Mutation Research, 247 (1991) 57-64 © 1991 Elsevier Science Publishers B.V. 0027-5107/91/$03.50 ADONIS 0027510791000648
57
MUT 04941
Diethyldithiocarbamate suppresses the plant activation of aromatic amines into mutagens by inhibiting tobacco cell peroxidase Michael J. Plewa, Shannon R. Smith and Elizabeth D. Wagner Institute for Environmental Studies, University of Illinois at Urbana-Champaign, Urbana, IL 61801 (U.S.A.) (Received 30 May 1990) (Revision received 10 August 1990) (Accepted 20 August 1990)
Keywords: Nicotiana tabacum; 2-Aminofluorene; m-Phenylenediamine; Mutation induction; Antimutagenicity; Plant cell/microbe coincubation assay
Summary Diethyldithiocarbamate is an antimutagen and repressed the activation of promutagens by plant systems. Earlier work implicated the involvement of tobacco cell (TX1) peroxidases in the plant cell activation of aromatic amines. We now present data that diethyldithiocarbamate represses the activation of 2-aminofluorene and m-phenylenediamine by inhibiting intracellular TX1 peroxidases under in vivo conditions. Concentrations of diethyldithiocarbamate that caused a 50% repression of TX1 cell activation of 2-aminofluorene and m-phenylenediamine also induced a 50% inhibition of TX1 cell peroxidase activity. Diethyldithiocarbamate in a concentration range between 25 and 500 /~M directly inhibited peroxidase activity in TX1 cell homogenates in a concentration-dependent manner. Similar results were observed with purified horseradish peroxidase. The kinetics of peroxidase activity were studied in homogenates from control cells and cells treated with 750/~M and 25 mM diethyldithiocarbamate. There was no significant difference among the K m values among the three groups with a mean ( _ standard error) K m of 2.58 + 0.23 raM. However, the Vmax differed from 4.02 to 2.12 nmoles tetraguaiacol/min//~g protein, in the control and in the 25 mM diethyldithiocarbamate treatment group, respectively. These data indicate that diethyldithiocarbamate is a non-competitive inhibitor of TX1 cell peroxidase.
Plant cells in suspension culture can activate promutagenic aromatic amines into mutagens as detected in Salmonella typhimurium (Plewa et al., 1983; Lhotka et al., 1987). The biochemical mechanisms of activation by tobacco cells have been examined. By using specific enzyme inhibitors we reported indirect evidence that peroxidases were involved in the activation of two aromatic amines (Wagner et al., 1989, 1990). Correspondence: Dr. Michael J. Plewa, Professor of Genetics, Institute for Environmental Studies, University of Illinois at Urbana-Champaign, 1101 West Peabody Drive, Urbana, IL 61801 (U.S.A.).
Peroxidase enzymes are ubiquitous in the plant kingdom. Peroxidases may catalyze N- or C-hydroxylation, N-sulfoxidation, N-acetylation, halogenation, dehalogenation or decarboxylation (Sandermann, 1982; Rojanapo et al., 1986). Peroxidative reactions normally proceed as shown below: peroxidase + H 202 ~ peroxidase-I peroxidase-I + AH 2 --* peroxidase-II + AH. peroxidase-II + AH 2 - , peroxidase + AH. 2AH. --* products
where peroxidase-I is designated as FeO 3+, peroxidase-II is designated as FeO 2+, H202 is the
58 stringent substrate for the peroxidase, and AH 2 is a non-specific substrate that functions as a hydrogen donor (Higashi, 1988). Diethyldithiocarbamate was an excellent inhibitor of the tobacco cell activation of 2-aminofluorene and m-phenylenediamine (Wagner et al., 1989, 1990). Diethyldithiocarbamate suppressed the plant activation of several promutagens by Arabidopsis and Tradescantia (Gichner and Veleminsky, 1984; Gichner et al., 1988). Diethyldithiocarbamate, a metal-chelating agent, is an inhibitor of superoxide dismutase (Heikkila, 1985). Depending on the mechanism of action, diethyldithiocarbamate exhibited either toxic or protective effects. Diethyldithiocarbamate potentiated oxygen toxicity as well as the DNA-damaging effects of hydrogen peroxide (Forman et al., 1980; Kleiman et al., 1990). Diethyldithiocarbamate was an efficient free-radical scavenger and inhibited lipid peroxidation (Lutz et al., 1973; Zanocco et al., 1989). The purpose of this research was to determine if diethyldithiocarbamate suppressed the tobacco cell activation of aromatic amines by inhibiting cellular peroxidases. Materials and methods
Preparation of titered TX1 cell suspension The protocols for the preparation of the plant cell suspension have been published (Plewa et al., 1988). The TX1 cells were washed in M X medium, a modified liquid culture medium lacking plant growth hormone. The fresh weight of the cell suspension was adjusted to a final concentration of 100 m g / m l in MX medium.
Preparation of TX1 cell homogenate For the determination of protein content and peroxidase activity, a TX1 cell homogenate was prepared. The washed and titered TX1 cell suspension was placed on ice, disrupted with a PolyTron homogenizer for 45 sec and centrifuged at 15000 × g for 2 rain. An aliquot of the supernatant was frozen at - 8 0 ° C for subsequent protein analysis. The other portion was kept on ice and immediately analyzed for peroxidase activity.
Assay for protein concentration The Bio-Rad protein assay (Bradford, 1976) was used to determine the protein content of TX1 cells. The microassay with a range of 1-25 #g p r o t e i n / m l was used. Lyophilized bovine 7globulin was chosen for the protein standard curves. The conditions for the analysis of protein content were described by Smith et al. (1989).
Chemicals Horseradish peroxidase (CAS No. 9003-99-0), guaiacol (CAS No. 90-05-1) and diethyldithiocarbamic acid, sodium salt (CAS No. 20624-25-3) were purchased from Sigma Chemical Co. (St. Louis, MO). Hydrogen peroxide (CAS No. 772284-1) was purchased from Fisher Scientific (Pittsburgh, PA). The standardized plasma protein bovine "t-globulin (Standard I) and Bio-Rad Dye Reagent Concentrate were purchased from BioRad Chemical Division (Richmond, CA).
Plant cells Long-term
suspension
cultures
of
tobacco
(Nicotiana tabacum), cell line TX1, were maintained by inoculating 3 g fresh weight from 7-day cultures into 100 ml of MX medium, a modified liquid culture medium of Murashige and Skoog (1962). The cultures were grown at 2 8 ° C in the dark with shaking at 150 rpm.
Determination of peroxidase activity To determine peroxidase activity, we measured the oxidation of guaiacol to tetraguaiacol by monitoring the change in absorbance at 470 nm (Maehly and Chance, 1954). The stoichiometry of the assay reaction is given below: 4 guaiacol + 4 H202 --~ 1 tetraguaiacol + 8 H 2 0 Peroxidase activity in 3-day and 7-day TX1 cells was analyzed in a reaction volume of 3 ml containing 50 m M potassium phosphate buffer, pH 7, 1 ml of a 0.3% H202 solution, 1 ml of a 1% guaiacol solution, and 25 /~1 of the TX1 cell homogenate. The cuvettes used as blanks were identical except that M X - medium was substituted for the TX1 cell homogenate. After the addition of the last component (guaiacol), the cuvette was quickly inverted twice, and the reaction was timed. Peroxidase activity was measured over a 5-min
59 time period using a model 552A Perkin-Elmer double-beam spectrophotometer. 3-5 independent replicates were conducted for each measurement within each experiment. Because of the high amount of peroxidase activity in 7-day cells, the amount of the H202 solution was reduced to 100 /~1 for all subsequent experiments with diethyldithiocarbamate. Results
E E
4-00
-
T
T
-1-95~ConfidenceLirnits ~ 300
~- 200
g
rn i
100
0
M - 5 0 mM), suppressed the plant activation of 2aminofluorene and m-phenylenediamine. A concentration of 1 mM diethyldithiocarbamate resulted in a 50% reduction of TX1 cell activation (Wagner et al., 1989, 1990). To determine if diethyldithiocarbamate was impeding plant activation of these promutagens by specifically inhibiting peroxidases, we analyzed the peroxidase activity in
TX1 cells that were exposed to a wide range of diethyldithiocarbamate concentrations under the identical conditions used to measure the inhibition of TX1 cell activation. This approach insured that the genetic and biochemical parameters of the parallel experimental designs could be directly compared. The concentrations of diethyldithiocarbamate used in the in vivo experiments were not toxic to the TX1 cells (Wagner et al., 1989, 1990). As illustrated in Fig. 3, TX1 cells that were exposed in vivo to 250/~M diethyldithiocarbamate had a significant reduction in peroxidase activity from that of the untreated cells. Approximately one-half of TX1 cell peroxidase activity was inhibited by diethyldithiocarbamate concentrations between 750 /~M and 2.5 mM. These same concentrations caused a 50% reduction in TX1 cell activation of 2-aminofluorene and m-phenylenediamine (Wagner et al., 1989, 1990). The reduction in peroxidase activity directly correlates with the suppression of the TX1 cell activation of these two aromatic amines. A significant concentration-dependent reduction in peroxidase activity was observed by adding diethyldithiocarbamate directly to TX1 cell homogenates (Fig. 4). These data confirm that diethyldithiocarbamate can specifically inhibit a TX1 cell peroxidase under in vitro conditions. The inhibitory effect of diethyldithiocarbamate was measured with a well defined enzyme, purified horseradish peroxidase (Fig. 5). Diethyldithiocarbamate significantly inhibited purified horseradish peroxidase at concentrations above 50 #M. The nature of the inhibition of TX1 cell peroxidase by diethyldithiocarbamate was examined (Table 1). The data from the Michaelis-Menten graph are represented in a Lineweaver Burk plot (Fig. 7). From 3 independent experiments, the K m value for the control cells was 2.79_+ 0.50 mM. The K m value for the cells treated with 750 /,M and 25 mM diethyldithiocarbamate was 2.31 _+ 0.27 mM and 2.65 _+ 0.62 mM, respectively. The mean K m value for all the groups was 2.58 _+ 0.23 mM. The K m values among the control and treated groups did not differ significantly while the Vmax values were different (Table 1). These data indicate that diethyldithiocarbamate is a non-competitive inhibitor of TX1 cell peroxidase.
63 0.9
o
#-
o.6
E ~
0.3
0.0
-0.8
-0.3
'
'
0.3
0.8
I / S (mM)
Fig. 7. A double-reciprocal plot of the enzyme kinetics presented in Fig. 6.
In conclusion, the TX1 cell activation of 2aminofluorene and m-phenylenediamine is completely inhibited by diethyldithiocarbamate. We conclude that the primary mechanism of the TX1 cell activation of these promutagens is via a peroxidase pathway in which the promutagens serve as the hydrogen donor. The data presented here demonstrate that diethyldithiocarbamate is a potent non-competitive inhibitor of TX1 cell peroxidase activity and that one mechanism for the antimutagenic effect of diethyldithiocarbamate is through its inhibition of cellular peroxidases.
Acknowledgments This research was supported in part by U.S. Environmental Protection Agency Grant R-815008 and by U.S. Air Force Office of Scientific Research Grant AFOSR-88-0336. Funds from the Institute for Environmental Studies at the University of Illinois are gratefully acknowledged.
References Ames, B.N., E.G. Gurney, J.A. Miller and H. Bartsch (1972) Carcinogens as frameshift mutagens: metabolites and derivatives of 2-acetylaminofluorene and other aromatic amine carcinogens, Proc. Natl. Acad. Sci. (U.S.A.), 69, 3128-3132. Ames, B.N., H.O. Kammen and E. Yamasaki (1975) Hair dyes are mutagenic: identification of a variety of mutagenic ingredients, Proc. Natl. Acad. Sci. (U.S.A.), 72, 2423-2427. Boyd, J.A., and T.E. Eling (1984) Evidence for a one-electron mechanism of 2-aminofluorene oxidation by prostaglandin H synthase and horseradish peroxidase, J. Biol. Chem., 259, 13885-13896.
Boy& J.A., D.J. Harvan and T.E. Eling (1983) The oxidation of 2-aminofluorene by prostaglandin endoperoxide synthetase, J. Biol. Chem., 258, 8246-8254. Bradford, M.M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding, Anal. Biochem., 72, 248-254. Chance, B., and A.C. Maehly (1955) Assay of catalases and peroxidases, in: S. Colowick and N. Kaplan (Eds.), Methods in Enzymology, Vol. 2, Academic Press, New York, pp. 764-775. Forman, H.J., J.L. York and A.B. Fisher (1980) Mechanism for the potentiation of oxygen toxicity by disulfiram, J. Pharmacol. Exp. Ther., 212, 452-455. Gichner, T., and J. Veleminsky (1984) Inhibition of dimethylnitrosamine-induced mutagenesis in Arabidopsis thaliana by diethyldithiocarbamate and carbon monoxide, Mutation Res., 139, 29-33. Gichner, T., J. Veleminsky and R. Rieger (1988) Antimutagenic effects of diethyldithiocarbamate towards maleic hydrazide- and N-nitrosodiethylamine-induced mutagenicity in the Tradescantia mutagenicity assay, Biol. Plant., 30, 14-19. Heikkila, R.E. (1985) Inactivation of superoxide dismutase by diethyldithiocarbamate, in: R.A. Greenwald (Ed.), Handbook of Methods for Oxygen Radical Research, CRC Press, Boca Raton, FL, pp. 387-390. Higashi, K. (1988) Metabolic activation of environmental chemicals by microsomal enzymes of higher plants, Mutation Res., 197, 273-288. Higashi, K., K. Ikeuchi, Y. Karasaki and M. Obara (1983) Isolation of immunochemically distinct form of cytochrome P-450 from microsomes of tulip bulbs, Biochem. Biophys. Res. Commun., 115, 46-52. Higashi, K., K. Ikeuchi, M. Obara, Y. Karasaki, H. Hirano, S. Gotoh and Y. Koga (1985) Purification of a single major form of microsomal cytochrome P-450 from tulip bulbs (Tulipa gesneriana L.), Agric. Biol. Chem., 49, 2399-2405. Kawanishi, T., Y. Ohno, A. Takahashi, A. Takanaka, Y. Kasuya and Y. Omori (1985) Relation between hepatic microsomal metabolism of N-nitrosamines and cytochrome P-450 species, Biochem. Pharmacol., 34, 919-924. Kleiman, N.J., R.-R. Wang and A. Spector (1990) Hydrogen peroxide-induced DNA damage in bovine lens epithelial cells, Mutation Res., 240, 35-45. Kuwahara, T., H. Shigematsu and T. Omura (1988) Purification of an aromatic amine-dependent NAD(P)H oxidase from tomato fruit and its characterization as a peroxidase, Agric. Biol. Chem., 52, 2597-2603. Lhotka, M.A., M.J. Plewa and J.M. Gentile (1987) Plant activation of m-phenylenediamine by tobacco, cotton, and carrot cell suspension cultures, Environ. Mol. Mutagen., 10, 79-88. Lotlikar, P.D., and K. Zaleski (1975) Ring- and N-hydroxylation of 2-acetamidofluorene by rat liver reconstituted cytochrome P-450 enzyme system, Biochem. J., 150, 561-564. Lutz, L.M., E.A. Glende and R.O. Recknagel (1973) Protection by diethyldithiocarbamate against carbon tetrachloride
64 lethality in rats and against carbon tetrachloride induced lipid peroxidation in vitro, Biochem. Pharmacol., 22, 17291734. M~ider, M., and C. Walter (1986) De-novo synthesis and release of peroxidases in cell suspension cultures of Nicotiana tabacurn L., Planta, 169, 273-277. Maehly, A.C., and B. Chance (1954) The assay of catalases and peroxidases, in: D. Glick (Ed.), Methods of Biochemical Analysis, Vol. 1, Interscience, New York, pp. 357-424. Miller, E.C., and J.A. Miller (1969) Studies on the mechanism of activation of aromatic amine and amide carcinogens to ultimate carcinogenic electrophilic reactants, Ann. N.Y. Acad. Sci., 163, 731-750. Murashige, T., and F. Skoog (1962) A revised medium for rapid growth and bioassays with tobacco cultures, Physiol. Plant., 15, 473-497. Plewa, M.J., D.L. Weaver, L.C. Blair and J.M. Gentile (1983) The activation of 2-aminofluorene by cultured plant cells, Science, 219, 1427-1429. Plewa, M.J., E.D. Wagner and J.M. Gentile (1988) The plant cell/microbe coincubation assay for the analysis of plantactivated promutagens, Mutation Res., 197, 207-219. Rojanapo, W., P. Kupradinun, A. Tepsuwan, S. Chutimataewin and M. Tanyakaset (1986) Carcinogenicity of an oxidation product of p-phenylenediamine, Carcinogenesis, 7, 1997-2002. Sandermann Jr., H. (1982) Metabolism of environmental chem-
icals: a comparison of plant and liver enzyme systems, in: E.J. Klekowski Jr. (Ed.), Environmental Mutagenesis, Carcinogenesis, and Plant Biology, Vol. I, Praeger, New York, pp. 1-32. Smith, S.R., M.M. Verdier, E.D. Wagner and M.J. Plewa (1989) Protein content of tobacco cells in relation to the plant activation of m-phenylenediamine and 2-aminofluorene, Environ. Mol. Mutagen., 14 (15), 188-189. Takahama, U. (1989) A role of hydrogen peroxide in the metabolism of phenolics in mesophyll cells of Vicia faba L.. Plant Cell Physiol., 30, 295-301. Wagner, E.D., J.M. Gentile and M.J. Plewa (1989) Effect of specific monooxygenase and oxidase inhibitors on the activation of 2-aminofluorene by plant cells, Mutation Res.. 216, 163-178. Wagner, E.D., M.M. Verdier and M.J. Plewa (1990) The biochemical mechanisms of the plant activation of promutagenic aromatic amines, Environ. Mol. Mutagen., 15, 236244. Watanabe, T., T. Hirayama and S. Fukui (1989) Phenazine derivatives as the mutagenic reaction product from o- or m-phenylenediamine derivatives with hydrogen peroxide, Mutation Res., 227, 135-145. Zanocco, A.L., R. Pavez, L.A. Videla and E.A. Lissi (1989) Antioxidant capacity of diethyldithiocarbamate in a metal independent lipid peroxidative process, Free Radical Biology and Medicine, 7, 151-156.
57
MUT 04941
Diethyldithiocarbamate suppresses the plant activation of aromatic amines into mutagens by inhibiting tobacco cell peroxidase Michael J. Plewa, Shannon R. Smith and Elizabeth D. Wagner Institute for Environmental Studies, University of Illinois at Urbana-Champaign, Urbana, IL 61801 (U.S.A.) (Received 30 May 1990) (Revision received 10 August 1990) (Accepted 20 August 1990)
Keywords: Nicotiana tabacum; 2-Aminofluorene; m-Phenylenediamine; Mutation induction; Antimutagenicity; Plant cell/microbe coincubation assay
Summary Diethyldithiocarbamate is an antimutagen and repressed the activation of promutagens by plant systems. Earlier work implicated the involvement of tobacco cell (TX1) peroxidases in the plant cell activation of aromatic amines. We now present data that diethyldithiocarbamate represses the activation of 2-aminofluorene and m-phenylenediamine by inhibiting intracellular TX1 peroxidases under in vivo conditions. Concentrations of diethyldithiocarbamate that caused a 50% repression of TX1 cell activation of 2-aminofluorene and m-phenylenediamine also induced a 50% inhibition of TX1 cell peroxidase activity. Diethyldithiocarbamate in a concentration range between 25 and 500 /~M directly inhibited peroxidase activity in TX1 cell homogenates in a concentration-dependent manner. Similar results were observed with purified horseradish peroxidase. The kinetics of peroxidase activity were studied in homogenates from control cells and cells treated with 750/~M and 25 mM diethyldithiocarbamate. There was no significant difference among the K m values among the three groups with a mean ( _ standard error) K m of 2.58 + 0.23 raM. However, the Vmax differed from 4.02 to 2.12 nmoles tetraguaiacol/min//~g protein, in the control and in the 25 mM diethyldithiocarbamate treatment group, respectively. These data indicate that diethyldithiocarbamate is a non-competitive inhibitor of TX1 cell peroxidase.
Plant cells in suspension culture can activate promutagenic aromatic amines into mutagens as detected in Salmonella typhimurium (Plewa et al., 1983; Lhotka et al., 1987). The biochemical mechanisms of activation by tobacco cells have been examined. By using specific enzyme inhibitors we reported indirect evidence that peroxidases were involved in the activation of two aromatic amines (Wagner et al., 1989, 1990). Correspondence: Dr. Michael J. Plewa, Professor of Genetics, Institute for Environmental Studies, University of Illinois at Urbana-Champaign, 1101 West Peabody Drive, Urbana, IL 61801 (U.S.A.).
Peroxidase enzymes are ubiquitous in the plant kingdom. Peroxidases may catalyze N- or C-hydroxylation, N-sulfoxidation, N-acetylation, halogenation, dehalogenation or decarboxylation (Sandermann, 1982; Rojanapo et al., 1986). Peroxidative reactions normally proceed as shown below: peroxidase + H 202 ~ peroxidase-I peroxidase-I + AH 2 --* peroxidase-II + AH. peroxidase-II + AH 2 - , peroxidase + AH. 2AH. --* products
where peroxidase-I is designated as FeO 3+, peroxidase-II is designated as FeO 2+, H202 is the
58 stringent substrate for the peroxidase, and AH 2 is a non-specific substrate that functions as a hydrogen donor (Higashi, 1988). Diethyldithiocarbamate was an excellent inhibitor of the tobacco cell activation of 2-aminofluorene and m-phenylenediamine (Wagner et al., 1989, 1990). Diethyldithiocarbamate suppressed the plant activation of several promutagens by Arabidopsis and Tradescantia (Gichner and Veleminsky, 1984; Gichner et al., 1988). Diethyldithiocarbamate, a metal-chelating agent, is an inhibitor of superoxide dismutase (Heikkila, 1985). Depending on the mechanism of action, diethyldithiocarbamate exhibited either toxic or protective effects. Diethyldithiocarbamate potentiated oxygen toxicity as well as the DNA-damaging effects of hydrogen peroxide (Forman et al., 1980; Kleiman et al., 1990). Diethyldithiocarbamate was an efficient free-radical scavenger and inhibited lipid peroxidation (Lutz et al., 1973; Zanocco et al., 1989). The purpose of this research was to determine if diethyldithiocarbamate suppressed the tobacco cell activation of aromatic amines by inhibiting cellular peroxidases. Materials and methods
Preparation of titered TX1 cell suspension The protocols for the preparation of the plant cell suspension have been published (Plewa et al., 1988). The TX1 cells were washed in M X medium, a modified liquid culture medium lacking plant growth hormone. The fresh weight of the cell suspension was adjusted to a final concentration of 100 m g / m l in MX medium.
Preparation of TX1 cell homogenate For the determination of protein content and peroxidase activity, a TX1 cell homogenate was prepared. The washed and titered TX1 cell suspension was placed on ice, disrupted with a PolyTron homogenizer for 45 sec and centrifuged at 15000 × g for 2 rain. An aliquot of the supernatant was frozen at - 8 0 ° C for subsequent protein analysis. The other portion was kept on ice and immediately analyzed for peroxidase activity.
Assay for protein concentration The Bio-Rad protein assay (Bradford, 1976) was used to determine the protein content of TX1 cells. The microassay with a range of 1-25 #g p r o t e i n / m l was used. Lyophilized bovine 7globulin was chosen for the protein standard curves. The conditions for the analysis of protein content were described by Smith et al. (1989).
Chemicals Horseradish peroxidase (CAS No. 9003-99-0), guaiacol (CAS No. 90-05-1) and diethyldithiocarbamic acid, sodium salt (CAS No. 20624-25-3) were purchased from Sigma Chemical Co. (St. Louis, MO). Hydrogen peroxide (CAS No. 772284-1) was purchased from Fisher Scientific (Pittsburgh, PA). The standardized plasma protein bovine "t-globulin (Standard I) and Bio-Rad Dye Reagent Concentrate were purchased from BioRad Chemical Division (Richmond, CA).
Plant cells Long-term
suspension
cultures
of
tobacco
(Nicotiana tabacum), cell line TX1, were maintained by inoculating 3 g fresh weight from 7-day cultures into 100 ml of MX medium, a modified liquid culture medium of Murashige and Skoog (1962). The cultures were grown at 2 8 ° C in the dark with shaking at 150 rpm.
Determination of peroxidase activity To determine peroxidase activity, we measured the oxidation of guaiacol to tetraguaiacol by monitoring the change in absorbance at 470 nm (Maehly and Chance, 1954). The stoichiometry of the assay reaction is given below: 4 guaiacol + 4 H202 --~ 1 tetraguaiacol + 8 H 2 0 Peroxidase activity in 3-day and 7-day TX1 cells was analyzed in a reaction volume of 3 ml containing 50 m M potassium phosphate buffer, pH 7, 1 ml of a 0.3% H202 solution, 1 ml of a 1% guaiacol solution, and 25 /~1 of the TX1 cell homogenate. The cuvettes used as blanks were identical except that M X - medium was substituted for the TX1 cell homogenate. After the addition of the last component (guaiacol), the cuvette was quickly inverted twice, and the reaction was timed. Peroxidase activity was measured over a 5-min
59 time period using a model 552A Perkin-Elmer double-beam spectrophotometer. 3-5 independent replicates were conducted for each measurement within each experiment. Because of the high amount of peroxidase activity in 7-day cells, the amount of the H202 solution was reduced to 100 /~1 for all subsequent experiments with diethyldithiocarbamate. Results
E E
4-00
-
T
T
-1-95~ConfidenceLirnits ~ 300
~- 200
g
rn i
100
0
M - 5 0 mM), suppressed the plant activation of 2aminofluorene and m-phenylenediamine. A concentration of 1 mM diethyldithiocarbamate resulted in a 50% reduction of TX1 cell activation (Wagner et al., 1989, 1990). To determine if diethyldithiocarbamate was impeding plant activation of these promutagens by specifically inhibiting peroxidases, we analyzed the peroxidase activity in
TX1 cells that were exposed to a wide range of diethyldithiocarbamate concentrations under the identical conditions used to measure the inhibition of TX1 cell activation. This approach insured that the genetic and biochemical parameters of the parallel experimental designs could be directly compared. The concentrations of diethyldithiocarbamate used in the in vivo experiments were not toxic to the TX1 cells (Wagner et al., 1989, 1990). As illustrated in Fig. 3, TX1 cells that were exposed in vivo to 250/~M diethyldithiocarbamate had a significant reduction in peroxidase activity from that of the untreated cells. Approximately one-half of TX1 cell peroxidase activity was inhibited by diethyldithiocarbamate concentrations between 750 /~M and 2.5 mM. These same concentrations caused a 50% reduction in TX1 cell activation of 2-aminofluorene and m-phenylenediamine (Wagner et al., 1989, 1990). The reduction in peroxidase activity directly correlates with the suppression of the TX1 cell activation of these two aromatic amines. A significant concentration-dependent reduction in peroxidase activity was observed by adding diethyldithiocarbamate directly to TX1 cell homogenates (Fig. 4). These data confirm that diethyldithiocarbamate can specifically inhibit a TX1 cell peroxidase under in vitro conditions. The inhibitory effect of diethyldithiocarbamate was measured with a well defined enzyme, purified horseradish peroxidase (Fig. 5). Diethyldithiocarbamate significantly inhibited purified horseradish peroxidase at concentrations above 50 #M. The nature of the inhibition of TX1 cell peroxidase by diethyldithiocarbamate was examined (Table 1). The data from the Michaelis-Menten graph are represented in a Lineweaver Burk plot (Fig. 7). From 3 independent experiments, the K m value for the control cells was 2.79_+ 0.50 mM. The K m value for the cells treated with 750 /,M and 25 mM diethyldithiocarbamate was 2.31 _+ 0.27 mM and 2.65 _+ 0.62 mM, respectively. The mean K m value for all the groups was 2.58 _+ 0.23 mM. The K m values among the control and treated groups did not differ significantly while the Vmax values were different (Table 1). These data indicate that diethyldithiocarbamate is a non-competitive inhibitor of TX1 cell peroxidase.
63 0.9
o
#-
o.6
E ~
0.3
0.0
-0.8
-0.3
'
'
0.3
0.8
I / S (mM)
Fig. 7. A double-reciprocal plot of the enzyme kinetics presented in Fig. 6.
In conclusion, the TX1 cell activation of 2aminofluorene and m-phenylenediamine is completely inhibited by diethyldithiocarbamate. We conclude that the primary mechanism of the TX1 cell activation of these promutagens is via a peroxidase pathway in which the promutagens serve as the hydrogen donor. The data presented here demonstrate that diethyldithiocarbamate is a potent non-competitive inhibitor of TX1 cell peroxidase activity and that one mechanism for the antimutagenic effect of diethyldithiocarbamate is through its inhibition of cellular peroxidases.
Acknowledgments This research was supported in part by U.S. Environmental Protection Agency Grant R-815008 and by U.S. Air Force Office of Scientific Research Grant AFOSR-88-0336. Funds from the Institute for Environmental Studies at the University of Illinois are gratefully acknowledged.
References Ames, B.N., E.G. Gurney, J.A. Miller and H. Bartsch (1972) Carcinogens as frameshift mutagens: metabolites and derivatives of 2-acetylaminofluorene and other aromatic amine carcinogens, Proc. Natl. Acad. Sci. (U.S.A.), 69, 3128-3132. Ames, B.N., H.O. Kammen and E. Yamasaki (1975) Hair dyes are mutagenic: identification of a variety of mutagenic ingredients, Proc. Natl. Acad. Sci. (U.S.A.), 72, 2423-2427. Boyd, J.A., and T.E. Eling (1984) Evidence for a one-electron mechanism of 2-aminofluorene oxidation by prostaglandin H synthase and horseradish peroxidase, J. Biol. Chem., 259, 13885-13896.
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