Sister chromatid exchanges produced by imipramine and desipramine in mouse bone marrow cells treated in vivo

Sister chromatid exchanges produced by imipramine and desipramine in mouse bone marrow cells treated in vivo

Toxicology Letters 132 (2002) 123– 129 www.elsevier.com/locate/toxlet Sister chromatid exchanges produced by imipramine and desipramine in mouse bone...

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Toxicology Letters 132 (2002) 123– 129 www.elsevier.com/locate/toxlet

Sister chromatid exchanges produced by imipramine and desipramine in mouse bone marrow cells treated in vivo R. Paniagua-Pe´rez a, E. Madrigal-Bujaidar b,*, C.S. Reyes a, G.J. Pe´rez a, M.O. Velasco a, D. Molina b a

Centro Nacional de Rehabilitacio´n S.S., Escuela Nacional de Ciencias Biolo´gicas, I.P.N. Carpio y Plan de Ayala, Sto Tomas, cp 11340, Mexico D.F., Mexico b Laboratorio de Gene´tica, Escuela Nacional de Ciencias Biolo´gicas, I.P.N. Carpio y Plan de Ayala, Sto Tomas, cp 11340, Mexico D.F., Mexico Received 7 December 2001; received in revised form 14 February 2002; accepted 14 February 2002

Abstract Imipramine and desipramine are two widely used tricyclic antidepressants which have shown conflicting results in regard to their in vitro genotoxic evaluation. The aim of this investigation was to determine the capacity of these compounds to induce in vivo sister-chromatid exchanges (SCEs) in mouse bone marrow cells. For each compound, the animals were organized in five groups constituted by five individuals. They were intraperitoneally (ip) administered with the test substances as follows: a negative control group treated with 0.4 ml of distilled water, a positive control group administered with cyclophosphamide (70 mg/kg), three groups treated with imipramine (7, 20 and 60 mg/kg), and three other groups treated with desipramine (2, 20 and 60 mg/kg). The general procedure included the subcutaneous implantation to each mouse of a 5-bromodesoxyuridine tablet (45 mg), and 1 h later, the administration of the chemicals involved. Twenty-one hours after the tablet implantation, the mice received colchicine, and 3 h later their femoral bone marrow was obtained in KCL, fixed, and stained with the Hoechst– Giemsa method. The results showed that both compounds were SCE inducers, starting from the second tested dose. The response of these compounds was dose-dependent, and showed that the highest tested dose increased about four times the SCE control level. The cellular proliferation kinetics was not affected by the chemicals, and the mitotic indexes were slightly diminished with the highest dose. These results indicate an in vivo genotoxic potential for both chemicals, and suggest that it is pertinent to follow their evaluation in other models. © 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Imipramine; Desipramine; SCEs; Mouse bone marrow

1. Introduction

* Corresponding author. Fax: + 52-5-396-3503. E-mail address: [email protected] Madrigal-Bujaidar).

(E.

Depression is a complex disease characterized by disturbances of the mind that may be expressed as irritability, insomnia, fatigue, agitation, psychomotor alterations, feelings of guilt and in-

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adequacy, concentration disturbances, and a suicidal tendency. The disease may originate from exogenous or endogenous factors (Corcos et al., 2000). A number of substances are known to produce beneficial physiologic or psychologic effects in the afflicted individuals. Among these chemicals, imipramine and desipramine are two widely used tricyclic antidepressants. However, they are also used for the treatment of other alterations such as enuresis, anxiety, bulimia, anorexia, alcoholism, menopause and neuralgia (Hulen and Healn, 1983; Gram et al., 1976). Imipramine (Fig. 1) is a tricyclic dibenzacepine compound derived from iminodibenzil; it differs from phenotiazine only in that it has an ethylenic band instead of a sulphur molecule, a difference that produces a central ring with seven members, and confers the stereochemical properties to the molecule (Quay and Moore, 1997). The therapeutic action of the compound is mainly inhibiting the neuronal recapture of monoamines such as noradrenaline and serotonin, in addition to potentiating the activity of catecolamines liberated in the reactive sites of the brain (Kessles, 1988; Amsterdam and Mendeis, 1980). Desipramine (Fig. 1) is a chemical produced during the imipramine metabolism as a demethylated derivative and may reach concentrations which are comparable with those of the original molecule (Blackwell, 1978). Its pharmacologic action is similar to the behavior described for imipramine; furthermore, it probably blocks the adenylyl cyclase activity, an action which may interfere in the coupling of monoamine with the noradrenergic receptor (Prichard and Greenberg, 1988).

Concerning the genotoxic potential of imipramine, several studies have revealed the following negative effects: no increase in the revertant rate of strains TA1537, TA98, and TA100 of Salmonella thyphimurium (Balbi et al., 1980); no alteration in the rate of non-disjunction and crossover in diploid strains of Aspergillus nidulans (Bignami et al., 1974), no effect in the rate of sex-linked recessive lethals in Drosophila (Filppova et al., 1975); the absence of chromosome aberrations in cultured human lymphocytes (Fu and Jarvik, 1977) as well as in bone marrow cells of rats administered in vivo with a single or multiple intraperitoneal (ip) injections (Bishun et al., 1974). Nevertheless, this medication has been found positive in the Ames S. thyphimurium test (strain TA100, with enzymatic activation) as well as in the Bacillus subtilis rec assay (Kawachi et al., 1980). Furthermore, it disrupts the DNA architecture, and decreases the DNA and the RNA synthesis in the polytene chromosomes of Sciara coprophila (Schmidt et al., 1970). Finally, it increases the frequency of sister chromatid exchanges (SCEs) and chromosomal aberrations in human lymphocyte cultures (Saxena and Ahuja, 1988), and the genotoxic damage using the wing somatic and recombination test in Drosophila (van Schaik and Graf, 1991). In the case of desipramine, not only are reports on its genotoxic potential very few, but they also show conflicting results: no response was detected in a study applying the Salmonella/microsome test (strains TA1534, TA1537, TA98 and TA100) (Kawachi et al., 1980), while positive data was found using the somatic mutation and recombination test (SMART) in Drosophila (van Schaik and Graf, 1991).

Fig. 1. Chemical structures of imipramine (A) and desipramine (B).

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In spite of the extensive use of such medication there is no clear definition in regard to their genotoxic capacity; the literature shows heterogeneous data and almost a lack of in vivo studies. Therefore, the genotoxic evaluation of imipramine and desipramine using various in vivo endpoints is underway in our laboratory at present. In this report we determined that both chemicals are SCE inducers in mouse bone marrow cells.

2. Material and methods

2.1. Chemicals and animals Hoechst 33258, 5-bromodeoxyuridine (BrdU), colchicine, imipramine and desipramine were purchased from Sigma Chemicals (St. Louis, MO, USA). Cyclophosphamide was obtained from Sanfer S.A. (Mexico City), and the Giemsa stain was obtained from Merck (Mexico City). Sodium citrate, sodium chloride, potassium phosphate and sodium phosphate, the salts to prepare the buffers, were purchased from Baker S.A. (Mexico City). The animals were obtained from the National Institute of Hygiene. They were male mice (NIH) of approximately 25 g of weight, which were kept in polypropylene cages at 23 °C and permitted to freely consume food (Purina) and tap water.

2.2. Genotoxicity procedure The evaluation of both medicaments followed a similar procedure. Initially, a lethal dose 50 by the ip route was obtained (Lorke, 1983). The result for imipramine was 118 mg/kg and for desipramine was 77 mg/kg. These data as well as the daily therapeutic doses utilized in humans (from 10 to 300 mg for imipramine, and from 5 to 250 mg for desipramine) were considered to establish the experimental doses for the assay: 7, 20 and 60 mg/kg for imipramine and 2, 20 and 60 mg/kg for desipramine. The SCE assay was made according to the methodology described earlier (Perry and Wolf,

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1974; Madrigal-Bujaidar and Sa´ nchez Sa´ nchez, 1991). A 45 mg BrdU tablet was 70% coated with paraffin and subcutaneously implanted to five groups of mice with five individuals each. One hour later, three of these groups received an ip injection with the selected doses of each medicament; another group (the negative control) was ip administered with 0.4 ml of distilled water, and the last group (the positive control), was ip injected with 70 mg/kg of cyclophosphamide. Twenty-one hours after these inoculations, the mice were ip injected with 5 mg/kg of colchicine to stop cell division and 3 h later they were killed by cervical dislocation. The epiphysis of each femur was cut and the bone marrow dispersed in a solution of KCl (0.075 M) at 37 °C; the cell suspension was left in the hypotonic solution for 30 min, centrifuged at 1500 rpm and fixed twice in a mixture of methanol– acetic acid (3:1). The cell suspension was dropped onto ethanol covered slides and slightly flamed; finally, the chromosomes were stained by the Hoechst–Giemsa method (Perry and Wolf, 1974; Madrigal-Bujaidar and Sa´ nchez Sa´ nchez, 1991). The microscopic analysis per mouse was carried out as follows: 30 second-division metaphases to determine the frequency of SCEs, 1000 cells to determine mitotic indexes (MI) and 100 cells to establish the cellular proliferations kinetics (CPK). Based on the CPK values, we obtained the average generation time (AGT) which was equal to 24/(M1 + 2M2 + 3M3)100. M1, M2 and M3 corresponded to the number of cells in first, second and third cellular division, respectively. The statistical analysis was made with an ANOVA test followed by the Student’s t-test.

3. Results The frequency of SCEs induced by imipramine is shown in Table 1. The low dose administered (7 mg/kg) did not increase the number of SCEs with respect to the value of the negative control group; however, the two high doses produced a genotoxic effect. With 60 mg/kg the increase

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Table 1 Effect of imipramine in mouse bone marrow cells. Sister chromatid exchanges (SCEs), average generation time (AGT), and mitotic index (MI). Agent

Distilled water Imipramine Imipramine Imipramine CP

Dose (mg/kg)

1.5 7 20 60 70

SCE X( 9 SD

1.52 9 0.12 1.7590.16 3.5790.41a 6.429 0.07a 6.439 0.42a

CPK (%) M1

M2

M3

33 36 37 30 33

51 50 53 61 58

16 14 10 9 9

AGT X( 9 SD

MI X( 9 SD

12.45 9 0.23 12.46 9 0.14 12.48 9 0.22 12.38 9 0.30 12.47 9 0.36

5.94 90.19 5.98 90.26 5.70 90.18 5.73 90.30a 4.909 0.08a

CP=cyclophosphamide. M1, M2 and M3 correspond to the frequency of metaphases in first, second and third cellular division. AGT=24/(1M1+2M2+3M3) 100. a Statistically significant difference with respect to the control value, ANOVA and Student’s t-tests (P =0.05).

over the control level was 4.9 SCEs, which correspond to 322% and was very close to the level obtained with cyclophosphamide. A linear regression analysis of the data obtained shows a correlation coefficient (r) equal to 0.974 (Y = 1.475 + 8.422e− 2X), indicating a dose-dependent response induced by imipramine (Fig. 2). The MI and the AGT produced by the compound are also shown in Table 1. With respect to the first parameter, the chemical produced a cytotoxic effect with only the highest dose tested, which inhibited the MI 21% with respect to the control mean. The CPK was characterized by the number of mitosis in M1, in M2, and in M3, which was very close to the rate observed in the control mice; these results produced a homogeneous AGT value in the experiment (between 12.45 and 12.48 h) The SCE results obtained with desipramine are shown in Table 2. A similar effect to the one described for imipramine was noted: an insignificant increase with the low dose, but a positive SCE elevation with the two highest doses, reaching a maximum increment of 4.48 SCEs (294%) above the basal value. The regression analysis showed an r of 0.989 (Y = 1.284 +7.7160e − 2X), indicating a dose-dependent response (Fig. 2). The MI showed no modification in relation to the control level with the two low doses; however, the dose of 60 mg/kg induced a 20% MI decrease. Finally, the AGT observed with desipramine varied between 12.45 and 13.05 h, showing no statistical differences among the data obtained in the experiment.

4. Discussion Medication in most cases may produce secondary health effects of variable degree, which in some cases may be a serious human health hazard. This potential damage is of particular concern with respect to compounds used for long periods and/or during pregnancy. The antidepressants studied are medicaments that may be continuously consumed for 6 months or longer, with a possible repetition of the treatment (Frommer et al., 1987). Also, there have been reports showing collateral health effects, mainly on the cardiovascular system (Fasoli et al., 1981; Burrows and Vohra, 1986), although other types of alterations have been described as well: for example, myoclonus, sexual dysfunction, and hyponatremia (Black and Kilzich, 1994; Karp, 1994; Colgate, 1993). Besides, the development of mammary cancer and pheochromocytoma has been described (Nemecek and Backfire, 1994; Ferguson, 1994), as well as a few cases of neonatal adaptation impairment and withdrawal syndrome when administered in the third trimester of pregnancy (Webster, 1973; Bares, 2000). On the other hand, it is known that therapeutic drugs may produce genotoxic damage by a direct interaction with DNA or after their metabolic transformation, and that by establishing their genotoxic level it is possible to propose preventive measures (Farber, 1987). Imipramine and desipramine have been clinically used for more than 35 years, yet surprisingly enough there is an almost lack of in vivo mam-

Fig. 2. Regression analyses obtained with the SCE frequencies induced by imipramine (A) and desipramine (B) in mouse bone marrow cells.

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malian studies to evaluate their effect on the genetic material. Our results confirm the usefulness of the SCE evaluation to detect genotoxicity; they show a dose-dependent effect produced by both compounds, and a clear SCE increase with the two high doses in an acute model. However, other studies should be performed to establish the precise risk for humans. In this context, it is pertinent to mention that the maximum daily dose recommended in humans is about 300 mg, an amount which is in the non-significant, low range used in our assay; however, one should keep in mind the usual long term treatment in humans, as well as the uncertainty in the efficacy of the DNA repair and detoxification systems to remove the genotoxic damage. Our positive result on imipramine does not coincide with the absence of damage found by Bishun and Williams measuring chromosomal aberrations in rat (Bishun et al., 1974). The difference may be related with the following factors: the distinct endpoint and organism studied, as well as the omission of a marking to identify chromosomes in first cellular division by the mentioned authors, which could give rise to the scoring of cells with fewer alterations in subsequent mitotic divisions. The genotoxic effect produced by the antidepressants has been related with the presence of an N atom in position 5 of the heterocyclic sevenmembered ring of the molecule (van Schaik and Graf, 1991). This observation coincides with the genotoxic findings in Drosophila produced by

chemicals closely related to imipramine (clomipramine, lofepramine, and mianserine), and with the absence of damage produced by the six-membered compounds maprotiline, and chlorpromazine (van Schaik and Graf, 1993). Besides, the chemical structure of the antidepressants includes two potentially dangerous components related with mutagenic and carcinogenic events, and particularly with the formation of SCEs: one of this components is the aromatic ring, and the other the nitro group (Bradley et al., 1981; Weintein, 1988). The latter may be transformed into nitroso compounds, which in turn may form alkylating molecules. Imipramine is metabolised by demethylation into desipramine, and to a lesser extent by hydroxylation into 2-hydroxyimipramine. Desipramine is metabolised by hydroxylation into 2-hydroxydesipramine. At the end of the biotransformation process, at least seven metabolites are formed (Zeugin et al., 1990), chemicals whose modifications with respect to the initial molecule are mainly located in the aromatic ring; therefore, it may be assumed that the nitro group is quantitatively more relevant for the SCE formation. Nevertheless, in light of the reported positive effect produced by imipramine and desipramine, as well as of a recent paper showing DNA damage by imipramine in cultured C6 rat glioma cells (Slamon et al., 2001), it is pertinent to extend the studies so as to clearly determine their mechanism of action.

Table 2 Effect of desipramine in mouse bone marrow cell. Sister chromatid exchanges (SCEs), average generation time (AGT), and mitotic index (MI). Agent

Distilled water Desipramine Desipramine Desipramine CP

Dose (mg/kg)

1.5 2 20 60 70

SCE X( 9 SD

1.52 9 0.12 1.6390.14 2.5490.21a 6.009 0.35a 6.739 0.32a

CPK (%) M1

M2

M3

33 32 37 39 32

51 58 49 45 57

16 10 14 16 11

AGT X( 9 SD

MI X( 9 SD

12.45 9 0.23 12.34 9 0.17 12.65 9 0.11 12.41 9 0.34 12.71 9 0.23

5.43 90.37 5.60 90.29 5.58 9 0.28 5.36 90.39a 4.28 90.46a

CP=cyclophosphamide. M1, M2 and M3 correspond to the frequency of metaphases in first, second and third cellular division. AGT=24/(1M1+2M2+3M3) 100. a Statistically significant difference with respect to the control value, ANOVA and Student’s t-tests (P =0.05).

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