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Sister-chromatid Exchanges Induced by Disulfiram in Bone Marrow and Spermatogonial Cells of Mice Treated In Vivo
Food and Chemical Toxicology 37 (1999) 757±763
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Research Section
Sister-chromatid Exchanges Induced by Disul®ram in Bone Marrow and Spermatogonial Cells of Mice Treated In Vivo E. MADRIGAL-BUJAIDAR1*, N. VELAZQUEZ-GUADARRAMA1, P. MORALES-RAMIREZ2, M. T. MENDIOLA2, A. LAGUNAS MARTIÂNEZ3 and G. CHAMORRO4 1 Laboratorio de GeneÂtica, Escuela Nacional de Ciencias BioloÂgicas, I.P.N., 2Instituto Nacional de Investigaciones Nucleares, 3Facultad de Ciencias QuõÂ mico-BioloÂgicas, UAG, MeÂxico and 4Laboratorio de ToxicologõÂ a Preclinica, E.N.C.B.
(Accepted 12 January 1999) AbstractÐDisul®ram is a widely used drug to treat alcoholism due to its capacity to inhibit the metabolism of acetaldehyde; however, its genotoxic potential is not well known. Thus, the aim of this investigation was to determine whether the chemical may induce sister-chromatid exchanges (SCEs) in an in vivo study using mouse bone marrow and spermatogonial cells. We used doses of 200, 400 and 800 mg/ kg body weight and compared the obtained data with the values determined in a negative control group as well as with a positive control group (cyclophosphamide, 50 mg/kg). The results in both systems indicated a weak genotoxic response by the chemical. In the case of bone marrow, a signi®cant SCE level was achieved only with the high tested dose, but in spermatogonial cells the three doses tested showed a signi®cant dierence with respect to the negative control. No signi®cant alterations in the mitotic index or in the cell proliferation kinetics were observed in somatic cells. Concerning the eect of cyclophosphamide, an increase in the level of SCEs was observed in both types of cells, reaching more than three times the values obtained in their respective control groups. # 1999 Elsevier Science Ltd. All rights reserved Keywords: disul®ram; sister-chromatid exchanges; mouse somatic and germ cells. Abbreviations: AGT = average generation time; BrdU = bromodeoxyuridine; CPK = cellular proliferation kinetics; DAME = diethylthiocarbamate acid methyl ester; DIS = disul®ram; MI = mitotic index; SCE = sister chromatid exchange.
INTRODUCTION
Alcohol abuse and alcohol dependence are human actions determined by complex genetic factors, as well as by lifestyle, environment and personality. The disease aects the organism in many forms, some of which are related with liver disease, brain damage, acute pancreatitis, degenerative changes of the heart and skeletal muscle, reproductive disorders, increased risk of cancer and teratogenic damage (Dufour and Fe Caces, 1993; Michaelis and *Corresponding author at: Lab. de GeneÂtica. Escuela Nacional de Ciencias BioloÂgicas, IPN. Carpio y Plan de Ayala S/N. Col., Sto. TomaÂs C.P. 11340 MeÂxico, D. F.
Michaelis, 1994). The importance of alcoholism in human health as well as its impact on socioeconomical aspects can be visualized if we consider that approximately 10% of the general population are excessive drinkers, with the male:female ratio varying from 3:1 to 5:1 in Western societies, where the annual per capita consumption in people aged 15 yr and above lies between 8 and 16 litres of pure alcohol (Hardt, 1992; Wells et al., 1991). In the United States, deaths related to alcohol consumption may represent nearly 3 million years of potential life lost to full life expectancy, and for the year 1990, the economic cost to society for alcohol abuse and alcohol dependence was estimated at $98.6 billion (Dufour and Fe Caces, 1993).
0278-6915/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved. Printed in Great Britain PII S0278-6915(99)00061-7
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E. Madrigal-Bujaidar et al.
One of the strategies used to attack alcohol intoxication is pharmacotherapy. This may be applied to treat alcohol withdrawal syndromes or to reduce or deter alcohol consumption; in the latter case, three general types of therapeutic approaches can be used: alcohol-sensitizing drugs, alcohol-conditioning drugs, and drugs that directly reduce alcohol consumption (Bohn, 1993). Our study concerns the genotoxicity of disul®ram (tetraethylthioperoxydicarbonic diamide) (DIS), a chemical belonging to the group of alcohol-sensitizing drugs, for which properties against alcoholism were suggested more than 40 years ago. DIS is an irreversible inhibitor of the liver aldehyde dehydrogenase, the NAD-dependent enzyme which oxidizes the ®rst metabolite of ethanol, acetaldehyde, and converts it to acetic acid mainly by the action of the aldehyde dehydrogenase mitocondrial isozyme (class 2) (Hellander, 1993). Acetaldehyde is a toxic compound involved in lipid peroxidation, generation of active-free radicals, inactivation of various enzymes or binding to proteins or other cell constituents; therefore, rapid elimination of the chemical from the interior of the cell is essential (Johansson, 1992). A daily DIS dose of 150±700 mg given to alcoholics has shown positive results to reduce or deter the consumption of alcoholic beverages due to the disul®ram±ethanol reaction, which increases the level of blood acetaldehyde. This, in turn, produces a number of unpleasant symptoms which may include ¯ushing of the skin, due to cutaneous vasodilatation, hypotention due to diastolic blood pressure, re¯ex tachycardia, tachypnoea, a sensation of warmth, palpitations, anxiety, headache, nausea and vomiting. The intensity and duration of the eects correlate with the time course of the blood acetaldehyde concentration, but there can be appreciable inter-individual and intraindividual variation in the acetaldehyde-mediated reaction (Petersen, 1992). DIS has been described as a chemical with low toxicity, since the minimal fatal dose in rabbits was determined to be 3 g/kg by the oral route, and several acute toxicity studies in mammals were also concordant (Petersen, 1992); however, a chronic study in rats showed that the daily administration of 250 mg/kg produced ataxia, and calci®ed masses in the cerebellum and basal ganglia (Fitzhugh et al., 1952). Cytotoxic eects of DIS have also been observed in rodent and human cell lines as well as in human lymphocytes, probably due to its eect on the decrease of DNA synthesis (Cohen and Robbins, 1990; Hellander and Lindahl-Kiessling, 1991). The chemical is considered safe concerning carcinogenic and teratogenic eects; however, embryotoxicity and fertility deterioration in rats have been described (Salgo and Oster, 1974), as well as the generation of zebra®sh embryos with
abnormalities in the anteroposterior axis (Costaridis et al., 1996). Concerning genotoxic studies, DIS has been shown to be mutagenic in the L51784 mouse lymphoma cell forward assay (McGregor et al., 1991). In this report, the compound was positive starting from 0.01 mg/ml, while at higher dose levels up to 2 mg/ml, the mutagenic response gradually diminished. Considering this result, as well as the widespread use of DIS, the aim of the present investigation was to determine whether disul®ram may increase the level of sister-chromatid exchanges (SCEs) in bone marrow and spermatogonial cells of mice treated in vivo.
MATERIAL AND METHODS
Chemicals and animals DIS was purchased from Sigma Chemicals Co. (St Louis, MO, USA) and dissolved in corn oil, a compound also obtained from Sigma. The DIS test doses used were 200, 400 and 800 mg/kg body weight, and these were based on a previous LD50 performed according to the method of Lorke using the oral route (Lorke, 1983). In this study we obtained a result of 1113 mg/kg; therefore, our high DIS dose corresponded to 71.8% of its LD50. 5bromodeoxyuridine (BrdU) (Hoechst 33258), and colchicine were obtained from Sigma Chemical Co. and dissolved in distilled water. Cyclophosphamide was purchased from Laboratorios Sanfer S.A. (MeÂxico), and dissolved in distilled water. Activated charcoal was purchased from Sigma, Giemsa stain from Merck-MeÂxico, S.A. and trypsin from Difco Laboratories (USA). Finally, Lab Rodent Diet 5001 pellets were used for mouse feeding. Male mice (NIH) with a mean weight of 28 g were obtained from the National Institute of Hygiene. They were kept in polypropylene cages in groups of ®ve animals each at a mean temperature of 238C, fed with standard food, and permitted to drink tap water ad lib. Pellet consumption was suspended 6 hr before beginning the experiment. Somatic cell study The selected DIS doses were administered orally to lots of ®ve animals, and 6 hr later the mice were injected ip with an aqueous suspension of BrdU previously adsorbed to activated charcoal (1.5 mg/ kg of body weight) (Madrigal-Bujaidar et al., 1997; Morales-Ramirez et al., 1984). The negative control was treated orally with 0.3 ml corn oil at the time of DIS administration, and the positive control animals were injected ip (cyclophosphamide, 50 mg/kg) 30 min before BrdU administration. 22 hr after the BrdU administration the animals were injected ip with colchicine (3.5 mg/g) and after 3 hr they were sacri®ced with CO2. The femurs were dissected and the bone marrow extracted in KCl 0.075 M at 378C,
SCEs induced by disul®ram in mice
and the cells were resuspended and left for 30 min; then they were ®xed three times with methanol±acetic acid (3:1) and ®nally cell spreading was made on iced slides followed by a slight ¯aming. The ¯uorescence-plus Giemsa method used for the dierential staining of sister chromatids has been described before (Madrigal-Bujaidar et al., 1997; MoralesRamirez et al., 1992), and consisted in the following basic steps: the cells were stained in Hoescht 33258 for 60 min; the slides were washed in tap water after which they were dried at 608C for 15 min; then they were buered with a citrate phosphate, pH 7.0 and exposed to black light for 60 min. After being washed and dried as before, the cells were immersed for 20 min at 608C in a saline citrate solution, washed and stained with a 4% Giemsa solution made in a phosphate buer, pH 6.8, for 10 min. The microscopic observations included an SCE scoring made in 30 second-division mitosis per mouse, the mitotic index determined in 1000 cells per animal, and the cell proliferation kinetics (CPK) established in 100 cells per mouse. In the latter case, the frequency of cells in ®rst, second, and third cellular division (M1, M2 and M3, respectively) was used to obtain the average generation time (AGT) with the equation AGT = 22/ (M1 + 2M2 + 3M3) 100 (Ivett and Tice, 1982). The statistical evaluation of the data obtained was initially made with a one-way ANOVA test followed by a Student's t-test. Germ cell study The experimental doses of DIS were administered to the animals by oral intubation, and 6 hr later the mice were injected ip with an aqueous suspension of BrdU (1.3 mg/kg) adsorbed to activated charcoal (Morales-Ramirez et al., 1984). Corn oil was administered orally to control animals at the time of DIS inoculation, and positive control animals were treated ip with 50 mg cyclophosphamide/kg 12 hr after BrdU injection; this time is usually chosen in germ cell studies considering the short half-life of the chemical (Palitti et al., 1982). Colchicine (7.5 mg/kg) was injected sc after 53 hr of BrdU administration, and 3 hr later the animals were killed by cervical dislocation. Then, the testes were dis-
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sected, the tunica albuginea separated from them, and the seminiferous tubules ®nely cut in phosphate buered saline (PBS) and washed three times in the same solution. The tissue was incubated for 5 min at 378C in a trypsin solution made in ethylenediaminetetraacetic acid (EDTA) without calcium or magnesium, and then a tissue disaggregation was made by passing it through a ®ne pore needle several times. The suspension obtained was centrifuged and washed three times in EDTA. Finally, the spermatogonial cells were treated with KCl 0.075 M at 378C for 5 min and ®xed in methanol±acetic acid (3:1) at least twice. The preparation of the slides as well as the staining procedure were done according to the method previously explained for bone marrow cells. The slides were observed microscopically to determine the frequency of SCEs by evaluating 30 second-division mitosis per mouse. The data obtained were statistically analysed with a one-way ANOVA and the Student's t-tests. RESULTS
The study to determine the genotoxic eect of DIS in bone marrow cells showed a dose-dependent response. Although the SCE increase was statistically signi®cant only with the high tested dose (800 mg/kg), a doubling of the basal level was not reached even with this dose; on the contrary, cyclophosphamide caused strong genotoxic damage (Table 1). The cumulative frequency of cells v. SCE number in bone marrow is presented in Fig. 1, showing no important dierences between the curves obtained in the control group and those obtained in the groups treated with dierent doses of DIS. A clear dierence was observed in the curve of the positive control group (cyclophosphamide, 50 mg/kg). In spermatogonial cells, the increase in the number of SCEs was statistically signi®cant in all three doses tested with respect to the control level. The values obtained with the two low doses were higher then those found in somatic cells in relation to their respective control data. However, the results did not correspond to a dose±response relationship, nor to a duplication of the basal level. The positive con-
Table 1. Frequency of sister-chromatid exchanges in bone marrow and spermatogonial cells of mice treated with disul®ram Bone marrow cells
Spermatogonial cells
Dose (mg/kg)
Agent
SCE X2 SD
INC
SCE X2 SD
INC
0 200 400 800 50
Corn oil Disul®ram Disul®ram Disul®ram Cyclophosphamide
2.2120.30 2.42 20.40 2.58 20.20 *3.1820.40 *7.4320.20
0.21 0.37 0.97 2.25
1.83 20.02 *2.25 20.10 *2.63 20.27 *2.44 21.49 *6.10 20.80
0.42 0.80 0.60 4.27
Statistically signi®cant dierence with respect to control level. One-way ANOVA and Student's t-tests; P = 0.05. The scoring was made in ®ve animals per lot and 30 cells per animal. INC = SCE increase (treated±control).
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Fig. 1. Cumulative frequency of cells with respect to SCE per cell in bone marrow. Mice treated with 0 (w), 200 ( ), 400 (W) and 800 (T) mg disul®ram/kg, and 50 mg cyclophosphamide/kg as positive control (Q).
trol (cyclophosphamide) showed a strong response, although weaker than that observed in somatic cells (Table 1). In Fig. 2 the curves of cumulative frequency of cells v. frequency of SCE indicate that DIS increases the frequency of cells with a high SCE number with respect to the control group, that is, in the control group only 20% of cells have two or more SCEs, and in the treated groups this percentage lies between 40 and 50%. The curve of the positive control group indicates a clear dierence from the untreated control. An interesting point is that spermatogonia seems to be less sensitive than bone marrow to SCE induction by cyclophosphamide, that is, in bone marrow no cells were observed with an SCE frequency lower than four, while in spermatogonia this group of cells represents 60%. Besides, in germ cells only a few cells have more than eight SCEs, while in bone marrow these cells are approximately 20%. With regard to the mitotic index, in the study performed in bone marrow cells we observed no statistical dierences between all tested groups, although a 2.3% decrease was noted in the animals treated with the high dose, and a dose-decrease behaviour was observed with all doses. This suggest a mild cytotoxic eect of the chemical. In relation to the cellular proliferation kinetics, a certain increase in the number of M1 cells was observed in mice treated with the highest dose of DIS, suggesting a delay in the cell proliferation; this alteration produced a signi®cant dierence with respect to control data concerning the average generation time value. However, the accumulation of M1 cells by cyclophosphamide was substantially greater than that obtained with the other treatments also producing an important cell-cycle delay as re¯ected by the obtained AGT value (Table 2).
DISCUSSION
Sister-chromatid exchanges correspond to physical exchange of DNA double helices between sister chromatids. They occur spontaneously in low numbers, but dramatically increase when cells are treated with DNA-damaging agents. This observation has been determinant in the extensive use of the method for genotoxic evaluation, moreover because SCE scoring has been of great value in the screening of weak genotoxic agents (Lindahl-Kiessling, et al., 1989). It is important to study the genotoxic eect in dierent tissues because besides the well established dierences in response, the health implications could also be dierent. Implications on genetic risk inferred from studies carried out in germ cells are particularly relevant. The results obtained in the present study indicate that DIS has a weak genotoxic eect in both somatic and germ cells, this conclusion corresponds to the SCE level obtained which did not reach a doubling of the control value, as well as the reduced SCE induction in comparison with that obtained with the positive control. The observed genotoxic eect induced by DIS may have several explanations. One of these refers to the direct or indirect induction of oxidative stress by the chemical, as suggested by Delmaestro and Tombetta (1995), who detected accumulation of copper, increased production of malondialdehyde and increased activity of glutathione peroxidase in rats treated orally with the compound. On the other hand, the metabolism of the chemical is a very complex process, and one or more of its metabolites may participate in the genotoxic damage, although this aspect has been poorly explored. DIS is rapidly reduced to diethylthiocarbamate, and in later steps to some active metabolites such as diethylthiocarba-
SCEs induced by disul®ram in mice
761
Fig. 2. Cumulative frequency of cells with respect to SCE per cell in spermatogonia. Mice treated with 0 (w), 200 ( ), 400 (W) and 800 (T) mg disul®ram/kg and 50 mg cyclophosphamide/kg as positive control (Q).
mic acid methyl ester (DAME ), this compound is active by injection, it possesses a more rapid eect and higher potency than DIS, and causes enzymatic inhibition in vivo and in vitro (Petersen, 1992). Smethyl N,N-diethylthiocarbamate sulfone, a logical metabolite of S-methyl N,N-diethylthiocarbamate sulfoxide, also has similar properties as the mentioned for DAME (Mays et al., 1995). Finally, two of the last chemicals formed in the DIS metabolic pathway are diethylamine and carbon disul®de; the latter is known to be a neurotoxin which is highly reactive with several macromolecules and is particularly relevant to this discussion because it possesses the capacity to increase the rate of structural chromosomal aberrations (Le and Fu, 1996). Another probable explanation for the detected genotoxic damage may be related to the acetaldehyde fraction of endogenous origin that has been detected in subjects not drinking ethanol. Several sources from which it may come have been proposed, including pyruvate and aminoacid metabolism, endogenous ethanol formation or protein degradation (Ericksson, 1987; Hellander, 1993). In human sub-
jects and in rats under DIS treatment, markedly elevated levels of acetaldehyde have been found (Eriksson, 1985). In this context it is worthwhile stressing that acetaldehyde has been described as an inducer of SCEs in vitro and in vivo (Hellander and Lindahl-Kiessling, 1991; Korte and Obe, 1981), a capacity which seems to increase in the presence of aldehyde dehydrogenase inhibitors such as 1-aminocyclopropanol (Hellander and Lindahl-Kiessling, 1991). DIS ingestion is useful in the treatment of alcohol dependence, but the drug should be given primarily to well-motivated patients, who must also be carefully monitored and given support (Kristenson, 1992; O'Farrel et al., 1995). However, the drug may occasionally produce adverse reactions in the organism (also including interactions with other chemicals), leading to health deterioration in various organs. The reported damage is related to hepatic, neurological, skin and psychiatric reactions in decreasing order of frequency, although other organs may be aected (Paulsen et al., 1992). The causes of some disorders are being understood, for
Table 2. Mitotic index ( MI ) and cellular proliferation kinetics ( CPK ) in bone marrow cells of mice administered with disul®ram CPK Dose (mg/kg)
Agent
M1
M2
M3
AGT h
MI %
0 200 400 800 50
Corn oil Disul®ram Disul®ram Disul®ram Cyclophosphamide
26.0 27.4 33.6 38.6 62.2
61.2 59.4 57.6 50.0 33.6
12.8 11.2 8.8 11.2 4.2
12.17 12.24 12.56 *12.78 *15.49
8.96 8.6 7.86 6.66 *2.6
Statistically signi®cant dierence with respect to control level. One-way ANOVA and Student's t-tests; P = 0.05. The scoring for CPK data was made in 100 cells per mouse, and for MI in 1000 cells per mouse. Average generation time (AGT) = 22/(M1 + 2M2 + 3M3) 100, where M1, M2 and M3 correspond to the frequency of cells in ®rst, second and third cell division.
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example, the neurological damage seems to be mediated by a neurotransmitter metabolism alteration which includes abnormal activity of serotonine, dopamine and tryptophan (Beck et al., 1995; Nagendra et al., 1993; Vaccari et al., 1996). The organic damage is mainly related to the DIS-alcohol reaction, which seems to be characterized by a highly interindividual variability; in severe cases, cytolitic hepatitis with fatal evolution has been reported, as well as the death of a patient showing a corrosion lesion in the lower oesophagus and stomach due to a violent chemical reaction in situ, in that study, an acetaldehyde blood concentration of 41 mg/litre was determined (Health et al., 1992). Considering these variable responses to DIS, our ®ndings con®rm the importance of being cautious with the use of this chemical treatment and of including proper clinical and periodical chemical monitoring of the patients under DIS therapy. This suggestion agrees with a histological study in DIStreated rats also administered with ethanol, which showed a decrease in the content of zymogen granules in acinar cells, and the appearance of intraplasmic vacuolization which seemed to have lipid inclusions (Honda et al., 1995). This is also in accordance with a report by Wicht et al. (1995) who found that in patients under DIS administration but without liver disease, the acetaldehyde level was higher than in those with liver disease, a ®nding which suggests that clinical symptoms do not necessarily re¯ect the intensity of the chemical reaction between both compounds. REFERENCES
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Research Section
Sister-chromatid Exchanges Induced by Disul®ram in Bone Marrow and Spermatogonial Cells of Mice Treated In Vivo E. MADRIGAL-BUJAIDAR1*, N. VELAZQUEZ-GUADARRAMA1, P. MORALES-RAMIREZ2, M. T. MENDIOLA2, A. LAGUNAS MARTIÂNEZ3 and G. CHAMORRO4 1 Laboratorio de GeneÂtica, Escuela Nacional de Ciencias BioloÂgicas, I.P.N., 2Instituto Nacional de Investigaciones Nucleares, 3Facultad de Ciencias QuõÂ mico-BioloÂgicas, UAG, MeÂxico and 4Laboratorio de ToxicologõÂ a Preclinica, E.N.C.B.
(Accepted 12 January 1999) AbstractÐDisul®ram is a widely used drug to treat alcoholism due to its capacity to inhibit the metabolism of acetaldehyde; however, its genotoxic potential is not well known. Thus, the aim of this investigation was to determine whether the chemical may induce sister-chromatid exchanges (SCEs) in an in vivo study using mouse bone marrow and spermatogonial cells. We used doses of 200, 400 and 800 mg/ kg body weight and compared the obtained data with the values determined in a negative control group as well as with a positive control group (cyclophosphamide, 50 mg/kg). The results in both systems indicated a weak genotoxic response by the chemical. In the case of bone marrow, a signi®cant SCE level was achieved only with the high tested dose, but in spermatogonial cells the three doses tested showed a signi®cant dierence with respect to the negative control. No signi®cant alterations in the mitotic index or in the cell proliferation kinetics were observed in somatic cells. Concerning the eect of cyclophosphamide, an increase in the level of SCEs was observed in both types of cells, reaching more than three times the values obtained in their respective control groups. # 1999 Elsevier Science Ltd. All rights reserved Keywords: disul®ram; sister-chromatid exchanges; mouse somatic and germ cells. Abbreviations: AGT = average generation time; BrdU = bromodeoxyuridine; CPK = cellular proliferation kinetics; DAME = diethylthiocarbamate acid methyl ester; DIS = disul®ram; MI = mitotic index; SCE = sister chromatid exchange.
INTRODUCTION
Alcohol abuse and alcohol dependence are human actions determined by complex genetic factors, as well as by lifestyle, environment and personality. The disease aects the organism in many forms, some of which are related with liver disease, brain damage, acute pancreatitis, degenerative changes of the heart and skeletal muscle, reproductive disorders, increased risk of cancer and teratogenic damage (Dufour and Fe Caces, 1993; Michaelis and *Corresponding author at: Lab. de GeneÂtica. Escuela Nacional de Ciencias BioloÂgicas, IPN. Carpio y Plan de Ayala S/N. Col., Sto. TomaÂs C.P. 11340 MeÂxico, D. F.
Michaelis, 1994). The importance of alcoholism in human health as well as its impact on socioeconomical aspects can be visualized if we consider that approximately 10% of the general population are excessive drinkers, with the male:female ratio varying from 3:1 to 5:1 in Western societies, where the annual per capita consumption in people aged 15 yr and above lies between 8 and 16 litres of pure alcohol (Hardt, 1992; Wells et al., 1991). In the United States, deaths related to alcohol consumption may represent nearly 3 million years of potential life lost to full life expectancy, and for the year 1990, the economic cost to society for alcohol abuse and alcohol dependence was estimated at $98.6 billion (Dufour and Fe Caces, 1993).
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One of the strategies used to attack alcohol intoxication is pharmacotherapy. This may be applied to treat alcohol withdrawal syndromes or to reduce or deter alcohol consumption; in the latter case, three general types of therapeutic approaches can be used: alcohol-sensitizing drugs, alcohol-conditioning drugs, and drugs that directly reduce alcohol consumption (Bohn, 1993). Our study concerns the genotoxicity of disul®ram (tetraethylthioperoxydicarbonic diamide) (DIS), a chemical belonging to the group of alcohol-sensitizing drugs, for which properties against alcoholism were suggested more than 40 years ago. DIS is an irreversible inhibitor of the liver aldehyde dehydrogenase, the NAD-dependent enzyme which oxidizes the ®rst metabolite of ethanol, acetaldehyde, and converts it to acetic acid mainly by the action of the aldehyde dehydrogenase mitocondrial isozyme (class 2) (Hellander, 1993). Acetaldehyde is a toxic compound involved in lipid peroxidation, generation of active-free radicals, inactivation of various enzymes or binding to proteins or other cell constituents; therefore, rapid elimination of the chemical from the interior of the cell is essential (Johansson, 1992). A daily DIS dose of 150±700 mg given to alcoholics has shown positive results to reduce or deter the consumption of alcoholic beverages due to the disul®ram±ethanol reaction, which increases the level of blood acetaldehyde. This, in turn, produces a number of unpleasant symptoms which may include ¯ushing of the skin, due to cutaneous vasodilatation, hypotention due to diastolic blood pressure, re¯ex tachycardia, tachypnoea, a sensation of warmth, palpitations, anxiety, headache, nausea and vomiting. The intensity and duration of the eects correlate with the time course of the blood acetaldehyde concentration, but there can be appreciable inter-individual and intraindividual variation in the acetaldehyde-mediated reaction (Petersen, 1992). DIS has been described as a chemical with low toxicity, since the minimal fatal dose in rabbits was determined to be 3 g/kg by the oral route, and several acute toxicity studies in mammals were also concordant (Petersen, 1992); however, a chronic study in rats showed that the daily administration of 250 mg/kg produced ataxia, and calci®ed masses in the cerebellum and basal ganglia (Fitzhugh et al., 1952). Cytotoxic eects of DIS have also been observed in rodent and human cell lines as well as in human lymphocytes, probably due to its eect on the decrease of DNA synthesis (Cohen and Robbins, 1990; Hellander and Lindahl-Kiessling, 1991). The chemical is considered safe concerning carcinogenic and teratogenic eects; however, embryotoxicity and fertility deterioration in rats have been described (Salgo and Oster, 1974), as well as the generation of zebra®sh embryos with
abnormalities in the anteroposterior axis (Costaridis et al., 1996). Concerning genotoxic studies, DIS has been shown to be mutagenic in the L51784 mouse lymphoma cell forward assay (McGregor et al., 1991). In this report, the compound was positive starting from 0.01 mg/ml, while at higher dose levels up to 2 mg/ml, the mutagenic response gradually diminished. Considering this result, as well as the widespread use of DIS, the aim of the present investigation was to determine whether disul®ram may increase the level of sister-chromatid exchanges (SCEs) in bone marrow and spermatogonial cells of mice treated in vivo.
MATERIAL AND METHODS
Chemicals and animals DIS was purchased from Sigma Chemicals Co. (St Louis, MO, USA) and dissolved in corn oil, a compound also obtained from Sigma. The DIS test doses used were 200, 400 and 800 mg/kg body weight, and these were based on a previous LD50 performed according to the method of Lorke using the oral route (Lorke, 1983). In this study we obtained a result of 1113 mg/kg; therefore, our high DIS dose corresponded to 71.8% of its LD50. 5bromodeoxyuridine (BrdU) (Hoechst 33258), and colchicine were obtained from Sigma Chemical Co. and dissolved in distilled water. Cyclophosphamide was purchased from Laboratorios Sanfer S.A. (MeÂxico), and dissolved in distilled water. Activated charcoal was purchased from Sigma, Giemsa stain from Merck-MeÂxico, S.A. and trypsin from Difco Laboratories (USA). Finally, Lab Rodent Diet 5001 pellets were used for mouse feeding. Male mice (NIH) with a mean weight of 28 g were obtained from the National Institute of Hygiene. They were kept in polypropylene cages in groups of ®ve animals each at a mean temperature of 238C, fed with standard food, and permitted to drink tap water ad lib. Pellet consumption was suspended 6 hr before beginning the experiment. Somatic cell study The selected DIS doses were administered orally to lots of ®ve animals, and 6 hr later the mice were injected ip with an aqueous suspension of BrdU previously adsorbed to activated charcoal (1.5 mg/ kg of body weight) (Madrigal-Bujaidar et al., 1997; Morales-Ramirez et al., 1984). The negative control was treated orally with 0.3 ml corn oil at the time of DIS administration, and the positive control animals were injected ip (cyclophosphamide, 50 mg/kg) 30 min before BrdU administration. 22 hr after the BrdU administration the animals were injected ip with colchicine (3.5 mg/g) and after 3 hr they were sacri®ced with CO2. The femurs were dissected and the bone marrow extracted in KCl 0.075 M at 378C,
SCEs induced by disul®ram in mice
and the cells were resuspended and left for 30 min; then they were ®xed three times with methanol±acetic acid (3:1) and ®nally cell spreading was made on iced slides followed by a slight ¯aming. The ¯uorescence-plus Giemsa method used for the dierential staining of sister chromatids has been described before (Madrigal-Bujaidar et al., 1997; MoralesRamirez et al., 1992), and consisted in the following basic steps: the cells were stained in Hoescht 33258 for 60 min; the slides were washed in tap water after which they were dried at 608C for 15 min; then they were buered with a citrate phosphate, pH 7.0 and exposed to black light for 60 min. After being washed and dried as before, the cells were immersed for 20 min at 608C in a saline citrate solution, washed and stained with a 4% Giemsa solution made in a phosphate buer, pH 6.8, for 10 min. The microscopic observations included an SCE scoring made in 30 second-division mitosis per mouse, the mitotic index determined in 1000 cells per animal, and the cell proliferation kinetics (CPK) established in 100 cells per mouse. In the latter case, the frequency of cells in ®rst, second, and third cellular division (M1, M2 and M3, respectively) was used to obtain the average generation time (AGT) with the equation AGT = 22/ (M1 + 2M2 + 3M3) 100 (Ivett and Tice, 1982). The statistical evaluation of the data obtained was initially made with a one-way ANOVA test followed by a Student's t-test. Germ cell study The experimental doses of DIS were administered to the animals by oral intubation, and 6 hr later the mice were injected ip with an aqueous suspension of BrdU (1.3 mg/kg) adsorbed to activated charcoal (Morales-Ramirez et al., 1984). Corn oil was administered orally to control animals at the time of DIS inoculation, and positive control animals were treated ip with 50 mg cyclophosphamide/kg 12 hr after BrdU injection; this time is usually chosen in germ cell studies considering the short half-life of the chemical (Palitti et al., 1982). Colchicine (7.5 mg/kg) was injected sc after 53 hr of BrdU administration, and 3 hr later the animals were killed by cervical dislocation. Then, the testes were dis-
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sected, the tunica albuginea separated from them, and the seminiferous tubules ®nely cut in phosphate buered saline (PBS) and washed three times in the same solution. The tissue was incubated for 5 min at 378C in a trypsin solution made in ethylenediaminetetraacetic acid (EDTA) without calcium or magnesium, and then a tissue disaggregation was made by passing it through a ®ne pore needle several times. The suspension obtained was centrifuged and washed three times in EDTA. Finally, the spermatogonial cells were treated with KCl 0.075 M at 378C for 5 min and ®xed in methanol±acetic acid (3:1) at least twice. The preparation of the slides as well as the staining procedure were done according to the method previously explained for bone marrow cells. The slides were observed microscopically to determine the frequency of SCEs by evaluating 30 second-division mitosis per mouse. The data obtained were statistically analysed with a one-way ANOVA and the Student's t-tests. RESULTS
The study to determine the genotoxic eect of DIS in bone marrow cells showed a dose-dependent response. Although the SCE increase was statistically signi®cant only with the high tested dose (800 mg/kg), a doubling of the basal level was not reached even with this dose; on the contrary, cyclophosphamide caused strong genotoxic damage (Table 1). The cumulative frequency of cells v. SCE number in bone marrow is presented in Fig. 1, showing no important dierences between the curves obtained in the control group and those obtained in the groups treated with dierent doses of DIS. A clear dierence was observed in the curve of the positive control group (cyclophosphamide, 50 mg/kg). In spermatogonial cells, the increase in the number of SCEs was statistically signi®cant in all three doses tested with respect to the control level. The values obtained with the two low doses were higher then those found in somatic cells in relation to their respective control data. However, the results did not correspond to a dose±response relationship, nor to a duplication of the basal level. The positive con-
Table 1. Frequency of sister-chromatid exchanges in bone marrow and spermatogonial cells of mice treated with disul®ram Bone marrow cells
Spermatogonial cells
Dose (mg/kg)
Agent
SCE X2 SD
INC
SCE X2 SD
INC
0 200 400 800 50
Corn oil Disul®ram Disul®ram Disul®ram Cyclophosphamide
2.2120.30 2.42 20.40 2.58 20.20 *3.1820.40 *7.4320.20
0.21 0.37 0.97 2.25
1.83 20.02 *2.25 20.10 *2.63 20.27 *2.44 21.49 *6.10 20.80
0.42 0.80 0.60 4.27
Statistically signi®cant dierence with respect to control level. One-way ANOVA and Student's t-tests; P = 0.05. The scoring was made in ®ve animals per lot and 30 cells per animal. INC = SCE increase (treated±control).
760
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Fig. 1. Cumulative frequency of cells with respect to SCE per cell in bone marrow. Mice treated with 0 (w), 200 ( ), 400 (W) and 800 (T) mg disul®ram/kg, and 50 mg cyclophosphamide/kg as positive control (Q).
trol (cyclophosphamide) showed a strong response, although weaker than that observed in somatic cells (Table 1). In Fig. 2 the curves of cumulative frequency of cells v. frequency of SCE indicate that DIS increases the frequency of cells with a high SCE number with respect to the control group, that is, in the control group only 20% of cells have two or more SCEs, and in the treated groups this percentage lies between 40 and 50%. The curve of the positive control group indicates a clear dierence from the untreated control. An interesting point is that spermatogonia seems to be less sensitive than bone marrow to SCE induction by cyclophosphamide, that is, in bone marrow no cells were observed with an SCE frequency lower than four, while in spermatogonia this group of cells represents 60%. Besides, in germ cells only a few cells have more than eight SCEs, while in bone marrow these cells are approximately 20%. With regard to the mitotic index, in the study performed in bone marrow cells we observed no statistical dierences between all tested groups, although a 2.3% decrease was noted in the animals treated with the high dose, and a dose-decrease behaviour was observed with all doses. This suggest a mild cytotoxic eect of the chemical. In relation to the cellular proliferation kinetics, a certain increase in the number of M1 cells was observed in mice treated with the highest dose of DIS, suggesting a delay in the cell proliferation; this alteration produced a signi®cant dierence with respect to control data concerning the average generation time value. However, the accumulation of M1 cells by cyclophosphamide was substantially greater than that obtained with the other treatments also producing an important cell-cycle delay as re¯ected by the obtained AGT value (Table 2).
DISCUSSION
Sister-chromatid exchanges correspond to physical exchange of DNA double helices between sister chromatids. They occur spontaneously in low numbers, but dramatically increase when cells are treated with DNA-damaging agents. This observation has been determinant in the extensive use of the method for genotoxic evaluation, moreover because SCE scoring has been of great value in the screening of weak genotoxic agents (Lindahl-Kiessling, et al., 1989). It is important to study the genotoxic eect in dierent tissues because besides the well established dierences in response, the health implications could also be dierent. Implications on genetic risk inferred from studies carried out in germ cells are particularly relevant. The results obtained in the present study indicate that DIS has a weak genotoxic eect in both somatic and germ cells, this conclusion corresponds to the SCE level obtained which did not reach a doubling of the control value, as well as the reduced SCE induction in comparison with that obtained with the positive control. The observed genotoxic eect induced by DIS may have several explanations. One of these refers to the direct or indirect induction of oxidative stress by the chemical, as suggested by Delmaestro and Tombetta (1995), who detected accumulation of copper, increased production of malondialdehyde and increased activity of glutathione peroxidase in rats treated orally with the compound. On the other hand, the metabolism of the chemical is a very complex process, and one or more of its metabolites may participate in the genotoxic damage, although this aspect has been poorly explored. DIS is rapidly reduced to diethylthiocarbamate, and in later steps to some active metabolites such as diethylthiocarba-
SCEs induced by disul®ram in mice
761
Fig. 2. Cumulative frequency of cells with respect to SCE per cell in spermatogonia. Mice treated with 0 (w), 200 ( ), 400 (W) and 800 (T) mg disul®ram/kg and 50 mg cyclophosphamide/kg as positive control (Q).
mic acid methyl ester (DAME ), this compound is active by injection, it possesses a more rapid eect and higher potency than DIS, and causes enzymatic inhibition in vivo and in vitro (Petersen, 1992). Smethyl N,N-diethylthiocarbamate sulfone, a logical metabolite of S-methyl N,N-diethylthiocarbamate sulfoxide, also has similar properties as the mentioned for DAME (Mays et al., 1995). Finally, two of the last chemicals formed in the DIS metabolic pathway are diethylamine and carbon disul®de; the latter is known to be a neurotoxin which is highly reactive with several macromolecules and is particularly relevant to this discussion because it possesses the capacity to increase the rate of structural chromosomal aberrations (Le and Fu, 1996). Another probable explanation for the detected genotoxic damage may be related to the acetaldehyde fraction of endogenous origin that has been detected in subjects not drinking ethanol. Several sources from which it may come have been proposed, including pyruvate and aminoacid metabolism, endogenous ethanol formation or protein degradation (Ericksson, 1987; Hellander, 1993). In human sub-
jects and in rats under DIS treatment, markedly elevated levels of acetaldehyde have been found (Eriksson, 1985). In this context it is worthwhile stressing that acetaldehyde has been described as an inducer of SCEs in vitro and in vivo (Hellander and Lindahl-Kiessling, 1991; Korte and Obe, 1981), a capacity which seems to increase in the presence of aldehyde dehydrogenase inhibitors such as 1-aminocyclopropanol (Hellander and Lindahl-Kiessling, 1991). DIS ingestion is useful in the treatment of alcohol dependence, but the drug should be given primarily to well-motivated patients, who must also be carefully monitored and given support (Kristenson, 1992; O'Farrel et al., 1995). However, the drug may occasionally produce adverse reactions in the organism (also including interactions with other chemicals), leading to health deterioration in various organs. The reported damage is related to hepatic, neurological, skin and psychiatric reactions in decreasing order of frequency, although other organs may be aected (Paulsen et al., 1992). The causes of some disorders are being understood, for
Table 2. Mitotic index ( MI ) and cellular proliferation kinetics ( CPK ) in bone marrow cells of mice administered with disul®ram CPK Dose (mg/kg)
Agent
M1
M2
M3
AGT h
MI %
0 200 400 800 50
Corn oil Disul®ram Disul®ram Disul®ram Cyclophosphamide
26.0 27.4 33.6 38.6 62.2
61.2 59.4 57.6 50.0 33.6
12.8 11.2 8.8 11.2 4.2
12.17 12.24 12.56 *12.78 *15.49
8.96 8.6 7.86 6.66 *2.6
Statistically signi®cant dierence with respect to control level. One-way ANOVA and Student's t-tests; P = 0.05. The scoring for CPK data was made in 100 cells per mouse, and for MI in 1000 cells per mouse. Average generation time (AGT) = 22/(M1 + 2M2 + 3M3) 100, where M1, M2 and M3 correspond to the frequency of cells in ®rst, second and third cell division.
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example, the neurological damage seems to be mediated by a neurotransmitter metabolism alteration which includes abnormal activity of serotonine, dopamine and tryptophan (Beck et al., 1995; Nagendra et al., 1993; Vaccari et al., 1996). The organic damage is mainly related to the DIS-alcohol reaction, which seems to be characterized by a highly interindividual variability; in severe cases, cytolitic hepatitis with fatal evolution has been reported, as well as the death of a patient showing a corrosion lesion in the lower oesophagus and stomach due to a violent chemical reaction in situ, in that study, an acetaldehyde blood concentration of 41 mg/litre was determined (Health et al., 1992). Considering these variable responses to DIS, our ®ndings con®rm the importance of being cautious with the use of this chemical treatment and of including proper clinical and periodical chemical monitoring of the patients under DIS therapy. This suggestion agrees with a histological study in DIStreated rats also administered with ethanol, which showed a decrease in the content of zymogen granules in acinar cells, and the appearance of intraplasmic vacuolization which seemed to have lipid inclusions (Honda et al., 1995). This is also in accordance with a report by Wicht et al. (1995) who found that in patients under DIS administration but without liver disease, the acetaldehyde level was higher than in those with liver disease, a ®nding which suggests that clinical symptoms do not necessarily re¯ect the intensity of the chemical reaction between both compounds. REFERENCES
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