Lymphocyte proliferation kinetics and sister-chromatid exchanges in individuals treated with metronidazole

It

Fundamental and Molecular Mechanisms of Mutagenesis

ELSEVIER

Mutation Research 305 (1994) 133-137

Lymphocyte proliferation kinetics and sister-chromatid exchanges in individuals treated with metronidazole Guillermo Elizondo a,b, Regina Montero a, Jorge E. Herrera b,c, Enrique Hong b, Patricia Ostrosky-Wegman a , , a Instituto de Investigaciones Biom~dicas, UNAM, P.O. Box 70228, Ciudad Universitaria, 04510 M~xico D.F., Mexico b Departamento de Farmacologfa y Toxicologla, CINVESTAI/,, Mdxico D.F., Mexico c Departamento de la Investigacidn Clfnica y Biora~dica, Hospital Miguel Silva, Morelia, Michoacdn, Mexico

(Received 15 January 1993) (Revision received 1 September 1993)

Abstract

Metronidazole, an effective agent for the treatment of protozoan infections, is frequently used in developing countries. However, the employment of this drug has been questioned in view of its mutagenicity in bacteria and carcinogenicity in mice. A genotoxic study was carried out in which cellular proliferation kinetics and the frequency of sister-chromatid exchanges were determined in human peripheral blood lymphocytes from 12 individuals treated with therapeutic doses of metronidazole. No effect was observed on mitotic index with the treatment, although a significant increase was found in three individuals after treatment. No increase of sister-chromatid exchanges was detected. The rate of lymphocyte proliferation kinetics showed an increase after the metronidazole treatment in all patients, indicating a possible immunostimulatory action. Key words: Human lymphocytes; Cell proliferation; Sister-chromatid exchange; Metronidazole

1. Introduction

Metronidazole is the drug of choice for the treatment of T r i c h o m o n a vaginalis (Cosar and Julou, 1959), E n t a m o e b a histolytica (Powell et al., 1966) and Giardia lamblia (Khambatta, 1971). It is also used as a chemotherapeutic agent due to its specific effect on anaerobic microorganisms

* Corresponding author.

(Tally et al., 1975), and it has been proposed as a cytotoxic and radiosensitizer drug specific for hypoxic tumor cells (Stone and Withers, 1974; Foster, 1976). Metronidazole has been reported to interact, bind and damage D N A (LaRusso et al., 1977; Edwards, 1977; Knight et al., 1978; Ludlum et al., 1988; Martelli et al., 1990). The drug per se and its metabolites have shown mutagenic activity in bacteria (Rosenkranz and Speck, 1975; Legator et al., 1975; Speck et al., 1976; Connor et al., 1977).

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134

G. Elizondo et aL /Mutation Research 305 (1994) 133-137

In orally treated Chinese hamsters, a higher frequency of sister-chromatid exchanges (SCE) was detected in intestinal cells (Neal and Probst, 1984). Moreover, metronidazole has been reported to induce lung tumors and lymphomas in Swiss male and female mice (Rustia and Shubik, 1972). Conversely, neither metronidazole nor its metabolites (commercially obtained) induced gene mutations in mammalian cells (Dayan et al., 1982), chromosomal aberrations (Lambert et al., 1979; Lambert and Lindblad, 1980; Hartley-Asp et al., 1981) or SCE (Lambert and Lindblad, 1980; Mahood and Wilson, 1981) in human cells in vitro. Furthermore, metronidazole did not induce chromosomal aberrations in blood lymphocytes (Mitelman et al., 1980) or in bone marrow cells (Salamanca et al., 1980) of patients. The contradictions between bacterial mutagenicity, the lack of effects on mammalian and human cells, the potential carcinogenicity in the animal bioassay, in addition to its wide use for infectious diseases, led us to evaluate the effects of metronidazole therapeutic doses on lymphocyte proliferation in healthy volunteers, measured as mitotic index (MI), cell proliferation kinetics (CPK) and replication index (RI). In addition, the frequency of SCE was determined.

coded and sent by air (1 h) to the laboratory where they were cultured. Heparinized blood samples were cultured at 37°C in RPMI 1640 medium (Flow Lab.) supplemented with Hepes, L-glutamine (2 raM) and non-essential amino acids (10 mM, Gibco). 5Bromodeoxyuridine (32 /zM, Sigma) and 0.2 ml phytohemagglutinin (Microlab) were added at the beginning of culture. Cells were harvested after 72 h. 2 /zg of colcemid (Microlab) was added to the culture 2 h before harvesting (Rojas et al., 1992). Flame-dried preparations of chromosomes were stained in Hoechst 33258 (0.5/zg/ml) for 30 min, then were exposed to black light (1.5-2.0 h) and stained for 5 min in a 2% Giemsa (Merck) solution in phosphate buffer, pH 6.8 (Perry and Wolff, 1974). The analysis of SCE was performed in 25 consecutive second-division metaphases, all with 46 centromeres. To determinate the CPK, the proportions of first-, second- and third-division cells were scored in 100 consecutive metaphases from 72-h cultures. RI was calculated according to the formula: RI = M1 + 2(M2) + 3(M3)/100 by Ivett and Tice (1982). The paired t test was used for the proportion of M1, M2, M3, SCE, RI and MI.

2. Materials and methods 3. Results and discussion

Twelve healthy men 18-25 years of age and weighing 55-65 kg participated in the study after giving informed consent. The investigation protocol was approved by the Ethics and Research Committees of the Miguel Silva Hospital, as well as the Public Health authorities of Morelia, Michoacfin. The subjects did not smoke and had not received any drug for at least 4 weeks. Nor had they been exposed to any known mutagen or carcinogen in the last 3 months before and during the study. Each subject received therapeutic doses of metronidazole (500 mg p.o.), 3 times a day for 10 days. Blood samples were taken on the first day of treatment before giving the first dose, and on the last day after the last dose. The samples were

Grouped data did not reveal a significant effect on mitotic index with the treatment (Table 1), although there was a significant increase in three individuals (Fig. 1; individuals 7, 11 and 12). These increases double two times the standard

Table 1 Mean values of mitotic index (MI) and replication index (RI) before and after metronidazole treatment MI

Mean + SD • P < 0.05.

RI

Before

After

Before

After

0.041 0.017

0.053 0.012

2.409 0.170

2.608 * 0.09

G. Elizondo et al. / Mutation Research 305 (1994) 133-137 % 300

Change

Table 3 Effects of therapeutical doses of metronidazole on lymphocyte proliferation kinetics

i n M.I.

~-

Donor

200

100 "

'

i

i

r

i

,

Donors

Fig. 1. Metronidazole effects on the mitotic index of lymphocytes from healthy donors treated with the medicament. Values are represented as the percent increment or decrement with respect to the sample before treatment.

deviation of the group and are greater than the temporal variations found in healthy donors (Table 2); we suggest that although there may be temporal variability, the significant increase observed in the three mentioned cases might be attributed to a greater susceptibility to the drug treatment. The physiological and pathological significance of the individual susceptibility needs to be further evaluated. The effects of metronidazole on lymphocyte proliferation kinetics are shown in Table 3. A faster CPK was observed in 10 individuals after treatment, and significant differences were obtained on RI and CPK, when data were analyzed by group (Tables 1 and 3, p <0.05). These changes in proliferation could be due to an accelerated stimulation process or to a decrease in cell cycle duration; in any case they would mean an effect on the immunological cell response.

Table 2 Mitotic index variations in time. Three samples from healthy donors Time

135

Donors A

B

C

D

E

F

0 15 days 1 month

0.024 0.021 0.026

0.019 0.028 0.033

0.025 0.043

0.031 0.025 0.029

0.037 0.041

0.034 0.035 0.037

Mean + SD

0.023 0.002

0.026 0.007

0.034 0.012

0.028 0.003

0.039 0.002

0.035 0.001

1 2 3 4 5 6 7 8 9 10 11 12

" Mean +SD

M1

M2

M3

Before

After

Before

After

Before

After

7 10 5 10 9 10 11 5 10 15 14 40

13 3 9 4 6 4 3 4 6 10 8 7

37 39 32 30 38 33 45 29 28 38 42 26

36 27 27 20 27 25 24 24 28 23 26 30

56 51 63 60 53 57 44 66 62 47 44 34

51 70 64 76 67 71 73 72 66 67 66 63

35.58 2.1

26.41" 1.9

53.08 2.7

67.18" 1.8

12.16 2.6

6.41" 0.8

* P < 0.05.

Increased lymphocyte proliferation in vivo produced by other imidazole derivatives, such as levamizole (Renoux and Renoux, 1971; Merluzzi et al., 1975) and cimetidine (Avella et al., 1978), has been reported. Furthermore, the use of metronidazole (Begg et al., 1974; Stone and Withers, 1974; Rauth and Kaufman, 1975; Foster et al., 1976), levamizole (Renoux and Renoux, 1972; Chirigos et al., 1973; Johnson et al., 1975; Renoux et al., 1976) and cimetidine (Gifford et al., 1981; Osband et al., 1981; Kikuchi et al., 1985) in tumor immunotherapy has been proposed, since they increase the immune response of mice against malignant tumors. A possible explanation for the observed effects on lymphocyte proliferation produced by metronidazole is that it could interact with histamine receptors of lymphocytes in a similar way as cimetidine does. Cimetidine is an antagonist of histamine-2 type receptor, which induces lymphocyte proliferation by blocking the histamine-induced release of suppressor factor (Griswold et al., 1984; Sahasrabudhe et al., 1987) and promoting the synthesis of interferon-T and interleukin-2 (Dohlsten et al., 1987). Moreover, the possible interaction of metronidazole with histamine receptors is suggested by its reported antiulcer effect (Gupta et

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G. Elizondo et al. / Mutation Research 305 (1994) 133-137

Table 4 Values of sister-chromatid exchanges before and after treatment Donor

Before

After

1 2 3 4 5 6 7 8 9 10 11 12

5.04 4.96 4.88 4.68 4.64 4.88 4.60 4.56 4.32 4.56 4.32 4.76

4.56 4.96 4.88 4.36 4.40 4.72 4.80 4.52 4.48 5.96 4.64 4.72

Mean + SD

4.68 0.23

4.75 0.42

al., 1976) and its ability to enhance the contractile activity of the histaminergic agonist (Winbery and Barker, 1985). With respect to a genotoxic activity, metronidazole did not induce changes either in the mean SCE frequency or in its dispersion among cells (Table 4). However, studies on the hprt assay are in progress to determine its mutagenicity in the same blood samples used for this study.

4. Acknowledgement We thank L. Herrera for his critical review.

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