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Orthophenylphenol mutagenicity in a human cell strain
Mutation Research, 156 (1985) 123-127
123
Elsevier MTR 00958
Orthophenylphenol mutagenicity in a human cell strain Hideko Suzuki *, Nobuo Suzuki 1,**, Mieko Sasaki and Kogo Hiraga Department of Toxicology, Tokyo Metropolitan Research Laboratory of Public Health, 24-1, Hyakunincho 3 chome, Shinjuku-kt6 Tokyo 160 (Japan) and I Department of Microbiology, School of Medicine, Chiba University,lnohana, Chiba 280 (Japan) (Received 24 October 1984) (Accepted 30 October 1984)
Summary Orthophenylphenol (OPP), a widely used fungicide, induced ouabain-resistant (OuaR) mutants in a ultraviolet (UV)-sensitive human RSa cell strain and the frequencies increased in a dose-related fashion. OPP was a more potent mutagen than UV at doses related to equal survival. These results suggest that OPP has a mutagenic activity and that further experiments on this chemical are warranted.
Orthophenylphenol (OPP) and its sodium salt (OPP-Na) are broad spectrum antimicrobials with a variety of applications, including the protection of edible crops from fungal contamination. OPP is reportedly neither teratogenic in rats nor mutagenic in mice (Kaneda et al., 1978). In the Ames Salmonella test, OPP and OPP-Na were not mutagenic (Kojima and Hiraga, 1978). However, Hanada (1977) found that OPP is positive in the rec-assay with Bacillus subtilis and mutagenic for E. coli and Salmonella typhimurium. Moreover, OPP-Na was found to be carcinogenic in rats (Hiraga and Fujii, 1981). The detection of mutations in DNA-repair-deficient human cells may be a useful tool for the mutagenic screening of suspect environmental agents (Stich et al., 1973). Cells from xeroderma pigmentosum (classical XP) patients, with hypersensitivity to UV killing and defects in DNA-repair (Cleaver, 1970; Lehmann et al., 1975) are reported to show enhanced frequencies of UV-induced mutations, although at equally cytotoxic * To whom requests for reprints should be addressed. ** Present address: Department of Biochemistry, School of Medicine, Chiba University, Inohana, Chiba 280 (Japan).
doses, the frequencies in classical XP strains are approximately equal to those in normal strains (Maher and McCormick, 1976). However, classical XP cells are difficult to obtain and to handle. Thus, we attempted to detect other strains with a high UV-sensitivity and poor capability of DNArepair synthesis, from already established cell lines (Suzuki and Fuse, 1981; Suzuki et al., 1982). In the present work, a human cell strain, RSa, with a high UV-sensitivity and low DNA-repair activity (Suzuki and Fuse, 1981; Suzuki, 1984a) was used and we attempted to determine whether or not OPP is mutagenic, in comparison with the mutagenicity of UV. Materials and methods
Chemicals OPP was obtained from Tokyo Kasei Co., Ltd., Tokyo. The compound was dissolved in cold 100% ethanol immediately before use and within 20 min was placed in individual culture dishes. The final concentration of ethanol in the medium was less than 0.5%. Ouabain was purchased from Sigma Chemical Co., St. Louis, MO.
0165-1218/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)
124
Cell culture RSa, a human cell strain, was derived from the human embryo (Suzuki and Fuse, 1981; Suzuki, 1984a). The cells were grown in Eagle's minimum essential medium (Gibco, Grand Island, NY) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Pel-freeze Biologicals, Rogers, AR), penicillin (100 units/ml), and streptomycin (100 /~g/ml) in a humidified atmosphere of 5% CO2 in air at 37°C. FBS of the same lot number was used. Cells were removed for subculturing with 0.25% trypsin in calcium- and magnesium-free phosphate-buffered saline (PBS). When cloning efficiency and mutation frequency were examined, the FBS concentration in the culture medium was increased to 15%.
Mutagenicity assay Cells were plated at a density of 8 x 105 cells/100-mm culture dish; attachment to the dishes was evident ate24 h and the preparations were then irradiated, as described (Suzuki and Fuse, 1981) or treated with OPP. In the case of the latter treatment, the culture medium was replaced with serum-free medium containing appropriate concentrations of OPP. After 24 h, the medium with OPP was removed and the cells were washed twice with PBS. The plates were then replenished with culture medium and incubated at 37°C for various expression times. When the plates became confluent before the end of the expression period, the cells were trypsinized and replated at a low cell density to continue growth in culture medium. At the end of the expression period, cells were trypsinized and replated for the determination of cytotoxicity and mutagenicity. Cytotoxicity was determined by plating 1.5 x 103 cells/100-mm culture dish (5 dishes for each point). After 10 days, the colonies were fixed and stained with a 30% methanol-water solution containing 0.2% (w/v) methylene blue. Colonies containing 50 or more cells were counted and survival was expressed as a percentage of solvent control. The cloning efficiencies of the control cells averaged 6-9%. For mutagenicity, cells were plated at a density of 5 X 10 4 cells/100-mm culture dish (6 dishes for each point) in medium containing 10- 7 M ouabain.
This cell density has been found to be optimal for the recovery of mutants. After 7 days the medium was changed, and then 7 days later the cultures were fixed and stained as described above. Colonies containing 50 or more cells were counted. The mutant frequency was determined by dividing the total number of mutant colonies by the total number of cells plated, corrected by the cloning efficiency and expressed as mutants/104 cells. Results were expressed as the mean of values obtained from 2 independent experiments.
Characterization of ouabain-resistant (Oua R) colonies Colonies obtained in mutagenicity assay were obtained by punching out each colony with a glass stamp and were characterized. Cells were grown in drug-free medium until a suitable number had been achieved (usually 20 generations of growth), and then tested for their resistance to ouabain by measuring their growth in different concentrations of ouabain (Mankovitz et al., 1974) and for their N a + / K + ATPase activities. Under these conditions, parental RSa cells had less than 0.1% of growth in 10 -7 M ouabain when compared to growth in ouabain-free medium. Cells with over a 10% growth in 10 -7 M ouabain relative to that in the ouabain-free plate were considered to be phenotypically resistant. N a + / K + ATPase activities in membrane preparations of Oua R and parental cells were measured, essentially by the method of Robbins and Baker (1977). Briefly, cells were suspended in 1 mM Tris-EDTA, pH 7.4, treated with sodium deoxycholate and centrifuged at 10 000 × g for 10 min at 4°C. The supernatant fractions were further centrifuged at 77000 x g for 14 h at 4°C, and the pellet was subjected to Dounce homogenization. The activities of N a + / K + ATPase in the homogenized membrane preparations were measured at 37°C for 10 min with a standard medium containing 30 mM histidine (adjusted to pH 7.1 with Tris), 100 mM NaC1, 10 mM KC1, 5 mM MgC12, and 1 mM ['/-32p]ATP (13 Ci/mmole, New England Nuclear). Experiments in which the effect of ouabain was tested were performed after preincubation of the membrane preparations with ouabain for 15 min at 37°C. The reaction was terminated by addition of ice-cold medium con-
125 taining 100 m M HC1, 1 m M orthophosphate, .1 m M pyrophosphate, 0.6 mg of bovine serum albumin and 120 mg of activated charcoal. Specific activity of N a + / K + ATPase was expressed as /~moles of released P i / m g protein of the homogenized preparation/h. Protein determinations were according to the method of Lowry et al. (1951) using bovine serum albumin as a standard. Results and discussion
-
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;
50
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20
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Fig. 1 shows the influence of mutation expression time on the frequency of mutants induced by UV at doses of 6 and 9 J / m 2. For both doses, the mutant frequency reached a maximum after 2 days and then declined slowly. Therefore, the period of expression selected for following experiments was 2 days. Fig. 2 shows the effects of UV on survival and mutant frequencies of RSa cells. The mutant frequencies increased with increasing doses of UV light. With 9 J / m 2 UV irradiation, the mutant frequency increased sharply ( 5 9 . 5 0 u a R mutants per 10 4 survivors). The mutant frequency in the unirradiated RSa cells was 2.0 Oua R mutants per 10 4 survivors. Thus, RSa cells seem to have a high mutability. RSa cells were next tested for the inducibility of Oua R mutants and susceptibility to cytotoxicity by OPP. The curves of mutation expression time at concentrations of 20 and 3 0 / t g / m l OPP are de-
40
g 2o
O(
I
3
I
I
6 UV ( J i m 2 )
9
Fig. 2. Cytotoxicityand mutagenicityof UV in RSa cells. picted in Fig. 1. The optimal expression time was 2 days for both concentrations of OPP, as was the case for UV. With the 2 days expression time, OPP treatment induced a linear increase in the frequency of Oua R mutants over the tested concentration range (15-30 # g / m l ) and concomitant decrease of the cloning ability, as shown in Fig. 3.
loo 1
-I
150
20
150
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> 100 ¢o
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IE
.
50
~E
0
1
2
3
4
Expression Time (days)
Fig. 1. Expression time for the induction of OuaR mutants in RSa cells by UV (n, 6 J/m2; I, 9 J/m 2) and OPP (z~, 20 ~g/ml; A, 30 ~g/ml).
I
OI
o
I
10 20 OPP ( u g / m l )
I
30
Fig. 3. Cytotoxicityand mutagenicityof OPP in RSa cells.
126 =
150
• -~
100
~
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I
100
I
I
I
l
50 Survival
I
I
20
Fig. 4. Equitoxic comparison of OuaR mutants induced by OPP and UV.
With a concentration of 30 ~tg/ml, the mutant frequency was approximately 100 times of controls ( 1 . 5 0 u a R mutants per 104 survivors). An equitoxic comparison of mutant frequencies in RSa cells after treatment with UV and with OPP is depicted in Fig. 4. OPP appeared more efficient than UV in inducing mutants in RSa cells, at doses giving equal survival. 20 colonies, both spontaneously and UV- or OPP-induced, were tested for ouabain resistance (Oua R) after growth in the absence of the selective agent. Using the criteria described in Materials and methods section, all the colonies appeared phenotypically resistant (10-80% survival) (data not shown). When the dose of ouabain required to inhibit growth to 10% was determined, the colonies were 13-700 times more resistant than were the parental cells (7 × 10 -8 M). Resistance to ouabain is probably the result of an alteration of membrane N a + / K ÷ ATPase, to the extent that it is no longer sensitive to ouabain (Baker et al., 1974). The Oua R and parental cells were assayed for their membrane N a + / K + ATPase in the absence and presence of ouabain, and activities of the enzyme in cells of each colony were obtained from 4 independent experiments. In the absence of ouabain, the activity of N a + / K + ATPase in membrane preparations of cells of each Oua g clone was similar to that of the parental cells: in the range 3 . 8 _ 0.7 (S.E.) /~moles P i / m g protein/h. In the
presence of 10 -6 M ouabain, N a + / K ÷ ATPase activity in parental cells remained 40 + 5% (S.E.), when compared with that of controls not exposed to ouabain. In concurrent experiments, cells of an Oua R clone, had 80 + 5% (S.E.) of the control N a + / K ÷ ATPase activity. Further, in the presence of 10 - 3 M ouabain, the activity in the parental cells was 30 + 4% (S.E.) of the controls, while that in the Oua R clone cells was 50 + 2% (S.E.). Similar results were obtained for the other Oua R clones in comparison to the parental cells. Thus, the ATPase activities in the Oua g cells examined were less sensitive to ouabain inhibition than was that of the parental ceils. In the present study, we showed that UV had a mutagenic activity on human RSa cells. It seems likely that RSa cells have a high mutability (mutant frequency is in the order of 1 0 - 4 ) , because, in HeLa cells, we found no evidence of Oua R cells, under the present assay conditions. Why RSa cells have a high mutability remains to be determined. In E. coli strain which is highly sensitive to UV killing and defective in excision repair, damages to D N A induce error-prone repair leading to mutation (Witkin, 1976). UV-sensitive RSa cells have a poor capability of DNA-repair synthesis, in comparison with various human cell strains (Suzuki and Fuse, 1981; Suzuki, 1984a; Suzuki et al., 1984b). DNA-repair synthesis is thought to monitor so-called excision repair (Suzuki and Fuse, 1981; Suzuki, 1984a). Thus, in the RSa cells, DNA alteration induced by UV might induce error-prone repair, and result in elevating the frequency of mutants. It has been suggested that OPP binds to DNA (Ushiyama et al., 1983). Furthermore, one of the metabolites of OPP, phenylhydroquinone, reportedly causes dose-related chromosome damages in cultured Chinese hamster CHO-K1 cells (Yoshida et al., 1981). Therefore, OPP may damage DNA, as is the case for UV. If so, such OPP effects may result in elevating the frequency of mutants in OPP-treated RSa cells. As mentioned above, OPP has been found to be mutagenic in tests using bacteria. However, there have been no reports on the mutagenicity of OPP on mammalian cells. To our knowledge, this is the first report of OPP-induced mutants in human cells.
127
Acknowledgement W e t h a n k M. O h a r a ( K y u s h u U n i v . ) for r e a d i n g the manuscript.
References Baker, R.M., D.M. Brunette, R. Mankovitz, L.H. Thompson, G.F. Whitmore, L. Siminovitch and J.E. Till (1974) Ouabain-resistant mutants of mouse and hamster cells in culture, Cell, 1, 9-21. Cleaver, J.E. (1970) DNA repair and radiation sensitivity in human (xeroderma pigmentosum) cells, Int. J. Rad. Biol., 18, 557-565. Hanada, S. (1977) Studies on food additives diphenyl and o-phenylphenol from standpoint of public health, Part (2) toxicological studies of diphenyl and o-phenylphenol, Nagoya-shiritsu Daigaku Igakkai Zasshi, 28, 983-995 (in Japanese). Hiraga, K., and T. Fujii (1981) Induction of turnouts of the urinary system in F344 rats by dietary administration of sodium o-phenylphenate, Food Cosmet. Toxicol., 19, 303-310. Kaneda, M., S. Teramoto, A. Shingu and Y. Shirasu (1978) Teratogenicity and dominant-lethal studies with o-phenylphenol, J. Pestic. Sci., 3, 365-370. Kojima, A., and K. Hiraga (1978) Mutagenicity of citrus fungicides in the microbial system, Annu. Rept. Tokyo Metr. Res. Lab. P.H., 29-2, 83-85 (in Japanese). Lehmann, A.R., S. Kirk-Bell, C.F. Arlett, M.C. Paterson, P.H.M. Lohman, E.A. de Weerd-Kastelein and D. Bootsma (1975) Xeroderma pigmentosum cells with normal levels of excision repair have a defect in D N A synthesis after UVirradiation, Proc. Natl. Acad. Sci. (U.S.A.), 72, 219-223. Lowry, O.H., N.J. Rosebrough, A. Farr and R.I. Randall (1951) Protein measurement with the folin phenol reagent, J. Biol. Chem., 193, 265-275.
Maher, V.M., and J.J. McCormick (1976) Effect of DNA repair on the cytotoxicity and mutagenicity of UVoirradiation and of chemical carcinogens in normal and xeroderma pigmentosum cells, in: J.M. Yuhas, R.W. Tennat and J.B. Regan (Eds.), Biology of Radiation Carcinogenesis, Raven, New York, pp. 129-145. Mankovitz, R., M. Buchwald and R.M. Baker (1974) Isolation of ouabain-resistant human diploid fibroblasts, Cell, 3, 221-226. Robbins, A.R., and R.M. Baker (1977) (Na,K)ATPase activity in membrane preparations of ouabain-resistant HeLa cells, Biochemistry, 16, 5163-5168. Stich, H.F., R.H.C. San and Y. Kawazoe (1973) Increased sensitivity of xeroderma pigmentosum cells to some chemical carcinogens and mutagens, Mutation Res., 17, 127-137. Suzuki, N., and A. Fuse (1981) A UV-sensitive human clonal cell line, RSa, which has low repair activity, Mutation Res., 64, 133-145. Suzuki, N., J. Nishimaki and T. Kuwata (1982) Characterization of a UV-resistant strain, uvr-10, established from a human clonal cell line, RSb, with high sensitivity to UV, 4NQO, MNNG and interferon, Mutation Res., 106, 357-376. Suzuki, N. (1984a) A UV-resistant mutant without an increased repair synthesis activity, established from a UV-sensitive human clonal cell line, Mutation Res., 125, 55-63. Suzuki, N., T. Kojima, T. Kuwata, J. Nishimaki, Y. Takakubo and T. Miki (1984b) Cross-sensitivity between interferon and UV in human cell strains; IF r, HEC-1 and CRL1200, Virology, 135, 20-29. Ushiyama, K.,'J¢~Kabashima, F.'Nagai, H. Ichikawa, I. Kano and T. Nakao (1983) Interactions of orthophenylphenol with DNA in vitro in the presence of liver microsomes, Seikagaku, 55-8, 1018 (in Japanese). Witkin, E.M. (1976) Ultraviolet mutagenesis and inducible DNA repair in Escherichia coli, Bacteriol. Rev,, 40, 869-907. Yoshida, S., M. Sasaki, T. Nakao and K. Hiraga (1981) Chromosome preparations from CHO-K1 cells used nunc plate, Annu. Rept. Tokyo Metr. Res. Lab. P.H., 32-2, 111-114.
123
Elsevier MTR 00958
Orthophenylphenol mutagenicity in a human cell strain Hideko Suzuki *, Nobuo Suzuki 1,**, Mieko Sasaki and Kogo Hiraga Department of Toxicology, Tokyo Metropolitan Research Laboratory of Public Health, 24-1, Hyakunincho 3 chome, Shinjuku-kt6 Tokyo 160 (Japan) and I Department of Microbiology, School of Medicine, Chiba University,lnohana, Chiba 280 (Japan) (Received 24 October 1984) (Accepted 30 October 1984)
Summary Orthophenylphenol (OPP), a widely used fungicide, induced ouabain-resistant (OuaR) mutants in a ultraviolet (UV)-sensitive human RSa cell strain and the frequencies increased in a dose-related fashion. OPP was a more potent mutagen than UV at doses related to equal survival. These results suggest that OPP has a mutagenic activity and that further experiments on this chemical are warranted.
Orthophenylphenol (OPP) and its sodium salt (OPP-Na) are broad spectrum antimicrobials with a variety of applications, including the protection of edible crops from fungal contamination. OPP is reportedly neither teratogenic in rats nor mutagenic in mice (Kaneda et al., 1978). In the Ames Salmonella test, OPP and OPP-Na were not mutagenic (Kojima and Hiraga, 1978). However, Hanada (1977) found that OPP is positive in the rec-assay with Bacillus subtilis and mutagenic for E. coli and Salmonella typhimurium. Moreover, OPP-Na was found to be carcinogenic in rats (Hiraga and Fujii, 1981). The detection of mutations in DNA-repair-deficient human cells may be a useful tool for the mutagenic screening of suspect environmental agents (Stich et al., 1973). Cells from xeroderma pigmentosum (classical XP) patients, with hypersensitivity to UV killing and defects in DNA-repair (Cleaver, 1970; Lehmann et al., 1975) are reported to show enhanced frequencies of UV-induced mutations, although at equally cytotoxic * To whom requests for reprints should be addressed. ** Present address: Department of Biochemistry, School of Medicine, Chiba University, Inohana, Chiba 280 (Japan).
doses, the frequencies in classical XP strains are approximately equal to those in normal strains (Maher and McCormick, 1976). However, classical XP cells are difficult to obtain and to handle. Thus, we attempted to detect other strains with a high UV-sensitivity and poor capability of DNArepair synthesis, from already established cell lines (Suzuki and Fuse, 1981; Suzuki et al., 1982). In the present work, a human cell strain, RSa, with a high UV-sensitivity and low DNA-repair activity (Suzuki and Fuse, 1981; Suzuki, 1984a) was used and we attempted to determine whether or not OPP is mutagenic, in comparison with the mutagenicity of UV. Materials and methods
Chemicals OPP was obtained from Tokyo Kasei Co., Ltd., Tokyo. The compound was dissolved in cold 100% ethanol immediately before use and within 20 min was placed in individual culture dishes. The final concentration of ethanol in the medium was less than 0.5%. Ouabain was purchased from Sigma Chemical Co., St. Louis, MO.
0165-1218/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)
124
Cell culture RSa, a human cell strain, was derived from the human embryo (Suzuki and Fuse, 1981; Suzuki, 1984a). The cells were grown in Eagle's minimum essential medium (Gibco, Grand Island, NY) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Pel-freeze Biologicals, Rogers, AR), penicillin (100 units/ml), and streptomycin (100 /~g/ml) in a humidified atmosphere of 5% CO2 in air at 37°C. FBS of the same lot number was used. Cells were removed for subculturing with 0.25% trypsin in calcium- and magnesium-free phosphate-buffered saline (PBS). When cloning efficiency and mutation frequency were examined, the FBS concentration in the culture medium was increased to 15%.
Mutagenicity assay Cells were plated at a density of 8 x 105 cells/100-mm culture dish; attachment to the dishes was evident ate24 h and the preparations were then irradiated, as described (Suzuki and Fuse, 1981) or treated with OPP. In the case of the latter treatment, the culture medium was replaced with serum-free medium containing appropriate concentrations of OPP. After 24 h, the medium with OPP was removed and the cells were washed twice with PBS. The plates were then replenished with culture medium and incubated at 37°C for various expression times. When the plates became confluent before the end of the expression period, the cells were trypsinized and replated at a low cell density to continue growth in culture medium. At the end of the expression period, cells were trypsinized and replated for the determination of cytotoxicity and mutagenicity. Cytotoxicity was determined by plating 1.5 x 103 cells/100-mm culture dish (5 dishes for each point). After 10 days, the colonies were fixed and stained with a 30% methanol-water solution containing 0.2% (w/v) methylene blue. Colonies containing 50 or more cells were counted and survival was expressed as a percentage of solvent control. The cloning efficiencies of the control cells averaged 6-9%. For mutagenicity, cells were plated at a density of 5 X 10 4 cells/100-mm culture dish (6 dishes for each point) in medium containing 10- 7 M ouabain.
This cell density has been found to be optimal for the recovery of mutants. After 7 days the medium was changed, and then 7 days later the cultures were fixed and stained as described above. Colonies containing 50 or more cells were counted. The mutant frequency was determined by dividing the total number of mutant colonies by the total number of cells plated, corrected by the cloning efficiency and expressed as mutants/104 cells. Results were expressed as the mean of values obtained from 2 independent experiments.
Characterization of ouabain-resistant (Oua R) colonies Colonies obtained in mutagenicity assay were obtained by punching out each colony with a glass stamp and were characterized. Cells were grown in drug-free medium until a suitable number had been achieved (usually 20 generations of growth), and then tested for their resistance to ouabain by measuring their growth in different concentrations of ouabain (Mankovitz et al., 1974) and for their N a + / K + ATPase activities. Under these conditions, parental RSa cells had less than 0.1% of growth in 10 -7 M ouabain when compared to growth in ouabain-free medium. Cells with over a 10% growth in 10 -7 M ouabain relative to that in the ouabain-free plate were considered to be phenotypically resistant. N a + / K + ATPase activities in membrane preparations of Oua R and parental cells were measured, essentially by the method of Robbins and Baker (1977). Briefly, cells were suspended in 1 mM Tris-EDTA, pH 7.4, treated with sodium deoxycholate and centrifuged at 10 000 × g for 10 min at 4°C. The supernatant fractions were further centrifuged at 77000 x g for 14 h at 4°C, and the pellet was subjected to Dounce homogenization. The activities of N a + / K + ATPase in the homogenized membrane preparations were measured at 37°C for 10 min with a standard medium containing 30 mM histidine (adjusted to pH 7.1 with Tris), 100 mM NaC1, 10 mM KC1, 5 mM MgC12, and 1 mM ['/-32p]ATP (13 Ci/mmole, New England Nuclear). Experiments in which the effect of ouabain was tested were performed after preincubation of the membrane preparations with ouabain for 15 min at 37°C. The reaction was terminated by addition of ice-cold medium con-
125 taining 100 m M HC1, 1 m M orthophosphate, .1 m M pyrophosphate, 0.6 mg of bovine serum albumin and 120 mg of activated charcoal. Specific activity of N a + / K + ATPase was expressed as /~moles of released P i / m g protein of the homogenized preparation/h. Protein determinations were according to the method of Lowry et al. (1951) using bovine serum albumin as a standard. Results and discussion
-
~o(~
;
50
"
20
6O ea
a.
o~
Fig. 1 shows the influence of mutation expression time on the frequency of mutants induced by UV at doses of 6 and 9 J / m 2. For both doses, the mutant frequency reached a maximum after 2 days and then declined slowly. Therefore, the period of expression selected for following experiments was 2 days. Fig. 2 shows the effects of UV on survival and mutant frequencies of RSa cells. The mutant frequencies increased with increasing doses of UV light. With 9 J / m 2 UV irradiation, the mutant frequency increased sharply ( 5 9 . 5 0 u a R mutants per 10 4 survivors). The mutant frequency in the unirradiated RSa cells was 2.0 Oua R mutants per 10 4 survivors. Thus, RSa cells seem to have a high mutability. RSa cells were next tested for the inducibility of Oua R mutants and susceptibility to cytotoxicity by OPP. The curves of mutation expression time at concentrations of 20 and 3 0 / t g / m l OPP are de-
40
g 2o
O(
I
3
I
I
6 UV ( J i m 2 )
9
Fig. 2. Cytotoxicityand mutagenicityof UV in RSa cells. picted in Fig. 1. The optimal expression time was 2 days for both concentrations of OPP, as was the case for UV. With the 2 days expression time, OPP treatment induced a linear increase in the frequency of Oua R mutants over the tested concentration range (15-30 # g / m l ) and concomitant decrease of the cloning ability, as shown in Fig. 3.
loo 1
-I
150
20
150
o
'~ lOG c~
&
> 100 ¢o
&
IE
.
50
~E
0
1
2
3
4
Expression Time (days)
Fig. 1. Expression time for the induction of OuaR mutants in RSa cells by UV (n, 6 J/m2; I, 9 J/m 2) and OPP (z~, 20 ~g/ml; A, 30 ~g/ml).
I
OI
o
I
10 20 OPP ( u g / m l )
I
30
Fig. 3. Cytotoxicityand mutagenicityof OPP in RSa cells.
126 =
150
• -~
100
~
5o
I
100
I
I
I
l
50 Survival
I
I
20
Fig. 4. Equitoxic comparison of OuaR mutants induced by OPP and UV.
With a concentration of 30 ~tg/ml, the mutant frequency was approximately 100 times of controls ( 1 . 5 0 u a R mutants per 104 survivors). An equitoxic comparison of mutant frequencies in RSa cells after treatment with UV and with OPP is depicted in Fig. 4. OPP appeared more efficient than UV in inducing mutants in RSa cells, at doses giving equal survival. 20 colonies, both spontaneously and UV- or OPP-induced, were tested for ouabain resistance (Oua R) after growth in the absence of the selective agent. Using the criteria described in Materials and methods section, all the colonies appeared phenotypically resistant (10-80% survival) (data not shown). When the dose of ouabain required to inhibit growth to 10% was determined, the colonies were 13-700 times more resistant than were the parental cells (7 × 10 -8 M). Resistance to ouabain is probably the result of an alteration of membrane N a + / K ÷ ATPase, to the extent that it is no longer sensitive to ouabain (Baker et al., 1974). The Oua R and parental cells were assayed for their membrane N a + / K + ATPase in the absence and presence of ouabain, and activities of the enzyme in cells of each colony were obtained from 4 independent experiments. In the absence of ouabain, the activity of N a + / K + ATPase in membrane preparations of cells of each Oua g clone was similar to that of the parental cells: in the range 3 . 8 _ 0.7 (S.E.) /~moles P i / m g protein/h. In the
presence of 10 -6 M ouabain, N a + / K ÷ ATPase activity in parental cells remained 40 + 5% (S.E.), when compared with that of controls not exposed to ouabain. In concurrent experiments, cells of an Oua R clone, had 80 + 5% (S.E.) of the control N a + / K ÷ ATPase activity. Further, in the presence of 10 - 3 M ouabain, the activity in the parental cells was 30 + 4% (S.E.) of the controls, while that in the Oua R clone cells was 50 + 2% (S.E.). Similar results were obtained for the other Oua R clones in comparison to the parental cells. Thus, the ATPase activities in the Oua g cells examined were less sensitive to ouabain inhibition than was that of the parental ceils. In the present study, we showed that UV had a mutagenic activity on human RSa cells. It seems likely that RSa cells have a high mutability (mutant frequency is in the order of 1 0 - 4 ) , because, in HeLa cells, we found no evidence of Oua R cells, under the present assay conditions. Why RSa cells have a high mutability remains to be determined. In E. coli strain which is highly sensitive to UV killing and defective in excision repair, damages to D N A induce error-prone repair leading to mutation (Witkin, 1976). UV-sensitive RSa cells have a poor capability of DNA-repair synthesis, in comparison with various human cell strains (Suzuki and Fuse, 1981; Suzuki, 1984a; Suzuki et al., 1984b). DNA-repair synthesis is thought to monitor so-called excision repair (Suzuki and Fuse, 1981; Suzuki, 1984a). Thus, in the RSa cells, DNA alteration induced by UV might induce error-prone repair, and result in elevating the frequency of mutants. It has been suggested that OPP binds to DNA (Ushiyama et al., 1983). Furthermore, one of the metabolites of OPP, phenylhydroquinone, reportedly causes dose-related chromosome damages in cultured Chinese hamster CHO-K1 cells (Yoshida et al., 1981). Therefore, OPP may damage DNA, as is the case for UV. If so, such OPP effects may result in elevating the frequency of mutants in OPP-treated RSa cells. As mentioned above, OPP has been found to be mutagenic in tests using bacteria. However, there have been no reports on the mutagenicity of OPP on mammalian cells. To our knowledge, this is the first report of OPP-induced mutants in human cells.
127
Acknowledgement W e t h a n k M. O h a r a ( K y u s h u U n i v . ) for r e a d i n g the manuscript.
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