Journal of Life Sciences Volume 5 Number (6)
J LS
Journal of Life Sciences
Volume 5, Number 1, January 2011 (Serial Number 33)
Contents
Research Papers
1 The 2+ Human P
5B -ATPase ATP13A2 Is Not a Ca Transporting Pump
Felicitas de Tezanos Pinto, Gerardo Raul Corradi and Hugo Pedro Adamo
7 The Role of Pumpkin Extraction on the Liver and Kidney of Mice Previously Treated with
Haloperidol during Lactation
Samira Omar Balubaid
15 The Sorption Isosteric Heats of Rough Rice in China
Xingjun Li, Zidan Wu and Hui Lu
22 Alterations of Antioxidative Enzymes Activities and Induction of Lipid Peroxidation in Germinating Wheat Seeds Subjected to Cadmium Stress
Surjendu Kumar Dey
29 Effect of Se-S Cooperated Application on the Mineral Content and Nutrition Quality of Garlic (Allium Sativum L.)
Huanxiu Li, Changquan Wang, Bing Li, Zesheng Yan and Yangxia Zheng
35 Effect of Deficit Irrigation at Different Growth Stages on Wheat Growth and Yield
Seyed Abdolreza Kazemeini and Mohsen Edalat
39 Biological Status of Indus River Dolphin (Platanista Minor Owen) in Indus River, Northern Pakistan
Farzana Perveen, Sardar Azher Mehmood, Shabbir Ahmed and Zia Ur Reman
48 Biochemical Composition and Nutritional Value of the Muscle Tissue of Yellowback Seabream, Evynnis Tumifrons, from the East China Sea
Lianjun Xia, Qiqun Cheng, Jianxue Lu, Junli Hou, Jian Xin and Min Liu
53 Legionella from Environmental and Clinical Homes for the Mentally Disabled and Comparison of Their Sequence Types
Annalisa Bianchi, Marina Tesauro, Michela Consonni, Fabrizio Pregliasco and Maria Gabriella Galli
Methods and Techniques
59 Detection of the Relationship between Imipenem Susceptible and Non-Susceptible Clinical Isolates of Acinetobacter Baumannii by Repetitive Element PCR-Mediated DNA Fingerprinting in an Egyptian Hospital
Soheir Helal, Mona M.A. Haleim and Maha Gaafar
66 Assessment of Soil Quality Using Microarthropod Communities Under Different Land System: A Case Study in the Mid-Hills of Central Nepal
Farida Begum, Roshan Man Bajracharya, Subodh Sharma and Bishal K. Sitaula
74 Synthesis and Surface-Active Properties of Carboxymethylcellulose Esters Obtained by Microwave Assisted Transesterification of Vinyl Laurate
Vladimíra Tomanová, Iva Sroková and Vlasta Sasinková
Journal of Life Sciences 5 (2011) 1-6
The Human P 2+
5B -ATPase ATP13A2 Is Not a Ca
Transporting Pump
Felicitas de Tezanos Pinto, Gerardo Raul Corradi and Hugo Pedro Adamo IQUIFIB-Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Junín 956, 1113 Buenos Aires, Argentina
Received: May 30, 2010 / Accepted: July 22, 2010 / Published: January 30, 2011.
Abstract: The human gene ATP13A2 has been proposed to code for an ATP powered ion transporter of the P 5B subfamily. Mutations of the human gene ATP13A2 were found to underlie an autosomal recessive form of early-onset parkinsonism (PD) with pyramidal degeneration and dementia. The ion transported by the ATP13A2 pump is not known, but several studies have shown that the
P 5 -ATPases influence the homeostasis of intracellular Ca 2+ , and thus it has been suggested that they transport Ca 2+ . In order to evaluate this possibility Chinese hamster ovary (CHO) cells stably expressing the human ATP13A2 protein have been obtained and the Ca 2+ transport activity of ATP13A2 was assessed by measuring the ATP-dependent uptake of Ca 2+ into microsomal vesicles. As a positive control vesicles containing the human plasma membrane Ca 2+ pump (PMCA) were used. No significant differences were found between vesicles containing the ATP13A2 protein and the control. Moreover, Ca 2+ was unable to induce the formation of the P-ATPase acylphosphate intermediate in vesicles containing the expressed ATP13A2. These results favor the idea that the ATP13A2 does not transport Ca 2+ .
Key words: P 5B -ATP13A2, calcium uptake, CHO cells overexpression.
Abbreviations: PMCA: plasma membrane Ca 2+ pump; CHO cells: Chinese hamster ovary cells.
1. Introduction phosphorylation site (DKTGTLT) found in all P-type ATPases, and the main difference between them
The P-type superfamily of ion pumps includes resides in the predicted sixth transmembrane segment transporters energized by the hydrolysis of ATP that (M6) which is presumably involved in forming the ion transport inorganic cations and other substrates across binding site. The predominant M6 sequence motif of cell membranes. These P-type ATPases are the P 5A pumps is PP(D/E)LPxE, while the members of characterized by the formation of a phosphorylated the P 5B subfamily have a conserved PP(A/V)LPAx intermediate during their reaction cycle. They are sequence motif. Five genes named present in prokaryotes and eukaryotes, and in the basis ATP13A1-ATP13A5 that belong to this group of of their conserved core sequences they have been P 5 -ATPases have been identified in humans. Only
classified into five subfamilies termed P 1 -P 5 or type I-V.
ATP13A1 belongs to the subgroup A and the others to The most poorly understood P-type ATPases are those the subgroup B [2]. The study of these P 5 -ATPases has of the P 5 subfamily, which are expressed only in
a great interest since they were linked with several eukaryotes. The P 5 -ATPases have been divided in two neurologic disorders. Loss of function mutations of the
subfamilies termed P 5A and P 5B based on protein
human gene ATP13A2 was found to underlie an alignments [1]. Both subfamilies harbor the autosomal recessive form of early-onset parkinsonism
with pyramidal degeneration and dementia Corresponding autor: Felicitas de Tezanos Pinto, Ph.D.,
(Kufor-Rakeb syndrome) [3]. Furthermore the research fields: biochemistry, cellular and molecular biology.
E-mail: ftpinto@qb.ffyb.uba.ar. interruption by inversion of the long arm of
2 The Human P -ATPase ATP13A2 Is Not a Ca 2+ 5B Transporting Pump
chromosome 3 in the human gene ATP13A4 was found nitrocellulose filters, Millipore; immunochemicals, in patients with autism spectrum disorder (ASD) and
Invitrogen, Molecular Probes, Vector Laboratories specific language impairment (SLI) [4].
and Amersham Biosciences; and reagents for cell
culture, thapsigargin, and other chemicals, Sigma. The and recent publications suggest that they affect the
The ion specificity of the P 5 -ATPases is unknown,
expression vector pcDNA3.1 carrying the V5-tagged intracellular level of different cations [5-8]. The yeast
human ATP13A2 cDNA was a generous gift of Drs. P 5A -ATPase Cod1p, in collaboration with the Golgi
Alfredo Ramirez and Christian Kubisch, Institute of Ca 2+ pump Pmr1p, was suggested to supply calcium to
Human Genetics, University of Bonn, Germany. the yeast ER. However, the function of Cod1p in
2.2 Protein Expression and Isolation of Cellular cellular Ca 2+ homeostasis is not equivalent to, nor
Membranes
redundant with that of Pmr1p. Moreover, it was shown Stable CHO cell lines expressing the recombinant
that the phenotype of mutant yeast cells lacking Cod1p PMCA were described previously [12]. CHO cells (cod1 ∆) could be partially suppressed by exogenous were lipofected with the expression vector pcDNA3.1 calcium [9]. Deletion of Cod1p alone did not affect carrying the V5-tagged human ATP13A2 cDNA using cellular calcium level, but deletion of both Cod1p and lipoafectamine 2000 transfection reagent (Invitrogen) Pmr1p produced a synergistic increase in the according to the manufacturer’s protocol. To express intracellular calcium level compared with pmr1 ∆ alone in a stable form the recombinant ATP13A2, the [5]. In addition Cod1p has been shown to be involved
transfected CHO cells were split into dishes of 10 cm in mechanisms that depend on the ER Ca in diameter 24 h post-transfection. 24 hours later the concentration like glycosylation of proteins in the
transfected cells were cultured in a selective DMEM secretory pathway, protein insertion orientation, and
medium supplemented with antibiotics and 10% of regulation of HMG-CoA reductase degradation [5,
9-11]. The idea of Ca 2+
dialyzed fetal calf serum containing the antibiotic
pumping P 5 -ATPases is
G418 at a final concentration of 600 μg/ml. After 3 favored by a recently publication which showed that weeks about 6-8 of the resulting colonies were cloned the over-expression of human ATP13A4 in COS-7
and expanded, and the expression of the pump was cells increased the intracellular calcium level [8].
investigated by immunoblotting. The crude Here, the ability of the human ATP13A2 to transport
microsomal membrane fractions and the erythrocyte Ca has been examined under conditions which are
inside-out membranes were prepared by the procedure optimal for the function of other well known Ca of Enyedi et al. [13] and Sarkadi et al. [14], pumps. For this purpose CHO cells were stably
respectively. Protein concentration was estimated by transfected with the ATP13A2 cDNA, and clones
means of the Bio-Rad protein assay, with bovine expressing the ATP13A2 protein were isolated. It was serum albumin as a standard. The amount of the found that the expressed ATP13A2 not only lacks any
expressed protein was estimated by quantitation of the Ca transport activity but also unable to promote the band intensity using the Gel Pro Analyzer 3.0 program formation of the catalytic phosphoenzyme from ATP. (version 3.1 for Windows TM , Media Cybernetics).
2. Material and Methods
2.3 Detection of the Human ATP13A2 Protein
2.1 Materials For immunofluorescence, the stable transfected
Reagents were purchased from the following cells were cultured on glass multiwell plates for 24-48
h. The cells were then washed twice with PBS and Sciences; Immobilon transfer membranes and fixed in 4% formaldehyde in PBS for 15 min on ice.
companies: 45 Ca and [ γ- 32 P]ATP, PerkinElmer Life
The Human P -ATPase ATP13A2 Is Not a Ca 2+ 5B Transporting Pump
After three washes with PBS the cells were
2.5 Detection of the Phosphorylated Intermediate permeabilized with 0.15% Tween 20 in PBS (PBST)
The phosphorylation reaction was carried out at for 5 min on ice. The recombinant ATP13A2 was
4 ℃ in a medium containing 30 g of microsomal detected with antibody to V5 (Invitrogen) at a dilution
protein, 160 mM KCl, 25 mM Tris-HCl (pH 7.0 at of 1:500 in PBST by incubation over night at 4 ℃.
4 ℃) and 4 μM thapsigargin to obtain a full inhibition After washing the cells three times with PBST, the
of SERCA pump in a reaction volume of 0.25 ml; anti-V5 was labeled by Zenon Alexa Fluor 568 mouse
0.15 mM CaCl 2 was added when indicated. The IgG2a (Molecular Probes) according to the reaction was initiated by the addition of 1 μM
manufacturer’s instructions for two hours at room [ γ- 32 P]ATP and terminated after 1 min with 15 μl of a temperature. Then the cells were washed three times
solution containing 100% trichloroacetic acid. The with PBST and after a single wash with PBS the cells
precipitated proteins were dissolved in sample buffer were covered by 50 μl of PBS and observed in a
and separated by SDS-PAGE in a 7% acrylamide gel confocal microscope FluoView 1000 (Olympus, Japan)
according to Sarkadi et al. [14]. After drying the gel, with an appropriate Alexa Fluor 568 filter.
they were exposed to a storage phosphor screen for 1 SDS-PAGE and immunoblotting were carried out
day and imaged using a Storm 840 Optical Scanner. as described previously [15]. Proteins were
electrophoresed on a 7.5% acrylamide gel according
3. Results
to Laemmli [16] and subsequently transferred to
3.1 Expression of the Recombinant ATP13A2 Protein Millipore Immobilon membranes. The membranes
were incubated over night at 4 ℃ with V5 monoclonal CHO cells were transfected with the pcDNA3.1 antibody (Invitrogen) according to the manufacturer’s
expression vector carrying the human ATP13A2 protocol. For staining, biotinylated anti-mouse cDNA and stable clones were selected by their
immunoglobulin G and avidin-streptoavidin resistance to the antibiotic G418. Immunofluorescence peroxidase conjugate were used.
experiments showed that the isolated clones
successfully expressed the ATP13A2 protein (Fig. 1a). Transport Assay
2.4 Ca 2+
Microsomal membranes from transfected cells were Ca 2+ uptake assays were performed as described
isolated and submitted to SDS-PAGE and previously [12]. The reaction mixture contained 100
immunobloting. The expressed ATP13A2 had the mM KCl, 50 mM Tris-HCl (pH 7.3 at 37 ℃), 5 mM
expected migration according to its predicted size of NaN 3 , 0.1 μM thapsigargin, 4 μg/ml oligomycin, 20
129 kDa, and judged by the intensity of the bands, it mM sodium phosphate, 1.5 mM ATP, 95 μM EGTA,
accounted for about 1% of the microsomal protein
(Fig. 1b). Thus, the expression level of ATP13A2 was the desired concentration of free Ca 2+ . The free
2.5 mM MgCl 45
2 and CaCl 2 (labeled with Ca) to give
similar to that reached by other P-type ATPases in the concentrations of Ca 2+ were calculated using the
same expression system [18].
program of Fabiato and Fabiato [17]. Vesicles (10 μg
3.2 Ca 2+ Transport Assays
of protein) were preincubated at 37 ℃ for 5 min, and In order to assess the Ca the reaction was initiated by the addition of ATP. The 2+ transport activity of the
45 reaction was finished after 5 min by filtering the 2+ expressed ATP13A2 the ATP dependent Ca
transport into microsomal vesicles was measured as a by the vesicles was determined by counting in a 2+ function of increasing Ca concentrations. As a positive
samples through a 0.45 μm filter. The 45 Ca taken up
scintillation counter. 2+ control, the Ca transport activity of the plasma membrane Ca 2+ ATPase (PMCA) was simultaneously
4 The Human P 5B -ATPase ATP13A2 Is Not a Ca 2+ Transporting Pump
Fig. 1 Expression of the recombinant ATP13A2 pump.
(a) Fluorescence microscopy to visualize ATP13A2 expression in transfected CHO cells. The left panels show CHO cells transfected with the empty vector pcDNA3.1 (upper panels) or with the expression vector pcDNA3.1 carrying the V5-tagged human ATP13A2 cDNA (bottom panels) incubated with an antibody to V5 revealed with Zenon Alexa Fluor 568 mouse IgG2a secondary antibody. The transmittance image of each one is shown in the right panels. (b) Immunoblot of microsomes from CHO cells transfected with cDNA encoding the V5-tagged human ATP13A2. For protein estimation, a V5-tagged Ag + /Cu + ATPase (V5-CopA) of 86 kDa purified from the extremophile organism Archeaglobus fulgidus was used. The amount of protein loaded is indicated at the top of each lane. The proteins were separated by SDS-PAGE and transferred to polyvinylidene difluoride membranes; finally they were detected with the V5 antibody as described under “Material and Methods”.
120 CHO
ATP13A2
h4PMCAxb
Fig. 2 Ca 2+ dependence of the Ca 2+ transport of recombinant ATP13A2 as compared with that of PMCA.
Ca 2+ uptake by microsomal vesicles was measured at different free Ca 2+ concentrations for 5 min at 37 ℃ as described under “Material and Methods”. The kinetic behavior of the recombinant PMCA (h4PMCAxb, filled triangles) is similar to the PMCA of erythrocytes IOVs (ePMCA, empty triangles). The activity of the recombinant ATP13A2 (empty circles) not differ from that obtained by the endogenous PMCA from CHO cells transfected with the empty vector (filled circles). Data points are the average of three to five experiments. The lines are the best fit to the data given by the Hill equation.
The Human P -ATPase ATP13A2 Is Not a Ca 2+ 5B Transporting Pump
measured using either inside out vesicles (IOVs) from contrast the phosphorylation pattern of membrane erythrocytes, or microsomal vesicles from CHO cells
vesicles containing the recombinant ATP13A2 was stable expressing the human PMCA isoform 4xb
similar to that of control membranes, indicating that (h4PMCAxb). As shown in Fig. 2, the activity of the
no phosphorylation attributable to the ATP13A2 PMCA enzyme gradually increased with the protein had occurred. Thus, the expressed ATP13A2
concentration of Ca 2+ , reaching half maximal activity was not activated by Ca to form the phosphorylated
at about 1 2+ μM of free Ca . By contrast, the Ca intermediate.
uptake activity of microsomes expressing the
4. Discussion
recombinant ATP13A2 was not significantly different from that of microsomal vesicles from CHO control
The P 5 -ATPases have been associated with the cells transfected with the empty vector indicating that 2+ homeostasis of intracellular Ca and this fact has led to
in these conditions no uptake of Ca 2+ was associated the idea that they are Ca transporters. Here, it was with the expressed ATP13A2.
investigated calcium as a possible substrate of the human ATP13A2 enzyme by comparing the Ca 2+ transport
3.3 Phosphorylation Reaction activity and Ca 2+ -dependent phosphoenzyme formation
The first step of the catalytic cycle of all well 2+ of ATP13A2 with that of the related Ca specific
2 -ATPase PMCA. Neither of the two Ca -dependent dependent transfer of the γ−phosphate from ATP to
characterized P-type Ca 2+ pumps involves the Ca P
activities were detected in the recombinant ATP13A2. the enzyme to form a phosphoenzyme intermediate.
These results suggest that although P 5 -ATPases influence As shown in Fig. 3 a strong band was observed when
many Ca 2+ related functions, Ca 2+ is not the substrate microsomes carrying the recombinant PMCA were
transported by the human P 5 -ATP13A2. Mutations in the phosphorylated with ATP in the presence of Ca 2+ .
ATP13A2 gene underlay an autosomal recessive form of Likewise, a band corresponding to the PMCA
early onset parkinsonism [3]. Expression of ATP13A2 in phosphoenzyme was observed in erythrocyte IOVs. In
animal models of PD is sufficient to rescue neurodegeneration associated with α-synuclein (α-syn)
aggregation [19], which is relevant to this study since it was used the same construct for the expression of ATP13A2 and thus it implies that no additional factors are needed in order to observe the biological response.
Because it has been hypothesized that P 5A and P 5B pumps may have different ion specificities [20], the lack
of Ca 2+ dependent activities of P
5B -ATP13A2 should not
be generalized to the whole P5 subfamily. Moreover, Phosphoenzyme formation was carried out as described under
Fig. 3 Formation of the phosphorylated intermediate.
recently it was reported that yeasts lacking Ypk9p, the “Material and Methods”. Lane 1, recombinant ATP13A2 with
2+ but Ca 2+ ; lane 2, recombinant ATP13A2 without Ca 2+
yeast ATP13A2 homolog, were unaffected by Ca
PMCA from erythrocyte IOVs with Ca 2+ (ePMCA); lane 4, were more sensitive to high concentrations of cadmium, recombinant PMCA with Ca 2+ (h4PMCAxb); lane 5, control
; lane 3,
manganese, nickel and selenium [7]. Yet another study empty pcDNA3.1 vector with Ca 2+ ; lane 6, pcDNA3.1
has shown that the manganese toxicity can be alleviated membranes as in lane 5, but the phosphorylation was carried
by the expression of the Ypk9p protein. On the basis of in the absence of thapsigargin allowing the visualization of the
SERCA phosphoenzyme; lane 7, control empty pcDNA3.1 these results it has been proposed that the yeast Ypk9p
vector without Ca 2+ . functions as a manganese transporter to protect cells from
6 The Human P -ATPase ATP13A2 Is Not a Ca 2+ 5B Transporting Pump
excess Mn 2+ exposure [19]. On the other hand, it was YPK9p the orthologue of human ATP13A2, Biochem. Biophys. Res. Commun. 383 (2) (2009) 198-202.
[8] J. Vallipuram, J. Grenville, D.A. Crawford, The CATP-5 of Caenorhabditis elegans is responsible for the
recently published that the deletion of the P 5B -ATPase
E646D-ATP13A4 mutation associated with autism reveals a defect in calcium regulation, Cell. Mol. Neurobiol. 30 (2)
tolerant phenotype seen in the presence of the toxic
(2010) 233-246.
spermidine analog norspermidine, raising the possibility [9] S.R. Cronin, A. Khoury, D.K. Ferry, R.Y. Hampton, that the polyamines are the substrates transported by Regulation of HMG-CoA reductase degradation requires the P-type ATPase Cod1p/Spf1p, J. Cell Biol. 148 (5) (2000) P 5B -ATPases [21].
915-924.
C. Suzuki, Y.I. Shimma, P-type ATPase spf1 mutants show a
Acknowledgments
novel resistance mechanism for the killer toxin SMKT, Mol. Microbiol. 32 (4) (1999) 813-823.
The authors thank Drs. Alfredo Ramirez and Christian [11] D.J. Tipper, C.A. Harley, Yeast genes controlling responses to topogenic signals in a model transmembrane protein, Mol.
Kubisch for supplying vector pcDNA3.1 carrying the
Biol. Cell. 13 (2002) 1158-1174.
V5-tagged human ATP13A2 cDNA and Luis M. [12] H.P. Adamo, M.E. Grimaldi, M.I. García Arguinzonis, Bredeston for the generous gift of the purified V5-tagged Deletions in the N-terminal segment of the plasma membrane Ca 2+ pump impair the expression of a correctly folded
Ag + /Cu ATPase. This work was supported in part by the functional enzyme, Biochemistry 39 (2000) 14893-14899. University of Buenos Aires (UBA, Grant B604), by the
A. Enyedi, A.K. Verma, A.G. Filoteo, J.T. Penniston, A highly active 120 kDa truncated mutant of the plasma Consejo Nacional de Investigaciones Científicas y
membrane Ca 2+ pump, J. Biol. Chem. 268 (1993) Técnicas (CONICET, Grant PIP 112-200801-02022),
10621-10626.
B. Sarkadi, A. Enyedi, Z. Foldes-Papp, G. Gardos, and by Agencia Nacional de Promoción Científica y
Molecular characterization of the in situ red cell membrane Tecnológica (ANPCyT, Grant BID PICT 2007-00702).
calcium pump by limited proteolysis, J. Biol. Chem. 261 (1986) 9552-9557.
References
F. de Tezanos Pinto, H.P. Adamo, Deletions in the acidic lipid-binding region of the plasma membrane Ca 2+ pump, A
[1] K.B. Axelsen, M.G. Palmgren, Evolution of substrate mutant with high affinity for Ca 2+ resembling the acidic specificities in the P-type ATPase superfamily, J. Mol. Evol.
lipid-activated enzyme, J. Biol. Chem. 277 (2002) 46 (1) (1998) 84-101.
12784-12789.
[2] P.J. Schultheis, T.T. Hagen, K.K. O’Toole, A. Tachibana, [16] U.K. Laemmli, Cleavage of structural proteins during the C.R. Burke, D.L. McGill, et al., Characterization of the P5
assembly of the head of bacteriophage T4, Nature 227 (1970) subfamily of P-type transport ATPases in mice, Biochem.
680-685.
A. Fabiato, F. Fabiato, Calculator programs for computing [3]
Biophys. Res. Commun. 323 (3) (2004) 731-738.
A. Ramirez, A. Heimbach, J. Grundemann, B. Stiller, D. the composition of the solutions containing multiple metals Hampshire, L.P. Cid, et al., Hereditary parkinsonism with
and ligands used for experiments in skinned muscle cells, J. dementia is caused by mutations in ATP13A2, encoding a
Physiol. 75 (1979) 463-505.
lysosomal type 5 P-type ATPase, Nat. Genet. 38 (10) (2006)
C.I. Cura, G.R. Corradi, D.E. Rinaldi, H.P. Adamo, High 1184-1191.
sensibility to reactivation by acidic lipids of the recombinant [4] 2+ D.A. Kwasnicka-Crawford, A.R. Carson, W. Roberts, A.M. human plasma membrane Ca -ATPase isoform 4xb purified
Summers, K. Rehnstrom, I. Jarvela, et al., Characterization of from Saccharomyces cerevisiae, Biochimica et Biophysica a novel cation transporter ATPase gene (ATP13A4)
Acta (BBA) - Biomembranes 1778 (12) (2008) 2757-2764. interrupted by 3q25-q29 inversion in an individual with
A.D. Gitler, A. Chesi, M.L. Geddie, K.E. Strathearn, S. language delay, Genomics 86 (2) (2005) 182-194.
Hamamichi, K.J. Hill, et al., Alpha-synuclein is part of a [5] S.R. Cronin, R. Rao, R.Y. Hampton, Cod1p/Spf1p is a P-type
diverse and highly conserved interaction network that ATPase involved in ER function and Ca 2+ homeostasis, J.
includes PARK9 and manganese toxicity, Nat. Genet. 41 (3) Cell Biol. 157 (2002) 1017-1028.
(2009) 308-315.
[6] M.K. Jakobsen, L.R. Poulsen, A. Schulz, P. Fleurat-Lessard,
A.B. Møller, T. Asp, P. Bach Holm, M.G. Palmgren, A. Møller, S. Husted, et al., Pollen development and
Phylogenetic analysis of P 5 P-type ATPases, a eukaryotic fertilization in Arabidopsis is dependent on the MALE
lineage of secretory pathway pumps, Mol. Phylogenet. Evol . GAMETOGENESIS IMPAIRED ANTHERS gene
46 (2007) 619-634.
encoding a Type V P-type ATPase, Genes & Dev. 19 (2005)
A. Heinick, K. Urban, S. Roth, D. Spies, F. Nunes, O. 2757-2769.
Phanstiel IV, et al., Caenorhabditis elegans P 5B -type ATPase [7] K. Schmidt, D.M. Wolfe, B. Stiller, D.A. Pearce, Cd 2+ , Mn 2+ ,
CATP-5 operates in polyamine transport and is crucial for Ni 2+ and Se 2+ toxicity to Saccharomyces cerevisiae lacking
norspermidine-mediated suppression of RNA interference, The FASEB Journal 24 (2010) 206-217.
Journal of Life Sciences 5 (2011) 7-14
The Role of Pumpkin Extraction on the Liver and Kidney of Mice Previously Treated with Haloperidol during
Lactation
Samira Omar Balubaid Department of Biology, King Abdul Aziz University, Jeddah, Saudi Arabia
Received: June 20, 2010 / Accepted: November 03, 2010 / Published: January 30, 2011.
Abstract: The present study was initiated to investigate the effect of haloperidol on the histological structures of the liver and kidney of mice and their infants. Pumpkin was used to inhibit the toxic effects of this drug. Mothers, directly after delivery from the first day to the weaning age at the 21 day were divided into four groups: the first group (control), the second group supplied with (1 mg haloperidol /kg /B.W./day), the third group was supplied with (1 ml pumpkin extraction /kg /B.W./day), the fourth group was supplied with (1 ml pumpkin /Kg/B.W.). The histological examination of the haloperidol treated mothers revealed its bad condition in compare with the other groups where there was absence of natural architecture of the liver and complete disintegration of the renal tubules. Also, the same symptoms were recorded in the liver and kidney of the infants. An obvious improvement in the structures of liver and kidney in mothers and infants as well as the recovery of most tissues to their normal appearance after the treatment with the mixture of haloperidol and pumpkin in compare with the haloperidol-group and pumpkin-group also showed a normal structure as the control-group.
Key words: Liver, kidney, mice, pumpkin, lactation.
1. Introduction resulted in an increase in alkaline phosphatase. R. Baldessarini et al. observed that haloperidol had a
Depression is a psychological state which is serious effect on the quantity of milk from mothers differentiated from the other known disturbances by through its effect on liver and bile secretion [4]. They consistent disordered mode with disturbed sleep, loss also found that the treatment by using this drug for a of appetite, losing of self confidence, reduced activity long period lead to a reduction in the sexual desire in and weak memory [1]. Depression is more common in both sexes. While in case of pregnancy and lactation, women than men. The different types of depression for there were no records for distorted embryos or infants. ladies were mentioned during ovulation, pregnancy, The absorption, distribution and secretion of delivery bleeding and at the post-ovulation time. haloperidol-14c in mice after muscular injection or Haloperidol is the most used drug as anti-depression forced feeding, showed that after one hour, the highest during pregnancy and lactation, and there is a close level of this drug was recorded in the blood, plasma and relationship between depression and changes in in tissues of lung, pancreas, kidney, liver and spleen, hormones of females [2]. Treatment of pregnant also similar elevation in the placenta of mice embryos, women with antidepressed drug during the last months while the drug levels in the milk were variable and of pregnancy, may lead to distorted embryos [3] and similar to those levels in the plasma after muscular
injection [5].
Corresponding author: S. Balubaid, Ph.D., associate Prof., Some nervous disorders and malformations in the research field: embryology. E-mail: dr.s_balubaid@hotmail.com.
8 The Role of Pumpkin Extraction on the Liver and Kidney of Mice Previously Treated with Haloperidol
during Lactation
spinal cord have been detected in pregnant mice and a tissue and the oxidative potential initiated by garlic significant increase in the prolactine secretion after
[19]. The pumpkin seed oil can prevent changes in injection with haloperidol [6].
plasma lipids and blood pressure associated with In a histological study carried by Ref. [7] the testes
inadequate estrogen availability [20]. The pumpkin of mice showed necrosis, decreasing in spermatozoa
extracts p5 and p6 had high content of total phenolics number and vacuolation of most germ cells. Also
and antioxidant activity coupled to moderate to high clozapine drug caused a disorder on the histological
alpha-glucosidase and angiotensin converting enzyme structure of liver, kidney and retina of eyes of the chick
inhibitory activities and has the potential to reduce embryos [8], an inhibition of CYP2B activity in liver [9]
hyperglycemia-induced pathogenesis and also and the drug effect the glomerulus and infiltration
associated complication linked to cellular oxidation process in kidney.
stress [14].
Pumpkin (Cucurbita pepo) is an important medical According to M. Makni et al. [21] who found that plant, which contains a large amount of water (94% of
pumpkin seed had anti-atherogenic and its weight) and carotene which converts to vitamin A in
hepatoprotective effect because pumpkin seed are rich the body [10]. According to the chemical and physical
source of an unsaturated fatty acids, antioxidant and properties of this plant, it is recommended as a good
fibers.
caloric diet [11]. F. Bion et al. recorded that pumpkin It is therefore of interest to examine the effect of usage did not change the mophometric measurements
haloperidol on histological structure of liver and of different organs as liver, kidney, gonads, spleen and
kidney in mothers’ mice during lactation and their brain of mice [12]. The oral daily dose of pumpkin seed
infants, also to investigate the role of pumpkin as a extraction to mice did not affect blood glucose level,
protective agent against the haloperidol urea, creatinin, total protein, uric acid and blood cells
histopathological alterations.
number [13]. On the other hand [14], revealed that the
2. Materials and Methods
usage of this plant may inhibit diabetes and it also associated with the reduction of the cellular oxidation
Female Swiss albino mice were obtained from potential and blood pressure. This plant contains a
experimental animal unit at King Fahd Medical higher level of D-chiroinositol which decreases
Research Center. The female animals during lactation glucose level in the blood, increases the hepatic
were housed in individual cages with their infants at a glycogen, increases insulin level and total hemoglobin
temperature 20-22 ℃ and were fed a standard Purina of diabetes patients [15]. Therefore, this plant is
chow and water.
considered as a good treatment for diabetes, as it Ten female mice were used in each group. The composed of polysaccharide and protein [16]. The
average weight per experimental animal was 200 g. extracted protein from this plant has an essential role in
The female mice during lactation were divided into 4 decreasing the harmful effects of protein malnutrition
groups.
[17]. Such protein leads to an elevation in the level of (1) The first group was untreated and used as a hepatic enzymes and a decreasing in the toxic effect of
control.
the carbon tetra-oxide because it contains (2) The second group was supplied with 1 mg anti-oxidative compound [18]. The extraction from the
haloperidol /kg B.W./daily. It was obtained from Swiss green leaves of pumpkin were used in treatment of
life (medical manufacturing) during the whole period many diseases and due to its higher antioxidative
of lactation from the 1st day to the weaning age at the activity it can remove the cellular toxicity of hepatic
21st day.
The Role of Pumpkin Extraction on the Liver and Kidney of Mice Previously Treated with Haloperidol
during Lactation
(3) Animals of group three were given daily the same
A–Mothers:
dose of haloperidol and fed with 1 ml pumpkin
(1) Liver
extraction /kg B.W. at the same period [15]. In some regions, it can be seen a dilation and (4) The fourth group was given the pumpkin
congestion, which is due to the disintegration of the extraction daily at the same dose level for the same
hepatic cells. This disintegration is represented by period.
appearance of fatty vacuoles inside cells (lipid At the end of lactation (21 days) liver and kidney
infiltration) and disorder in the liver architecture that specimens from each of the four groups were fixed in
could be recognized by the disappearance of the neutral formalin. Also, infants were dissected at
characteristic strands of hepatocyte (Fig. 2). In other 7-14-21 days after birth and specimens from liver and
regions, disintegration in the wall of the portal and kidney were fixed in neutral formalin, embedded in
central veins, disintegration of nuclei and cellular paraffin. The sections were stained with Hematoxylin
membranes of the hepatocyte and fibrosis of the portal and Eosin [22].
regions could be noticed compared with other three groups especially the fourth group (pumpkin).
3. Results
(2) Kidney
3.1 Changes in Weight Histological investigation of kidney from mothers treated with the drug 1 mg/kg B.W. for 21 days
Data in Table 1 showed a highly significant decrease indicated a complete dissociation of the proximal (P < 0.05) in the infant body weights in the group tubules. This disassociation is represented by treated with Haloperidol drug only as compared with disintegration of the lining cells and their nuclei. Some control group. Infant body weights of Haloperidol + tubules appeared completely vacuolated (degenerated) pumpkin group showed significant increase than and most of cells disappeared. Sections also show Haloperidol-group. The fourth group which treated separation and swelling of the lining cells of the distal with Pumpkin showed a significant increase in body tubules. Moreover, separation of the tubular cells from weight as compared with control group during the three their basement membrane with a partial or complete weeks of the experiment. plugging of the tubular cavities were also recorded. A
3.2 Histological Changes reduction in the intertubular connective tissues with a large vacuoles, partitioning of glomerulus, reduction of
3.2.1 Group Treated with Pumpkin Only epithelial layer, disorder in the granular layer, dilation Tested sections which treated with pumpkin only in of urinary cavity (Fig. 3) and malformation of liver and kidney for both mothers and infants showed collecting tubules in the medulla were recorded, healthy appearance (Fig. 1) as compared with control compared with other three groups especially the fourth group.
group (pumpkin).
3.2.2 Group Treated with the Drug (Haloperidol)
Table 1 Indicated the effect of haloperidol, haloperidol + pumpkin and pumpkin on the infant weights during the experiment in compare with control group.
Weeks
Third (Mean ± S.E * ) Control Haloperidol
Groups
First (Mean ± S.E * )
Second (Mean ± S.E * )
30.01 ± 0.5744 * S.E = Standard Error of Mean.
10 The Role of Pumpkin Extraction on the Liver and Kidney of Mice Previously Treated with Haloperidol during Lactation
B–Infants (1) Liver Sections of liver at one week age, showed serious
disintegration of the hepatic cells represented by the separation and disruption of these cells in the tissue, rapture of cellular wall, karyolitic nuclei with appearance of lipid filtration and tissue fibrosis (Fig. 4). While at two weeks age, a decrease in the lipid filtration and cellular disintegration were observed. On the other hand, after three weeks, sections of liver
1 appeared pale stained with oedema and a disorder in
Fig. 1 Light micrograph of kidney section from infants of
the structural architecture was recorded. This disorder
21 days after delivery from pumpkin treated mothers
was due to the separation of hepatic cells which lead to
showing normal kidney structure ( H & E 40 x).
enlargement of the hepatic acini and presence of fatty vacuoles in hepatic cells (Fig. 5), malformation of portal
Fig. 2 Light micrograph of liver from haloperidol treated Fig. 4 Light micrograph of liver section from infants of 7 mothers showing disappearance of the characteristic
days after delivery from haloperidol treated mothers strands of hepatocyte deformed hepatocytes (arrows) (E& H
showing decayed hepatocytes (H) appearance of fat filtration 100 x).
(Unclear) and tissue fibrosis (arrow) (H & E 100 x).
Fig. 3 Light micrograph of kidney from haloperidol Fig. 5 Light micrograph of liver section from infants of 21 treated mothers (1 ml/kg B.W.) showing rare intertubular
days after delivery from haloperidol treated mothers connective tissue (arrow), decayed proximal (PT) and distal
showing congestion of blood sinusoid (BV) and oedema (DT) tubules and deformed glomerulus(G) (H &E 100x).
(arrow) in the tissue(H & E 100 x).
The Role of Pumpkin Extraction on the Liver and Kidney of Mice Previously Treated with Haloperidol
during Lactation
regions, dilation of blood vessels and decreasing in the bile ductules number, compared with other three groups especially the fourth group (pumpkin).
(2) Kidney Investigation of kidney sections at 7-14 days age
showed that the cortex is smaller when compared with medulla, and it have a relatively lower number of
BV
glomerulus compared with the control group. Sections also showed a reduction in glomerulus, epithelial disintegration, enlargement of the urinary cavity, a
complete disintegration of proximal and distal tubules
Fig. 7 Light micrograph of kidney from infants of 21 days
with cellular infilteration (Fig. 6). After three weeks of after delivery from haloperidol treated mothers showing
congestion (BV) and partitioning and atrophy of glomerulus
delivery, sections of kidney showed congestion inside
(circle) (H & E40 x).
the tissue, interruption of the inner wall of renal arteries, by the following histological observations, normal
swelling of proximal and distal tubules cells and appearance of renal tubules, normal appearance, size
adhesion of their nuclei with basement membrane, and number of glomerulus and back to the normal size partitioning and atrophy of glomerulus mainly at cortex and structure of intertubular connective tissue (Fig. 8).
(Fig. 7), while these damages were less obvious in the For liver, sections showed a resistance to the toxicity of
medulla as compared with control or compared with the drug and appeared structurally normal. The hepatic
the other three groups especially the fourth group strands arranged normally, the portal regions were
(pumpkin). recovered, oedema disappeared, the fatty filtration was
3.2.3 Treated Group with the Drug (Haloperidol) retarded and hepatic cells returned their normal and Pumpkin
structure (Fig. 9). And also tissues for infants of three Generally, there were obvious improvements in the
weeks’ age. While for one and two weeks age, there kidney and liver sections of mothers and infants as well
were some limited regions still suffered from the as the regain of normal appearance of most tissues.
harmful influence of the drug, compared with other These improvements could be summarized for kidney
three groups especially the fourth group (pumpkin).
4. Discussion
The results of the present study indicated that haloperidol administered to mice during lactation
decreased the infants’ body weight in comparison with control, while treatment with pumpkin only, showed a
marked increase in infant’s body weight. These results were in agreement with those obtained by R. Holson et al. [23], who concluded that all nervous drugs led to retardation of growth due to a reduction in the DNA
6 formation, protein contents. Also, J. Zhang et al. [24]
Fig. 6 Light micrograph of kidney from infants of 14 days after delivery from haloperidol treated mothers showing
mentioned that injection of pregnant mice with
shrinked glomerulus (G) decayed proximal (circle) and
haloperidol lowered the weight of infants of two weeks
distal (square) tubules (H & E 40 x).
age in comparison with control.
12 The Role of Pumpkin Extraction on the Liver and Kidney of Mice Previously Treated with Haloperidol
during Lactation
The present histological damages in tissues of infants indicated that the drug reaches them from mothers through milk. Haloperidol affects the quantity of milk from mothers through its action on the rate of prolactin hormone secretion [4].
The results obtained in this work showed that haloperidol have a damage effect on mice kidney of mothers and infants and this was supported by A. Patel [27] who mentioned that the anti-nervous drug
8 (haloperidol) to mice during lactation caused
Fig. 8 Light micrograph of kidney from haloperidol and
embryonic distortion. This effect may be explained by
pumpkin treated mothers showing of the normal tissue
the deficiency in DNA formation [23], delay in the cell
structure (H & E 40x).
division [27] and its direct and indirect effects on the enzymatic activity of cell. This might lead to a disorder in the cellular functional activity and disintegration of cellular cytoplasmic processes in kidney. Another possibility of the effect of the drug (haloperidol) might
be through its direct influence on the cell membrane as it had a great ability to dissolve in lipids, and this in turn distorted the nervous impulses through the cell membrane and the cellular metabolism.
Therefore, the damage of epithelium of renal tubules
9 and hepatic cells, dilation of the blood vessels,
Fig. 9 Light micrograph of liver section from haloperidol
increasing of the cell size and presence of oedema
and pumpkin treated mothers showing resume normal tissue structure (H & E 10 x).
around cells might refer to the increasing in the permeability of cell membrane and changing in the
The present data demonstrated that treatment of
metabolic mechanisms [8].
mice with haloperidol during lactation can led to The present investigation demonstrated that changes in liver’s and kidney’s weights in mothers and
pumpkin extraction reduced the toxic effect of infants. Before, R. Sommi et al. [25] showed that
haloperidol on liver and kidney tissues of female mice medium and high doses of hydrochloride alfloxetine,
and their infants and this might be due to its highly (anti-depression drug) led to an elevation on the
content of B-carotein, which agreed with M. Makni cellular hepatic disintegration and an increase in the
[21] who discussed the antioxidant and fatty precipitation in hepatic cells. The toxic impact of
hepatoprotective effect of the active groups treated the anti-depressed drug resulted in critical with pumpkin. B-carotene had been proved to be a
morphological and functional changes in the hepatic powerful antioxidant and profound protective actions lipid associated with several disintegrated changes
against tumor [28] because there was increasing where female mice exposed to antinervous drug during
interest in the role of antioxidant vitamins like pregnancy especially in time of organs formation or
B-carotene in neutralizing free radicals and overtly embryonic growth, showed retardation in growth with
aggressive oxygen species [29]. B-Carotene was functional distortion of some organs [26].
among the most efficient substance known for
The Role of Pumpkin Extraction on the Liver and Kidney of Mice Previously Treated with Haloperidol
during Lactation
quenching the excitation energy of single oxygen and [6] A. Jurand, L. Martin, Teratogenic potential of two also for trapping certain organic free radicals. It had a neurotropic drugs, haloperidol and dextromoramid, tested on mouse embryos, Teratology 42 (1) (1990) 45-54.
direct inhibitory effect on liver microsomal enzymes [7] S. Khalifa, Effect of camels urine and milk, honey bee [10], thus offering another mechanism of its anticancer
with nigela sativa mixture and ginger on the toxic nature. Pumpkin also was considered to be one of the
potentials of haloperiol antipsychotie agents on fertility in most effective anti-oxidative [18] that increases the the male albino rat, J. Toxico. 34 (2006) 119-129. [8] B. Abd El-Magid, F. Ghamdy, Effect of haloperidol
rate of hepatic enzymes alkaline phosphatase (ALP), (Hadoldecanoso) anti-depression drug on the development
glutamate oxaloacetate transaminase (GOT), and of some organs in chick embryo, Ph.D. Girl’s College of glutamate pyruvate transaminase (GPT) and decrease
Science, Uni. of King Abdul Aziz, Jeddah, Saudi Arabia, the toxic effect [17], which agreed with the findings of
2007. [9] R. Tacke, F. Popp, B. Muller, A. Theis, C. Burschka, A.
Ref. [30] in which Nardostachys jatamansi (an aqueous Hamacher, et al., Sila-Haloperidol, asilicon analogue of
root extract) reversed the haloperidol-induced the dopamine (D(2))-Receptor antagonist Haloperidol: catalepsy in rats for its antioxidant and anticataleptic
synthesis, pharmacological properties, and metabolic fate, effects.
Chem. Med. Chem. 11 (1) (2008) 152-164. [10] H. Basha, Prophetic Medicine, 2nd ed., Elswady lib., 1993,
It is concluded that pumpkin extract was effective in
Jeddah.
preventing the toxic effect of haloperidol on liver and [11] J. Salgado, M. Takashima, Chemical and biological kidney of female mice during lactation, and their
characterization of meal and protein isolates from infants. Much additional studies were needed before
pumpkin seed (Cucurbita moschate), Arch Latinoam Nutr. the authors might confidently make recommendations 42 (4) (1992) 443-450. [12] F. Bion, D. Pessoa, M. Lapa, A. Campos, N. Antunes, S.
regarding dietary pumpkin in the prevention of toxic Lopes, The use of amultimix as dietary supplement: Study effect of the anti-depressive drugs.
in rats, Arch Latinoam Nutr. 47 (3) (1997) 242-247. [13] A. De Queiroz-Neto, M. Mataqueiro, A. Santana, A.
Acknowledgments
Alessi, Toxicological evaluation of acute and subacute oral administration of Cucurbita maxima seed extracts to
The author is grateful to Dr. Awatef M. Ali, Assi. rats and swine, J. Ethnopharmacol. 43 (1) (1994) 45-51. Prof. of histology and cell biology, Alexandria
[14] Y. Kwon, E. Apostolidis, Y. Kim, K. Shetty, Eealth University for her kind revision of the manuscript.
benefits of traditional corn, beans, and pumpkin: In Vitro studies for hyperglycemia and hypertension management,
References
Med. Food 10 (2) (2007) 226-235. [15] T. Xia, Q. Wang, D-chiro-inositol found in Cucurbita
[1] B. Barentt, Postnatal depression “failure thrive”, Mother ficifolia (Cucurbitaceae) fruit extracts plays the Medicine of Australia 33 (3) (1990) 64-72. hypoglycemia role in streptozocin diabetic rats, Pharm. [2] M. Sagud, L. Hotujac, A. Mihalijevic-Peles, M. Pharmacol. 58 (11) (2006) 1527-1532. Jacovljevic, Gender difference in depression, Cell [16] L. Quanhong, F. Caili, R. Yukui, H. Guanghui, C. Tongyi, Autropol. 26 (2002) 149-154. Effect of protein-bound polysaccharide isolated from [3] H. Nurnberg, Treatment of mania in the last sex monthes pumpkin on insulin in diabetic rats, Plant Foods Hum. of pregnancy, Hosp. Community Psychiatry 31 (2) (1980)
122-126.
Nutr. 60 (1) (2005) 13-16. [17] C. Nkosi, A. Opoku, S. Terblanche, Effect of pumpkin
[4] R. Baldessarini, F. Tarazi, Drugs and the treatment of psychiatric disorders (Psychosis and Mania), Chapter 19
seed (Cucurbita pepo) protein isolate on the activity levels and 20 in Goodman and Gilmans, The Pharmacological
of certain plasma enzymes in CC14-induced liver injury in Basis of Therapeutics Mc Graw-Hill, Medical Publishing
low-protein fed rats, Phytother. Res. 19 (4) (2005) Division, 10th ed., 2001, pp. 485-520.
341-345.
[5] H. Miyazaki, Y. Matsunaga, K. Nambu, Y. Oh-e, Y. [18] C. Nkosi, A. Opoku, S. Terblanche, Antioxidative effect Yoshida, M. Hashimot, Disposition and metabolism of
of pumpkin seed (Cucurbita pepo) protein isolate in [14c] haloperidol in rats, Arzneimittelfrschung 36 (3)
CC14-induced liver injury in low-protein fed rats, (1986) 443-452.
Phytother. Res. 20 (11) (2006) 935-940.
14 The Role of Pumpkin Extraction on the Liver and Kidney of Mice Previously Treated with Haloperidol during Lactation
[19] G. Oboh, Hepatoprotective property of ethanolic and offspring, Neurotoxicol Teratol. (18) (1996) 49-57. aqueous extracts of fluted pumpkin (Telfairia occidentalis)
[25] R. Sommi, M. CRISMON, C. Bowden, Fluoxetine: A leaves against garlic-induced oxidative stress, J. Med.
serotonin specific second generation antidepressant, Food 8 (4) (2005) 560-563.
otology functional effect on the offspring after parental [20] M. Gossel-Williams, K. Lyttle, T. Clarke, M. Gardner, O.
drug exposure, Pharmacotherapy 7 (1987) 1-15. Simon, Supplementation with pumpkin seed oil improves
[26] F. Lu, “Basic Toxicology” Fundamental Target Organs plasma lipid profile and cardiovascular outcomes of
and Riskassessment, 2nd ed, New York, Washington, female nonovariectomized and ovariectomized
Philadelphia, London, 1991, pp. 133-144. Sprague-dawley rats, Phytother. Res. (22) (2008) 873-877.
[27] A. Patel, Neurobiological aspects of functional [21] M. Makni, H. Fetoui, N. Gargouri, M. Garoui el, H. Jaber,
teratogenesis: cell proliferation and development of J. Makni, T. Boudawara, N. Zeghal, Hypolipidemic
certain nurotransmitters systems, in: T. Fujii, P.M. Adams, andhepatoprotective effects of flax and pumpkin seed
(Eds.), Functional Tera, 1987, pp. 133-144. mixture rich in Omega-3 and Omega-6 fatty acids in