Vitamin C mediated protection on cisplatin induced mutagenicity in mice

Mutation Research 421 Ž1998. 139–148

Vitamin C mediated protection on cisplatin induced mutagenicity in mice Anirudha Giri 1, Dimos Khynriam, Surya B. Prasad

)

Cell and Tumor Biology Lab, Department of Zoology, School of Life Sciences, North-Eastern Hill UniÕersity, Shillong 793 022, India Received 12 March 1998; revised 30 June 1998; accepted 30 June 1998

Abstract In present studies the development of chromosomal aberrations, micronuclei in bone marrow cells and sperm head abnormalities were used as mutagenic bioassay in Swiss albino mice treated with cisplatin alone or ascorbic plus cisplatin. It was noted that in the combined treated hosts the frequency of all the mutagenic parameters were always significantly less than that treated with cisplatin alone. These findings suggest a protective role of ascorbic acid against cisplatin induced mutagenic potentials. Interestingly, in combined treated hosts glutathione ŽGSH. level in bone marrow cells increased significantly which may suggest a possible mechanism of ascorbic acid mediated protection against cisplatin induced mutagenic potentials in the hosts. q 1998 Elsevier Science B.V. All rights reserved. Keywords: Cisplatin; Vitamin C; Mutagenicity; Chromosomal aberration; Micronucleus; Sperm-shape abnormality

1. Introduction Cisplatin Ž Cis-dichlorodiammineplatinum-II. is a potent chemotherapeutic agent which has gained a widespread use against various malignant tumors in different experimental animals w1,2x and in a variety of human malignancies w3x. Many of the biological properties and effects of cisplatin have been well documented w4,5x with numerous reports indicating that the cellular DNA could be the primary target in its anticancer activity w6,7x. However, the therapeutic efficacy of cisplatin is limited due to the development of drug resistance w8x and major side effect, ) Corresponding author. Tel.: q91-364-250105ŽO.r250093ŽR.; Fax: q91-364-250076r250108 1 Present address: Assam University, PO Box No. 63, Silchar788001, India.

nephrotoxicity w9x. Furthermore, its mutagenic potentiality has been shown in bacteria w10,11x as well as in mammalian cells w12–14x. An increased carcinogenic risk with the development of secondary malignancies in animalsrpatients treated with cisplatin has also been reported w15,16x. In an attempt to overcome these impediments, the development of many new cisplatin analogues w17x or the use of cisplatin in combination with other agents w18x have been tried with different degrees of success. Vitamin C Žascorbic acid., an essential nutrient and active reducing agent, is involved in numerous biological effects w19–21x. The chemopreventiver therapeutic role of vitamin C against cancers have been widely reported w22,23x. However, the definite role of vitamin C in cancer treatment still remains controversial w23x. Ascorbic acid at a nontoxic concentration, in combination with certain pharmaco-

0027-5107r98r$ - see front matter q 1998 Elsevier Science B.V. All rights reserved. PII: S 0 0 2 7 - 5 1 0 7 Ž 9 8 . 0 0 1 5 8 - 4

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logical agents produces a synergistic or additive effect on the growth inhibition of tumor cells in culture in vitro w24x and in vivo w25,26x. Considering the reports on mutagenic potentials and carcinogenic risks in using therapeutic dose of cisplatin, earlier we used the subtherapeutical dose of cisplatin with ascorbic acid against murine ascites Dalton’s lymphoma and an effective sequence dependent synergistic antitumor activity was noted w26x leading to tumor regression with significant increase in the host survivals. These findings evoked the idea of evaluating the effectiveness of ascorbic acid on cisplatin induced mutagenicity in Dalton’s lymphoma bearing mice under same experimental conditions. Development of chromosomal aberrations, micronuclei in bone marrow cells and sperm shape abnormality were used as parameters for mutagenic bioassay. The results of the present studies suggest a definite protective role of ascorbic acid in cisplatin induced mutagenicity in the hosts.

2. Materials and methods 2.1. Animals and tumor maintenance Swiss albino mice colony has been maintained in the laboratory under conventional conditions with food pellets ŽAmrut Laboratory animal feeds, Delhi. and water ad libitum. Ascites Dalton’s lymphoma is being maintained in mice by intraperitoneal Ži.p.. transplantations of 1 = 10 7 cells per animal Ž0.25 ml vol. in phosphate buffered saline, PBS, 0.15 M NaCl, 0.01 M sodium phosphate buffer, pH 7.4. in inbred male or female mice of 9–10 weeks age. Tumor transplanted hosts usually survive for 20–24 days. As the cisplatin treatment is commonly used against malignancy in the hosts, only the tumorous animals of both sexes were used for the experiments. 2.2. Chemicals Cisplatin was provided as gift from Professor C.L. Litterst of NIH, USA. L-ascorbic acid Žvitamin C. was purchased from HiMedia laboratories, Mumbai. Colchicine and sodium citrate were acquired from SRL, Mumbai while Giemsa’s stain was from Glaxo, Mumbai. Reduced glutathione, 5,5X-dithio-

bis-2-nitrobenzoic acid ŽDTNB. were bought from Sigma, USA. All other chemicals used in the experiment were of analytical grade. Glass double distilled water was always used in preparation of different reagentsrsolutions. Cisplatin was thoroughly mixed in 0.89% NaCl in darkness 10–15 min before use. 2.3. Chromosomal analysis As our earlier studies have suggested the effective chemotherapeutic combination of ascorbic acid Ž0.5%, through drinking water. with subtherapeutical dose of cisplatin Ž4 mgrkg body wt.. against Dalton’s lymphoma w26x, same combination was used in present studies also. Tumor transplanted animals were divided into four groups consisting of 5–6 animals each, according to randomized block design. The day of tumor transplantation was taken as day 0. One group of mice were administered with single dose of cisplatin Ži.p., 4 mgrkg body wt.. on the 10th day post-tumor transplantation. The second group of mice were given ascorbic acid Ž0.5%; 8.4–9.5 mg dayy1 animaly1 . orally through drinking water from the first day of tumor transplantation and on the 10th day post-tumor transplantation, animals were injected with cisplatin Ži.p., 4 mgrkg body wt... In the third group of mice serving as control, same volume of 0.89% NaCl was injected while the fourth group of mice were given ascorbic acid only. After 24, 48, 72 and 96 h of cisplatin treatment Ži.e., on 11, 12, 13 and 14th day of tumor transplantation. mitotic arrest was initiated 1.5 h prior to sacrificing the animal by injection of colchicine Ži.p., 4 mgrkg body wt... Bone marrow cells were collected from humerous and femur by flushing in warm Ž378C. sodium citrate Ž1%. solution with a hypodermic syringe, aspirated and incubated at 378C for 10 min. The material was then centrifuged Ž1000 rpm for 5 min. and fixed in acetic acidrmethanol Ž1:3.. Finally, before preparation of slides, fixed material was again centrifuged and resuspended in a small volume of fixative by gentle flushing until a cloudy suspension resulted. Two to three drops of this suspension were dropped on a clean slide previously chilled in 50% ethanol, burnt on a flame for a while, air-dried and stained on the following day for 1 h with Giemsa Ž5 ml of stock stain q4 ml methanol q91 ml phosphate buffer, pH

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6.8., washed and mounted in DPX. One hundred good metaphase spreads were examined per animal. Chromosomal aberrations were classified into the general categories of breaksrgaps, exchanges and sister chromatid unions. Gaps have not been considered for statistical analysis of the data due to their controversial genetic significance w27x.

erythrocytes, yellowishrorange colour. and immature white cells Žreddish-blue. were analysed for the presence of micronuclei ŽMN.. Only rounded bodies approximately one-fifth to one-twentieth the size of the main nucleus, lying closer to the nucleus and possessing a staining intensity similar to that of the main nucleus, were scored as micronuclei.

2.4. Micronucleus assay

2.5. Sperm head abnormality assay

The animals were sacrificed by cervical dislocation 30 h after the single dose of cisplatin. For each treatment 3–4 animals were used in different groups as designed in Section 2.3. Bone marrow smears and staining were done following the method of Schmid w28x with slight modifications. Briefly, both the femora were removed and the bone marrow was flushed out into a centrifuge tube with 1% sodium citrate solution Ž208C. from a syringe. The bone marrow cells were dispersed by gentle pipetting and collected by centrifugation at 1000 rpm for 5 min at 48C. The cell pellet was resuspended in a small volume of 5% fetal calf serum in PBS. A drop of this suspension was smeared on a clean slide, air-dried, fixed in absolute methanol for 15 min and stained the following day with May Grunwald-Giemsa. Two thousand each of polychromatic erythrocytes ŽPCEs, immature erythrocytes, pink to purple in colour., normochromatic erythrocytes ŽNCEs, mature

The male mice were divided into three groups consisting of 3–4 animals each. First group of mice were treated with single dose of cisplatin Ž4 mgrkg body wt.. on the 10th day post-tumor transplantation. Second group of mice were orally given ascorbic acid Ž0.5% in drinking water from the second day post-tumor transplantation. and the cisplatin was administered on the 10th day. In the third group of mice serving as controls same volume of 0.89% NaCl was injected. After 10 days of cisplatin treatment, the cauda epididymis of the mice under different experimental conditions was removed and placed in physiological saline. It was minced into pieces with scissors and then left undisturbed for 20 min for the diffusion of spermatozoa. The spermatozoa were spread on microscopic slides, air-dried, fixed in absolute methanol for 15 min and stained with 1% aqueous eosin-Y on the following day. Five hundred sperms from each animal were examined for the

Table 1 Percentage of bone marrow chromosomal aberrations Exchanges

Isochromatid Breaks Gaps

SCU

Totala Mean " S.D.

0.33 0.33 12.00 8.50 5.60 1.75

0.16 0.15 5.33 3.75 2.80 2.25

– – 2.34 2.00 0.40 0.50

– – 4.00 1.75 0.21 0.25

– – 1.66 2.50 2.00 0.24

0.66 " 0.51 0.61 " 0.50 59.66 " 5.02 27.75 " 3.59 b 12.40 " 1.67 c 7.27 " 1.25d

7.00 4.00 2.33 0.66

3.33 3.64 2.65 1.01

2.33 1.01 0.66 0.40

3.32 1.00 2.00 0.34

0.66 3.01 1.34 0.32

54.65 " 7.02 17.33 " 2.51b 7.32 " 2.30 c 3.31 " 0.57 d

Treatment

Total no. of metaphases

Chromatid Breaks

Gaps

Control Only Vit C Cisplatin 24 h Cisplatin 48 h Cisplatin 72 h Cisplatin 96 h

600 600 400 500 400 300

0.16 0.14 50.33 19.50 7.21 4.01

Vit C q Cisplatin 24 h Cisplatin 48 h Cisplatin 72 h Cisplatin 96 h

300 300 300 300

48.34 9.33 2.65 1.34

SCU s Sister chromatid unions. a Values are excluding gaps. Student’s t-test, n s 3–6. Results with same superscripts differ significantly from each other. b p F 0.02; c p F 0.05; d p F 0.02.

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was administered on the 10th day following tumor transplantation. Fourth group of mice used as controls were kept without ascorbic acid and cisplatin. After one day of cisplatin treatment, group of mice was sacrificed, blood was collected and serum was separated. The sera collected at different experimental conditions were used for ascorbic acid estimation using the method of Omaye et al. w30x. 2.7. Glutathione (GSH) estimation

Fig. 1. Graph showing the comparative changes in the number of abnormal metaphase spreads in the population of bone marrow cells treated with cisplatin alone or vit C Žascorbic acid. plus cisplatin in vivo.

abnormalities in sperm head shapes following the criteria as close as possible to those established by Wyrobek and Bruce w29x. 2.6. Serum ascorbic acid estimation The animals were divided into four groups consisting of 4 mice each, according to randomized block design. The day of tumor transplantation was taken as day 0. The first group of mice were treated with cisplatin Ži.p. 4 mgrkg body wt.. on the 10th day of tumor transplantation. Second group of mice were orally given vitamin C Ž0.5% ascorbic acid in drinking water, from the second day post tumor transplantation.. Third group of mice were given ascorbic acid in the same fashion and the cisplatin

Glutathione was determined as non-protein ŽNPSH. and total sulfhydryl ŽTSH. contents in liver and bonemarrow cells of mice under different experimental conditions Žuntreated normal mice, tumorous mice, tumorous mice treated with cisplatin alone or treated with vitamin C plus cisplatin, as per details given in Section 2.6. using the method of Sedlak and Lindsay w31x. Briefly, tissues were homogenized in 0.02 M EDTA, pH 4.7. For determination of NPSH, the homogenate Ž500 ml. was precipitated with 10% trichloroacetic acid Ž500 ml., centrifuged and the supernatant Ž800 ml. 1.6 ml of Tris–EDTA buffer Ž0.4 M, pH 8.9. was added before the addition of Ellman’s reagent Ž10 mM 5,5X-dithio-bis-2-nitrobenzoic acid in methanol, 25 ml.. TSH was determined by adding the homogenate or pure reduced from of glutathione Ž100 ml. to 0.2 M Tris–EDTA buffer Ž1 ml, pH 8.2. and 0.02 M EDTA, pH 4.7 Ž0.9 ml. followed by 20 ml of Ellman’s reagent. After 30 min of incubation at room temperature, the absorbance was read at 412 nm in Beckman DU-640 spectrophotometer. Samples for TSH determinations were centrifuged before absorbance of supernatants were taken.

Table 2 Percentage of micronuclei ŽMN. in bone marrow cells Žvalues are mean " S.D.. Treatment

PCEs

NCEs

Nucleated cells

Control Cisplatin Ž8 mgrkg. Cisplatin Ž4 mgrkg. Vitamin C Vitamin C q Cisplatin Ž4 mgrkg.

0.13 " 0.05 1.26 " 0.04 c 0.56 " 0.14 b,c 0.16 " 0.05 0.30 " 0.05 b

0.06 " 0.04 0.53 " 0.13 0.46 " 0.05d 0.13 " 0.04 0.23 " 0.05d

0.16 " 0.06 0.50 " 0.10 0.30 " 0.05a 0.10 " 0.05 0.16 " 0.04 a

Student’s t-test, n s 3–4. Results with same superscripts differ significantly from each other. a p F 0.05; b,c ,d p F 0.01.

A. Giri et al.r Mutation Research 421 (1998) 139–148

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2.20"0.36 9.93"1.70 ) 6.60"0.56 )

3.1. Chromosomal aberrations

21 92 61

2 7 5

1 9 6

– 2 1

Chromosomal aberration ŽCA. analysis in bonemarrow cells of male and female mice showed no variation in the results. Table 1 presents the results of aberration analysis in mitotic chromosomes of bone marrow cells. Both chromatid and isochromatid type gaps, chromatid deletions that include breaks, sister chromatid unions forming rings, and exchanges forming dicentrics were observed in treated series. Chromatid breaks and gaps occurred more frequently. Total number of aberrations ŽTable 1. as well as the percent abnormal metaphases ŽFig. 1. decreased appreciably with the time of treatment from 24 h to 96 h in both the treated groups and a comparative analysis at corresponding periods of treatment revealed that total abnormal metaphases were always significantly less in vitamin C plus cisplatin treated group of mice than those receiving cisplatin alone ŽFig. 1..

C P, cisplatin, Vit C, vitamin C. Students t-test; ns 3–4. ) Significantly different from each other, pF 0.05.

5 21 13 66 298 198 Control C P Ž10 days. Vit CqC P Ž10 days.

37 167 112

Abnormal sperms Treatment

Table 3 Incidence of sperm head abnormality

Amorphus

Banana

Hooked

Triangular

Dwarf

Double headed

% of abnormal sperms mean"S.D.

3. Results

Fig. 2. Histogram showing the reduction in cisplatin induced genotoxic damage in presence of ascorbic acid. CA s chromosomal aberrations; MNs micronuclei; SA ssperm abnormality.

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3.2. Micronucleus (MN) assay In comparison to the controls, the frequency of MN was increased in cisplatin treated mice with the maximum MN being induced in PCEs ŽTable 2.. However, in the group of mice treated with ascorbic acid plus cisplatin combination, the frequency of micronucleated cells decreased significantly as compared to that of the group treated with cisplatin alone. Treatment of mice with 0.5% ascorbic acid through drinking water did not induce any significant variation in the incidence of MN as compared to the control values ŽTable 2.. 3.3. Sperm head abnormality Various forms of sperm heads, i.e., banana shaped, dwarf, triangular, amorphous, hooked etc were readily recognized. Analysis of these abnormal sperm shapes showed that overall amorphous types Ž56%. and hooked heads Ž31%. were more prevalent in different groups than banana-like heads Ž7%., triangular Ž2.5%. and dwarf heads Ž2.8%.. Cisplatin treatment of mice for 10 days resulted a significant increase in sperm abnormalities ŽTable 3.. Compara-

Table 4 Total ŽTSH. and Non-protein ŽNPSH. glutathione concentration Žmmolrg. in tissues of the mice under different experimental conditions Tissues

Treatment

TSH mean "S.D.

NPSH mean "S.D.

Liver

Normal Žcontrol. Tumor bearing Žcontrol. Cisplatin Vitamin C qcisplatin

11.72"1.50

4.20"0.80

11.91"1.06

4.50"0.16 a

11.64"1.88 12.15"0.86

3.85"0.20 a,b 4.32"0.21b

Normal Žcontrol. Tumor bearing Žcontrol. Cisplatin Vitamin C qCisplatin

3.10"0.28

0.44"0.02

3.28"0.17 f

0.46"0.01d

2.67"0.25 c,f 3.85"0.35 c

0.38"0.02 d,e 0.45"0.03 e

Bone marrow

Cisplatin Ž4 mgrkg body wt.. was given for 24 h on the 10th day and vitamin C Žascorbic acid, 0.5%. in drinking water from the first day after tumor transplantation. Statistical analysis: Student’s t-test, ns 4, results with same superscripts differ significantly from each other. c,d pF 0.01; a,f pF 0.02; b,e pF 0.05.

tive analysis of sperm head abnormalities in mice treated with cisplatin alone and ascorbic acid plus cisplatin showed significant decrease in abnormalities in the later group of mice ŽTable 3., thus they also depicted the similar trend as noted for chromosomal aberrations and incidence of MN. The overall protective function of ascorbic acid on cisplatin induced genotoxic effects observed in the form of CA, MN and sperm head abnormality has been presented in Fig. 2. 3.4. Serum ascorbic acid Fig. 3. Histogram showing the changes in serum ascorbic acid content under different experimental conditions. Ns normal mice; TBs tumor bearing mice; Vt s tumor bearing mice supplemented with 0.5% ascorbic acid in drinking water from the first day after tumor transplantation; CPs tumor bearing mice treated with cisplatin Ž4 mgrkg body wt..; VtqCPs tumor bearing mice treated with ascorbic acid and cisplatin. Statistical analysis: Student’s t-test, ns 4; results with same superscripts differ significantly from each other, a: pF 0.05; b,c,d: pF 0.01; e: pF 0.02.

The results of the serum ascorbic acid measurement showed that ascorbic acid level decreased in tumor bearing mice Ž7.11 mgrml. as compared to that of normal mice Ž10.38 mgrml.. Cisplatin treatment of the tumor bearing hosts for one day did not alter the ascorbic acid level significantly. However, in the group of tumor bearing mice receiving 0.5%

A. Giri et al.r Mutation Research 421 (1998) 139–148

ascorbic acid only or combined treatment of 0.5% ascorbic acid plus cisplatin Ž4 mgrkg body wt.. showed significant increase in serum ascorbic acid concentration Ž13.44 and 11.05 mgrml respectively. ŽFig. 3.. 3.5. GSH leÕels A significant decrease in non-protein ŽNPSH. thiol contents of bone marrow and liver was noted after cisplatin treatment for 24 h. However, the NPSH and total glutathione ŽTSH. increased significantly in the bone marrow of vitamin C plus cisplatin treated mice as compared to the group of mice treated with cisplatin alone ŽTable 4..

4. Discussion The results of the present investigations show the protective effect of vitamin C Žascorbic acid, AA. on cisplatin induced mutagenic effects in murine system. Development of chromosomal aberrations ŽCA., micronuclei ŽMN. and sperm head abnormality have been commonly used as sensitive biological indicator in the mutagenic bioassays of a drug. In present study the development of all these mutagenic parameters ŽTables 1–3. were seen after cisplatin treatment in vivo and it supports earlier findings of its genotoxic properties w10,14x. The chromosomal aberration pattern revealed that chromatid breaks and gaps occurred more frequently. The total number of aberrant metaphases ŽFig. 1. as well as CA ŽTable 1. were noticed to be highest at 24 h of treatment which decreased appreciably during later periods. It has been reported that chemicals in general produce the highest frequency of aberrations in rodents 24 h after single exposure, which roughly coincides with the normal length Ž22–24 h. of the cell cycle w32x. However, decrease in aberrations during later periods could be due to various possible reasons such as death of damaged cells, the clearance of drug from the body, post-replication repair process etc. In fact, an involvement of post-replication repair process in cisplatin induced DNA damage has been established w33x. However, as compared to cisplatin treated animals, the abnormal metaphases were always less Ž35 to 52%. in cisplatin plus AA treated group at corre-

145

sponding time points ŽFig. 1.. Total chromosomal aberrations also decreased appreciably at 48–96 h in AA plus cisplatin treated hosts ŽTable 1.. These findings in turn suggest a definite protective role of AA on chromosomal aberrations induced by cisplatin. Micronuclei ŽMN. are cytoplasmic chromatin masses with the appearance of small nuclei that arise from chromosome fragments of intact whole chromosomes lagging behind at the anaphase stage of cell division w34x and they can be easily recognized in the cytoplasm of immature polychromatic erythrocytes w28x. The incidence of MN was increased in cisplatin treated mice ŽTable 2.. Giavini et al. w35x reported a dose-related increase of MN in the blastocysts treated with anticancer drugs, i.e., cis-platinum, cyclophosphamide and adriamycin. The comparative analysis of the frequency of MN in the mice treated with cisplatin alone and AA plus cisplatin also showed similar trend as noted for CA, i.e., the frequency of micronucleated cells decreased significantly in the later group ŽTable 2.. However, the occurrence of comparatively lower frequency of MN than CA could be due to the fact that the fate of chromosomal fragments are uncertain so far their segregation in MN is concerned. Apart from that all fragments do not necessarily form visible MN w36x. Mice treated with cisplatin for 10 days developed various sperm head abnormalities ŽTable 3.. This suggests that cisplatin could reach the germ line cells and indicates its potentiality as germ cell mutagen also. Cisplatin has been reported to cause sertoli cell dysfunction w37x and embryotoxicity w35x in rats. An analysis of sperm head abnormality in mice treated with cisplatin as a single agent and in combination with ascorbic acid showed a significant decrease in abnormality in the combined treated group ŽTable 3. which also depicts the similar protective role of AA as observed for CA and MN. It has been reported that dietary AA protects human sperm from endogenous oxidative DNA damage that could affect sperm quality w38x. Thus, the results of all the three mutagenic parameters in present studies showing the significant reduction in cisplatin induced genotoxic damage in presence of ascorbic acid clearly suggest the protective role of ascorbic acid Žvitamin C. ŽFig. 2. on cisplatin’s mutagenic potentials. In fact, the ability of ascorbic acid to confer marked protection

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to the animals against many toxic chemical agents and heavy metals have been described w19x. Ascorbic acid mediated inhibition of bacterial Ž Salmonella typhimurium induced TA 100. mutagenicity induced by N-methyl-N X-nitro-N-nitrosoguanidine w39x and rat mutagenicity induced by the alkylating agent, Nethyl-N-nitrosourea ŽENU. w40x has been reported. Cisplatin also behaves like an alkylating agent so the similar modulation mechanism might be involved. Since GSH has been reported to be implicated in the metabolism of cisplatin w41x, the GSH contents were measured in bone marrow and liver of mice under different experimental conditions. The results from this part of experiment suggest the possible involvement of cellular GSH as a mechanistic step in ascorbic acid mediated protection against cisplatin induced mutagenicity. It was seen that cisplatin treatment for 24 h resulted a significant decrease in bone marrow GSH ŽTable 4. with the development of mutagenic effects ŽTables 1 and 2.. A decrease in bone marrow GSH noted after cisplatin treatment could lead to less protective mechanism in bone marrow cells and thereby developing more cisplatin induced mutagenic effects. In fact, enhanced cisplatin toxicity by a decrease in tissue GSH levels has been noticed w42x. However, in the group of mice combined treated with AA and cisplatin showing decreased mutagenic effects and a significant increase in bone marrow TSH and NPSH ŽTable 4. suggests the definite significance of GSH also. It could be the elevated level of GSH to protect the cells against cisplatin induced mutagenicity. Ascorbic acid and GSH are effective water-soluble cytoplasmic antioxidants participating in cellular protection against oxidative stress and toxic agents w43x. A protective role of ascorbic acid in 2,4-dichlorophenol induced teratogenicrcarcinogenic toxicity along with significantly increased liver ascorbic acid and GSH levels has also been reported w44x. Johnston et al. w45x showed that vitamin C supplementation to healthy adults elevated red blood cell GSH. In present studies as expected the serum AA level increased significantly in combined treated hosts ŽFig. 3. but the increase in bone marrow GSH seems to be of special significance as far as the incidence of various mutagenic effects in the bone marrow is concerned. Lowered mutagenic frequencies observed in the bone marrow of hosts treated with cisplatin

and cysteine Žprecursor of GSH. simultaneously Žunpublished results. support this hypothesis. It has been suggested that GSH and ascorbate pools in cells may interact with each other, producing cooperative antioxidative protective effects w46x. The cooperation between AA and GSH in various protective effects have been reviewed by Meister w47x where it was reported that giving ascorbate to animals led to increase GSH levels. Thus, the present studies clearly indicate a protective role of ascorbic acid against cisplatin induced mutagenicity and suggest the possible cooperative involvement of GSH also in its protective function.

Acknowledgements The authors are grateful to Dr. R.K. Bhola of Gauhati University for providing ascites Dalton’s lymphoma. The financial support was provided by North-Eastern Hill University.

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