Genotoxicity of nitric oxide produced from sodium nitroprusside

Mutation Research 413 Ž1998. 121–127

Genotoxicity of nitric oxide produced from sodium nitroprusside Weici Lin ) , Hongwei Xue, Shengying Liu, Yiqin He, Juanling Fu, Zongcan Zhou

1

Department of Toxicology, Beijing Medical UniÕersity, Beijing 100083, China Received 19 February 1997; revised 5 January 1998; accepted 8 January 1998

Abstract Induction of mutation and micronucleus ŽMN. formation by nitric oxide ŽNO. was investigated in mammalian cells using sodium nitroprusside ŽSNP. as a drug donor of NO. Results showed that the concentration of NOy 2 in the tested solution rose according to time- and concentration-exposure to SNP. The treatment of SNP Ž0.5–8 m molrml with S9 or 2–8 m molrml without S9. induced a concentration-dependent increase in the mutation frequency at the gpt gene locus in g12 cells and caused a 13- ŽyS9. to 25- ŽqS9. fold increase above the background level at the highest concentration. A statistically significant increase in the number of micronucleated binucleated cells ŽMNBN. was also observed in treated groups. MNBN‰, MN‰ and the proportion of the multiple micronuleated cells increased in a concentration-dependent manner in the concentration range of SNP Ž0.5–4 m molrml with S9 or 2–8 m molrml without S9.. Our results indicate that SNP, an NO releasing drug, is genotoxic in g12 cells. q 1998 Elsevier Science B.V. Keywords: Nitric oxide; Sodium nitroprusside; g12 cell; Genotoxicity

1. Introduction Nitric oxide ŽNO. has become one of the most important molecules in the area of biological and medical investigation in recent years. Research on NO has mainly focused on its function as a messenger in immunological reactions. It has been verified that during an inflammatory response, nitric-oxide synthase ŽNOS. is induced and NO is produced in large quantities in macrophages, Kupffer cells, hepatocytes and other cells w1,2x. Epidemiological investi)

Corresponding author. Tel: q86-10-62091531; fax: q86-1062015681. 1 E-mail: [email protected]

gations show that certain chronic inflammations are related to carcinogenesis w2x. Several reports have indicated that NO is mutagenic in vitro. Exposure to low concentrations of NO, alone or in combination with NO 2 , results in significantly enhanced mutation in Salmonella typhimurium strain TA1535. NO is also a more effective mutagen than NO 2 w3x. NO-releasing compounds, including sperm ine – NO complex, NaŽO 2 N2 –NET2 . and glyceryl trinitrate, are mutagenic to S. typhimurium strain TA1535 and almost all of the analyzed mutants contained C ™ T transitions in the his G46 ŽCCC. target codon w4,5x. Mutagenicity of NO has also been shown in mammalian cells. When TK6 human lymphoblastoid cells were

1383-5718r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 1 3 8 3 - 5 7 1 8 Ž 9 8 . 0 0 0 1 4 - X

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W. Lin et al.r Mutation Research 413 (1998) 121–127

treated by directly introducing NO gas to the medium, cells were mutated at both HPRT and TK loci, DNA strands were broken and purine bases were deaminated in a concentration dependent manner w6x. NO could induce OUAr mutation in rat lung cells after exposure in vivo to NO w7x. Hepatocellular carcinoma was seen beginning at 78 weeks of age in male F344 rats receiving dietary glyceryl trinitrate from 8 weeks of age w8x. Consequently Beckman w9x and Schmidt et al. w10x have suggested that NO has a double-edged role in specialized tissues and cells. This means that NO is not only an important bioregulatory agent but may also be an endogenous cytotoxin, mutagen andror carcinogen. In this paper, we used a slow-releasing NO donor, sodium nitroprusside, to determine the gpt gene locus mutation and micronucleus formation in the g12 cell line. Regarding these two endpoints, there have been no previously-published data on NOmediated genotoxic effects. Our results indicate that NO and SNP are genotoxic in g12 cells.

2. Material and methods 2.1. Cell culture The g12 cell line was obtained from Dr. Catherine B. Klein ŽNew York University Medical Center, Nelson Institute of Environmental Medicine, NY.. The cells were routinely grown in Ham’s medium F12 ŽGIBCO; Grand Island, NY. supplemented with 5% newborn calf serum ŽGIBCO., 100 IUrml penicillin and 100 m grml streptomycin at 378C in a humid atmosphere containing 5% CO 2 . 2.2. Measurement of nitrite (NO2y) concentration As an oxidation product of NO liberated from SNP, NOy 2 was spectrophotometrically determined at 546 nm according to the Griess reaction w11x with a calibration curve using known concentrations of sodium nitrite. The measurements were performed at different incubation times Ž0, 0.5, 1, 3, and 24 h. with a single concentration Ž0.3 m molrml. of SNP ŽSuzhou Tiaoyuan Chemical, China, approximately 99%. and after 1 h of treatment with several desired

concentrations Ž0, 0.5, 1, 2, 4 and 8 m molrml. of SNP Žsee Sections 2.3 and 2.4.. Results were expressed as net amounts of NOy 2 per ml. 2.3. Mutagenicity assay at gpt locus The mutagenicity assay was conducted according to the replating technique, described by Klein and Rossman w12x. In brief, g12 cells were seeded in triplicate at 3 = 10 2 cells in each 24 cm2 tissue culture flask with 5 ml culture medium for the determination of toxicity and in duplicate at 8 = 10 4 cells in each 40 cm2 flask with 8 ml culture medium for the determination of mutant fraction. After 4 h of incubation, the attached cells of treated groups were exposed to the designed concentrations of SNP, freshly prepared in Hank’s Balanced Salt Solution ŽHBSS. without phenol red, for 1 h at 378C in capped flasks in the dark. The cells of untreated controls were also incubated in HBSS Žno SNP. for an equal time. The positive control was 0.5 m grml of N-methyl-N X-nitro-N-nitrosoguanidine ŽMNNG; Sigma, St. Louis, MO. without S9 or 2.5 m grml of benzoŽ a.pyrene ŽBŽ a.P; Sigma. with S9. The liver S9 was obtained from male Wistar rats treated with Aroclor 1254. After treatment, the tested solution was removed from each flask and the NOy 2 concentration in the solution was immediately determined. In order to obtain the exact absorption value by nitrite, the zero point was checked with HBSS without phenol red. The presence of S9 did not affect the absorption measurement, because there was a control containing S9 and its absorption was zero. The flasks were rinsed with HBSS and replenished with fresh medium. Following a 7-day phenotypic expression period, cells from each treatment were reseeded in selective medium containing 10 m grml 6thioguanine ŽSigma, 6-TG F12. in 10 replicates at 2 = 10 5 cellsrflask for mutant selection and in nonselective medium ŽF12. in triplicate at 2 = 10 2 cellsrdisk Ž f 33 mm. for the determination of plating efficiency. For toxicity and plating efficiency, colonies in nonselective medium were scored by staining after 1 week. Mutant colonies in 6-TG F12 were stained and scored after 12 days. Plating efficiencies were calculated by dividing the number of colonies by the number of cells reseeded in each

W. Lin et al.r Mutation Research 413 (1998) 121–127

group. The mutant fraction was calculated according to the formula: Mutant fraction the number of 6 – TG t mutant colonies s

plating efficiency= 2 = 10 6

A three-fold increase in mutant fraction over the background level was regarded as a positive result. Statistical evaluations were made by means of analysis of Poisson random variables for comparing mutant fraction of each treatment group with that of control. The correlation coefficients Ž r . between the mutant fraction and the concentration of NOy 2 were calculated. 2.4. Micronucleus assay The MN assay was performed according to the procedure of Keshava et al. w13x and Rodilla et al. w14x. First, about 10 6 g12 cells were seeded in every 15 cm2 flask with 3 ml culture medium and allowed to attach and grow for 24 h, then exposed to various concentrations of SNP with or without S9 in HBSS. MNNG and BŽ a.P were used as the positive controls. Three or four replicate cultures were included for each treatment group. An hour after exposure, SNP was removed, NOy 2 concentration in the solution was measured and flasks were immediately rinsed and the freshly prepared medium containing 6 m grml cytochalasin B ŽCYB; Sigma. was added. After 18 h of post-treatment incubation, the medium was decanted and the cells, dislodged with trypsin– EDTA, were collected and centrifuged at 1000 rpm for 5 min. The supernatant was removed and the pellet was resuspended in the remaining solution. The cells were treated with hypotonic solution Ž0.075 M KCl. at 378C for 4 min and fixed with freshly prepared fixative Žmethanol:acetic acid s 3:1. for 5 min and recentrifuged. The fixation step was repeated once. Finally, the cell pellet was resuspended in 0.5 ml of fixative Žmethanol:acetic acid s 97:3. and 2–4 drops of cell suspension were dropped onto a cold, wet slide. Following air drying at room temperature, the cells were stained with 0.1% Giemsa and scored by using a microscope. For calculation of nuclear division index, 400 cells per treatment group were scored for the presence of one, two or more

123

than two nuclei. For micronucleus analysis, 1000 binucleated cells ŽBN. of each treatment group were scored for the presence of one, two or more than two micronuclei ŽMN.. The adopted morphological criteria regarding MN scoring in BN cells were like those described by Keshava et al. w13x and Rodilla et al. w14x. Nuclear division index ŽNDI. was calculated according to the formula: NDI s 1 N q Ž 2 = 2 N . q Ž 4 = ) 2 N . r400 cells scored where N is the number of nuclei. The frequencies of micronucleated binucleated cells ŽMNBN. were analyzed by x 2 test. Correlation coefficients were calculated for both MNBN and NDI with NOy 2 content in the solution tested.

3. Results 3.1. NO2y formation NOy 2 concentrations were determined in order to reflect the production of NO after exposure to SNP. An increase in NOy 2 concentration in the solution Table 1 NOy 2 concentration in treatment solution after exposure to 0.3 m molrml SNP ŽqS9. for different time Time Žh. NOy 2 a

.a

Žnmolrml

0

0.5

1.0

3.0

24.0

0

1.60

2.41

4.81

27.56

Mean of duplicate samples.

Table 2 NOy 2 concentration in treatment solution after exposure to various concentrations of SNP for 1 h Concentration of SNP Ž m molrml.

Ž . a,b NOy 2 concentration nmolrml yS9 qS9

0.0 0.5 1.0 2.0 4.0 8.0

0 ND c ND c 2.64"1.24 6.86"1.10 13.77"1.37

a

0 5.50"0.82 12.53"1.73 25.55"2.45 56.14"3.90 69.00"1.75

Mean"standard deviation Ž8–9 replicate samples.. ryS 9 s 0.9980 Ž P - 0.01., rqS9 s 0.9563 Ž P - 0.01.. c Not determined.

b

W. Lin et al.r Mutation Research 413 (1998) 121–127

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Table 3 Mutagenic effect of the NO donor SNP at the gpt locus in g12 cells Concentration of SNP a Ž m molrml.

S9

Survival efficiency Ž%.

Reseeding plating efficiency Ž%.

Number of 6-TG r mutant b

Mutant fractionr10 6 cells

0 2 4 8 MNNG c 0 0.5 1 2 8 BŽ a.P d

y y y y y q q q q q q

100 94.40 79.31 60.36 82.83 100 90.20 81.18 78.43 38.82 89.41

107.2 149.7 92.0 59.5 122.0 128.0 95.3 88.0 81.3 88.5 91.8

10 8 53 75 171 11 9 31 81 196 119

4.66 2.97 28.80)) 63.03)) 70.08)) 4.30 4.72 17.61)) 49.80)) 110.73)) 64.79))

Fold of the negative control y 6.18 13.52 15.04 1.10 4.10 11.58 25.75 15.07

a

g12 cells were treated with SNP for 1 h. 6-Thioguanine resistant mutant. X c N-methyl-N -nitro-N-nitrosoguanidine Žused at a final concentration of 0.5 m grml.. d BenzoŽ a.pyrene Žused at a final concentration of 2.5 m grml.. )), P - 0.01. b

was observed after exposure to single concentration Ž0.3 m molrml. of SNP ŽqS9. with increase of time ŽTable 1.. A dose-dependent increase in NOy 2 concentration was also observed within the dose range of 0.5–8.0 m molrml of SNP in the mutagenicity assay and the micronucleus assay. The elevation of NOy 2 concentration was more obvious in the presence of S9 ŽTable 2..

The mutant fraction was 4 or 6 times of the background at the concentration of 1 m molrml SNP with S9 or 4 m molrml SNP without S9. At the highest tested concentration Ž8 m molrml SNP., the mutant fraction rose to 13 times Žwithout S9. and 25 times Žwith S9. of the solvent control value. In addition, there was a positive correlation between the mutant fraction and NOy concentration Ž ryS9 s 0.9779, 2 rqS 9 s 0.9875, P - 0.05 for both..

3.2. NO-induced mutation at gpt locus 3.3. NO-induced MN formation It was observed that a substantial concentrationdependent decrease in relative survival and a dosedependent increase in mutant fraction at gpt locus in g12 cells were induced by SNP treatment ŽTable 3..

As SNP concentration increased, the proportion of binucleated cells and the NDI in g12 cells decreased, and when the tested solution contained S9 mixture,

Table 4 Effect of the NO donor SNP on the micronucleus formation in g12 cells without S9 Concentration of SNP Ž m molrml.

Cell cycle kineticsr400 cells 1N 2N )2 N

NDI

MNBN cellsr1000 BN cells 1MN 2MN ) 2MN

MNBN Ž‰.

MN Ž‰.

0 2.0 4.0 8.0 MNNG a

174 " 17 209 " 33 217 " 34 301 " 23 357 " 8

1.59 " 0.04 1.49 " 0.08 1.46 " 0.06 1.26 " 0.06 1.13 " 0.02

22 " 6 30 " 2 45 " 4 56 " 3 98 " 19

24.5 " 7.2 34.7 " 2.5 54.5 " 0.7)) 75.5 " 0.7)) 131.7 " 16.1))

27.3 " 8.8 44.3 " 5.0 67.0 " 4.2 101.0 " 1.4 180.0 " 8.7

222 " 40 189 " 33 180 " 35 98 " 21 39 " 7

4"1 3"1 4"1 2"2 5"1

3"2 2"2 7"2 14 " 2 20 " 5

0 2"2 3"1 6"0 13 " 8

NDI, nuclear division index; N, number of nuclei; BN, binucleated cells; MN, micronucleus; MNBN, micronucleated binucleated cells. X a N-methyl-N -nitro-N-nitrosoguanidine Žfinal concentration 2.5 m grml.. )), P - 0.01.

W. Lin et al.r Mutation Research 413 (1998) 121–127

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Table 5 Effect of the NO donor SNP on the micronucleus formation in g12 cells with S9 Concentration of SNP Ž m molrml.

Cell cycle kineticsr400 cells 1N 2N )2 N

NDI

MNBN cellsr1000 BN cells MNBN Ž‰. 1MN 2MN ) 2MN

MN Ž‰.

0 0.5 1.0 2.0 4.0 BŽ a.P a

206 " 12 307 " 10 338 " 13 356 " 5 374 " 3 333 " 10

1.54 " 0.03 1.25 " 0.03 1.17 " 0.03 1.12 " 0.02 1.07 " 0.01 1.19 " 0.02

24 " 9 40 " 3 53 " 5 69 " 2 92 " 8 75 " 5

25.7 " 9.7 50.0 " 11.5 90.3 " 13.7 119.0 " 13.1 167.7 " 5.5 116.3 " 10.6

184 " 14 90 " 10 59 " 13 42 " 5 25 " 1 63 " 11

10 " 4 3"1 3"0 2"2 1"1 4"2

1"1 3"2 10 " 3 12 " 2 17 " 2 17 " 2

0 2"1 4"3 7"3 8"1 2"2

24.7 " 9.5 40.0 " 12.3) 67.0 " 6.2)) 88.0 " 2.6)) 117.3 " 5.7)) 93.7 " 4.7))

NDI, nuclear division index; N, number of nuclei; BN, binucleated cells; MN, micronucleus; MNBN, micronucleated binucleated cells. a BenzoŽ a.pyrene Žfinal concentration 2.5 m grml.. ), P - 0.05. )), P - 0.01.

the decline of these values was more obvious ŽTables 4 and 5.. These data indicated that SNP treatment manifested cytotoxicity and induced cell cycle delay. The clear dose-dependent increase in the frequencies of micronuclei and micronucleate binucleated ŽMNBN. cells with increasing SNP concentrations was also observed. In the frequency of MNBN cells, there was a statistically significant increase over the control at the concentrations of 4 and 8 m molrml SNP in the absence of S9 and the lowest effective mutagenic concentration was 0.5 m molrml SNP in the presence of S9. There are negative correlations between NDI and NOy 2 concentrations in the treatment solution Ž ryS 9 s y0.9876, rqS9 s y0.9366, P - 0.01 for both. and positive correlations between MNBN‰ and NOy 2 concentration in the treatment solution Ž ryS 9 s 0.9706, rqS9 s 0.9834, P - 0.05 for both.. In addition, it was also noted that the proportion of binucleated cells with multiple micronuclei Ž2 and ) 2MN. increased more obviously at the high concentration of SNP than at the low concentration of SNP.

4. Discussion Sodium nitroprusside is a peripheral vasodilator and can be used clinically for the treatment of hypertensive emergencies, for ventricular unloading in acute congestive heart failure, and after myocardial infarction. SNP contains a nitrosonium ŽNOq. ion, thus NO will be released when the solution of SNP is irradiated with visible light. SNP also can be

progressively decomposed to release a large amount of NO in solution in the dark or in the presence of various reducing agents including cysteine, other thiols and cytochrome P450 with an NADPH-regenerating system on the following equation w15,16x. Fe Ž CN . 5NO NOy 2

2y

q H 2 O ™ Fe Ž CN . 5H 2 O

2y

q NO

is the product of oxidation of NO w17,18x, so the increase of NOy 2 concentration can be regarded as the indicator of the production of NO. In this work it was shown that NOy concentration 2 increased in SNP concentration- and time-dependent manner. We found that mutation at the gpt locus and micronucleus formation in g12 cells were induced by SNP. The lowest mutagenic concentration for the two end points tested was 4 m molrml SNP without S9 and 0.5 or 1.0 m molrml SNP with S9, respectively. These concentrations were equivalent to 5–12 nmolrml NO in practical measurement. Meanwhile, within the concentration range tested, the positive correlations were shown between the elevation of NOy 2 concentration in treatment solution and the increase in mutant fraction at gpt locus or the increase in the frequency of MNBN cells. SNP can be decomposed to NO and CNy. NO in aqueous solution containing oxygen is primarily oxiŽ . w x dized to NOy 2 nitrite . Fehsel et al. 19 have pointed out that cyanide ions ŽCNy. did not induce DNA damage. Although sodium nitrite, the possible nitric product of SNP, was mutagenic in S. typhimurium strain TA1535 w20x, it was negative or unconfirmedly negative in bone marrow micronucleus assay and

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W. Lin et al.r Mutation Research 413 (1998) 121–127

was unresolved in carcinogenicity tests in rodents w21,22x. There also was no available information about specific locus mutation w23–25x. Isuda and Kato have showed that nitrite could cause the chromosome aberration of hamster cells and the lowest effective concentration was 10 mM w26x. But in our Ž paper the concentration of NOy 2 was much lower 69 m M at the highest concentration of SNP with S9.. So it is likely that the primary cause leading to genotoxicity in g12 cells is not nitrite but the excessive accumulation of NO produced from SNP. We believe that this paper is the first one to report that micronuclei may be induced by NO. We also noted that the proportion of MNBN cells with multiple micronuclei Ž2 and ) 2. after treatment with SNP was higher than that of the control. Recent studies have verified that NO elicits concentrationand time-dependent DNA single-strand breaks in treated TK6 human lymphoblasts, intact NIH3T3 cells and isolated nuclei from NIH3T3 cells w6,27x. We presume that micronuclei could be formed from the fragments of DNA single-strand broken by NO during DNA replication. Our results are in agreement with Nguyen et al., who showed that TK6 human lymphoblastoid cells were mutated 15- to 18-fold above background level at both the hprt and tk gene loci when extraneous NO gas was directly introduced into the medium at the concentration of 22 m molrml w6x. However, our research is more similar to the situation of chronic rise and excessive accumulation of NO under the pathological state. It has been reported that macrophages might produce p mol NO per cell per hour over days w6x. In our experiment, NO was gradually delivered from SNP and the cells were exposed to a constant barrage of NO, so that the genotoxic damage in mammalian cells could be induced by NO at a much lower concentration Ž5–12 nmolrml. than in the previous other report Ž5.5–22 m molrml. w6x. It is known that NO has genotoxicity, so it might be a potential carcinogen Žinitiator. for human. Epidemiological studies suggested that several human cancers, such as hepatocellular carcinoma and gastric cancer, were associated with chronically elevated NO in infections w2x. Animal experiments showed that high-dose single boluses of glyceryl trinitrate, a donor of NO, had no detectable influence on tumor yield in rats, but could induce

hepatocellular carcinoma after prolonged feeding w8x. Therefore, further investigation is necessary for the potential carcinogenicity of NO and its possible mechanism of initiation and promotion.

Acknowledgements This work was supported by the National Foundation of Natural Sciences of China ŽNo. 39310602..

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