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Effects of fluphenazine hydrochloride on the bonemarrow cells of swiss mice
237 Mutation Research,
89 (1981)
ElsevierlNorth-Holland
K. PRABHAKAR Department
237-240
Biomedical Press
.O and M. SANJEEVA RAO
of Genetics,
&mania
University, Hyderabad-500
007 (India)
(Received 15 July 1980) (Revision received 12 Qecember 1980) (Accepted 16 December 1980)
Summary The possible mutagenic effects induced by flunhenazine hydrochloride in bone-marrow cells of mice were evaluated by the micronucleus test. Mice were treated orally and intraperitoneally with the drug at dose of 55, 110 and 165 mg/kg and 21.25, 42.50 and 63.75 mg/kg resp. Bone-marrow preparations were made, and the slides were analysed for the presence of ~~ronuclei in develop~g e~thro&y~s. Fluphenaz~e hy~ochlo~de induced a signifi sant dose-dependent increase of micronuclei in polyc~omatic erythrocytes by oral administration, whereas only a marginal increase was observed at 42.50 and 63.75 mg/kg dose levels by intraperitoneal administration. Further, fluphenazine hydrochloride induced bone-marrow depletion at all dose levels with oral administration only.
Tranq~l~zers, a group of drugs introduced as psychotherapeutic agents, are being extensively used in human medicine to allay anxiety and tension. Among tranq~lizers, ~hlorprornaz~i~ and related pheno~~i~~e groups are used in the treatment of psychiatric disorders and in the treatment of nausea and vomiting (Jarvik, 1970). Several million people have been treated with these drugs since their introduction into medicine. It was reported in t,he available literature that high incidences of chromosome gaps and breaks were observed in human leucocyte cultures treated with perphenazine, chlorpromazine and trifluoperazine (Nielsen et al., 1969; Jenkim, 19710). Trifluoperazine induced a high frequency of reversion mutations in S. ~~~~~~~~~~~ G-46 (Wenkata Ramanaiah et al., 1980). Another phenothi~~e derivative, ~~uproma~ine, induced ehromosomal breakage in kidney cells of the rat igloo (Green et al., ~9?0~~ dominant lethal mutations in :mniee(Petersen and Legator, 1973) and abnor0165-1218/81/0000-0000/!$02.~0
0 Elsevier/North-Holland
Biomedical Press
rr,a,ities in the shape of the mouse sperm-head (personal communication). l~luphenazi~e hydrochloride, a phenothiazine compound, is mutagenic in the host-mediated assay with S. ty~~jj~u~iurn G-46 in mice (Prabhakar Rao and Sanjeeva Rao, 1980a) and it causes X-linked recessive lethal mutations in Drosophila melan~gaster (Prabbakar Rao and Sanjeeva Rao, 198Ob), Further, it also produces ~lo~~inant lethal mutations and cytogenetic effects on male-germ cells cf mice (Prabhakar Rao, 1978). The micronucleus test in mouse bone marrow is a sensitive test system in vivo fBr cytogeneti~~y active agents (Maier and Schmid, 1976). This test system, was used to evaluate the cytogenetic effects of fluphenazine in mouse bone-marrow cells, and the results are reported herein. aterial and methods Random-bred Swiss mice, aged 7-8 weeks and weighing 23-27 g, were used. Fluphe~l~ine hy~ochloride ~Ana~nsol) was pur~ha~d in tablet form from M/s. Sarabhai Chemicals, Baroda, India. The tablets were dissolved in sterile distilled water or physiological saline for oral and intraperitoneal administration, The doses administered were 55, 110 and 165 mg/kg orally and 21.25, 42.50 and 63.75 mg/kg in~ape~tone~ly. The doses administered ranged from % LDsO to % LDsO (Table 1). The tranquilizer was given in 2 equal doses separated by 24 h, and the animals were killed 30 h after the initial exposure. Bonemarrow smears were made according to Schmid {1976), stained in MayGrunwald-Giemsa and mounted in EuparaI. 6 animals were used per treatment; 2000 polychromatic erythrocytes and the corresponding number of normochromatic erythrocytes were scored per animal for the presence of micronuclei. The incidence of the micronuclei in the treated series compared with controls was carried out by using the approximate t test adopted from Goldstein (1965), assuming that the occurrence of micronuclei follows a Poisson dist~bution.
TABLE
1
MUTAGENIC OF MICE
EFFECTS
OF FLUPHENAZINE
HYDROCHLORIDE
ON THE BONE-MARROW
TISSUE
_-. Dose (me/kg)
Gontrol (oral) 55 (oral) 110 (oral) 165 (oral) Control (i.p.) 21.25 (i.p.> 42.50 (i.p.) 63.75 (i.p.)
Number of animals
PCE scored
13 020 11390’ 11620 13. 584 12 13 13 13
940 322 244 469
MN-PCE
MN-NCE
Total MN
(o/oo f
(‘bo 1
fO/oo1
3.5 6.8* 8.6* 14.7*
0.9 1.8
4.5 8.6 11.6 17.9 4.3 5.3 5.9 6.7
3.2 4.7 5.3* 5.3*
3.0 3.3 1.0 6.6 0.6 1.4
PCE/NCE
1.01 0.47 0.36 0.37 0.94 1.05 0.96 1.11
i.p., intraperitoneal; MN-PCE, micronucleated polychromatic erythrocytes (per 1000 PCE scored): MNNCE, micronucleated normochromatic erythrocytes (per 1000 PCE scored): Total-MN. total micronucleated erythrocytes (per 1000 PCE scored). * Significant at P < 0.05.
esullts and discussion
The results show that, in the series treated by oral. doses cf ~uphe~azin~, there was a significant dose-dependent increase in the frequency of micronucleated polychromatic erythrocytes (Table 1). However, there was only a rn~~n~ increase in microi~ucleated poly~hrom~ti~ erythro~yte~ in the bonemarrow ceLs of mice given an intraperitoneal injection of fluphcnazine. Further, fluphenazine produced a marginal increase of micronuclei in normochromatic ery~rocy~s when given orally. But it did not induce any effect in the frequency of micronuclei in normochromatic erythrocytes by intraperitoneal administration (Table 1). The difference in the induction of micronuclei in polychromatic and normochromatic erythrocytes between oral and intraperitone~ treatments may be attributed to the differences in the mode of administration and the amount of drug administered and probably also the amount of drug reaching the target cells. Fu~her, these differences may also be due to me~bo~c activation of the drug by bacteria or their chemical modification by stomach acids. There was a striking depletion of bone-marrow cells in all dose groups when ~uphenazine was administered orally, but no effect was observed by intraperitoneal administration. These dose groups yielded a gradual decrease of the ratio of polychromatic to normochromatic erythrocytes. Probably this effect may also be explained on the basis of the mode of administration and toxic effects induced by the drug in mice. Not much published literature is available regarding the possible cytogenetic effects on the bone-marrow cells with the phenothiazine group of tranquillizers in the micronucleus test. Flupben~ine produced c~omQsome re~~gements in male germ cells of mice (Prabhakar Rae, 1978). The results obtained in the present investigation show that fluphenazine is capable of producing a high incidence of micronuclei in polychromatic erythrocytes in the bone-marrow cells of treated mice. This effect may be due to the clastogenic and/or spindlepoison effect of the chemical.
We acknowledge the encouragement the period of experimentation.
and help of Prof.
02% Reddi
during
References Goldstein, A. (1965) Biostatistics - An introductory Text, Macm~, New York. ‘3~. 117-124. Green, S., K. Palmer and MS. Legator (1970) In vitro cytogenetic investigation of calcium cyckmate, cyclohexylamine and triflupromazine, Food Cosmet. Toxic& 8. 617623. Jarvik, M.E. (1970) Drugs used in the treatment of psychiatric disorders, in: L.S. Goodman pnd A. @Iman (Eds.), The PhannacologicaI Basis of Therapeutics, 4th Edn., MaemiUan. New York. PP. 151203. Jenkins, E. (1970) Phenothiacines and chromosome. damage, Cytologia. 35, 552-560. Maier, P., and W. Schmid (1976) Ten model mutagens evaluated by the micronucleus test, Mutation Res., 40. 32&-338. Nielsen, J., U. F&&&h promasine,
ami T. Tsuboi
perphenazine
(1969)
aru;d lysergide,
Chromosome
abnormalities
Brit. Med. J., 3.634-636.
in patients
treated
with Cb)Or-
240 Petersen, K.W., and V.S. Legator (1973)
Dominant
J mice, Mu’tation Res., 17, 87-92. Prabhakar Rae, K. (1978) Investigations
on the Genetic
lethal
effects Effects
and Mice, PhD. Thesis, Osmania University. Prabhakar Rae, K., and M. Sanjeeva. Rao (1980a) Mutagenic in mice by host-mediated assay, IRCS Med. Sci.. 8. 717-718. Prabhakar Rao, K.. and M. Sanjeeva Rao (1980b) Mutagenic Drosophila mcIanogaster. Toxicol. Lett.. 7. 161-164.
of triflupromazine
in hybrid C!, D, F, /
Induced
by Tranquilizers
in Drosophila
potential
of fluphenazine
hydrochloride
effects
of fluphenazine
hydrochloride
in
Schmid. W. (1976) The micronucleus test for cytogenetic analysis, in: A Hollaender (Ed.), Chemical Mutagens, Principles and Methods for their Detection, Vol. 4, Plenum, New York, pp. 31-53. Venkata Ramanaiah. T.. S. Devaki Nandan, K. Prabhakar Rao and M. Sanjeeva Rao (1980) Mutagenicity of Eskazine (trifhioperazine) with bacterial systems. Indian J. Hered., 12.21-24.
89 (1981)
ElsevierlNorth-Holland
K. PRABHAKAR Department
237-240
Biomedical Press
.O and M. SANJEEVA RAO
of Genetics,
&mania
University, Hyderabad-500
007 (India)
(Received 15 July 1980) (Revision received 12 Qecember 1980) (Accepted 16 December 1980)
Summary The possible mutagenic effects induced by flunhenazine hydrochloride in bone-marrow cells of mice were evaluated by the micronucleus test. Mice were treated orally and intraperitoneally with the drug at dose of 55, 110 and 165 mg/kg and 21.25, 42.50 and 63.75 mg/kg resp. Bone-marrow preparations were made, and the slides were analysed for the presence of ~~ronuclei in develop~g e~thro&y~s. Fluphenaz~e hy~ochlo~de induced a signifi sant dose-dependent increase of micronuclei in polyc~omatic erythrocytes by oral administration, whereas only a marginal increase was observed at 42.50 and 63.75 mg/kg dose levels by intraperitoneal administration. Further, fluphenazine hydrochloride induced bone-marrow depletion at all dose levels with oral administration only.
Tranq~l~zers, a group of drugs introduced as psychotherapeutic agents, are being extensively used in human medicine to allay anxiety and tension. Among tranq~lizers, ~hlorprornaz~i~ and related pheno~~i~~e groups are used in the treatment of psychiatric disorders and in the treatment of nausea and vomiting (Jarvik, 1970). Several million people have been treated with these drugs since their introduction into medicine. It was reported in t,he available literature that high incidences of chromosome gaps and breaks were observed in human leucocyte cultures treated with perphenazine, chlorpromazine and trifluoperazine (Nielsen et al., 1969; Jenkim, 19710). Trifluoperazine induced a high frequency of reversion mutations in S. ~~~~~~~~~~~ G-46 (Wenkata Ramanaiah et al., 1980). Another phenothi~~e derivative, ~~uproma~ine, induced ehromosomal breakage in kidney cells of the rat igloo (Green et al., ~9?0~~ dominant lethal mutations in :mniee(Petersen and Legator, 1973) and abnor0165-1218/81/0000-0000/!$02.~0
0 Elsevier/North-Holland
Biomedical Press
rr,a,ities in the shape of the mouse sperm-head (personal communication). l~luphenazi~e hydrochloride, a phenothiazine compound, is mutagenic in the host-mediated assay with S. ty~~jj~u~iurn G-46 in mice (Prabhakar Rao and Sanjeeva Rao, 1980a) and it causes X-linked recessive lethal mutations in Drosophila melan~gaster (Prabbakar Rao and Sanjeeva Rao, 198Ob), Further, it also produces ~lo~~inant lethal mutations and cytogenetic effects on male-germ cells cf mice (Prabhakar Rao, 1978). The micronucleus test in mouse bone marrow is a sensitive test system in vivo fBr cytogeneti~~y active agents (Maier and Schmid, 1976). This test system, was used to evaluate the cytogenetic effects of fluphenazine in mouse bone-marrow cells, and the results are reported herein. aterial and methods Random-bred Swiss mice, aged 7-8 weeks and weighing 23-27 g, were used. Fluphe~l~ine hy~ochloride ~Ana~nsol) was pur~ha~d in tablet form from M/s. Sarabhai Chemicals, Baroda, India. The tablets were dissolved in sterile distilled water or physiological saline for oral and intraperitoneal administration, The doses administered were 55, 110 and 165 mg/kg orally and 21.25, 42.50 and 63.75 mg/kg in~ape~tone~ly. The doses administered ranged from % LDsO to % LDsO (Table 1). The tranquilizer was given in 2 equal doses separated by 24 h, and the animals were killed 30 h after the initial exposure. Bonemarrow smears were made according to Schmid {1976), stained in MayGrunwald-Giemsa and mounted in EuparaI. 6 animals were used per treatment; 2000 polychromatic erythrocytes and the corresponding number of normochromatic erythrocytes were scored per animal for the presence of micronuclei. The incidence of the micronuclei in the treated series compared with controls was carried out by using the approximate t test adopted from Goldstein (1965), assuming that the occurrence of micronuclei follows a Poisson dist~bution.
TABLE
1
MUTAGENIC OF MICE
EFFECTS
OF FLUPHENAZINE
HYDROCHLORIDE
ON THE BONE-MARROW
TISSUE
_-. Dose (me/kg)
Gontrol (oral) 55 (oral) 110 (oral) 165 (oral) Control (i.p.) 21.25 (i.p.> 42.50 (i.p.) 63.75 (i.p.)
Number of animals
PCE scored
13 020 11390’ 11620 13. 584 12 13 13 13
940 322 244 469
MN-PCE
MN-NCE
Total MN
(o/oo f
(‘bo 1
fO/oo1
3.5 6.8* 8.6* 14.7*
0.9 1.8
4.5 8.6 11.6 17.9 4.3 5.3 5.9 6.7
3.2 4.7 5.3* 5.3*
3.0 3.3 1.0 6.6 0.6 1.4
PCE/NCE
1.01 0.47 0.36 0.37 0.94 1.05 0.96 1.11
i.p., intraperitoneal; MN-PCE, micronucleated polychromatic erythrocytes (per 1000 PCE scored): MNNCE, micronucleated normochromatic erythrocytes (per 1000 PCE scored): Total-MN. total micronucleated erythrocytes (per 1000 PCE scored). * Significant at P < 0.05.
esullts and discussion
The results show that, in the series treated by oral. doses cf ~uphe~azin~, there was a significant dose-dependent increase in the frequency of micronucleated polychromatic erythrocytes (Table 1). However, there was only a rn~~n~ increase in microi~ucleated poly~hrom~ti~ erythro~yte~ in the bonemarrow ceLs of mice given an intraperitoneal injection of fluphcnazine. Further, fluphenazine produced a marginal increase of micronuclei in normochromatic ery~rocy~s when given orally. But it did not induce any effect in the frequency of micronuclei in normochromatic erythrocytes by intraperitoneal administration (Table 1). The difference in the induction of micronuclei in polychromatic and normochromatic erythrocytes between oral and intraperitone~ treatments may be attributed to the differences in the mode of administration and the amount of drug administered and probably also the amount of drug reaching the target cells. Fu~her, these differences may also be due to me~bo~c activation of the drug by bacteria or their chemical modification by stomach acids. There was a striking depletion of bone-marrow cells in all dose groups when ~uphenazine was administered orally, but no effect was observed by intraperitoneal administration. These dose groups yielded a gradual decrease of the ratio of polychromatic to normochromatic erythrocytes. Probably this effect may also be explained on the basis of the mode of administration and toxic effects induced by the drug in mice. Not much published literature is available regarding the possible cytogenetic effects on the bone-marrow cells with the phenothiazine group of tranquillizers in the micronucleus test. Flupben~ine produced c~omQsome re~~gements in male germ cells of mice (Prabhakar Rae, 1978). The results obtained in the present investigation show that fluphenazine is capable of producing a high incidence of micronuclei in polychromatic erythrocytes in the bone-marrow cells of treated mice. This effect may be due to the clastogenic and/or spindlepoison effect of the chemical.
We acknowledge the encouragement the period of experimentation.
and help of Prof.
02% Reddi
during
References Goldstein, A. (1965) Biostatistics - An introductory Text, Macm~, New York. ‘3~. 117-124. Green, S., K. Palmer and MS. Legator (1970) In vitro cytogenetic investigation of calcium cyckmate, cyclohexylamine and triflupromazine, Food Cosmet. Toxic& 8. 617623. Jarvik, M.E. (1970) Drugs used in the treatment of psychiatric disorders, in: L.S. Goodman pnd A. @Iman (Eds.), The PhannacologicaI Basis of Therapeutics, 4th Edn., MaemiUan. New York. PP. 151203. Jenkins, E. (1970) Phenothiacines and chromosome. damage, Cytologia. 35, 552-560. Maier, P., and W. Schmid (1976) Ten model mutagens evaluated by the micronucleus test, Mutation Res., 40. 32&-338. Nielsen, J., U. F&&&h promasine,
ami T. Tsuboi
perphenazine
(1969)
aru;d lysergide,
Chromosome
abnormalities
Brit. Med. J., 3.634-636.
in patients
treated
with Cb)Or-
240 Petersen, K.W., and V.S. Legator (1973)
Dominant
J mice, Mu’tation Res., 17, 87-92. Prabhakar Rae, K. (1978) Investigations
on the Genetic
lethal
effects Effects
and Mice, PhD. Thesis, Osmania University. Prabhakar Rae, K., and M. Sanjeeva. Rao (1980a) Mutagenic in mice by host-mediated assay, IRCS Med. Sci.. 8. 717-718. Prabhakar Rao, K.. and M. Sanjeeva Rao (1980b) Mutagenic Drosophila mcIanogaster. Toxicol. Lett.. 7. 161-164.
of triflupromazine
in hybrid C!, D, F, /
Induced
by Tranquilizers
in Drosophila
potential
of fluphenazine
hydrochloride
effects
of fluphenazine
hydrochloride
in
Schmid. W. (1976) The micronucleus test for cytogenetic analysis, in: A Hollaender (Ed.), Chemical Mutagens, Principles and Methods for their Detection, Vol. 4, Plenum, New York, pp. 31-53. Venkata Ramanaiah. T.. S. Devaki Nandan, K. Prabhakar Rao and M. Sanjeeva Rao (1980) Mutagenicity of Eskazine (trifhioperazine) with bacterial systems. Indian J. Hered., 12.21-24.