The Journal of Basic & Applied Zoology (2016) 75,13–27

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The protective effect of against verapamil embryotoxicity during prenatal liver and kidney development of mice Mus musculus

Amin A. Seleem

Zoology Dept. Faculty of Science, Sohag University, Egypt

Received 29 September 2015; revised 18 February 2016; accepted 24 March 2016

KEYWORDS Abstract Verapamil is a calcium channel blocker that has been widely used in the treatment of car- Verapamil; diovascular abnormalities, hypertension and angina pectoris. The present study investigates the Bee venom; effect of bee venom against verapamil embryotoxicity, bee venom (BV) is characterized with anti- Heat shock ; , anti-inflammatory, anti-rheumatoid, pain-relieving and neuroprotective agents. The current BAK; study was carried out on 70 pregnant female mice which were divided into two main groups, the first Embryotoxicity main group divided into three subgroups, control, treated with single and twice dose daily of ver- apamil (40 mg/kg) that was treated from zero day of gestation to scarification of females at E10. The second main group that was treated from the seventh day of gestation was divided into four subgroups, control, treated with single dose daily of verapamil (40 mg/kg), injected with bee venom (150 lg/kg/BW) and treated with verapamil combined with bee venom, the females were sacrificed at E14 and E17. The results of this study showed that verapamil treated groups once or twice daily in the first main experiment showed abortion and resorption of uteri embryos. In the second main experiment, developing liver and kidney at E14 and E17 in verapamil treated group showed abnor- mal architecture of histological picture and alterations of immunohistochemical expression of heat shock and BAK that were associated with ultrastructure abnormalities at E17. Bee venom treated group showed the similar structure as control, verapamil combined with bee venom treated group exhibited amelioration against verapamil embryotoxicity. In conclusion, bee venom could be considered as a therapeutic agent and it has a curative effect against the of verapamil during development of liver and kidney. Ó 2016 The Egyptian German Society for Zoology. Production and Hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Introduction and regulation of blood pressure in hypertensive patients (Girvin and Johnston, 2004; Lipsitz et al., 2005; Pavlovic Calcium channel blockers (CCBs) or calcium antagonists are et al., 2004). Verapamil is an L-type voltage-dependent calcium among the most widely used drugs in cardiovascular medicine channel blocker, belongs to the phenylalkylamine group, and has been used in the treatment of angina pectoris, cardiovascu- Peer review under responsibility of The Egyptian German Society for lar abnormalities, hypertension, cardiac arrhythmia and coro- Zoology. nary vasospasm (Kumar and Hall, 2003; Grossman and http://dx.doi.org/10.1016/j.jobaz.2016.03.001 2090-9896 Ó 2016 The Egyptian German Society for Zoology. Production and Hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). 14 A.A. Seleem

Messerli, 2004; Hirota et al., 2004; Wu et al., 2004). Verapamil experiment. The studied pregnant females of the second main given either in vivo or in vitro has been shown to inhibit calcium groups were treated from the 7th day of pregnancy to sacrifi- uptake (Goligorsky et al., 1985) and any alterations in calcium cation at prenatal stages E14 and E17 that were divided into homeostasis can be quite toxic (Choi, 1992) where the concen- four subgroups (10 each), control, treated with single dose tration of cytosolic calcium is a key factor for control of many daily of verapamil (40 mg/kg), treated with bee venom subcu- diverse cellular and pathophysiological functions. Calcium taneously injected (150 lg/kg/BW) (Alvarez-Fischer et al., channel blockers have been shown in animal experiments to 2013) and treated with verapamil single dose combined with be able to induce teratogenic effects and increase the incidence bee venom subcutaneously injected. of embryolethality (Fukunishi et al., 1980; Cabov and Palka, 1984; Robert et al., 2011). Verapamil, is a classic chemo- Histological examination sensitizer which can enhance the antitumor effect in several cancer cells including neuroblastoma, lung and colorectal can- The whole embryos at prenatal stages E14 and E17 of studied cer (Ikeda et al., 1987; Wang et al., 2010; Koski et al., 2012). groups were fixated in carnoy’s fixative, dehydrated in ethyl Bee venom (BV) is the venom that store within their , cleared in methyl benzoate, toluene and mounted in venom sacs for self-defense which is also known as apitoxin, it paraffin wax. Serial section (7 l thick) of the selected tissues is extracted from honeybees and is commonly used in Oriental from livers and kidneys were stained by hematoxylin and eosin medicine to relieve pain and treat inflammatory diseases. Bee for general histology (Drury and Wallington, 1976). venom is known to be a very complex mixture of at least 18 active components including active , (a major Immunohistochemical investigation component of BV), apamin, adolapin (Kwon et al., 2005) and amines that contain histamine, catecholamines, polyamines, Sections of selected liver and kidney at E14 and E17 of the sec- melittin, and (Orsˇolic´ , 2012). Melittin is the ond main experiment were mounted on Superfrost/Plus glass principal component of BV and constitutes approximately slides. The slides were deparaffinized in xylene, rehydrated in 50% of its dry weight (Vogel, 1981). Bee venom is introduced serial alcohol and retrieved for re-antigenicity using 10 mM natural anticancer agents (Orsˇolic´ et al., 2003; Gajski and citrate buffer at pH6 at 100 °C in oven for an hour Garaj-Vrhovac, 2011), Anti-mutagenic, (Varanda et al., (Buchlowalow and Bocker, 2010). Sections were incubated 1999), anti-inflammatory (Nam et al., 2003), anti-nociceptive, with specific primary antibody against anti-HSP70 (rabbit (Kim et al., 2003), anti-rheumatoid arthritis (Kwon et al., polyclonal, spring, Bioscience, USA) and BAK (rabbit poly- 2005), pain-relieving (Kwon et al., 2001), neuroprotective clonal antibody, Santa Cruz, Biotechnology, INC., Germany), (Alvarez-Fischer et al., 2013) and immune modulatory activity for three hours at room temperature. Sections were then (Nam et al., 2005). The aim of the present study was to examine washed using phosphate buffer and incubated with secondary the ameliorative effect of bee venom against embryotoxicity of antibody (biotinylated goat anti-polyvalent HRP DAB detec- calcium channel blockers (verapamil) during development of tion system, Spring Bioscience, USA). Then sections were liver and kidney at prenatal stages E14 and E17. washed with phosphate buffer and then visualized by mix chromogen solution with DAB. Stained sections were mounted Material and methods with DPX mounting media and investigated under light micro- scope (Axio Scope, Zeiss, Germany). Chemicals Transmission electron microscope study Verapamil was obtained from Sigma–Aldrich Company USA. Bee venom of Apis mellifera was obtained from Faculty of Liver and kidney samples at E17 from the different subgroups Agriculture, Assuit University. of the second main experiment were fixated in 2.5% glu- taraldehyde (Mercer and Birbeck, 1972), then washed twice Animals and experimental design in distilled water, then post-fixed in 1% osmium tetroxide, then washed twice in distilled water and dehydrated in ascending series of ethanol. At the stage of clearing, samples were passed Adult female and male mice Mus musculus (weighting 25–30 g) in propylene oxide. In the infiltration process samples were were used. The animals were obtained from a closed random transferred to a mixture of propylene oxide with epon resin bred colony at Faculty of Science, Sohag University, Egypt. 1:1. The samples were then embedded in pure epon resin in The mice were maintained on food and water ad libitum and blocks at 60 °C overnight. Ultrathin sections were cut using housed in groups in isolated cages. The animals were acclima- ultratome (Reichert, Supernova, Germany) and were mounted tized for 2 weeks prior to usage. A male and two females were in grids, stained with uranyl acetate and lead citrate (Reynold, placed in one cage and left overnight. The presence of the vagi- 1963). Sections were examined and photographed under a Joel nal plug, next morning was considered as fertilization indica- Transmission Electron Microscopy (JEM 1010, Japan). tor, and was considered zero day of pregnancy. Seventy pregnant female were divided into two main groups, the first main group (primary experiment) that was treated from zero Results day of gestation to prenatal E10 was divided into three sub- groups (10 each), control, treated with single dose daily and Toxicological observation twice dose daily in the morning and in the evening of vera- pamil (40 mg/kg). The second main group (essential experi- The females of verapamil treated groups either once or twice ment) was designed after the results of the primary daily that were treated from zero day of gestation showed Protective effect of bee venom against verapamil embryotoxicity 15 blood in their vagina that has been considered the signs of were separated from each other with numerous nucleated red abortion at prenatal stage E10 as compared with the control blood cells (arrow, Plate 2B). Bee venom treated group (Pla- group. Also, the percentage of abortion and resorption of te 2C) showed similarity with control structure. Verapamil embryos in the twice treated groups (arrows, Plate 1C) was combined with bee venom group didn’t exhibit verapamil dis- higher than the once treated ones (arrows, Plate 1B) as com- turbances (Plate 2D). The developing liver of control group at pared with the control group (Plate 1A). Verapamil treated E17 consists of primitive hepatic cords of hepatocytes and pri- group that were treated from the seventh day of gestation in mary blood sinusoids (Plate 2E). Verapamil treated group the second main experiment showed resorbed embryos (arrow, (Plate 2F) showed loosely arranged hepatocytes, hemorrhage Plate 1E) at E14, bee venom treated group didn’t elucidate any (white arrows), aggregation of inflammatory cells (red arrow) resorbed embryos (Plate 1F), verapamil combined with bee and the majority of the apoptotic cells were identified as hep- venom group showed recovery of resorption effect of vera- atocytes based on their larger size with large eosinophilic cyto- pamil (Plate 1G) as compared to the control group (Plate 1D). plasm (black arrow). Bee venom treated group elucidated the same architectural pattern of control (Plate 2G), verapamil Histological, immunohistochemical and ultrastructural combined with bee venom treated group showed improvement investigation of liver of the dangerous effects of verapamil (Plate 2H). Immunohistochemistry staining of heat shock protein Hematoxylin and eosin staining of developing liver at E14 (HSP) expression in control group of developing liver at E14 showed that the control group had a normal architecture of (Plate 3A) and E17 (Plate 3E) showed faint staining. Vera- hematopoietic cells (Plate 2A). Verapamil treated group at pamil treated group at E14 (arrow, Plate 3B) and E17 (arrow, E14 showed loose arrangement of hematopoietic cells that Plate 3F) showed an increase in the expression of HSP. Bee

Plate 1 Photographs of uterus in the different groups of primary experiment at prenatal stage E10, control (A), verapamil groups daily treated with single (arrows, B) and twice (arrows, C) dose showed resorbed embryos. Uterus of the secondary experiment at prenatal stage E14 of different groups, control (D), verapamil treated group showed resorbed embryo (arrow, E), bee venom (F) and verapamil combined with bee venom (G) treated groups showed similar picture to control. 16 A.A. Seleem

Plate 2 Photomicrographs of H&E staining of developing liver at E14 showed the normal structure of hematopoietic (H) cells and blood vessel (BV) in control group (A), verapamil treated group exhibited loosely arranged hematopoietic cells (arrows, B). Bee venom (C) and verapamil combined with bee venom treated (D) groups didn’t exhibit verapamil disturbances. Developing liver of control group at E17 (E) showed hepatic strands (HS) and blood vessel (BV), verapamil treated group (F) elucidated loosely arranged hepatocytes, hemorrhage (white arrows), eosinophilic cytoplasm (black arrow) and aggregation of inflammatory cells (red arrow). Bee venom (G) and verapamil combined with bee venom (H) treated groups showed improvement of the dangerous effects of verapamil. Scale bar 10 lm. venom treated group didn’t reveal any increase in expression at treated group at E14 (arrows, Plate 4B) and E17 (arrows, Pla- E14 (Plate 3C) and E17 (Plate 3G). Verapamil combined with te 4F) as compared with the normal expression in the control bee venom treated group elucidate amelioration in the expres- group at E14 (Plate 4A) and E17 (Plate 4E). Bee venom treated sion of HSP at E14 (Plate 3D) and E17 (Plate 3H) as compared group exhibited similarity in BAK expression with control with verapamil treated group. Immunohistochemistry staining group at E14 (Plate 4C) and E17 (Plate 4G). Verapamil com- of BAK showed a reduction in the expression in the verapamil bined with bee venom treated group elucidated recovery of Protective effect of bee venom against verapamil embryotoxicity 17

Plate 3 Photomicrographs of heat shock protein immunostained of developing liver in control group at E14 (A) and E17 (E) showed faint staining. Highly marked increase was detected in the verapamil treated group at E14 (arrow, B) and E17 (arrow, F). Bee venom treated at E14 (C), E17 (G) and verapamil combined with bee venom group at E14 (D) and E17 (H) didn’t reveal any increase in heat shock protein expression. Scale bar 10 lm.

expression and became similar to control at E14 (Plate 4D) several morphological distortions as severe decline of endo- and E17 (Plate 4H). plasmic reticulum, fragmented and decomposed mitochondria, Ultrastructure of hepatocytes in developing liver of control shrunken nuclei (pyknosis), irregular nuclear membrane, group at E17 (Plate 8A) was characterized by the normal abnormal chromatin condensation on the nuclear membrane, architecture of nucleus, mitochondria, endoplasmic reticulum, small amounts of lipid droplets and vacuoles (V) of varying nuclear membrane and normal chromatin on the nuclear mem- sizes (arrows, Plate 8B). Bee venom treated group didn’t show brane. Hepatocytes of verapamil treated group at E17 showed any change in ultrastructure of hepatocytes at E17 (Plate 8C). 18 A.A. Seleem

Plate 4 Photomicrographs of BAK immunostained of developing liver in control group at E14 (A) and E17 (E) showed normal expression. Verapamil treated group at E14 (arrows, B) and E17 (arrows, F) showed reduction of BAK expression. Bee venom treated at E14 (C), E17 (G) and verapamil combined with bee venom at E14 (D) and E17 (H) groups exhibited similarity in expression with control group. Scale bar 10 lm.

Verapamil combined with bee venom treated group (Plate 8D) marked decrease in the carbohydrate content. Bee venom trea- exhibited amelioration in the abnormalities of verapamil ted group (Plate 9C) showed a similar distribution of carbohy- effects. drates to the control group. Verapamil combined with bee The normal distribution of carbohydrates content in PAS venom treated group (Plate 9D) exhibited recovery abnormal- staining was observed in the developing liver of control group ities of carbohydrate distribution that was induced by at E17 (Plate 9A), verapamil treated group (Plate 9B) showed a verapamil. Protective effect of bee venom against verapamil embryotoxicity 19

Plate 5 Photomicrographs of H&E stained in developing control kidney at E14 (A) and E17 (E) showed normal structure of glomerulus (G) and renal tubules (T). Verapamil treated group at E14 (B) and E17 (F) exhibited atrophy, shrinkage of renal corpuscle and disorganization of kidney tubules. Developing kidney in bee venom treated group at E14(C), E17 (G) and combined group of verapamil with bee venom at E14 (D) and E17 (H) showed similar structure of control. Scale bar 10 lm.

Histological, immunohistochemical and ultrastructural disorganization and degeneration of kidney tubules. Bee examination of kidney venom treated group showed a similar structure to control either at E14 (Plate 5C) or E17 (Plate 5G). The combined The histological picture of kidney in control group at E14 (Pla- group of verapamil with bee venom at E14 (Plate 5D) and te 5A) and E17 (Plate 5E) showed renal parenchyma, develop- E17 (Plate 5H) elucidated amelioration in the kidney ing of glomerulus and kidney tubules. Verapamil treated group parenchymatous organizations, glomerulus and tubules as at E14 (Plate 5B) and E17 (Plate 5F) showed atrophy and compared with verapamil effects. shrinkage of renal corpuscle, decrease in glomerular cellularity, Developing kidney of the control group at E14 (Plate 6A) showed faint expression of heat shock protein (HSP) in the 20 A.A. Seleem

Plate 6 Photomicrographs of heat shock protein immunostained of developing kidney in control group at E14 (A) and E17 (E). Highly marked increase in heat shock protein was detected in the verapamil treated group at E14 (arrows, B) and E17 (arrows, F). Bee venom treated group at E14 (C), E17 (G) and verapamil combined with bee venom treated group at E14 (D) and E17 (H) didn’t show any disturbance in heat shock protein expression. Scale bar 10 lm.

tubules and mesenchyme tissue that was increased at E17 (Pla- expression at E14 (Plate 6D) and E17 (Plate 6H). Developing te 6E). Elevation of HSP expression was detected in the devel- kidney of control group at E14 (Plate 7A) showed normal oping kidney tubules and mesenchyme tissue of the verapamil BAK expression that that was increased at E17 (Plate 7E). treated group at E14 (Plate 6B) and E17 (Plate 6F). Bee venom Verapamil treated group showed a decrease in BAK expression treated group didn’t reveal any change in HSP expression at at E14 (Pl7B) and E17 (Plate 7F). Bee venom treated group E14 (Plate 6C) and E17 (Plate 6G). Verapamil combined with exhibited similarity in BAK expression with control group at bee venom treated group elucidate amelioration in HSP E14 (Plate 7C) and E17 (Plate 7G). Verapamil combined with Protective effect of bee venom against verapamil embryotoxicity 21

Plate 7 Photomicrographs of BAK immunostained of developing kidney in control group at E14 (A) and E17 (E). Verapamil treated group at E14 (arrows, B) and E17 (arrows, F) showed reduction of BAK expression. Bee venom treated group exhibited similarity in expression with control group at E14 (C) and E17 (G). Verapamil combined with bee venom group treated group elucidated recovery of expression and return to control at E14 (D) and E17 (H). Scale bar 10 lm.

bee venom treated group exhibited recovery of BAK expres- that have long microvilli (Mv) forming the brush border. sion and became similar to control at E14 (Plate 7D) and PCT and DCT have large rounded nuclei (N) with normal E17 (Plate 7H). peripheral chromatin distribution, well defined nucleolus The ultrastructure of developing kidney tubules of control (NC), mitochondria (M) and normal thickness of basement group at E17 showed normal appearance of proximal convo- membrane (BM). In verapamil treated group, destruction luted tubule (PCT) (Plate 8E) and distal convoluted tubules and reduction in the number and size of apical microvilli of (DCT) (Plate 8I). PCT is lined by simple cuboidal epithelium PCT (arrows, Plate 8F) were noted. Intracellular vacuolations 22 A.A. Seleem

Plate 8 Transmission electron micrograph of transverse section of hepatocytes in control group at E17 (A) is characterized by the normal architecture of nucleus (N), nucleolus (NC), heterochromatin (HC), Euchromatin (EC), nuclear membrane (NM), mitochondria (M) and endoplasmic reticulum (ER). Verapamil treated group suffers from decline in the endoplasmic reticulum, fragmented mitochondria, shrunken nuclei, irregular nuclear membrane, abnormal chromatin condensation, large vacuoles (V) and small (arrows, B). Bee venom treated group didn’t show any change in ultrastructure of hepatocytes (C). Verapamil combined with bee venom group exhibited amelioration in the abnormalities of verapamil (D). Developing kidney tubules in control group at E17 contains proximal convoluted tubule (PCT) (E) and distal convoluted tubule (DCT) (I) with large rounded nuclei (N), nucleolus (NC), mitochondria (M) and basement membrane (BM). Verapamil treated group in PCT (F) and DCT (J) showed destruction of apical microvilli (arrows) in PCT and suffers from vacuoles (V), lysosomes (L), fragmented mitochondria (M), basement membrane thickening (BM), shrinking nucleus (N) with irregular shape and abnormal distribution of peripheral chromatin. Bee venom treated group didn’t show any alteration in ultrastructure of PCT (G) and DCT (K). Verapamil combined with bee venom group exhibited amelioration in the ultrastructure of PCT (H) and DCT (L). Magn. A-D 6000, E-L 4000. Protective effect of bee venom against verapamil embryotoxicity 23

Plate 9 Photomicrographs of PAS staining of developing control liver (A) and kidney (E) at E17 showed normal distribution of carbohydrates content that was marked decrease in verapamil treated group in liver (B) and kidney (F). Bee venom treated group showed normal distribution of PAS staining in liver (C) and kidney (G). Verapamil combined with bee venom treated group exhibited recovery in the abnormalities of carbohydrates distribution in the liver (D) and kidney (H). Scale bar 10 lm.

(V), lysosomal structures (L), fragmented and degenerated (Plate 8K). Verapamil combined with bee venom group exhib- mitochondria (M), thickening of basement membrane (BM), ited amelioration in the ultrastructure of proximal convoluted shrinking nucleus (N) with irregular shape and clumps with tubules (Plate 8H) and distal convoluted tubules (Plate 8L). abnormal distribution of peripheral chromatin were noted in The normal appearance of PAS staining in kidney of con- PCT (Plate 8F) and DCT (Plate 8J). Bee venom treated group trol group at E17 (Plate 9E) was observed in the basement at E17 didn’t show any alteration in ultrastructure of proximal membrane of Bowman capsule and the brush border of prox- convoluted tubules (Plate 8G) and distal convoluted tubules imal convoluted tubules. Verapamil treated group at E17 24 A.A. Seleem exhibited reduction in PAS staining of the basement mem- been carried out. BV is a well-established hepatoprotective brane of Bowman capsule and altered brush border of PCT agent and has the ability to circumvent the hepatocellular (Plate 9F). Bee venom treated group at E17 showed the similar toxicity induced by methotrexate (Darwish et al., 2013). Also, distribution of PAS staining (Plate 9G) as the control group. the radioprotective, antimutagenic, anti-inflammatory, antiar- Verapamil combined with bee venom treated group exhibited thritic, antihyperalgesic and antinociceptive effects of BV have recovery (Plate 9H) in the abnormalities of carbohydrate dis- been documented (Park et al., 2004; Roh et al., 2004; Son tribution that was induced by verapamil. et al., 2007; Elshater et al., 2014). In addition, BV has protec- tive effects on renal tubular injury (Clark et al., 1999; Hyunseong et al., 2013; Elshater et al., 2014). BV has been pro- Discussion ven to be a useful compound in potentiating the apoptotic kill- ing capacity of some chemotherapeutic agents such as The present study demonstrated the dangerous effects of vera- bleomycin in cancer cell lines (Orsˇolic´ , 2009; Jo et al., 2012). pamil that cause abortion and resorption of embryos either in Several studies have demonstrated that BV and/or melittin daily or twice treatment of verapamil (40 mg/kg) in the pri- have direct antitumor effects in vivo (Liu et al., 2002; mary experiment and cause histological and ultrastructure Orsˇolic´ , 2012; Russell et al., 2004) and in vitro on various types alterations during the development of liver and kidney at of cancerous cells, including liver (Liu et al., 2008), bone (Chu E14 and E17 in the secondary experiment. Previous studies et al., 2007), prostate, breast, cervical, and renal cancer cells have shown that verapamil at dose (60 mg/kg/day) may cause (Park et al., 2010, 2011). miscarriages via inhibiting implantation (Uslu et al., 2013). In Apamin is important component of bee venom (BV) and it addition, previous use of verapamil in the treatment of fetal has a potent to increases of intracellular calcium during an paroxysmal tachycardia (Wolff et al., 1980; Lilja et al., 1984; action potential (Bond et al., 2004). Apamin is possibly a cru- Truccone and Mariona, 1985) and during tocolytic therapy cial component in bee venom regarding its protective action (Strigl et al., 1981) coincided with the results of this study. that is due to a moderate and persistent elevation of cytosolic By the same token, verapamil causes neural tube closure calcium (Salthun-Lassalle et al., 2004). Another molecule in defects (Lee and Nagele, 1986), growth retardation, abnormal- bee venom might enhance the action of apamin called Melittin ities in forelimbs, heart, head and yolk sac circulation (Stein that has several interesting pharmacological properties as a et al., 1990). In addition, calcium channel blockers induce ter- result of its interaction with negatively changed phospholipids. atogenic effects of embryos (Fukunishi et al., 1980; Cabov and Also, it inhibits the activity of the Na/K-ATPase (Yang and Palka, 1984; Robert et al., 2011). Carrasquer, 1997; Lopina, 2001). Furthermore, the specific Calcium plays a crucial role in regulation of cellular pro- character of the major BV component melittin has an anti- cesses (Rasmussen, 1986). Endoplasmic reticulum of the liver calmodulin activity through which it forms a calcium- is the major intracellular calcium pool (Somilyo et al., 1985) dependent high-affinity complex of calmodulin and melittin and intracellular calcium homeostasis is maintained by cooper- (Tanaka et al., 1985). Other studies cited several mechanisms ation between cellular transport and the individual compart- of BV , including cell cycle alterations, effects on ments of the cell (Claret-Berthon et al., 1977; Carafoli, proliferation and/or growth inhibition and induction of apop- 1987). An impairment of the calcium-regulating systems is tosis or necrosis (Gajski and Garaj-Vrhovac, 2013). often associated with an uncontrolled increase in the cytosolic Verapamil treated group showed elevation in the expression calcium concentration (Lauterburg, 1987; Thurman et al., of heat shock proteins (HSPs) during development of liver and 1988; Okuda et al., 1992). Verapamil, a calcium channel kidney at E14 and E17 that may be attributed to HSPs that blocker has been shown to inhibit calcium uptake have been associated with cytoprotection during stress and (Goligorsky et al., 1985) where it act as ligands, binding may help to restore cellular integrity and promote recovery strongly to the cellular membranes and thereby inhibiting cal- in renal tubular cells exposed to ischemic injury (Aufricht cium influx (Bogin et al., 1981). Thus, verapamil is commonly et al., 1998; Emami et al., 1991; Van Why and Siegel, 1998). used as a calcium channel blocker for management of ischemic By the same token, Vicencio et al., 2003 demonstrated an heart diseases, supraventricular tachyarrhythmias, active increased activation of HSP in immature rat tubules exposed antianginal, antiarrhythmic, mitral stenosis, migraine prophy- to anoxia compared with mature tubules (Gaudio et al., laxis and antihypertensive (McCall, 1976; Johnston et al., 1998). Moreover, one feature of the protection mechanism of 1981; Opie, 2005; Dong-Yun et al., 2010). In addition, some HSP involves altered calcium ion homeostasis (Smith et al., studies have investigated the protective effect of verapamil 1995). On the contrary, verapamil treated group showed inhi- on the inflammatory response in the liver during endotoxic bition of BAK expression during liver and kidney development (Standiford et al., 1995; Cavaillon et al., 1992). neuroprotec- at E14 and E17 as compared to control. BAK is a member of tive in several acute neurotoxicity models (Liu et al., 2011) BCL-2 family, reduction of BCL-2 expression increases cell antiproliferative effect on tumor cell in vivo and in vitro death to perform the tissue morphogenesis during develop- (Schmidt et al., 1988 and Jian et al., 2007) and nephroprotec- ment or the organ functions (Maskey et al., 2009). At the same tive effect against lead, cadmium, mercuric chloride, cephalos- context, BCL-2 family proteins are inhibitors and inducers of porins, gentamycin toxicity (Watson et al., 1987; Sleijfer et al., cell death (Hardwick and Soane, 2013). In addition, BCL-2 1987; Ali et al., 2002; Zhang et al., 2013). was found to promote cell survival as opposed to promoting Bee venom treated group showed the similar histological cell proliferation (Vaux et al., 1988; Tsujimoto, 1989). More- and ultrastructure picture to control. Also, it showed protec- over, BAK controls apoptosis through the regulation of cal- tion properties against verapamil embryotoxicity at E14 and cium levels (Klee and Pimentel-Muinos, 2005). In conclusion, E17 during development of liver and kidney. 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