Increased sister chromatid exchange and chromosomal aberration frequencies in psychiatric patients receiving psychopharmacological therapy

Mutation Research 413 Ž1998. 169–175

Increased sister chromatid exchange and chromosomal aberration frequencies in psychiatric patients receiving psychopharmacological therapy M. Paola Bigatti

a,)

, Daniela Corona a , Carmine Munizza

b

a

b

Dipartimento di Biologia Animale e dell’Uomo, UniÕersita` di Torino, Torino, Italy Dipartimento di Psichiatria-Azienda Regionale 4-Centro Studi e Ricerche in Psichiatria, Torino, Italy Received 28 October 1997; revised 4 February 1998; accepted 4 February 1998

Abstract Combinations of various psychotropic drugs are often used, sometimes for long periods, in the treatment of various forms of psychiatric disorders. This paper evaluates the cytogenetic consequences of daily exposure to polytherapy with antianxiety, antipsychotic and antimaniacal drugs by determining chromosomal aberrations ŽCA. and sister chromatid exchange ŽSCE. in peripheral blood samples. The study was performed with a group of 36 psychiatric patients: 18 receiving long-term treatment with lithium carbonate, combined with benzodiazepines ŽBD. and antipsychotic agents ŽGroup A. and 18 treated with BD and antipsychotics ŽGroup B.. Among the latter, 7 patients had only been treated for 1 month ŽGroup B1 .. The results reveal a significant increase Ž p - 0.01. in cells with aberrations in the two groups of patients ŽA,B. compared to controls. Moreover, complex aberrations Ždicentrics, rearrangements, chromatid exchanges. had a frequency of 0.63% in patients receiving long-term treatment compared to 0.11% in controls, corresponding to the general spontaneous rate. The mean frequency of SCErcell and the percentage of cells with a high frequency of exchanges ŽHFC. also showed a highly significant difference compared to controls in both Group A and Group B. Group B1 Žpatients who only commenced treatment 1 month earlier. did not differ from the control group with regard to the frequency and type of chromosomal aberration or in relation to the mean frequency of SCErcell. No significant differences were detected between Groups A and B both of which showed similar frequencies of cells with aberrations, SCErcell and HFC. No correlations were observed in Group A between lithemia and the biological markers studied. q 1998 Elsevier Science B.V. Keywords: Lithium; Benzodiazepine; Neuroleptics; Genotoxicity; Lymphocyte; Human

1. Introduction The majority of pyschiatric disorders undergoing psychopharmacological treatment fall into the fol) Corresponding author. Dipartimento di Biologia Animale e dell’Uomo, Universita` di Torino, Via Accademia Albertina, 17 10123 Torino, Italy. Tel.: q39-011-8122374; fax: q39-0118124561; E-mail: [email protected]

lowing diagnostic categories: anxiety disorders, personality disorders, schizophrenic disorders and affective disorders. The latter also include both minor forms, lacking psychotic characteristics, and major forms-unipolar Ždepressive. or bipolar Žmanic-depressive.-with psychotic type phenomena. The longterm treatment of these disorders, and in particular schizophrenic and affective forms with psychotic symptoms, is very often based on combinations of

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 2 8 - X

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M.P. Bigatti et al.r Mutation Research 413 (1998) 169–175

drugs belonging to distinct categories w1x. The following main categories of drugs are used: -antianxiety and hypnotic agents, primarily benzodiazepines; -antipsychotic drugs Žin particular neuroleptics, of which the parent drug is chlorpromazine.; -antidepressant drugs; -mood-stabilizers, whose main representative is lithium carbonate. Even if the use of polytherapy is not advised in psychiatric literature, it nonetheless occurs frequently in clinical practice and these patients are exposed, often for prolonged periods, to a series of substances or metabolites which might individually possess a genotoxic potential and produce addictive effects. Many psychotropic drugs, like lithium, have been tested for their genotoxicity in various ‘test systems’, often with contradictory results. The group of pharmacological agents identified as benzodiazepines ŽBD., particularly in its most wellknown form diazepam, has been the subject of extensive studies regarding its genotoxicity. Negative results on human chromosomes were reported by Cohen et al. w2x, Staiger w3x, Zhurkov w4x, White et al. w5x, Husum et al. w6x and positive ones by Stenchever et al. w7,8x, and Van Bao et al. w9x. Increased chromosomal aberrations in cell cultures of Chinese hamster were highlighted by Lafy and Parry w10x following treatment with four structurally correlated benzodiazepines: diazepam, madazepam, midazolam and bromazepam. Diazepam was included as a suspect aneugen in a trial coordinated by the Commission of the European Communities ŽCEC. w11x. The results of the trial revealed that ‘in vitro’ the substance produces a mitotic andror meiotic arrest with induction of aneuploidy, whereas results were less homogeneous in laboratory animals. For review, see Marchetti et al. w12x and Giri and Banerjee w13x. With regard to the effects of neuroleptics ŽNL., in a recent review of the genotoxicity of chlorpromazine and related phenothiazines w14x, Gocke reports that cytogenetic tests in patients treated with phenothiazine derivatives do not allow unequivocal conclusions to be drawn. For example, Madle et al. w15x observed a 2.5 fold increase in exchange type aberrations in schizophrenic patients who were treated with perazine but also with other drugs. On the other hand, Crossen and Morgan w16x and Nielsen et al.

w17x found no increase in patients treated with chlorpromazine alone. With regard to lithium, although numerous studies report the teratogenic potential of this metal in invertebrates, fishes and mammals w18x it does not appear to represent a risk to man at therapeutic doses w19x. At chromosomal level, positive results have only been reported in a few studies or using experimental models. The authors refer to the works by Banduhn et al. w20x, Johnson w21x, Leonard et al. w22x for a critical review of the subject. Increases in the frequencies of SCE or chromosomal aberrations ŽCA. may be regarded as biological markers of personal exposure to dangerous chemical agents and certainly represent the first signs of DNA damage. The study of these markers is therefore one of the most sensitive and useful means of biomonitoring human exposure to genotoxic substances w23,24x. Furthermore, recent studies have revealed the existence of an association between the frequency of chromosomal aberrations in lymphocytes and the probability of tumor induction w25x. This paper reports a study performed with two groups of patients, one treated with NL and BD, and the other with NL, BD and lithium carbonate. SCE and CA analysis in peripheral blood lymphocytes were used to determine the consequences at the chromosomal level of daily exposure to a mixture of chemical substances whose genotoxic potential is still open to debate.

2. Materials and methods Peripheral blood samples were taken from 36 patients suffering mainly from bipolar affective disorders and receiving psychopharmacological treatment in two different psychiatric departments. Of these, 18 ŽGroup A. were treated with lithium carbonate, but were also receiving BD and NL. Eighteen patients ŽGroup B. were only treated with NL and BD. Patients in Group A received an average daily dose of lithium carbonate of 841.7 mgrday Ž300– 1200.. The majority of patients received therapeutic treatment of 900 mgrday. The mean level of

M.P. Bigatti et al.r Mutation Research 413 (1998) 169–175

lithemia 1 in the group was 0.64 mEqrl Ž0.3–0.97.. The mean duration of treatment was 3.3 years Ž6 months–10 years.; one of the subjects treated for six months had recommenced treatment after an interval of 2 years. When blood samples were taken, all patients in Group A were receiving treatment with BD andror NL. We were not always informed of the doses. The second group of patients was treated with NL and BD, and treatment generally consisted of the combination of these two classes of drugs, except for two patients who were treated with BD alone and two with NL alone. Seven patients in the second group ŽGroup B1 . had only been treated for a brief period Žapproximately one month. and the previous use of BD andror NL had been irregular. When blood samples were taken, the patients were receiving an average 6.8 mgrday of benzodiazepines and 330 mgrday of neuroleptics, expressed in chloropromazine equivalents. 2 The remaining 11 patients had received treatment for a mean period of 5.5 years Ž6 months–15 years.. The mean daily dose when blood samples were taken was 3.7 mgrday of BD and 430 mgrday of NL, expressed in chlorpromazine equivalents. Table 1 lists the benzodiazepines and neuroleptics prescribed to patients in the study sample. The patients examined were not employed in jobs that involved their exposure to genotoxic substances. Moreover, information regarding exposure to X-rays or vaccines during the period prior to blood sampling and smoking habits was also recorded. Controls were analyzed at the same time as those exposed to psychotropic drugs and included volun-

1

Given that lithium salts have a low therapeutic index the concentration of lithium ions in plasma or serum must be determined to guarantee the safe use of the drug which is also influenced by sodium plasma levels. Lithemia corresponds to the measurement of the plasma concentration of lithium ions in order to check the presence of therapeutic levels Ž0.5–1.2 mEqrl. and the absence of toxic concentrations Ž )1.2 mEqrl.. 2 Although the most commonly used antipsychotic drugs are deemed to have similar therapeutic effects, they nonetheless differ considerably in terms of strength; the different classes of drugs must therefore be compared using equivalent dosages, namely the relative strengths obtained using conversion factors which allow the equivalent to a standard dose of 100 mg of chlorpromazine to be calculated for each drug.

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Table 1 Benzodiazepines and neuroleptic drugs used for treatment and number of patients treated Group A no. Benzodiazepines Alprazolam Bromazepam Clordexmethyldiazepam Clotiazepam Flunitrazepam Diazepam Lorazepam Oxazepam Triazolam Neuroleptics Haloperidol Haloperidol decanoate Clopenthixol Chlorpromazine Clothiapine Fluphenazine decanoate Pimozide Promazine Tiapride

1 1

Group B no.

1 5 1 1 1 7 1

3 3

1

5

11 1 1 1 2 1 2 1

1

1

Patients in Group A also received treatment with lithium carbonate. The same subject might receive treatment with more than one of the active ingredients listed.

teers from the same geographical area who had no known history of exposure to psychopharmacological therapy and were matched, as far as possible, for sex, age and smoking habits. However, the number of smokers was slightly higher among patients: 53% compared to 43% ŽTable 2..

Table 2 Demographic characteristics of the 72 subjects studied Patients

Control group

Group A

Group B

Type Women Men

8 Ž44%. 10 Ž56%.

5 Ž28%. 13 Ž72%.

15 Ž42%. 21 Ž58%.

Smoking habits Non smokers Smokers Mean age Žrange.

9 Ž50%. 9 Ž50%. 42 Ž26–65.

8 Ž44%. 10 Ž56%. 37 Ž18–69.

21 Ž58%. 15 Ž42%. 38 Ž22–76.

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Table 3 Frequency of chromosomal aberrations in patients and controls

Number of subjects No of cells observed Gaps Chromatid breaks Chromatid exchanges Chromosome breaks Dicentrics Rearrangements Rings Chromosome pulverization Total aberrations No. of cells with aberrations

A Ž%.

B Ž%.

B1 Ž%.

Control group Ž%.

16 1580 29Ž1.83. 15Ž0.95. 3Ž0.19. 22Ž1.39. 3Ž0.19. 4Ž0.25. y 1 Ž0.06. 48Ž3.04. 47Ž2.97. a

10 928 17Ž1.83. 14Ž1.51. 3Ž0.32. 9Ž0.97. 2Ž0.21. 1Ž0.10. y y 29Ž3.12. 29Ž3.12.

7 700 6Ž0.86. 4Ž0.57. y 8Ž1.14. y y y y 12Ž1.71. 12Ž1.71.

27 2622 30Ž1.44. 24Ž0.91. y 18 Ž0.69. 2Ž0.08. y 1Ž0.04. y 44Ž1.68. 43Ž1.64.

Gaps were recorded but not counted. Rearrangement: abnormal monocentric chromosome. a p - 0.01 ŽChi-square test..

2.1. Cytogenic tests For each subject 0.5 ml of peripheral blood was cultured for 72 h in 6 ml of RPMI-1640 medium ŽGibco., supplemented with 25% of fetal calf serum, 3% of phytohemagglutinin M and antibiotics Ž100 Urml of penicillin and 50 m grml of streptomycin.. After a 24-h incubation, 5-bromodeoxyuridine ŽBrdU. was added to the cultures at a final concentration of 5 m grml. Colcemid Ž0.2 m grml. was added 1 h before harvesting. After a brief hypotonic treatment ŽKCl 0.075 M. cultures were routinely fixed Žmethanol and acetic acid 3:1. and air-dried preparations were made. Differential staining of sister-chromatid exchanges was carried out using a modified version of the FPG method w26x. The coded slides were scored by 2 investigators. With some exceptions, 100 cells, in first division, were examined for CA and 50, in second division,

for SCE analysis. The number of cells in the first ŽM 1 ., second ŽM 2 ., third and subsequent divisions ŽM 3 . were counted in 100 metaphases. The Proliferation Rate Index ŽPRI. ŽM 1 q 2M 2 q 3M 3r100. w27x was calculated for cell kinetic analysis. The sample size is not the same for the two biomarkers examined because in some cases it was not possible to evaluate both SCE and CA for each subject ŽTables 3 and 4.. 2.2. Statistical analysis SCE frequency analysis was performed using oneway variance analysis ŽANOVA. and Student’s t-test. In order to normalize SCE distribution, a square root transformation was applied. The percentages of high frequency cells ŽHFC. were calculated as suggested by Moore and Carrano w28x and analyzed using the Chi-square test. The

Table 4 Frequency of SCE, percentage of HFC, PRI in patients and controls No. of subjects A B B1 Controls a

18 11 6 29

No. of cells observed 830 469 190 1249

p - 0.01 ŽVariance analysis.; b p - 0.001 ŽChi-square test..

SCE" DS

HFC Ž%. a

7.16 " 1.65 7.22 " 2.02 a 4.52 " 1.35 4.80 " 1.55

b

11.40 12.79 b 0.53 3.76

PRI 1.78 1.96 2.00 2.25

M.P. Bigatti et al.r Mutation Research 413 (1998) 169–175

significance of the difference between CA was determined using the Chi-square test.

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Table 4 reports the mean frequency of SCE, the percentage of HFC and PRI. Again in this instance, a significant difference Ž p - 0.01. was found between controls and patients receiving long-term therapy, with the latter showing a percentage increase of approximately 50% in the mean SCE frequency. The critical number of HFC was determined in the control group using binomial distribution and a level of significance of 0.05 according to Moore and Carrano w28x. Twelve exchangesrcell was deemed as the cut-off point. The percentage of cells with a high exchange frequency tripled in patients in Groups A and B compared to controls Ž p - 0.001.. In Group A, the range of variation stretched from 0 to 20 high frequency cells per subject, and in Group B from 0 to 22. In the latter group, a cell with 40 isochromatid exchanges was counted in one patient. Group B1 did not differ from the control group with regard to mean SCE frequency and presented a limited percentage of HFC Ž0.53%.. The PRI diminished slightly in patients and revealed slightly slower proliferative kinetics compared to controls. Table 5 shows the mean SCE frequency, the percentage of HFC and the percentage of cells with aberrations observed in the control group and in patients Žin this case Groups A and B were analyzed together. divided according to smoking habits. The comparison between controls and patients always gave significant results for both SCE and CA. Within the control group statistical analysis showed that the percentage of cells with aberrations was significantly higher Ž p - 0.05. in smokers, whereas the mean SCE frequency did not differ significantly between smokers and non-smokers. On the other hand, within the patient group a significant increase was detected in smokers for both CA Ž p - 0.05. and SCE Ž p - 0.01.. The increase in the percentage of HFC was highly

3. Results Table 3 reports the different types of chromosomal aberrations found in the controls and patients examined. Owing to their ambiguous nature as chromosomal aberrations, the gaps, even if scored, were not considered when calculating total aberrations. From an analysis of these data it can be seen that the frequency of cells with aberrations almost coincides, in all groups, with the overall frequency of chromosomal aberrations given that cells with multiple aberrations were never observed. On the other hand, a significant increase Ž p - 0.01. in cells with aberrations was noted in the two groups of patients ŽA,B. compared to controls. On the contrary, the seven patients ŽGroup B 1 . who had only commenced treatment one month earlier did not differ significantly from the control group. No differences in the frequency of aberrations were detected between Groups A and B Ž3.04% in the former, 3.12% in the latter.. When the type of aberration was analyzed, it was found that complex aberrations Ždicentric, chromatid exchanges, stable chromosomal rearrangements. had a frequency of 0.63% in patients receiving long-term therapy, Groups A and B, without any marked differences between the groups, compared to 0.11% in controls which corresponds to the overall spontaneous rate Ž18%. w29x. Moreover, more than half of the patients Ž69%. carried these types of aberrations. Complex rearrangements were not observed in the small group of patients treated for a brief period. The phenomenon of phase S cell fusion with the formation of pulverized chromosomes was observed in one patient in Group A.

Table 5 Mean frequency of SCErcell, HFC and CA in patients ŽGroups A and B. and controls divided according to smoking habits SCErcell Control non-smokerrnon-smoking patients Control smokerrsmoking patients a

HFC a

4.52r6.34 5.11r7.38 a

CA b

3.62r12.50 3.94r13.75 b

p - 0.001 ŽStudent’s t-test.; b p - 0.001 ŽChi-square test.; c 0.02 - p - 0.05 ŽChi-square test..

1.09r2.14 c 2.22r3.72 c

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significant Ž p - 0.001. in patients, four times that of the respective controls. In the control group and patients, however, no significant difference was found between smokers and non-smokers with regard to the percentage of HFC, although the former presented slightly higher values: the increase was respectively 8.8% and 10%. No correlations were detected in Group A between lithemia and the biological markers examined.

HFC represents particularly damaged cells, probably a lymphocyte subpopulation with persisting genotoxic damage and they may be a good indicator of damage caused by chronic exposure w23x. In this study, HFC in patients receiving long-term therapy increased dramatically compared to controls; moreover, 82.8% of patients presented HFC, whereas in the control group the percentage of subjects with HFC was equivalent to 55.2.

4. Discussion

5. Conclusion

All patients included in our study sample received psychopharmacological therapy with benzodiazepines andror neuroleptics, as shown in Table 1. These two classes of drugs have similar sedative effects, but also very different mechanisms of activity and clinical effects. In addition to benzodiazepines and neuroleptics, 18 subjects also received daily treatment with lithium carbonate. This kind of polytherapy is widely used in the long-term treatment of the major psychiatric disorders; it is therefore difficult for a patient to be exposed to a single psychoactive drug Žor group of similar drugs, e.g., neuroleptics.. For this reason, studies in man concerning the genotoxic effects of individual benzodiazepine or neuroleptic drugs are rare and do not always exist w17x. The approach adopted in this study aimed to evaluate whether psychopharmacological treatment in general, consisting in the use of different active ingredients, even if classifiable into two main classes Žneuroleptics and benzodiazepines., can provoke damage at a chromosomal level and whether the addition of lithium might enhance the possible genotoxic effect. The results obtained show an overall and significant increase in both the biomarkers studied in patients receiving long-term treatment, whereas the seven patients who had only recently commenced therapy, even if treated with higher doses, at least of BD, did not differ from controls. Lithium carbonate treatment in combination with BD andror NL did not increase chromosomal damage given that SCE and CA levels and the type of aberration Žpresence of complex rearrangements. were virtually identical in both groups.

The relationships between smoking and SCE and between smoking and CA have been examined by several authors with discordant results w29–32x. In this study, the patients, divided according to smoking habits, showed significant increases compared to their respective controls for both biomarkers. While differences were detected between smokers and nonsmokers in the frequency of CA, but not SCE, in the control group, among the patients smokers presented a statistically significant increase in both cytogenetic ‘end-points’, perhaps indicating an additive effect between smoking and drugs. As was stated earlier, the patients examined in this study were exposed for a long period ŽGroups A and B. to more than one active pharmacological ingredient. This complex situation did not enable an association to be identified between the cytogenetic effects observed and exposure to a particular compound. However, the results obtained reiterate the need both to extend this type of study and to make a careful evaluation of the cost-benefit ratio of longterm psychopharmacological treatment, in particular for young subjects.

Acknowledgements We thank Dr. Antonino Bigatti for his advice in the early planning stages of this study and Prof. Laura Lamberti who has contributed to its development. We thank Drs. M. Rosa Longo for her collaboration and Elisabetta Cremona for technical help. The authors wish to thank the referees for their comments on the original version of this paper. This

M.P. Bigatti et al.r Mutation Research 413 (1998) 169–175

study has been supported by the Italian MURST Ž60% and 40% grants..

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