- Email: [email protected]
Dinitrochlorobenzene is genotoxic by sister chromatid exchange in human skin fibroblasts
Genetic Toxicology
ELSEVIER
Mutation Research 371 (1996) 105-108
Dinitrochlorobenzene is genotoxic by sister chromatid exchange in human skin fibroblasts Laurie D. D e L e v e 1 Department of Pharmacology, Di~ision of Clinical Pharmacology, Research Institute, Hospital for Sick Children, and Department of Pharmacology, UniL:ersit3'of Toronto, Toronto, Ont., Canada, M5G 1X8
Received 5 June 1996: revised 2 July 1996; accepted 30 July 1996
Abstract Dinitrochlorobenzene (DNCB) is clinically efficacious in the therapy of alopecia areata, but its use was limited when it was found to be mutagenic in the Ames test. However, there has been renewed interest in the immunomodulatory benefits of topically applied dinitrochlorobenzene in patients with human immunodeficiency virus and systemic lupus erythematosus. The current study examines the genotoxicity of dinitrochlorobenzene in human skin fibroblasts using sister chromatid exchange. Dinitrochlorobenzene caused a significant increase in sister chromatid exchange at concentrations ranging from 2.5 to 10 IxM. Thus, dinitrochlorobenzene is genotoxic in human skin fibroblasts at concentrations well below those used clinically. The potential for long-term toxicity from dinitrochlorobenzene will have to be weighed against the severity and prognosis of the diseases for which it is used. Keywords: Sister chromatid exchange; Dinitrochlorobenzene;Cell, cultured; Dermatologic agent
1. Introduction 1-Chloro-2,4-dinitrobenzene or dinitrochlorobenzene (DNCB) has been used in the treatment of severe alopecia areata and resistant viral warts. It fell into disfavor in 1979 when a number of studies demonstrated mutagenicity in the Ames test [1-5] with significant increases in mutagenicity at 3 - 1 0
Abbreviations: DNCB, dinitrochlorobenzene;SCE, sister chromatid exchange i USC School of Medicine, Div. of Gastrointestinal and Liver Diseases, 1333 San Pablo St-MMR 401, Los Angeles CA 90033, USA. Tel.: (213) 342-3248; Fax: (213) 342-3243; E-mail: [email protected].
txg DNCB per plate. Although it had been postulated that rnutagenic contaminants might contribute to part of its mutagenicity, Wilkerson et al. demonstrated that these contaminants do not add to the mutagenic effect of DNCB in the Ames test [4]. Because of the concerns about potential mutagenicity the use of DNCB was largely limited to experimental studies of allergic contact dermatitis. There has been a recent resurgence in interest in topical DNCB because of its ability to boost Thl response. This was initially examined in patients infected with human immunodeficiency virus (HIV) [6-8] but has now been applied to systemic lupus erythematosus [9]. Concerns regarding safety were discounted on several grounds [6,10]: the occurrence
0165-1218/96/$15.00 Copyright © 1996 Elsevier Science B.V. All rights reserved. PI1 S0165- 12 18(96)0009 1-2
106
L.D. DeLet,e / Mutation Research 371 (1996) 105-108
of false positives in Ames testing; the lack of carcinogenicity of DNCB in in vivo studies with rodents [11]; and the lack of toxicity in HIV patients. The goal of the current study was to examine the genotoxicity of dinitrochlorobenzene in human skin fibroblasts using sister chromatid exchange.
2. Materials and methods
Materials. All chemicals were obtained from the Sigma Chemical Corporation (St. Louis, MO). Human skin fibroblasts grown from biopsies from the forearm of normal human subjects were a kind gift from the Department of Genetics at the Hospital for Sick Children (Toronto, Ont., Canada). Sister chromatid exchange. Fibroblasts were plated at a density of 3,000 cells/cm 2 on slides placed in tissue culture dishes and were grown in alpha Minimum Essential Medium with 10% fetal calf serum. DNCB was dissolved in dimethyl sulfoxide and the final concentration of the solvent in the medium was 0.025%. The day after plating cells were exposed to DNCB for 2 h in a 37°C, CO 2 incubator. At the end of the incubation period the dishes were washed once with phosphate buffered saline and fresh medium with 10 txm bromodeoxyuridine was added. For the next 46 h, the dishes were kept in the dark in a 37°C, CO 2 incubator. Cells were arrested in metaphase by a 2-h incubation with 0.1 IxM colchicine and were then lysed with 0.075 M potassium chloride. Slides were fixed with methanol/glacial acetic acid (3: 1), incubated with 5 Ixg/ml bisbenzamide for 15 min and exposed to ultraviolet light (peak wavelength 335 rim) for 2 h. After a 35-rain incubation in 2 × standard saline citrate solution (standard saline citrate/0.3 M sodium chloride : 0.03 M trisodium citrate) at 65°C, slides were stained with a Gurr stain (2 ml of Giemsa stain in 50 ml phosphate buffer, pH 6.8). Since the inclusion of an $9 fraction has been shown by several other investigators to inhibit mutagenicity of DNCB in the Ames test by around 50%, no $9 fraction was added [I,4,5]. Cell proliferation. Baseline cell number was determined in a control plate at the beginning of the incubation. Inhibition of growth by DNCB is expressed as a percentage of the growth in control
cells. Cell number was determined by Coulter counter. Data analysis. All experiments were performed in duplicate and three experiments were done with each cell strain. Only metaphases in which all 46 chromosomes were present were used to count exchanges. For each determination, SCEs were counted in 20 metaphases. Since the distribution of SCE was found to be skewed, geometric means (logarithmic transformation) were used, and this restored a normal distribution to the values of both the treated and control populations. Dose-response curves were compared by two-way analysis of variance, and individual points were compared ~ posteriori by least significant difference.
3. Results and discussion The SCE assay requires cells to undergo two cell cycles in the presence of bromodeoxyuridine after exposure to the test compound. In the current study insufficient metaphases were found in two of the three cells strains at 10 I~M DNCB, so the SCE assay could only be performed at concentrations up to 7.5 p~M in those cells. In Table 1 the effect of DNCB on SCE are shown, and in Fig. 1 the percent increase in SCE relative to the solvent control is shown for the three fibroblasts cell strains. Comparison of the dose response curves between 2.5 and 7.5 ~M by two-way analysis of variance was significant at p < 0.005. The increase in the number of exchanges was significant compared to the control starting at 2.5 I~M ( p < 0.025), as well as at 5 and 7 p,M ( p < 0.0001) by the least significant difference test. Since extreme reductions in cell number have been shown to artificially increase SCEs [12], the effect of DNCB on cell proliferation is provided in Table 2. This is the first demonstration in human cells that DNCB is genotoxic. Sister chromatid exchange (SCE) is widely used as a useful and sensitive test of genotoxicity [ 13]. It is considered a good predictor of mutagenicity and carcinogenicity [14], although the exact mechanism of formation of SCEs is unknown. In a recent assessment of the literature by the U.S. Environmental Protection Agency's Gene-Tox Program [13], genotoxicity by SCE of a large number of
107
L D. DeLeve / Mutation Research 371 (1996) 105-108 Table 1 SCE frequencies (mean + SD) in solvent or dinitrochlorobenzene treated fibroblasts Cell strain
Exp. No.
O IxM
2.5 I~M
5 I~M
7.5 p,M
10 p.M
Control No. 1
1 2 3
9.38 5:0.01 7.46 5:0.36 8.92 _ 0.59
11.18 5:0.35 8.355 5:0.13 10.86 + 0.32
12.86 + 0.86 11.11 ___1.14 13.86 5:0.77
NT 11.36 5:0.44 15.47 5:0.42
21.90 + 0.27 11.82 + 0.06 16.92 5:0.27
Control No. 2
1 2 3
9.55 5:1.38 9.26 5:0.01 7.69 5:1.11
10.15 5:0.78 12.4 5:0.86 9.39 5:0.25
11.16 5:2.37 14.1 + 0.72 10.89 + 2.16
12.58 + 0.47 14.69 ___0.18 11.91 _+ 1.04
13.09 5:0.50
Control No. 3
1 2 3
6.32 + 0.27 7.65 + 0.76 7.55 + 0.97
8.2 + 0.32 10.39 + 0.36 9.63 5:0.71
10.86 + 0.86 11.24 5:0.41 11.22 5:0.83
13.44 + 0.33 11.92 + 1.94 13.56 5:0.25
15.12 + 0.40
SCE frequencies from 3 separate experiments in 3 individual control cell strains. NT, not tested; * insufficient data.
compounds was examined. In that report the magnitude of a positive response by SCE was graded by three criteria: a 3-point monotonic dose response, statistical significance at the highest dose with p < 0.001 and a doubling of the SCE frequency above the control. DNCB fits the first two criteria and, at 10lxM, falls just short of the third. Thus, DNCB induces SCE in human fibroblasts and does so in a very significant fashion. A 2-h exposure to DNCB induced a significant increase in SCE at 2.5 ~M or 0.00005%. When used
i: 2, °0- / /f~ t " 0
,
,
,
2.5 5 7.5 10 Dinitrochlorobenzene, FM
Fig. 1. Dinitrochlorobenzene-induced sister chromatid exchange. Each of the three tracings represents the increase in sister chromatid exchange in a normal human skin fibroblast cell strain expressed as the percent increase above the solvent control. Values are expressed as mean + SEM, n = 3. Control No. 1 ( • ) , control No. 2 (O), control No. 3 ( • ) .
clinically the dose used is between 0.1 and 2% for alopecia areata, 2% for systemic lupus erythematosus [9] and between 0.02 and 2% in HIV patients [7,8], used recurrently for prolonged periods of time. When applied topically, DNCB is rapidly absorbed with recovery of 53% of the drug and its metabolites in the urine [15]. Thus, the potential for genotoxic effects is not just limited to the skin. One study has been reported on carcinogenicity testing in rodents [16]. This study found that it was not carcinogenic when fed in large doses for up to 4 months. However, since the proposed clinical use is topical, it would perhaps be more pertinent to study the carcinogenicity of topically applied drug. Particularly in a drug that is less mutagenic in the presence of a metabolic activating system, the first pass effect of an orally administered drug may lead to a quite different outcome than topical application. HIV infection currently leads to a terminal illness, so that a beneficial drug with a potential genotoxic side effect may well be warranted. However, the Table 2 Cell proliferation as a measure of dinitrochlorobenzene toxicity Cell strain
2.5 I~M
5 I~M
7.5 p~M
10 [xM
Control No. 1 Control No. 2 Control No. 3
94 + 6 955-7.7 99+2
66 ± 2 63+8 90+8
63 + 2 4 8 + 10 91 + 7
53 + 9 4 0 + 10 76+ 1
Values are cell numbers at time of harvest (after 48 hour incubation) as a percentage of the cell number present in the solvent treated control.
108
L.D. DeLeve / Mutation Research 371 (1996) 105-108
risk-benefit analysis may be different in systemic lupus erythematosus depending on the individual patient. The possibility of long-term toxicity from DNCB will have to be weighed against the severity of the disease being treated and the benefits that might be achieved by the use of this compound.
Acknowledgements The author would like to thank the late Marilyn Cannon for her technical assistance and Dr. Stephen Spielberg for his support and advice. L.D.D. was supported by a Medical Research Council of Canada Fellowship.
References [1] Strobel, R. and G. R~Shrborn (1980) Mutagenic and cell transforming activities of 1-chlor-2,4-dinitrobenzene (DNCB) and squaric-acid-dibutylester (SADBE), Arch. Toxicol., 45, 307-314. [2] Summer, K.H. and W. GiSggelmann (1980) 1-Chloro-2,4-dinitrobenzene depletes glutathione in rat skin and is mutagenic in Salmonella typhimurium, Mutation Res., 77, 91-93. [3] Black, H.S., F.F. Castrow, II and J. Gerguis (1985) The mutagenicity of dinitrochlorobenzene, Arch. Dermatol., 121, 348-349. [4] Wilkerson, M.G., T.H. Connor and J.K. Wilkin (1988) Dinitrochlorobenzene is inherently mutagenic in the presence of trace mutagenic contaminants, Arch. Dermatol., 124, 396398. [5] Shimizu, M., Y. Yasui and N. Matsumoto (1983) Structural specificity of aromatic compounds with special reference to mutagenic activity in Salmonella typhimurium - a series of chloro- or fluoro-nitrobenzene derivatives, Mutation Res., 116, 217-238.
[6] Stricker, R.B., B.F. Elswood and D.I. Abrams (1991) Dendritic cells and dinitrochlorobenzene (DNCB): a new treatment approach to AIDS, Immunol. Lett., 29, 191-196. [7] Stricker, R.B., Y.S. Zhu, B.F. Elswood, C. Dumlao, J. Van Elk, T.G. Berger, J. Tappero, W.L. Epstein and D.D. Kiprov (1993) Pilot study of topical dinitrochlorobenzene (DNCB) in human immunodeficiency virus infection, Immunol. Lett., 36, 1-6. [8] Stricker, R.B., B.F. Elswood, B. Goldberg, C. Dumlao, J. Van Elk, J. Henry, E.E. Winger and W.L. Epstein (1994) Clinical and immunologic evaluation of HIV-infected patients with dinitrochlorobenzene, J. Am. Acad. Dermatol., 31,462-466. [9] Stricker, R.B., B. Goldberg and W.L. Epstein (1995) Immunological changes in patient with systemic lupus erythematosus with topical dinitrochlorobenzene, Lancet, 345, 1505-1506. [10] Stricker, R.B. and B. Goldberg (1995) Safety of topical dinitrochlorobenzene, Lancet, 346, 1293. [11] Weisburger, E.K., A.B. Russfield, F. Homburger, J.H. Weisburger, E. Roger, C.G. Van Dongen and K.C. Chu (1978) Testing of twenty one environmental aromatic amines or derivatives for long term toxicity and carcinogenicity, J. Environ. Pathol. Toxicol., 2, 325-356. [12] Galloway, S.M., D.A. Deasy, C.L. Bean, A.R. Kraynak, M.J. Armstrong and M.O. Bradley (1987) Effects of high osmotic strength on chromosome aberrations, sister-chromatid exchanges and DNA strand breaks, and the relation to toxicity, Mutation Res., 189, 15-25. [13] Tucker, J.D., A. Auletta, M.C. Cimino, K.L. Dearfield, D. Jacobson-Kram, R.R. Tice and A.V. Carrano (1993) Sisterchromatid exchange: second report of the Gene-Tox program, Mutation Res., 297, 101-180. [14] Abe, S. and M. Sasaki (1982) Sister chromatid exchange as an index of mutagenesis and/or carcinogenesis, in: A.A. Sandberg (Ed.), Sister chromatid exchange, Alan R. Liss,Inc., New York, pp. 461-514. [15] Feldmann, R.J. and H.I. Maibach (1970) Absorption of some organic compounds through the skin in man, J. Invest. Dermatol., 54, 399-404. [ 16] Shapiro, J. (1993) Topical immunotherapy in the treatment of chronic severe alopecia areata, Dermatol. Clin., 11, 611-617.
ELSEVIER
Mutation Research 371 (1996) 105-108
Dinitrochlorobenzene is genotoxic by sister chromatid exchange in human skin fibroblasts Laurie D. D e L e v e 1 Department of Pharmacology, Di~ision of Clinical Pharmacology, Research Institute, Hospital for Sick Children, and Department of Pharmacology, UniL:ersit3'of Toronto, Toronto, Ont., Canada, M5G 1X8
Received 5 June 1996: revised 2 July 1996; accepted 30 July 1996
Abstract Dinitrochlorobenzene (DNCB) is clinically efficacious in the therapy of alopecia areata, but its use was limited when it was found to be mutagenic in the Ames test. However, there has been renewed interest in the immunomodulatory benefits of topically applied dinitrochlorobenzene in patients with human immunodeficiency virus and systemic lupus erythematosus. The current study examines the genotoxicity of dinitrochlorobenzene in human skin fibroblasts using sister chromatid exchange. Dinitrochlorobenzene caused a significant increase in sister chromatid exchange at concentrations ranging from 2.5 to 10 IxM. Thus, dinitrochlorobenzene is genotoxic in human skin fibroblasts at concentrations well below those used clinically. The potential for long-term toxicity from dinitrochlorobenzene will have to be weighed against the severity and prognosis of the diseases for which it is used. Keywords: Sister chromatid exchange; Dinitrochlorobenzene;Cell, cultured; Dermatologic agent
1. Introduction 1-Chloro-2,4-dinitrobenzene or dinitrochlorobenzene (DNCB) has been used in the treatment of severe alopecia areata and resistant viral warts. It fell into disfavor in 1979 when a number of studies demonstrated mutagenicity in the Ames test [1-5] with significant increases in mutagenicity at 3 - 1 0
Abbreviations: DNCB, dinitrochlorobenzene;SCE, sister chromatid exchange i USC School of Medicine, Div. of Gastrointestinal and Liver Diseases, 1333 San Pablo St-MMR 401, Los Angeles CA 90033, USA. Tel.: (213) 342-3248; Fax: (213) 342-3243; E-mail: [email protected].
txg DNCB per plate. Although it had been postulated that rnutagenic contaminants might contribute to part of its mutagenicity, Wilkerson et al. demonstrated that these contaminants do not add to the mutagenic effect of DNCB in the Ames test [4]. Because of the concerns about potential mutagenicity the use of DNCB was largely limited to experimental studies of allergic contact dermatitis. There has been a recent resurgence in interest in topical DNCB because of its ability to boost Thl response. This was initially examined in patients infected with human immunodeficiency virus (HIV) [6-8] but has now been applied to systemic lupus erythematosus [9]. Concerns regarding safety were discounted on several grounds [6,10]: the occurrence
0165-1218/96/$15.00 Copyright © 1996 Elsevier Science B.V. All rights reserved. PI1 S0165- 12 18(96)0009 1-2
106
L.D. DeLet,e / Mutation Research 371 (1996) 105-108
of false positives in Ames testing; the lack of carcinogenicity of DNCB in in vivo studies with rodents [11]; and the lack of toxicity in HIV patients. The goal of the current study was to examine the genotoxicity of dinitrochlorobenzene in human skin fibroblasts using sister chromatid exchange.
2. Materials and methods
Materials. All chemicals were obtained from the Sigma Chemical Corporation (St. Louis, MO). Human skin fibroblasts grown from biopsies from the forearm of normal human subjects were a kind gift from the Department of Genetics at the Hospital for Sick Children (Toronto, Ont., Canada). Sister chromatid exchange. Fibroblasts were plated at a density of 3,000 cells/cm 2 on slides placed in tissue culture dishes and were grown in alpha Minimum Essential Medium with 10% fetal calf serum. DNCB was dissolved in dimethyl sulfoxide and the final concentration of the solvent in the medium was 0.025%. The day after plating cells were exposed to DNCB for 2 h in a 37°C, CO 2 incubator. At the end of the incubation period the dishes were washed once with phosphate buffered saline and fresh medium with 10 txm bromodeoxyuridine was added. For the next 46 h, the dishes were kept in the dark in a 37°C, CO 2 incubator. Cells were arrested in metaphase by a 2-h incubation with 0.1 IxM colchicine and were then lysed with 0.075 M potassium chloride. Slides were fixed with methanol/glacial acetic acid (3: 1), incubated with 5 Ixg/ml bisbenzamide for 15 min and exposed to ultraviolet light (peak wavelength 335 rim) for 2 h. After a 35-rain incubation in 2 × standard saline citrate solution (standard saline citrate/0.3 M sodium chloride : 0.03 M trisodium citrate) at 65°C, slides were stained with a Gurr stain (2 ml of Giemsa stain in 50 ml phosphate buffer, pH 6.8). Since the inclusion of an $9 fraction has been shown by several other investigators to inhibit mutagenicity of DNCB in the Ames test by around 50%, no $9 fraction was added [I,4,5]. Cell proliferation. Baseline cell number was determined in a control plate at the beginning of the incubation. Inhibition of growth by DNCB is expressed as a percentage of the growth in control
cells. Cell number was determined by Coulter counter. Data analysis. All experiments were performed in duplicate and three experiments were done with each cell strain. Only metaphases in which all 46 chromosomes were present were used to count exchanges. For each determination, SCEs were counted in 20 metaphases. Since the distribution of SCE was found to be skewed, geometric means (logarithmic transformation) were used, and this restored a normal distribution to the values of both the treated and control populations. Dose-response curves were compared by two-way analysis of variance, and individual points were compared ~ posteriori by least significant difference.
3. Results and discussion The SCE assay requires cells to undergo two cell cycles in the presence of bromodeoxyuridine after exposure to the test compound. In the current study insufficient metaphases were found in two of the three cells strains at 10 I~M DNCB, so the SCE assay could only be performed at concentrations up to 7.5 p~M in those cells. In Table 1 the effect of DNCB on SCE are shown, and in Fig. 1 the percent increase in SCE relative to the solvent control is shown for the three fibroblasts cell strains. Comparison of the dose response curves between 2.5 and 7.5 ~M by two-way analysis of variance was significant at p < 0.005. The increase in the number of exchanges was significant compared to the control starting at 2.5 I~M ( p < 0.025), as well as at 5 and 7 p,M ( p < 0.0001) by the least significant difference test. Since extreme reductions in cell number have been shown to artificially increase SCEs [12], the effect of DNCB on cell proliferation is provided in Table 2. This is the first demonstration in human cells that DNCB is genotoxic. Sister chromatid exchange (SCE) is widely used as a useful and sensitive test of genotoxicity [ 13]. It is considered a good predictor of mutagenicity and carcinogenicity [14], although the exact mechanism of formation of SCEs is unknown. In a recent assessment of the literature by the U.S. Environmental Protection Agency's Gene-Tox Program [13], genotoxicity by SCE of a large number of
107
L D. DeLeve / Mutation Research 371 (1996) 105-108 Table 1 SCE frequencies (mean + SD) in solvent or dinitrochlorobenzene treated fibroblasts Cell strain
Exp. No.
O IxM
2.5 I~M
5 I~M
7.5 p,M
10 p.M
Control No. 1
1 2 3
9.38 5:0.01 7.46 5:0.36 8.92 _ 0.59
11.18 5:0.35 8.355 5:0.13 10.86 + 0.32
12.86 + 0.86 11.11 ___1.14 13.86 5:0.77
NT 11.36 5:0.44 15.47 5:0.42
21.90 + 0.27 11.82 + 0.06 16.92 5:0.27
Control No. 2
1 2 3
9.55 5:1.38 9.26 5:0.01 7.69 5:1.11
10.15 5:0.78 12.4 5:0.86 9.39 5:0.25
11.16 5:2.37 14.1 + 0.72 10.89 + 2.16
12.58 + 0.47 14.69 ___0.18 11.91 _+ 1.04
13.09 5:0.50
Control No. 3
1 2 3
6.32 + 0.27 7.65 + 0.76 7.55 + 0.97
8.2 + 0.32 10.39 + 0.36 9.63 5:0.71
10.86 + 0.86 11.24 5:0.41 11.22 5:0.83
13.44 + 0.33 11.92 + 1.94 13.56 5:0.25
15.12 + 0.40
SCE frequencies from 3 separate experiments in 3 individual control cell strains. NT, not tested; * insufficient data.
compounds was examined. In that report the magnitude of a positive response by SCE was graded by three criteria: a 3-point monotonic dose response, statistical significance at the highest dose with p < 0.001 and a doubling of the SCE frequency above the control. DNCB fits the first two criteria and, at 10lxM, falls just short of the third. Thus, DNCB induces SCE in human fibroblasts and does so in a very significant fashion. A 2-h exposure to DNCB induced a significant increase in SCE at 2.5 ~M or 0.00005%. When used
i: 2, °0- / /f~ t " 0
,
,
,
2.5 5 7.5 10 Dinitrochlorobenzene, FM
Fig. 1. Dinitrochlorobenzene-induced sister chromatid exchange. Each of the three tracings represents the increase in sister chromatid exchange in a normal human skin fibroblast cell strain expressed as the percent increase above the solvent control. Values are expressed as mean + SEM, n = 3. Control No. 1 ( • ) , control No. 2 (O), control No. 3 ( • ) .
clinically the dose used is between 0.1 and 2% for alopecia areata, 2% for systemic lupus erythematosus [9] and between 0.02 and 2% in HIV patients [7,8], used recurrently for prolonged periods of time. When applied topically, DNCB is rapidly absorbed with recovery of 53% of the drug and its metabolites in the urine [15]. Thus, the potential for genotoxic effects is not just limited to the skin. One study has been reported on carcinogenicity testing in rodents [16]. This study found that it was not carcinogenic when fed in large doses for up to 4 months. However, since the proposed clinical use is topical, it would perhaps be more pertinent to study the carcinogenicity of topically applied drug. Particularly in a drug that is less mutagenic in the presence of a metabolic activating system, the first pass effect of an orally administered drug may lead to a quite different outcome than topical application. HIV infection currently leads to a terminal illness, so that a beneficial drug with a potential genotoxic side effect may well be warranted. However, the Table 2 Cell proliferation as a measure of dinitrochlorobenzene toxicity Cell strain
2.5 I~M
5 I~M
7.5 p~M
10 [xM
Control No. 1 Control No. 2 Control No. 3
94 + 6 955-7.7 99+2
66 ± 2 63+8 90+8
63 + 2 4 8 + 10 91 + 7
53 + 9 4 0 + 10 76+ 1
Values are cell numbers at time of harvest (after 48 hour incubation) as a percentage of the cell number present in the solvent treated control.
108
L.D. DeLeve / Mutation Research 371 (1996) 105-108
risk-benefit analysis may be different in systemic lupus erythematosus depending on the individual patient. The possibility of long-term toxicity from DNCB will have to be weighed against the severity of the disease being treated and the benefits that might be achieved by the use of this compound.
Acknowledgements The author would like to thank the late Marilyn Cannon for her technical assistance and Dr. Stephen Spielberg for his support and advice. L.D.D. was supported by a Medical Research Council of Canada Fellowship.
References [1] Strobel, R. and G. R~Shrborn (1980) Mutagenic and cell transforming activities of 1-chlor-2,4-dinitrobenzene (DNCB) and squaric-acid-dibutylester (SADBE), Arch. Toxicol., 45, 307-314. [2] Summer, K.H. and W. GiSggelmann (1980) 1-Chloro-2,4-dinitrobenzene depletes glutathione in rat skin and is mutagenic in Salmonella typhimurium, Mutation Res., 77, 91-93. [3] Black, H.S., F.F. Castrow, II and J. Gerguis (1985) The mutagenicity of dinitrochlorobenzene, Arch. Dermatol., 121, 348-349. [4] Wilkerson, M.G., T.H. Connor and J.K. Wilkin (1988) Dinitrochlorobenzene is inherently mutagenic in the presence of trace mutagenic contaminants, Arch. Dermatol., 124, 396398. [5] Shimizu, M., Y. Yasui and N. Matsumoto (1983) Structural specificity of aromatic compounds with special reference to mutagenic activity in Salmonella typhimurium - a series of chloro- or fluoro-nitrobenzene derivatives, Mutation Res., 116, 217-238.
[6] Stricker, R.B., B.F. Elswood and D.I. Abrams (1991) Dendritic cells and dinitrochlorobenzene (DNCB): a new treatment approach to AIDS, Immunol. Lett., 29, 191-196. [7] Stricker, R.B., Y.S. Zhu, B.F. Elswood, C. Dumlao, J. Van Elk, T.G. Berger, J. Tappero, W.L. Epstein and D.D. Kiprov (1993) Pilot study of topical dinitrochlorobenzene (DNCB) in human immunodeficiency virus infection, Immunol. Lett., 36, 1-6. [8] Stricker, R.B., B.F. Elswood, B. Goldberg, C. Dumlao, J. Van Elk, J. Henry, E.E. Winger and W.L. Epstein (1994) Clinical and immunologic evaluation of HIV-infected patients with dinitrochlorobenzene, J. Am. Acad. Dermatol., 31,462-466. [9] Stricker, R.B., B. Goldberg and W.L. Epstein (1995) Immunological changes in patient with systemic lupus erythematosus with topical dinitrochlorobenzene, Lancet, 345, 1505-1506. [10] Stricker, R.B. and B. Goldberg (1995) Safety of topical dinitrochlorobenzene, Lancet, 346, 1293. [11] Weisburger, E.K., A.B. Russfield, F. Homburger, J.H. Weisburger, E. Roger, C.G. Van Dongen and K.C. Chu (1978) Testing of twenty one environmental aromatic amines or derivatives for long term toxicity and carcinogenicity, J. Environ. Pathol. Toxicol., 2, 325-356. [12] Galloway, S.M., D.A. Deasy, C.L. Bean, A.R. Kraynak, M.J. Armstrong and M.O. Bradley (1987) Effects of high osmotic strength on chromosome aberrations, sister-chromatid exchanges and DNA strand breaks, and the relation to toxicity, Mutation Res., 189, 15-25. [13] Tucker, J.D., A. Auletta, M.C. Cimino, K.L. Dearfield, D. Jacobson-Kram, R.R. Tice and A.V. Carrano (1993) Sisterchromatid exchange: second report of the Gene-Tox program, Mutation Res., 297, 101-180. [14] Abe, S. and M. Sasaki (1982) Sister chromatid exchange as an index of mutagenesis and/or carcinogenesis, in: A.A. Sandberg (Ed.), Sister chromatid exchange, Alan R. Liss,Inc., New York, pp. 461-514. [15] Feldmann, R.J. and H.I. Maibach (1970) Absorption of some organic compounds through the skin in man, J. Invest. Dermatol., 54, 399-404. [ 16] Shapiro, J. (1993) Topical immunotherapy in the treatment of chronic severe alopecia areata, Dermatol. Clin., 11, 611-617.