In vivo and in vitro antigenotoxic effect of nordihydroguaiaretic acid against SCEs induced by methyl methanesulfonate

Mutation Research 419 Ž1998. 163–168

In vivo and in vitro antigenotoxic effect of nordihydroguaiaretic acid against SCEs induced by methyl methanesulfonate E. Madrigal-Bujaidar a

a,)

a , S. Dıaz , P. Revuelta ´ Barriga b, M. Cassani a, P. Marquez ´

b

Laboratorio de Genetica, Escuela Nacional de Ciencias Biologicas, IPN Carpio y Plan de Ayala, Sto. Tomas, ´ ´ ´ Mexico, D.F. cp 11340 b Laboratorio de Citogenetica, FESC UNAM, Mexico ´ Received 22 January 1998; revised 10 August 1998; accepted 27 August 1998

Abstract Nordihydroguaiaretic acid ŽNDGA. is a phenolic lignan which has shown to cause a variety of actions potentially useful for human health; therefore, in this investigation we determined its capacity for inhibiting the rate of sister chromatid exchanges ŽSCEs. induced by methyl methanesulfonate ŽMMS.. We tested the effect of 0.25, 0.50, 1.0, and 2.0 mM of NDGA on the damage exerted by 55 mM of MMS. Cultured human lymphocytes from two female donors were used for the experiment. The best result concerning its modulatory action was obtained with 1.0 mM of NDGA; with this dose the mean inhibitory index including both donors reached 68.2%. The values obtained for the mitotic and proliferative indexes were not significantly modified with respect to the basal data. We also used the mouse bone marrow in vivo system to evaluate the inhibitory effect of the chemical. In this study we tested 1.0, 6.0, and 11.0 mgrkg of NDGA intraperitoneally Ži.p.. administered 1 h before an i.p. injection of MMS Ž40 mgrkg.. The best inhibitory index in this model corresponded to the dose of 11 mgrkg of NDGA Ž86.9%.. The mitotic index and the average generation time showed no significant variation with respect to the control data. Our study established that NDGA produces antigenotoxic action in mammalian cells in vitro and in vivo. q 1998 Elsevier Science B.V. All rights reserved. Keywords: Nordihydroguaiaretic acid; Antigenotoxicity; Human lymphocytes; Mouse bone marrow

1. Introduction The ability of man and other animal species to resist the toxic effects of environmental agents is dependent on detoxification and antioxidant systems. In this context it is important to cope with genotoxic carcinogens that may react with DNA to originate genic and chromosome mutations. One of the suggested strategies for this purpose is to identify effective antimutagens and anticarcinogens and to in) Corresponding author. Fax: q52-5-3-96-35-03; E-mail: [email protected]

crease man’s exposure to them as a means of decreasing the incidence of cancer, a disease which may be related to lifestyle, eating habits or work activities w1x. Mutagen inhibitors have been described mainly in fruits and vegetables. These chemicals may be newly discovered antimutagenic plant extracts where the active principle is not yet known, or specific individual agents which are isolated from active extracts; in this case, these particular compounds may be chemically synthetized in a further step w2x. An example of this procedure is represented by nordihydroguaiaretic acid ŽNDGA., a phenolic lignan originally extracted

1383-5718r98r$ - see front matter q 1998 Elsevier Science B.V. All rights reserved. PII: S 1 3 8 3 - 5 7 1 8 Ž 9 8 . 0 0 1 2 8 - 4

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E. Madrigal-Bujaidar et al.r Mutation Research 419 (1998) 163–168

from the Larrea diÕaricata shrubs and used as an antioxidant in processed meat, dairy products and baked goods, and whose synthesis has produced excellent results since new chemical steps were introduced in the early 1970s w3x. More recently, the property of NDGA as an enzymatic inhibitor has been well established w4,5x. Likewise, its capacity to reduce toxicity and neurotoxicity in cultures of Chinese hamster V-79 cells, cultured hippocampal neurons or bladder damage in mice in vivo has been proved w6–8x, as well as its potential to experimentally interrupt a neurodegenerative pathway relevant to the pathophysiology of Alzheimer disease w9x. The chemical has also been claimed to inhibit both the initiation and the promotion phases in the carcinogenesis of breast and skin w10–14x; therefore, research concerning its antigenotoxic potential is mandatory. To our knowledge, there are only two studies in this area, and the Ames test is used in both. One of these reports shows the antimutagenic potency of the chemical in the strains TA100 and TA98, with and without S-9 homogenate; the authors found a significant effect of NDGA concerning the reduction of the damage produced by direct and indirect mutagens: methyl methanesulfonate, benzo Ž a.pyrene, 2-aminofluorene, and aflatoxin B 1. They also determined a dose-dependent inhibition by NDGA on the mutagenicity induced by the nitrosation products of methylurea w11x. The other study determined the antimutagenic potency of the chemical against the damage produced by benzoŽ a.pyrene in strain TA 98 with metabolic activation w15x. These data prompted us to develop genotoxic and antigenotoxic studies using mammalian cells. In a previous report, we demonstrated that NDGA is capable of increasing the rate of sister-chromatid exchanges ŽSCEs. both in vivo and in vitro w16x; in this report we show that NDGA is also an antigenotoxic agent against the sister-chromatid exchanges induced by methyl methanesulfonate. 2. Materials and methods

ŽBrdU., nordihydroguaiaretic acid, methyl methanesulfonate ŽMMS., Hoechst 33258, colchicine, and dimethyl sulfoxide ŽDMSO.. The culture medium, McCoy 5A, was obtained from In Vitro, and the salts used to prepare buffers were purchased from J.T. Baker. 2.2. Human lymphocyte cultures For the present study BrdU and MMS were dissolved in distilled water, and NDGA in a 2% mixture of DMSOrdistilled water. The peripheral blood of two female volunteers 27 years of age was obtained by venipuncture. These donors had not been exposed to X-rays or any other specific mutagen in the previous 3 months. Duplicate sterile bottles were prepared with 0.6 ml of whole blood, mixed with 8.5 ml of McCoy 5A culture medium, and 0.6 ml of phytohemagglutinin; no fetal calf serum was added. They were placed in the incubator at 378C for 24 h and then, BrdU Ž17 mgrml. was added to all cultures, as were MMS and NDGA in the appropriate doses, to finally establish the following cell cultures: a negative control with only BrdU, a positive control with MMS Ž55 mM., NDGA in doses of 0.25, 0.5, 1.0 and 2.0 mM, and finally, cultures with MMS Ž55 mM. plus NDGA in doses of 0.25, 0.5, 1.0 and 2.0 mM. After 48 h at 378C, colchicine Ž0.5 mgrml. was added for 1 h, the cells were harvested following the usual procedure, and then stained with the fluorescence-plus Giemsa Technique w17,18x. Slides were scored to evaluate the following points: Ž1. the frequency of SCEs in 30-s division mitoses per dose; Ž2. the mitotic index in 1000 cells per dose; and Ž3. the cell proliferation kinetics Ž100 cells per dose. expressed through the proliferative index ŽPI., as the rate of first ŽM1., second ŽM2. and third ŽM3. cellular division, where PI s 1M1 q 2M2 q 3M3r100. The results were evaluated statistically with a two-way ANOVA and the Student’s t-tests. Finally, an inhibitory index ŽII. was obtained with the formula II s1y

NDGA plus MMS treated cellsynegative control

=100

MMS treated cellsynegative control

2.1. Chemicals

2.3. Mouse bone marrow study

Most of the chemicals used in this investigation were purchased from Sigma: bromodeoxyuridine

For this study, MMS was dissolved in distilled water, and NDGA was dissolved in a 24% mixture

E. Madrigal-Bujaidar et al.r Mutation Research 419 (1998) 163–168

of DMSOrdistilled water. Initially, a lethal dose 50 of MMS by intraperitoneal route Ži.p.. was determined w19x. The obtained value was 177.48 mgrkg in male mice ŽNIH. weighing 25 g, kindly donated by the National Institute of Hygiene. For the genotoxic study, 5 mice per lot of the same strain indicated above were used. The animals were maintained at 238C, fed with a commercial diet ŽPurina. and permitted to freely consume food and tap water. According to the obtained LD50 and a pilot study, we used 40.0 mgrkg of MMS Žlot 1. to elevate the rate of SCEs, in order to make the appropriate comparisons with three groups of animals i.p. administered with 1.0, 6.0, and 11.0 mgrkg of NDGA plus 40.0 mgrkg of MMS Žlots 2, 3, 4.. The experiment also included three more groups of mice treated only with 1.0, 6.0 and 11.0 mgrkg of NDGA Žlots 5, 6, 7., and another lot considered the negative control was administered with the solvent Žlot 8.. The experimental procedure included a subcutaneous implantation of a BrdU tablet of 45 mg partially coated with paraffin w20x; 30 min later the i.p. administration of MMS to lot 1, and the administration of NDGA to lots 2, 3, 4, 5, 6, and 7; also, at this time the solvent was inoculated to lot 8. One hour later, animals from groups 5, 6, and 7 received an injection of MMS with the indicated dose. Twenty-

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one hours later the animals were i.p.-treated with colchicine for 3 h to stop cell division; then the mice were killed with CO 2 ; the bone marrow of both femurs was mixed with KCl 0.075 M at 378C for 30 min, and the cells were fixed in Clarke’s solution twice. After that, the cell suspension was dropped onto cold slides, and finally the staining was done according to the method mentioned earlier for the in vitro study. Microscopic observations were made to establish the rate of SCEs in 30 cells per mouse, the mitotic index in 1000 cells per animal, and the cell proliferation kinetics Ž100 cells per mouse., expressed as the average generation time ŽAGT. s 24r1M1q 2M2 q 3M3 = 100, where M1, M2 and M3 represent the rate of cells in first, second, and third cellular division. A statistical analysis was performed with a two-way ANOVA and the Student’s t-tests. Also, the II already indicated for the in vitro study was again applied in this experiment.

3. Results With respect to the in vitro SCE induction study, we found a similar pattern in both donors. This includes a significant increase produced by MMS, a dose-dependent increase when NDGA was adminis-

Table 1 Effect of nordihydroguaiaretic acid on the sister-chromatid exchanges ŽSCE., mitotic index ŽMI. and proliferative index ŽPI. induced by methylmethanesulfonate in cultured human lymphocytes Agent

Dose mM

Donor 1 SCE "

Control MMS NDGA NDGA NDGA NDGA NDGAq MMS NDGAq MMS NDGAq MMS NDGAq MMS

0 55 0.25 0.5 1.0 2.0 0.25 q 55 0.5 q 55 1.0 q 55 2.0 q 55

a

6.9 12.2 b 7.1a 7.5a 8.2 a 9.2 ab 8.4 a 8.2 a 8.4 a 9.6 ab

Donor 2 S.E. 0.43 0.75 0.46 0.41 0.59 0.54 0.58 0.48 0.57 0.48

Controls A 2% solution of DMSOrdistilled water. a Significant difference with respect to MMS. b Significant difference with respect to control. Two-way ANOVA and the Student’s t-test. P s 0.05.

II %

71.7 75.5 71.7 50.0

MI % 2.7 2.5 2.5 2.6 2.3 2.2 2.6 2.3 2.2 2.1

PI % 2.2 2.29 2.06 2.13 2.27 2.19 2.17 2.17 2.08 1.96

SCE " a

6.7 10.1b 7.5a 9.4 b 9.0 b 11.8 ab 8.1ab 9.0 b 7.9 a 11.1b

S.E. 0.50 0.60 0.45 0.57 0.69 0.46 0.46 0.65 0.43 0.45

II %

MI %

PI %

58.8 32.3 64.7 –

2.5 2.4 2.6 2.4 2.3 2.4 2.4 2.2 2.2 2.1

2.11 1.89 2.08 1.71 1.87 1.91 1.94 1.7 2.03 1.63

E. Madrigal-Bujaidar et al.r Mutation Research 419 (1998) 163–168

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Table 2 Effect of nordihydroguaiaretic acid on the sister-chromatid exchanges ŽSCE., mitotic index ŽMI. and average generation time ŽAGT. induced by methylmethanesulfonate in mouse bone marrow Agent

Dose

SCE Mean

"S.D.

DMSOq H 2 0 MMS NDGA NDGA NDGA NDGAq MMS NDGAq MMS NDGAq MMS

24% vrv 40 mgrkg 1.0 mgrkg 6.0 mgrkg 11.0 mgrkg 1.0 q 40 mgrkg 6.0 q 40 mgrkg 11.0 q 40 mgrkg

2.1a 6.0 b 2.3 a 3.3 ab 3.8 ab 5.2 ab 4.2 ab 2.6 a

0.04 0.34 0.38 0.38 0.18 0.21 0.34 0.27

II %

AGT h "

S.D.

MI % "

S.D.

18.5 44.8 86.9

12:29 12:25 11:54 11:16 12:05 12:38 11:48 12:11

0.26 0.54 0.53 0.66 0.43 0.30 0.64 0.60

4.2 4.8 4.8 4.7 4.1 5.3 4.3 4.1

3.5 2.1 1.9 3.4 2.4 3.0 2.9 2.5

a

Significant difference with respect to MMS. Significant difference with respect to negative control. ANOVA and Student’s t-test. P s 0.05. b

tered alone Žbut showing a statistical difference regarding the control with only the high dose., and a SCE decrease when NDGA was tested as inhibitor of the damage produced by MMS, a characteristic that seems to be lost with the high tested dose Ž2.0 mM.. The statistical analysis of pooled data from both donors showed no significant difference; however, the numerical data of the donors were different mainly due to a lower SCE level produced by MMS, as well as to a higher cell response to NDGA in donor 2 as compared to donor 1. This variation between the two individuals was reflected in a difference concerning the determined inhibitory index; thus, the pooled data of the three low doses gave an II mean of 73.0% for donor 1 and 51.9% for donor 2. The best range for the antigenotoxic expression was between 0.25 and 1.0 mM ŽTable 1.. As regards data on MI and AGT, the observed response showed no significant differences when the chemicals were tested alone or combined; however, a mild decrease of both parameters was noted when both compounds were combined using the high dose Ž2.0 mM of NDGA plus 55 mM of MMS. ŽTable 1.. With respect to the in vivo study, it was shown that 40 mgrkg of MMS produced an SCE elevation corresponding to about three times the basal level ŽTable 2.. An SCE dose-dependent increase by nordihydroguairaretic acid was observed in comparison with the SCE value determined in the control animals; however, this result was not statistically

significant. On the contrary, when NDGA was tested to determine its protective effect on the damage produced by MMS, a dose-dependent decrease in the rate of SCEs was obtained when the results were compared with the value determined for MMS alone; a linear regression analysis indicated a correlation coefficient equal to 0.98 Ž Y s y0.259 X q 5.604.. The statistical difference appeared with all three tested doses, but the highest II was obtained with 11.0 mgrkg of NDGA plus 40.0 mgrkg of MMS Ž86.9%.. Concerning the values obtained for AGT and MI, no important variations were observed in any of the combinations of the experiment; for AGT, the lowest and the highest results were 11.16 h and 12.38 h, and for the second parameter the same data points showed 4.1% and 5.3% ŽTable 2..

4. Discussion MMS is a monofunctional alkylating agent with both neoplastic and mutagenic activities. It alkylates DNA at the N y 7 position of guanine and the N y 3 position of adenine, although other minor reaction products may be formed, for example, 1-methylcytosine and 1-methyladenine w21x. All these changes may give rise to abnormal base pairing at DNA replication. In general, at least three types of DNA alterations have been attributed to the chemical: alkylation, depurination, and single strand breaks

E. Madrigal-Bujaidar et al.r Mutation Research 419 (1998) 163–168

w21x. Our results confirmed the genotoxicity of MMS by showing almost a duplication of the SCE rate in the in vitro assay and a triple increase in the in vivo study. The SCE endpoint has been related to the production of methylguanine lesions, which in turn have been related to tumor formation w22x. Also, the induction of SCEs by MMS has been studied in earlier experiments in human lymphocytes with strong positive results w23,24x. Recent attention has been focused on the identification of naturally occurring plant phenols as possible cancer quimopreventive agents. The studies are based on the qualities of a number of compounds including antioxidation, trapping of active oxygen species, ability to inhibit nitrosation, capacity to modulate enzymatic activities that participate in metabolic activation of carcinogens, and antigenotoxicity w25–27x. The antigenotoxic activity of NDGA seems to depend on the specific mutagen involved and on its biological characteristics. Consequently, the origin of such action may have several explanations; one of them refers to its participation as enzyme inhibitor in the arachidonic acid cascade as well as in the P450-dependent monooxigenases, probably because the binding of the hydroxy groups with the catalytic siteŽs. of the enzyme w4x. This inhibition may eliminate the bioactivation of promutagens acting in this way w4,5x. A second property of NDGA, which is related to its phenolic structure, concerns its antioxidative action, as well as its capacity for trapping free radicals; this prevents them from reaching the critical target sites. This action has been observed in a number of studies including the control of lipid oxidation of cooked ground pork, the reduction of cytotoxicity produced in vitro by hydrogen peroxide, the elimination of reactive oxygen species produced by the peptide B amyloid in the nervous system, or the beneficial effect of NDGA on inflammatory bowel diseases where a sustained release of oxyradicals have been mentioned w28–30x. Concerning our study with MMS as a mutagen, as suggested by Wang et al. w11x, the NDGA modulatory response may be related to its action as a molecular interceptor of the methyl groups that are the electrophilic species with which NDGA may react. Once this effect is confirmed, it would be interesting to determine in in vitro assays if the interaction between

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chemicals occurs in the culture media, in the cells, or in both. Also, to better understand the process it would be advisable to use different treatment times for the involved substances. Based on our previous study on the genotoxicity of NDGA w16x, in this experiment we applied doses corresponding to the low range of those used before, with the purpose of reducing the genotoxic damage, so as to better explore the usefulness of the chemical as an antimutagen. The selected doses seem to be adequate for our purpose, as we found an inhibitory effect with at least the three tested low doses in human lymphocytes, although the effect was markedly reduced with the high dose Ž2.0 mM.. Our antimutagenic results were evident in the whole organism with the three tested doses of NDGA, showing a better II value with the tested high dose Ž11 mgrkg.. This suggests that a higher amount of the chemical is required for the antigenotoxic activity to occur in vivo, a fact probably related to the participation of biotransformation and detoxification processes. As the AGT and MI values corresponding to this dose were not altered in relation to their respective control values, it seems worthwhile to test if higher doses may still inhibit the MMS damage. This would help to establish the antigenotoxic range of NDGA. In this context, it is worthwhile to recall that NDGA is a molecule that follows a redox cycle behavior, and that this characteristic is intimately related to its action as a genotoxicrantigenotoxic compound. In summary, in agreement with studies performed earlier in Salmonella typhimurium, we found an inhibitory effect of NDGA on the genotoxic damage produced by MMS in mammalian cells. This confirms the presence of a modulatory property of the chemical which may be eventually used for the benefit of man, but care should be taken concerning its application due to the characteristics of the compound which may, on the other hand, originate undesirable genotoxic andror histological effects.

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