 100
86 105
90 85 89
Sister chromatid conversion (colonies t S.D.) -
Sister chromatid conversion (His+/lO" cells t S.E.)
274 t 14 346 t 21 280 k 14 309 t 70 385 ? 22 481 t 16
495 f 25 573 35 250 f 13 346 2 79 512 k 29 670 f 23 I .SO f 0.59* 347 t 22 419 f 15 408 t 8 449 k 34 377 f 26 555 f 20 1.51 f 0.45** 280 f 6 295 f 17 284 t 12 322 t 27 366 f 5 316 t 3 3.29 f 1.45** 1396 ? 216 1603 t 241 1400 f 184 1875 t 172 1408 t 67 1862 t 290 3640 t 161 73.16 t 6.32*** 1325 t 568 579 ? 38 647 t 51 2318 t 237 1701 t 283 1571 f 122 2757 f 80 53.43 t 13.54***
*
208 1 3 208 t 8 242 t 5 2 3 6 t 18 250 t 17 368 t 13 269 f 6 245 t 14 287 12 278 23 298 t 4 270 3
* * *
86 36
147 t 23 161 2 24 154 t 20 165 t 15 150 4 7 167 t 26 138 t 6
I00 I30 I36 107 I28 I10 54
I10 f 47 62 t 4 73 t 6 206 t 21 180 t 30 143 f I I 125 t 4
100
96 I05
84 101
*
Gene conversion (colonies t S . D . ) 54 t 6 54 t 7 34 k 6 5 4 t 13 42 5 86 t 50
*
1084 1015
1096 t 102 I I10 f 72 I220 2300 76 1 713 729 719 653 645 5 3 f 16 52 t 8 53 t 7 48 t 5 50 t 4 41 t 8 22 f 2 90 t 7 9 2 2 13 I17 t I I 71 f 12 8 6 2 15 103 t 15 41 t 9
Gene conversion (ADE+/IO' cells t S.E.) 9.8 f 1.2 9.0 t 1 . 1 3.1 2 0.5 6.1 t 1.4 5.5 f 0.7 18.3 f 2.1 0.06 t 0.02** 181 205 141 2 13 211 f 14 I84 347 1.47 f 0.52* I59 171 144 I67 I60 151
-0.64 t 0.91 101 t 30
104t 15 9 6 2 13 110 t 12
93 t 8 91 t 18 118t I I 0.49 0.37 216 2 16 171 f 2 4 207 f 19 160 t 27 162 k 28 226 f 33 181 t 39 0.01 t 0.79
*
?The number of colony forming units (CFU) as well as the number of sister chromatid conversion colonies and gene conversion colonies t S.D. are shown. The frequencies (+S.E.) of sister chromatid conversion and gene conversion are calculated by means of linear regression analysis. *0.01 < P < 0.05; **0.001 < P < 0.01; ***P< 0.01.
(Fig. I ) . The reduction was approximately 20 revertants per nmol of 284. For comparison, the reduction in mutagenicity by the known antimutagenic compounds ellipticine and 9-hydroxyellipticineis also shown in Fig. 1 . In Figure 2, the modulating effect of 312 on the BaP mutagenicity with strains TA98 and TAIOO can be seen. A much less pronounced decrease in mutagenicity was seen with 3 12 compared to 284. The reduction is statistically significant only with tester strain TA 100. The frequency of SCE in CHO cells after treatment with five different nitrogen heterocycles is shown in Table 111. All nitrogen heterocycles caused a statistically significant increase in SCE in the CHO cells. The highest increase, 104 SCE/cell per pM, was seen with camptothecin and the lowest effects were seen with rutaecarpine and dehydrorutae-
carpine which gave an increase in SCE frequency of 0.2 or less. The tryptophan derivatives gave an increase of between 3 and 7 SCE per p M during these treatment conditions. The effect of 284 on the cytochrome P450-dependent EROD activity was tested using rat hepatocytes (Fig. 3). Compound 284 reduced the activity both in the absence of cAMP/IBMX and in the presence of cAMP/IBMX. The reduction was approximately 15.5 nmol/min per pM of 284 in the absence of CAMP. The dose response curve in the presence of CAMPhad a similar slope. In order to study the specific inhibition of cytochrome P450IA1 the mutagenic effects on BPD in XEM2 cells were studied in the presence of 284 and 312. In the preliminary experiments with the XEMd-Mz cells, expressing cytochrome P450IA2, a much lower effects was seen with
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Rannug et al. TAlOO
1 8 TA98
I
TABLE Ill. Sister Chromatid Exchange Frequency in CHO-cells After 2 or 4 hr Treatment With the Tryptophan Photoproducts 284 and 312 and Two Other Nitrogen Heterocvcles. RutaecarDine and 7.8-dihvdrorutaecar~inet
1000
Treatment time (hrl
Concentration o*M)
Mean frequency of SCE f SD
284
2
0 0.12 0.37 1.23
Slope 284
4
0
600
312
4
400
Slope Rutaecarpine
4
0 8. I 27.0 54.0
Slope Dehydrorutaecarpine
4
0 2.7 8.1 27.0
Slope Carnptothecin
4
9.02 f 0.83 I I .86 f 0.60 12.38 t 0.63 13.62 2 1.08 2.81 t 0.57***." 9.58 f 3.10 14.88 f 1.48 10.26 1.42 I I .32 2 0.47 11.14 f 0.89 12.00 2 0.41 6.50 f 2.76*." 8.60 t 2.46 10.40 f 3.15 12.18 f 3.37 12.10 f 1.86 0.06 f O.OI***." 8.46 t 2.42 I I .30 3.09b I I .57 f 3.48' 14.20 t 4.52 0.19 ? 0.03***." 8.98 f 0.77 12.12f 1.59 19.02 f 1.81 24.34 f 3.02 104.2 f I I .8***."
ComDound
800
rev./plate
T 200
0
I
I
0
5
10
15
20
nmol Fig. 2. Antimutagenic effects of photoproduct 3 12 in Salmonella r v p h imurium TAlOO and TA98 in the presence of 20 nmol BaP with 5 8 S9.
BPD. Therefore, a reduction in the effects in the presence of 284 and 3 12 was difficult to establish. For comparison, two other high affinity Ah receptor ligands, rutaecarpine and dehydrorutaecarpine, were tested simultaneously. As can be seen from Table IV, all compounds showed an antimutagenic effect in the XEM2 cells. No such decrease in mutagenicity was seen with 284 and 312 when tested together with the corresponding diol epoxide of BaP (data not shown).
DISCUSSION The results from the genotoxicity tests based on different genetic endpoints in both prokaryotic and eukaryotic systems indicate that the tryptophan photoproducts 284 and 3 12 do not induce point mutations. This conclusion receives further support from AHH induction experiments on rat hepatoma cells in vitro [Rannug and Rannug, 19891. In these experiments no increase in the number of 6-TG resistant cells could be observed after the 24 hr treatment with 284 (unpublished results). All the nitrogen heterocycles have also been tested for SCE in CHO cells in the absence of
I .23 0 0.03 0.11 0.21
0 0.015 0.072 0.144
Slope
*
*
?The topoisomerase I inhibitor camptothecin was used as a positive control. "No. of SCE's per FM 2 SE. '10 cells scored. '35 cells scored. *0.01 < P < 0.05; **0.001 < P < 0.01; ***P < 0.01.
an exogenous metabolizing system. Although a statistically significant dose dependent increase in SCEs could be noted, the effects of 284, 312, rutaecarpine, and dehydrorutaecarpine were low, at least one order of magnitude lower compared to the positive control camptothecin. Further experiments with somewhat higher treatment concentrations of 284 and 3 12 on strains XEM2 and XEMd-Mz also showed a small but statistically significant increase in the SCE frequencies (data not shown). It can therefore be concluded that these substances induce a small increase in the frequency of SCE in different mammalian cells and that this induction seems to be independent of cytochrome P450 activity. Together with the small increase in sister chromatid conversions in yeast the results point to a low but specific genotoxic potency, related to changes on the sister chromatid level only. Both substances are antimutagenic, at least with certain cytochrome P450-dependent mutagens such as BaP. The inhibition of EROD activity in rat hepatocytes shown with
Tryptophan Photoproducts Are Antimutagenic
295
284 as well as the antimutagenic effect in XEM2 cells of both 284 and 312 point to an inhibition of cytochrome P450IA 1 . Also the other two nitrogen heterocycles, rutaecarpine and dehydrorutaecarpine, showed the same effects as 284 and 3 12, i.e., a small increase in SCE and antimutagenic effects towards BPD in XEM2 cells. Our results indicate that within the group of nitrogen heterocycles there are some with quite unique biological properties. This can be exemplified by the most potent of the two tryptophan derivatives, i.e., 284. This substance has a high Ah receptor affinity, in fact, one order of magnitude higher than that of TCDD [Rannug et al., 1987; Rannug and Rannug, 19891. It induces AHH activity at concentrations one order of magnitude lower than (3-naphthoflavone, although the maximal extent of induction is not extremely P d P high [Rannug and Rannug, 19891. One possible explanation CO €0 CO N 53 el el of the modest induction potential can be that the compound occupies the enzyme and thereby inhibits the B(a)P-hydroxylase activity. This is further indicated by the inhibition of N N ‘u EROD activity and the antimutagenic effects towards BPD 0 in mammalian cells. The substance thus seems to inhibit the Fig. 3. Inhibition of ethoxyresorufin 0-deethylase (EROD) activity in Same cytochrome p450 isoenzyme that it induces. primary hepatocytes from male Sprague Dawley rats in the presence of the increased number of cells in three out of four experidifferent concentrations of photoproduct 284. The incubations were per- ments may indicate that the substance also may increase cell formed with or without the addition of N6,2’-O-dibutyryladenosine3’5’- survival or cell division.
z
T
5.
2.
8
cyclic monophosphate (CAMP)and 3-isobutyl-I-methyl-xanthine(IBMX)
TABLE IV. The Antimutagenic Effects in XEM2 Cells (hprt Locus) Towards 0.1 FM Benzo(a)pyrene-trans(-)7,8-dihydrodiol (BPD) of the Tryptophan Derived Compounds 284 and 312t Concentration Compound Experiment I 0 BPD BPD 284
+
BPD
BPD
+ Rutaecarpine
+ Dehydrorutaecarpine
(FM)
(a)
Frequency of 6-TG resistant cells x lo6
CEF
mutants
100 145
5 I73 25
83 70 93 82 77
102 130 134 119 97 147 179 130
1.0? 0.7 50.2 ? 5.0 5.2 f 1.9 0.2 2 0.4 4.4 1.7 1.6 f 1 . 1 0.8 f 0.8 3.0 2 1.2 3.0 f 1.9 0.8 f 0.8 2.4 f 2.1
100 82 I06 95 91 106 90 86 97 82 84
167 163 169 147 223 252 245 155 226 176 263
0.2 2 0.4 52.4 ? 3.4 7.6 -C 2.3 2.0 2 1.6 3.8 f 3.1 48.8 f 2.3 33.4 4.7 23.8 f 1.3 0 0 4.0 2.5
I I60 22 7 8 97 68 77 0 0 8
100
0.03 0.08 0.24 0.3 1.o 3.0 0.3 1.o 3.0
No. of
Survival
89 78 77 77 87
104
*
1
17 6 3 15 10
2 9
Experiment I1
0 BPD BPD
+ 284
BPD
+ 312
BPD
+ Dehydrorutaecarpine
0.03 0.08 0.24 0.003 0.01 0.03 0.33 1.o 3.0
*
*
tFor comparison the effects of the plant derived compounds rutaecarpine and 7,8-dehydrorutaecarpine are also shown.
296
Rannug et al.
The biological effects of these compounds must be studied in more detail, since the result so far indicate biologically important properties. It is reasonable to postulate that these naturally occurring heterocycles and tryptophan derivatives constitute an important class of biological signal substances involved not only in mutagenicity and carcinogenicity, but also in normal cell proliferation and cell differentiation.
ACKNOWLEDGMENTS We would like to thank Dr. I. Klasterska for valuable discussions and Mrs. Yvonne Eklund, Irja Hardling, MariAnn Westman, and Margareta Wihlborg for skillful technical assistance. This work was supported by grants from the Swedish Cancer Society and the Swedish Natural Science Research Council.
REFERENCES Agurell E. Cederberg H. Ehrenberg L. Lindahl-Kiessling K, Rannug U. Tornqvist M (1991): Genotoxic effects of ethylene oxide and propylene oxide: A comparative study. Mutat Res 250:229-237. Astrom A. DePierre JW ( 1986): Rat-liver microsomal cytochrome-P-450: Purification. characterization. multiplicity and induction. Biochim Biophys Acta 853:1-27. Backer LC, Allen JW, Harrington-Brock K, Campbell JA. DeMarini DM, Doerr CL, Howard DR, Klingerman AD, Moore MM (1990): Genotoxicity of inhibitors of DNA topoisomerases I (camptothecin) and II (m-AMSA) in vivo and in vitro. Mutagenesis 5541-547. Borkman R, Hibbard L, Dillon J (1986): The photolysis of tryptophan with 337.1 nm laser radiation. Photochem Photobiol43: 13-19. Creed D (1984): The photophysics and photochemistry of the near U V absorbing amino acids-I, Tryptophan and its simple derivatives. Photochem Photobiol 39537-562. Doemer J, Dogra S. Friedberg T. Monier S. Adesnik M, Glatt H. Oesch F (1988): Stable expression of rat cytochrome P-45011BI cDNA in Chinese hamster cells (V79) and metabolic activation of aflatoxin BI. Proc Natl Acad of Sci (USA) 855769-5773. Dogra S, Doemer J, Glatt H. Molders H. Sieged P, Friedberg T, Seidel A. Oesch F (1990): Stable expression of rat cytochrome P-4501AI
cDNA In (V79) Chinese hamster cells and their use in mutagenicity testing. Mol Pharmacol 37:608413. Gillner M. Bergman J. Cambillau C, Gustafsson J-A ( 1989): Interaction of rutaecarpine alkaloids with specific binding sites for 2.3.7.8-tetrachlorodibenzo-p-dioxin in rat liver. Carcinogenesis 10:651-654. Hogberg J. Kristofcrsson A ( 1977): A correlation between glutathione levels and cellular damage in isolated hepatocytes. Eur J Biochem 74:77-82. Lake BG, Paine AJ (1983): Lnduction of hepatic cytochrome P-450 and drug metabolism by metyrapone in the rat: Relevance to its effects in rat-liver cell culture. Xenobiotica 13:725-730. Lesca P. Lecointe P, Paoletti C, Mansuy D (1979): Ellipticines are potent inhibitors of microsomes-dependent chemical mutagenesis. Chem Biol Interact 25:279-287. Lim M. Liu LF. Jacobson-Kram D, Williams JR (1986): Induction of sister chromatid exchanges by inhibitors of topoisomerases. Cell Biol Toxicol2:485494. Maron DM, Ames BN (1983): Revised methods for the Salmonella niutagenicily test. Mutat Res I13:173-215. Perry P. Wolff S ( 1974):New giemsa method for the differential staining of sister chromatid exchange. Nature 251:15&158. Rannug A. Rannug U. Rosenkranz HS, Winquist L, Westerholm R. Agurell E, Grafstrom AK ( 1987): Certain photooxidized derivatives of tryptophan bind with very high affinity to the Ah receptor and are likely to be endogenous signal substances. J Biol Chem 262:1542215427. Rannug A. Rannug U (1989): UV-irradiation of tryptophan gives rise to AHH-inducing compounds with very high affinity for the Ah receptor. Cheniosphcre 18:1101-1106. Schiestl RH. Igarashi S. Hastings PJ (1988): Analyses of the mechanism for reversion of a disrupted gene. Genetics I19:237-247. Schiestl RH, Gietz RD, Mehta RD, Hastings PJ (1989): Carcinogens induce intrachromosomal recombination in yeast. Carcinogenesis 10: 1445-1455. Sjiigren M. Sjoberg U, Rannug U (1992): Mutagenicity of tryptophan photoproducts in the Ames Salmonella assay. Mutat Res (submitted). Zimmerman FK, Kern R, Rosenberger H ( 1975): A yeast strain for simultaneous detection of induced mitotic crossing over mitotic gene conversion and reverse mutation. Mutat Res 28:38 1-388.
Accepted by-
R.R. Tice
Certain Tryptophan Photoproducts Are Inhibitors of Cytochrome P450-Dependent Mutagenicity U. Rannug, E. Agurell, A. Rannug, and H. Cederberg Department of Genetics (U.R.) and Department of Genetic and Cellular Toxicology (E.A., H.C.), Wallenberg Laboratory, Stockholm University, Stockholm, and Department of Toxicology, National Institute of Occupational Health, Solna (A.R.),Sweden Two photoproducts, derived from UV-irradiation of the amino acid L-tryptophan and with high Ah (TCDD) receptor binding affinity, were tested for genotoxic and antimutagenic effects. The two indolo[3,2-b]carbazole derivatives, with the molecular weights of 284 and 312, respectively, were tested in Saccharomycescerevisiae strain D7 for mitotic gene conversion and reverse mutation and in strain RS112 for sister chromatid conversion and gene conversion. No significant (P > 0.05) genotoxic effects were found in strain D7, while strain RS112 showed a small but significant increase in the frequency of sister chromatid conversions. In Chinese hamster ovary (CHO) cells the two compounds induced a statistically significant but less than twofold increase in the frequency of sister chromatid exchanges (SCE). No mutations were detected when the compounds were tested in Sal-
monella typhimurium strains TA98 and TA100. However, both 284 and 312 acted as antimutagens on strain TA100+S9 in the presence of benzo(a)pyrene. The decrease in mutagenicity by the most potent compound 284 was 20 revertants/nmol. This effect could be explained by an inhibitory effect on the cytochrome P450dependent ethoxyresorufin O-deethylase (EROD) activity as seen in rat hepatocytes. The two compounds were also tested with hamster cells expressing rat cytochrome P-4501A1. The results support the conclusion that this cytochrome P-450 isozyme is inhibited by the tryptophan photoproducts. Similar results were also seen with two other high affinity Ah receptor ligands the quinazolinocarboline alkaloids rutaecarpine and dehydrorutaecarpine. 0 1992 Wiley-Liss, Inc.
Key words: Saccharomyces, Salmonella, HPRT-mutation, SCE, antimutagen, nitrogen heterocycles, EROD activity
INTRODUCTION Ultraviolet or near ultraviolet irradiation of the amino acid L-tryptophan gives rise to a considerable number of photoproducts. Although several of these compounds have been identified [Creed 1984; Borkman et al., 19861 they only constitute a minor fraction of all derivatives formed. As in the case of pyrolysis of tryptophan, mutagenic derivatives are also formed after UV photolysis [Sjogren et al., 19921. After UV irradiation of tryptophan solutions, however, at least two compounds which may be unique and of special interest in this context are formed. The compounds, which are indolo[3,2-b]carbazole derivatives, will be described elsewhere [Rannug, manuscript in preparation]. One of these compounds has a molecular weight of 284 and the molecular weight of the other is 3 12. Both compounds have a high affinity for the Ah or TCDD (2,3,7,8-tetrachlorodibenzo-para-dioxin) receptor. The affinity of compound 312 is in the same range as that of TCDD, while 284 shows an affinity at least one order of magitude higher [Rannug et al., 19871. 284 has also been shown to induce AHH activity in rat hepatoma cells in vitro [Rannug and Rannug, 19891. 0 1992 Wiley-Liss, Inc.
Both compound 284 and 3 I2 were tested for genotoxic effects and for modulating effects on cytochrome P450dependent metabolism and mutagenicity. The genotoxic effects were evaluated using different systems, both prokaryotic and eukaryotic, based on several genetic endpoints. The initial mutagenicity tests were carried out with Salmonella typhimurium strains TA98 and TA100. In addition, the compounds were studied in two different systems in yeast, Saccharomyces cerevisiae. One of the strains used, D7 has been developed by Zimmermann et al. [1975]. This strain is a multipurpose system for detection of various biological endpoints such as gene mutations, mitotic crossing-over and mitotic gene conversion. The other yeast strain, RSI 12, recently developed by Schiestl et al. [ 1988, 19891 is used to score intrachromosomal recombinational events. In RS 1 12 a plasmid containing an internal fragment of the HIS3 gene
Received March 5, 1992; revised and accepted July 20, 1992. Address reprint requests to U. Rannug, Dept. of Genetics, Wallenberg Lab., Stockholm University, S-106 91, Stockholm. Sweden.
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has been integrated at the genomic HIS3 site. This resulted in two copies of the HIS3 gene, one with a terminal deletion at the 3' end and the other with a terminal deletion at the 5' end. Reversion of this gene disruption can be obtained by mitotic sister chromatid conversion [Schiestl et al., 19881. Mitotic gene conversion at the adenine locus can also be detected in RS 112. The compounds 284 and 3 12 were tested for mitotic gene conversion and reverse mutation in Saccharomyces cerevisiae strain D7 and for mitotic sister chromatic conversion and mitotic gene conversion in strain RSI 12. The compounds were also tested for sister chromatid exchanges (SCE) and HPRT mutations in mammalian cells in vitro. The antimutagenic effects initially noted with Salmonella typhimurium in combination with benzo(a)pyrene, were studied further in mammalian cells. For this purpose the V79 Chinese hamster cells expressing specific rat cytochrome P450 isozymes, developed by Doehmer and coworkers [Doemer et al., 1988; Dogra et al., 19901, were chosen. The modifying effect of our test compounds on the induction of HPRT mutations by benzo(a)pyrene-frans-7,8diol (BPD) was studied in XEM2 cells and XEMd-Mz cells expressing cytochrome P4501A 1 and IA2, respectively. The effect on cytochrome P450IA-dependent metabolism, i.e., 0-deethylation of 7-ethoxyresorufin or EROD activity, was tested in the presence of compound 284 using primary hepatocyte suspensions from male Sprague-Dawley rats. The antimutagenic effects in Salmonella of the tryptophan photoproducts were compared with ellipticine and 9-hydroxyellipticine, known to be antimutagenic against benzo(a)pyrene in this system [Lesca et al,. 19791. In the mammalian systems the effects were compared with the structurally related plant alkaloid rutaecarpine and its derivative 7,8-dehydrorutaecarpine. Both these substances are, like compounds 284 and 312, high affinity Ah receptor ligands [Gillner et al., 1989; Rannug unpublished]. MATERIALS AND METHODS Chemicals
Compounds 284 and 312 were produced by UV-irradiation of tryptophan solutions as described by Rannug et al. [ 19871 and Sjogren et al. [ 19921. The purification procedure and chemical characterization of these two compounds will be described elsewhere [ Rannug, manuscript in preparation]. Ellipticine was purchased from Aldrich-Chemie GmbH & Co. K.G., Germany. 9-Hydroxyellipticine, rutaecarpine, and 7,8-dehydrorutaecarpine were synthesized and kindly provided by J. Bergman. Benzo(a)pyrene (BaP), N6,2'-0-dibutyryladenosine 3'5'-cyclic mono-phosphate (CAMP),3-isobutyl-1 -methyl-xanthine (IBMX), and camptothecin were purchased from Sigma Chemical Company (St Louis, MO). Benzo(a)pyrene-rrans-7.8-dihydrodiol (BPD) were purchased from NCI, Midwest Research Institute (Kansas City, MO).
Genotoxicity in Saccharomyces cerevisiae: Yeast strains.
The diploid S. cerevisiae D7 (MAT a/a ilvl-92/ilvl-92 trp5- 12ltrp5-27 cyhR2/CYHS2 ade2-40lade2- 1 19), kindly provided by F.K. Zimmermann and RS I 12 (MAT a/a ura3521ura3-52 leu2-3,I 12/leu2-A98 trp5-27lTRP5 arg4-31 ARG4 ade2-40lade2- 101 ilvl-91lILVl HtS3:: pRS61his3A200 lys2-801), kindly provided by R.H. Schiestl, were used. Genotoxicity tests.
The assays for gene conversion and reverse mutation were performed according to Zimmermann et al. [I9751 with minor modifications [Agurell et al., 1991). Yeast strain RS 1 12 was used in the assay for sisterchromatid conversion and gene conversion, which was performed according to Schiestl et al. [ 19891. The two compounds were dissolved in dimethylsulfoxide (DMSO). The test tubes were sealed and incubated for 17 h at 30°C with vigorous shaking. Mutagenicity Test in Salmonella typhimurium
The Salmonella typhimurium strains TA98 and TAlOO were kindly provided by B.N. Ames. The mutagenicity tests were performed both as plate incorporation tests and as preincubation tests [Maron and Ames, 19831. The tests for antimutagenic effects were carried out only as preincubation tests. In the latter case the following additions to the test tubes were made 30 min prior to incubation at 37°C: 0.1 ml of an overnight culture of the tester strain (TA98 or TAIOO); 0.5 ml of a S9-mix prepared from livers of Arochlor 1254 pre-treated male Sprague-Dawley rats; 30 ~1 DMSO or benzo(a)pyrene (BaP, 20 nmol/plate) dissolved in DMSO; and 40 1.p of a solution of the test compounds or the corresponding volume of solvent (DMSO). As positive controls, i.e., compounds with known antimutagenic effects, ellipticine and 9-hydroxyellipticine were used [Lesca et al., 19791. These compounds and 284 were tested at concentrations of 5, 10, and 20 nmol per plate and 312 at 4.5, 9, and 18 nmol per plate. After the preincubation, 2.5 ml soft agar was added to each tube and the contents thoroughly mixed and poured onto minimal agar medium [Maron and Ames, 19831. Histidine and biotin were added to the minimal medium instead of to the top agar. The plates were incubated for 48 hr and then the revertant colonies were counted on a Biotran I1 colony counter. Metabolism Studies
Primary hepatocytes were prepared from male SpragueDawley rats (ALAB, Sollentuna, Sweden) by the two step collagenase perfusion technique (Hogberg and Kristofersson, 19771. The rats, weighing 200 g, were maintained on ordinary lab chow (R3, ASTRA-Ewos, Sweden) and water
Tryptophan Photoproducts Are Antimutagenic 2000
1500
rev./plate
1000
500
0 0
2
4
6
B
10
12
14
16
18
20
Wl
Fig. 1. Antimutagenic effects of photoproduct 284 (m), ellipticine ( + ) and 9-hydroxyellipticine (A) in Sulmonellu typhimurium TA IOO+S9 in the presence of 20 nmol BaP.
ad lib. The enzymatic activity was measured as 7-ethoxyresorufin 0-deethylation, which is primarily catalyzed by cytochrome P4501Al [ h r o m and DePierre, 19861, essentially as described by Lake and Paine (1983). The 2 ml reaction mixture contained 50 mM Tris-HC1, 50 mM MgS04, 5 mM NADP+, 75 mM DL-isocitric acid, 5 U/ml isocitric dehydrogenase, and 5 pM 7-ethoxyresorufin. After preliminary dose-finding experiments, 0.02, 0.2, and 2.0 pM concentrations of 284 (in 5 pI DMSO) were chosen for the incubation. To one of the series a mixture of CAMPand IBMX was added. The reaction was started by the addition of 5 pM 7-ethoxyresorufin. The dealkylation was monitored spectrofluorometrically on a Perkin-Elmer fluorometer with excitation at 536 nm and emission at 582 nm. Genotoxicity Tests With Mammalian Cells
Sister chromatid exchanges (SCE) were studied in a UV sensitive strain of Chinese hamster ovary (CHO) cells. The experiments were basically carried out as described by Perry and Wolf [1974]. The cells were cultured in Ham’s F12 medium supplemented with glutamine, 10% newborn-calf serum and penicillin (10 IU/ml), fungizone (1.5 pg/ml), and streptomycin (10 pg/ml). The cells were reseeded 24 hr before treatment and the treatment time was 2 or 4 hr in 20 ml Hanks’ balanced salt solution (HBSS). The test com-
291
pounds were dissolved in DMSO and added in a volume of 100 or 200 pl. After the treatment period the cells were washed with HBSS and fresh F12 medium containing BrdUrd (0.01 mM) was added. The cells were then incubated in the dark for 25 hr. Two hours before harvest colcemid (0.1 pg/ml) was added. The cells were then washed with HBSS, trypsinized, treated hypotonically with 1% trisodium citrate for 15 min, fixed in ethano1:acetic acid (3:l), dropped onto ice-cold wet slides, and air dried. Sister chromatid differentiation was obtained with the fluorescence+Giemsa technique [Perry and Wolf, 19741. The SCE frequency was determined by counting 50 complete metaphase plates from each treatment concentration. In addition to 284 and 312 three other plant derived, nitrogen heterocycles, rutaecarpine, 7,8-dehydrorutaecarpine, and camptothecin were also tested for SCE induction. The latter compound is known to induce SCE both in vitro and in vivo [Lim et al., 1986; Backer et al., 19901. The two V79 derived cell lines, XEM2 expressing rat cytochrome P450IA 1 and XEMd-Mz expressing P450IA2, used in the HPRT mutagenicity tests, were kindly provided by Dr. J. Doehmer. The cells were maintained in DulbecCO’Smodified Eagle’s medium (DMEM) supplemented with 10% fetal calf serum according to Doehmer et al. [1988]. The medium was supplemented with Hepes (10 mM), penicillin (10 IU/ml), streptomycin (10 pg/ml), and fungizone (1.5 pg/ml). The day before treatment 25 cm2 flasks were inoculated with 750,000 cells in complete medium (DMEM). Before treatment the medium was changed to HBSS with Ca2+ and Mg2+ and the test substance, dissolved in DMSO, was added. The treatment time was 3 hr. After treatment the cells were washed with HBSS, trypsinized and 300,000 cells were added to 9 cm2 petri dishes in DMEM and reseeded every third day for a total expression time of nine days. After the expression time cells were plated in 6-thioguanine containing medium (6-TG, 7 pg/ml), 200,000 cells/plate, for determining the number of 6-TG resistant cells and in 6-TG free medium for calculating cloning efficiency (CEF) and mutation frequency. The cells were treated with 0.1 pM BPD in the absence or presence of the compounds 284 or 312. For comparison, rutaecarpine, and dehydrorutaecarpine were also tested for antimutagenic effects. Statistical Analyses
The genotoxicity tests, the antimutagenic tests and the EROD activities were analyzed by linear regression analyses with the following significance levels: n.s., P > 0.05; *0.01 < P < 0.05; **0.001 < P < 0.01; ***P < 0.001. The standard deviation is indicated in each figure.
RESULTS The results from the yeast assays are shown in Tables I and 11. From the tables it can be seen that neither 284 nor
292
Rannug et al.
TABLE 1. Genotoxicity Tests of Compounds 284, 312 and Camptothecin in Saccaromyces cerevisiae D7t Compound 284 Exp. 1
Slope 284 Exp. 2
Slope 312
Slope Camptothecin Exp. I
Slope Camptothecin Exp. 2
SloDe
Conc . (pM)
CFU t S.D.
61 + I I 59 + 7 65 + 7 103 + 20 . 78 2 8 117 2 10 0.36 2 0.08*** 29 2 4 0 17 +. 2 7.46 23 ? 4 18.66 24 2 8 37.32 27 c 6 74.65 149.30 31 + 6 0.09 + 0.03** 0 192 f 16 158 + 2 0.68 1.71 152 + 16 3.42 146 + 8 198 + 5 6.84 13.68 I84 + 7 1.63 f 1.10 0 157 + 14 176 + 18 0.12 0.34 169 + 12 I .06 155 + 6 3.19 146? 10 9.56 133 + 5 28.71 102 + 9 -2.21 ? 0.29*** 0 89 ? 3 0.12 80 + 8 0.34 YO + 2 103 t 2 1.06 3.19 80 + 9 9.56 84 + 9 35 c 5 28.71 - I .86 + 0.23***
0 7.46 18.66 37.32 74.65 149.30
%
100
97 I07 I69 I28 I92 100
59 79 83 93 128
Gene-conversion (colonies t S.D.1 38 2 36 + 43 t 792 79f 69t
3 3 7 II 13 15
12.5 c 0.9 12.4 + 1.0 13.4 + 2.0 15.4 f 2.0 20.3 + 3.4 9.9 + 0.9 -0.006 + 0.017 21 .0 2.5 10.0 + 4.6 21.3 + 6.1 4.5 + 2.4 21.9 2 3.0 6.8 f 0.7 -0.051 c 0.036 9.52 2 1.16 10.63 + 2.81 8.52 ? 0.74 8.07 f 1.07 12.10 f 1.53 9.12 + I .84 0.019 0.102 10.30 + 2.08 8.90 1.32 10.75 ? 1.48 11.56 + 0.97 14.52 + 3.14 29. I4 f 5.65 55.72 3. I6 1.623 + 0.067*** 19.66 + 0.00 14.23 f 1.84 13.28 2 2.92 16.61 + 2.80 24.17 + 1.00 43.35 + 2.11 98.57 + 1.43 2.916 + 0.064***
*
24 + 3 7 2 3 19 t 6 4 t 2 23 t 3 102 1
I00 83 79 76 103 96
73 + 9 67 f 18 52 + 5 47 2 6 9 6 f 12 6 7 + 14
I00 I12 I08 99 93 85 65
6 5 2 13 63 + 9 73 2 10 72 t 7 85 f 18 120 2 44 2 2 7 + 13
100
70 f 0 45 f 6 48t II 5 8 2 15 65 2 15 146 2 7 138 + 2
90 101 I I6 99 94 39
T R P + / I Ocells ~ f S.E.
* *
*
Reverse mutation (colonies t S.D.)
ILV+/lOh cells t S.E.
14 + 5 9 f 3 II 2 3 9 + 2 14 + 2 5+1
0.47 f 0.17 0.30 ? 0. I 1 0.34 ? 0.09 0. I8 t 0.04 0.35 f 0.06 0.08 ? 0.02 -0.002 + 0.001** 4.6 t 1.0 3.0 2 2.0 3.7 f 0.3 7.2 t 1.5 4.2 2 0.4 3.2 t 0.7 -0.007 f 0.007 I .46 f 0.20 2.68 t 1.00 1.13 f 0.17 1.46 t 0.09 1.73 + 1.03 2.13 t 0.37 0.035 t 0.033 1.01 t 0.28 1.87 2 0.78 2.81 t 0.58 0.70 + 0. I7 1.05 t 0.55 2.60 f 0.44 2.33 2 0.12 5.09 2 1.30** 2.01 f 0.81 1.99 f 0.33 1.34 f 0.21 1.73 2 0.62 2.13 f 0.59 1.41 f 0.32 5.60 2 1.15 0.129 f 0.018***
II r 2 4 2 3 7+1 14 3
*
9+1 10 + 2
22 + 3 34 + 13 14 + 2 17+ I 28 f 16 31 + 6 13t4 262 II 38 f 8 9 f 2 12 t 6 28 + 5 19t I 14 f 6 13 f 2 10+2 14 2 5 14 + 4 10 f 2 16 + 3
?The number of colony forming units (CFU) as well as the number of gene conversion colonies and reverse mutation colonies + S.D. are shown. The frequencies (fS.E.) of gene conversion and reverse mutation are calculated from the dose response curves by means of linear regression analysis. *0.01 < P < 0.05; **0.001 < P < 0.01; ***P < 0.001.
312 were found to be genotoxic in strain D7, but that 284 gave a decrease in the reverse mutation frequency which was statistically significant ( P < 0.01) in one of the two experiments (Table I). In strain RSI 12, however, both compounds gave a significant ( P < 0.05) increase in the frequency of sister chromatid conversion and 284 also gave a significant increase in the gene conversion frequency at the adenine locus. A 15-fold difference in the spontaneous background levels for the adenine locus in RS 1 12 was seen. This instability of the gene conversion rate at this locus has also been demonstrated by Schiestl et al. [ 19891, who noted a 10-fold difference in the spontaneous frequency between experiments. A variability in the results from the reverse mutation experiments was also seen and was most likely due to difficulties in counting the number of revertant colonies, since the size of the colonies in some cases was extremely
small. A small but significant increase in the number of colony forming units (CFU) was seen in three of the four experiments performed with 284 (Tables I, 11). Camptothecin gave much more pronounced effects. It gave a clear reduction in CFU ( P < 0.001) in both yeast strains. Camptothecin induced gene conversions, I .6-2.9 TRP+/106 cells per p M ( P < O.OOl), and reverse mutations 0.1-5.1 ILV'I lo6 cells per pM in strain D7. The frequency of sister chromatid conversions in strain RS 1 12 was increased significantly ( P < O.OOl), by camptothecin, corresponding to between 53 and 73 conversions/106 cells per p M . No reproducible statistically significant effects were noted in the two Salmone[lu tester strains TA98 and TA 100 with 284 or 312 (data not shown). In the combined mutagenicity tests on Salmonella with BaP a significant ( P < 0.001) reduction in the BaP mutagenicity was seen
293
Tryptophan Photoproducts Are Antimvtagenic
TABLE II. Genotoxicity Tests of Compounds 284, 312 and Camptothecin in Succuromyces cerevisiue RS112t
Conc. Compound
(uM)
284 Exp. I
0 7.46 18.66 37.32 74.65 149.30
Slope 284 Exp. 2.
Slope 312
Slope Camptothecin Exp. 1
Slope Camptothecin Exp. 2
SloDe
0 7.46 18.66 37.32 74.65 93.31
0 0.68 1.71 3.42 6.84 13.68 0 0.12 0.34 I .06 3.19 9.56 28.71 0 0.12 0.34 1.06 3.19 9.56 28.71
Survival (CFU t S.D.) ll0t II 121 f 17 224 f 20 179 t 16 150 t 70 1 4 4 t 10 0.00 t 0.22 120 f 10 9 9 t 15 119 f 19 105 f I 133 6 133 t 15 0.26 f 0.09* 192 f 26 1 6 6 t 13 202 f 14 173 2 14 163 t 15 171 f 7 - 1.34 t 0.97 105 f 12 101 f 13 l l 0 t 12 88 f 5 106 f 2 902 13 38 f I -2.24 t 0.25*** 83 t 4 108 f 12 113t 5 89 t 6 106 t 14 91 f 18 45 t 7 - 1.88 t 0.33***
*
%
100
I10 204 I63 I36 131 100
121 99 88 Ill Ill 100
86 105
90 85 89
Sister chromatid conversion (colonies t S.D.) -
Sister chromatid conversion (His+/lO" cells t S.E.)
274 t 14 346 t 21 280 k 14 309 t 70 385 ? 22 481 t 16
495 f 25 573 35 250 f 13 346 2 79 512 k 29 670 f 23 I .SO f 0.59* 347 t 22 419 f 15 408 t 8 449 k 34 377 f 26 555 f 20 1.51 f 0.45** 280 f 6 295 f 17 284 t 12 322 t 27 366 f 5 316 t 3 3.29 f 1.45** 1396 ? 216 1603 t 241 1400 f 184 1875 t 172 1408 t 67 1862 t 290 3640 t 161 73.16 t 6.32*** 1325 t 568 579 ? 38 647 t 51 2318 t 237 1701 t 283 1571 f 122 2757 f 80 53.43 t 13.54***
*
208 1 3 208 t 8 242 t 5 2 3 6 t 18 250 t 17 368 t 13 269 f 6 245 t 14 287 12 278 23 298 t 4 270 3
* * *
86 36
147 t 23 161 2 24 154 t 20 165 t 15 150 4 7 167 t 26 138 t 6
I00 I30 I36 107 I28 I10 54
I10 f 47 62 t 4 73 t 6 206 t 21 180 t 30 143 f I I 125 t 4
100
96 I05
84 101
*
Gene conversion (colonies t S . D . ) 54 t 6 54 t 7 34 k 6 5 4 t 13 42 5 86 t 50
*
1084 1015
1096 t 102 I I10 f 72 I220 2300 76 1 713 729 719 653 645 5 3 f 16 52 t 8 53 t 7 48 t 5 50 t 4 41 t 8 22 f 2 90 t 7 9 2 2 13 I17 t I I 71 f 12 8 6 2 15 103 t 15 41 t 9
Gene conversion (ADE+/IO' cells t S.E.) 9.8 f 1.2 9.0 t 1 . 1 3.1 2 0.5 6.1 t 1.4 5.5 f 0.7 18.3 f 2.1 0.06 t 0.02** 181 205 141 2 13 211 f 14 I84 347 1.47 f 0.52* I59 171 144 I67 I60 151
-0.64 t 0.91 101 t 30
104t 15 9 6 2 13 110 t 12
93 t 8 91 t 18 118t I I 0.49 0.37 216 2 16 171 f 2 4 207 f 19 160 t 27 162 k 28 226 f 33 181 t 39 0.01 t 0.79
*
?The number of colony forming units (CFU) as well as the number of sister chromatid conversion colonies and gene conversion colonies t S.D. are shown. The frequencies (+S.E.) of sister chromatid conversion and gene conversion are calculated by means of linear regression analysis. *0.01 < P < 0.05; **0.001 < P < 0.01; ***P< 0.01.
(Fig. I ) . The reduction was approximately 20 revertants per nmol of 284. For comparison, the reduction in mutagenicity by the known antimutagenic compounds ellipticine and 9-hydroxyellipticineis also shown in Fig. 1 . In Figure 2, the modulating effect of 312 on the BaP mutagenicity with strains TA98 and TAIOO can be seen. A much less pronounced decrease in mutagenicity was seen with 3 12 compared to 284. The reduction is statistically significant only with tester strain TA 100. The frequency of SCE in CHO cells after treatment with five different nitrogen heterocycles is shown in Table 111. All nitrogen heterocycles caused a statistically significant increase in SCE in the CHO cells. The highest increase, 104 SCE/cell per pM, was seen with camptothecin and the lowest effects were seen with rutaecarpine and dehydrorutae-
carpine which gave an increase in SCE frequency of 0.2 or less. The tryptophan derivatives gave an increase of between 3 and 7 SCE per p M during these treatment conditions. The effect of 284 on the cytochrome P450-dependent EROD activity was tested using rat hepatocytes (Fig. 3). Compound 284 reduced the activity both in the absence of cAMP/IBMX and in the presence of cAMP/IBMX. The reduction was approximately 15.5 nmol/min per pM of 284 in the absence of CAMP. The dose response curve in the presence of CAMPhad a similar slope. In order to study the specific inhibition of cytochrome P450IA1 the mutagenic effects on BPD in XEM2 cells were studied in the presence of 284 and 312. In the preliminary experiments with the XEMd-Mz cells, expressing cytochrome P450IA2, a much lower effects was seen with
294
Rannug et al. TAlOO
1 8 TA98
I
TABLE Ill. Sister Chromatid Exchange Frequency in CHO-cells After 2 or 4 hr Treatment With the Tryptophan Photoproducts 284 and 312 and Two Other Nitrogen Heterocvcles. RutaecarDine and 7.8-dihvdrorutaecar~inet
1000
Treatment time (hrl
Concentration o*M)
Mean frequency of SCE f SD
284
2
0 0.12 0.37 1.23
Slope 284
4
0
600
312
4
400
Slope Rutaecarpine
4
0 8. I 27.0 54.0
Slope Dehydrorutaecarpine
4
0 2.7 8.1 27.0
Slope Carnptothecin
4
9.02 f 0.83 I I .86 f 0.60 12.38 t 0.63 13.62 2 1.08 2.81 t 0.57***." 9.58 f 3.10 14.88 f 1.48 10.26 1.42 I I .32 2 0.47 11.14 f 0.89 12.00 2 0.41 6.50 f 2.76*." 8.60 t 2.46 10.40 f 3.15 12.18 f 3.37 12.10 f 1.86 0.06 f O.OI***." 8.46 t 2.42 I I .30 3.09b I I .57 f 3.48' 14.20 t 4.52 0.19 ? 0.03***." 8.98 f 0.77 12.12f 1.59 19.02 f 1.81 24.34 f 3.02 104.2 f I I .8***."
ComDound
800
rev./plate
T 200
0
I
I
0
5
10
15
20
nmol Fig. 2. Antimutagenic effects of photoproduct 3 12 in Salmonella r v p h imurium TAlOO and TA98 in the presence of 20 nmol BaP with 5 8 S9.
BPD. Therefore, a reduction in the effects in the presence of 284 and 3 12 was difficult to establish. For comparison, two other high affinity Ah receptor ligands, rutaecarpine and dehydrorutaecarpine, were tested simultaneously. As can be seen from Table IV, all compounds showed an antimutagenic effect in the XEM2 cells. No such decrease in mutagenicity was seen with 284 and 312 when tested together with the corresponding diol epoxide of BaP (data not shown).
DISCUSSION The results from the genotoxicity tests based on different genetic endpoints in both prokaryotic and eukaryotic systems indicate that the tryptophan photoproducts 284 and 3 12 do not induce point mutations. This conclusion receives further support from AHH induction experiments on rat hepatoma cells in vitro [Rannug and Rannug, 19891. In these experiments no increase in the number of 6-TG resistant cells could be observed after the 24 hr treatment with 284 (unpublished results). All the nitrogen heterocycles have also been tested for SCE in CHO cells in the absence of
I .23 0 0.03 0.11 0.21
0 0.015 0.072 0.144
Slope
*
*
?The topoisomerase I inhibitor camptothecin was used as a positive control. "No. of SCE's per FM 2 SE. '10 cells scored. '35 cells scored. *0.01 < P < 0.05; **0.001 < P < 0.01; ***P < 0.01.
an exogenous metabolizing system. Although a statistically significant dose dependent increase in SCEs could be noted, the effects of 284, 312, rutaecarpine, and dehydrorutaecarpine were low, at least one order of magnitude lower compared to the positive control camptothecin. Further experiments with somewhat higher treatment concentrations of 284 and 3 12 on strains XEM2 and XEMd-Mz also showed a small but statistically significant increase in the SCE frequencies (data not shown). It can therefore be concluded that these substances induce a small increase in the frequency of SCE in different mammalian cells and that this induction seems to be independent of cytochrome P450 activity. Together with the small increase in sister chromatid conversions in yeast the results point to a low but specific genotoxic potency, related to changes on the sister chromatid level only. Both substances are antimutagenic, at least with certain cytochrome P450-dependent mutagens such as BaP. The inhibition of EROD activity in rat hepatocytes shown with
Tryptophan Photoproducts Are Antimutagenic
295
284 as well as the antimutagenic effect in XEM2 cells of both 284 and 312 point to an inhibition of cytochrome P450IA 1 . Also the other two nitrogen heterocycles, rutaecarpine and dehydrorutaecarpine, showed the same effects as 284 and 3 12, i.e., a small increase in SCE and antimutagenic effects towards BPD in XEM2 cells. Our results indicate that within the group of nitrogen heterocycles there are some with quite unique biological properties. This can be exemplified by the most potent of the two tryptophan derivatives, i.e., 284. This substance has a high Ah receptor affinity, in fact, one order of magnitude higher than that of TCDD [Rannug et al., 1987; Rannug and Rannug, 19891. It induces AHH activity at concentrations one order of magnitude lower than (3-naphthoflavone, although the maximal extent of induction is not extremely P d P high [Rannug and Rannug, 19891. One possible explanation CO €0 CO N 53 el el of the modest induction potential can be that the compound occupies the enzyme and thereby inhibits the B(a)P-hydroxylase activity. This is further indicated by the inhibition of N N ‘u EROD activity and the antimutagenic effects towards BPD 0 in mammalian cells. The substance thus seems to inhibit the Fig. 3. Inhibition of ethoxyresorufin 0-deethylase (EROD) activity in Same cytochrome p450 isoenzyme that it induces. primary hepatocytes from male Sprague Dawley rats in the presence of the increased number of cells in three out of four experidifferent concentrations of photoproduct 284. The incubations were per- ments may indicate that the substance also may increase cell formed with or without the addition of N6,2’-O-dibutyryladenosine3’5’- survival or cell division.
z
T
5.
2.
8
cyclic monophosphate (CAMP)and 3-isobutyl-I-methyl-xanthine(IBMX)
TABLE IV. The Antimutagenic Effects in XEM2 Cells (hprt Locus) Towards 0.1 FM Benzo(a)pyrene-trans(-)7,8-dihydrodiol (BPD) of the Tryptophan Derived Compounds 284 and 312t Concentration Compound Experiment I 0 BPD BPD 284
+
BPD
BPD
+ Rutaecarpine
+ Dehydrorutaecarpine
(FM)
(a)
Frequency of 6-TG resistant cells x lo6
CEF
mutants
100 145
5 I73 25
83 70 93 82 77
102 130 134 119 97 147 179 130
1.0? 0.7 50.2 ? 5.0 5.2 f 1.9 0.2 2 0.4 4.4 1.7 1.6 f 1 . 1 0.8 f 0.8 3.0 2 1.2 3.0 f 1.9 0.8 f 0.8 2.4 f 2.1
100 82 I06 95 91 106 90 86 97 82 84
167 163 169 147 223 252 245 155 226 176 263
0.2 2 0.4 52.4 ? 3.4 7.6 -C 2.3 2.0 2 1.6 3.8 f 3.1 48.8 f 2.3 33.4 4.7 23.8 f 1.3 0 0 4.0 2.5
I I60 22 7 8 97 68 77 0 0 8
100
0.03 0.08 0.24 0.3 1.o 3.0 0.3 1.o 3.0
No. of
Survival
89 78 77 77 87
104
*
1
17 6 3 15 10
2 9
Experiment I1
0 BPD BPD
+ 284
BPD
+ 312
BPD
+ Dehydrorutaecarpine
0.03 0.08 0.24 0.003 0.01 0.03 0.33 1.o 3.0
*
*
tFor comparison the effects of the plant derived compounds rutaecarpine and 7,8-dehydrorutaecarpine are also shown.
296
Rannug et al.
The biological effects of these compounds must be studied in more detail, since the result so far indicate biologically important properties. It is reasonable to postulate that these naturally occurring heterocycles and tryptophan derivatives constitute an important class of biological signal substances involved not only in mutagenicity and carcinogenicity, but also in normal cell proliferation and cell differentiation.
ACKNOWLEDGMENTS We would like to thank Dr. I. Klasterska for valuable discussions and Mrs. Yvonne Eklund, Irja Hardling, MariAnn Westman, and Margareta Wihlborg for skillful technical assistance. This work was supported by grants from the Swedish Cancer Society and the Swedish Natural Science Research Council.
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Accepted by-
R.R. Tice
