Genotoxic Effect Of Calcium Channel Blockers

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Genotoxic Effect Of Calcium Channel Blockers

ABSTRACT: Calcium ions are vital in many biological processes including a variety of enzymatic reactions ,coupling of electrical activation to cellular secretion ,bone metabolism and sperm function.Calcium channel blockers (CCB) drugs inhibit calcium –evoked contractions in depolarized smooth muscles and produces vasodilation by blocking the entry of calcium . CCB drugs are used in treatment of hypertension ,angina and headaches. Less information is available regarding male reproductive dysfunction and the genotoxic effect of these drugs .There fore to assess the involvement of CCB in inducing infertility and genotoxicitywere investigated in this study. Male mice were orally administrated Ca+2 channel blockers nifedipine and ethosuximide for 15 days at dosages 40 or 100 mg/kg body weight respectivily and assayed for chromosome aberrations , sperm abnormalities and testis weight.CCB administration in mice causes increase in percentage of chromosome aberration and sperm shape change.Inaddition ,CCB treatment showed no change in testis weight.Inconclusion:There is evidence that CCB inducegenotoxicity and reduce fertility in mice.

Introduction Calcium ions are vital in many biological processes including enzymatic reactions, activation of excitable cells, coupling of electrical activation to cellular activation , haemostasis , bone metabolism and sperm functions, ca+2 is implicated in diverse cellular functions in both germ and somatic cells ( Steele et al. 1992 &Berridge et al. 2003 ) .

Calcium channel blockers (CCB) are drugs that inhibit calcium- evoked contractions in depolarized smooth muscles . CCB reduces active tone of the muscles and produce vasodilation by blocking the entry of calcium . CCB drugs are widely used in the treatment of hypertension , arrhythmias , angina , migraine and headaches .( 2004 ) McDonough et al &(2005) Godfraind .

The types of CCB based on the gating stimulus, are divided into ligand - dependent Ca+2 channels (LDCC) and voltage -dependent Ca+2 channels (VDCC), which identified in various cell types including testiculargerm cells andsomatic cells (1999) Goodwin et al.. VDCC is Classified into L- type Ca+2 channels and T-type Ca+2 channel . (2002 )Son et al . VDCC is demonstrated to be important in spermatogenesis and steroid genesis as required for germ cell development. (2002) Jagannathan et al .

Calcium ion was found to be a primary determinant of sperm cell function like progressive motility , capacitation , and a crosome reaction (1999) Westenbroek& Babcock . The presence of voltage – gated ca+2 channels in the mammalian sperm was reported by several studies (2000 )Wennemuth et al. & (2003) park et al..Drugs blocking calcium channel were suggested to reduce male fertility (. 2004)Trevion et al. Furthermore , recent studies mentioned that , CCB have reversible anti-fertility effect in male(.2011) Lee et al, &( 2011 ) Morakino et al.

However,a fundamental question is whether drugs blocking calcium channel have side effect that reduces male fertility and induce mutaginicity as some reports suggest that calcium ion mediates sperm functions and fertilization process (1993) Adeoya-Osiguwa&Fraser and( 2004) Trevino et al.

For this purpose, the aim of this study was designed to investigate the mutagenic effect of CCB in bone marrow and testiscells, and to know whether this effect is reversible or no.

Materials and methods : Animal and experimental design : Thirty male mice weighing 18-20 g were purchased from king AbdoulAziz university . Animals were housed in cages placed in an animal room with room temperature and lighting for 12 consecutive hours per day . Animals were fed a rodent chow and tap water ad libituim . Animals were divided into five groups with six mice per group . In group 1 ( Control ) distilled water was administrated orally for 15 days . In groups 2-3 , nifedipine and ethosuximide were dissolved in distilled water ad administrated orally for 15 days at dosages 40 or 100 mg/kg body weight respectively . In groups 4-5 mice were treated with drugs for 15 days and they kept for another 15 days for recovery .

Testicular sample : After sacrificed the testis was removed , washed with saline and cleared of excess fat . the weight of each were taken immediately .

Sperm abnormalities analysis : Sperm analysis for abnormalities was done on sample derived from caudaepididymis by conventional method. (2009 ) Markinyo. About 100 sperm cells were examined in each sample and classified as either normal or abnormal sperm .

Preparation of chromosomes :Both of control and treated animals were infected intraperitioneally with Colchicine 2 hrs before sacrificed .Methaphase chromosomes were prepared from bone marrow cells , according to. (1987) preston et al . The slides were stained with Giemsastain . Ten metaphases were scored for structural and numerical chromosomal abnormalities .The structural chromosomal aberration were represented with deletion , chromatid gaps , centromeric fusion , acentric fragment and ring chromosomes . The numerical aberrations were classified into chromosomal counts less or more than 40 chromosomes hypoploidy and polyploidy .

Results:

Effect of nifedipine and ethosuximide on chromosome changes:

Oral administration of nifedipine or ethosuximide induced high frequencies of structural and numerical chromosomal aberration as compared to the control level ( Table 1 -2 ).

Table (1) : Effect of nifedipine and ethosuximide on numericachromosomal aberration .

%

Total

poly

hypo

group \ ab.

16.6

10

0

10

control

56.6

34

10

24

Nifedipine

43.3

26

0

26

Ethosuximide

Table( 2 ): Effect of nifedipine and ethosuximide on structural chromosomal aberration

%

Total

Iso.G.

A.F.

Ring

Braek

C.F.

C.S.

Del.

Gap.

Group\ab.

10

6

0

0

0

0

2

3

1

0

Control

244

122

2

2

3

3

22

42

48

2

Nifedipine

264

132

1

2

2

2

21

56

44

4

Ethosuximide

The most abundant types of structural chromosomal aberrations as a result of treatment with nifedipine or ethosuximide were deletion ( Del. ) , centric separation ( C.S . ) and centromeric fusion ( C.F. ) . Besides ,numerical aberrations were observed in the form of hypoploidy and polyplody . Referring to tables( 1& 2 ) , the percentages of numerical chromosomal aberration under the effect of nifedipine were more effective than that caused by ethosuximide . With regards to structural chromosomal aberration ,ethosuximide was found to induce higher percentage than nifedipine .Fig (1-4)

Figure(1):percentage of numerical chromosome aberration of control and treated groups.

Figure(2):percentage of strctural chromosome aberration of control and treated groups.

20130515_095022-1.jpg

20130514_120058.jpg ( A ) ( B )

20130520_104738.jpg20130514_110820-1.jpg ( C ) ( D )

20130520_131352-1.jpg20130520_113105-1.jpg

( E ) ( F )

Figure(3): Metaphase spread of control and nifedipine showing normal chromosome and aberrated chromosomes after drugs treatment ,control(A) ,Ring and chromatid exchange (B),deletion (C, F), centric fusion (D,E).

PicsArt_1369344557944.jpg20130520_134652-1.jpgPicsArt_1369344898708.jpg ( A ) ( B )

PicsArt_1369338646646.jpgPicsArt_1369338810769.jpgPicsArt_1369343031169.jpg( C ) ( D )

( E ) ( F )

20130514_114631.jpgIMG-20130523-WA0034.jpg

( G ) ( H )

Figure(4): Metaphase spread of ethosuximidetraeated group showing,centric separation(A) ,Ring (B),deletion (C), a centric fragment(D), break(E), iso-gap (F).,cntric fusion(G), hypopliody(H) .

Effect of nifedipine and ethosuximide on testis weight . There are no changes could be recorded in the mean weight of the testis in all treated groups as compared to the control group ( Table (3), Figur(4)).

Table 3 : Effect of nifedipine and ethosuximide on testisweight .

Mean / gm

Group

0.11

Control

0.12

Nifedipine

1.12

Ethosuximide

Figure(4): Effect of nifedipine and ethosuximide on testis weight

Effect of nifedipine and ethosuximide on sperm abnormalities : Table (4) represents the percentages of abnormal sperm shape in nifedipine and ethosuximide treated groups . These percentages are increased in both treated groups as compared to control group . The percents of sperm shape changes under the effect of nifedipine were more than that recorded with ethosuximide . Broken sperms were found to be the most frequent type of sperm abnormalities . Fig (4&5)

%

Total

sperm abnormalities

Groups

Tail

Head

Broken sperm

6.16

37

3

7

27

Control

20

120

43

25

52

Nifedipine

10.16

61

11

24

26

Ethosuximide

Table (4) : Effect of nifedipine and ethosuximide on sperm shape abnormalities.

Figure(4):Effect of nifedipine and ethosuximide on sperm shape abnormalities.

20130520_110207-1.jpg

(A)

20130520_110724-1.jpg20130520_111952-1.jpg20130516_152414-1.jpg20130512_143004-1.jpg ( I ) ( I I )

( III ) ( IV )

20130520_115208-1.jpg ( B )

PicsArt_1369342623934.jpg

( D)

IMG-20130520-WA0007.jpgIMG-20130520-WA0010.jpg

( I ) ( II )

20130520_110028-1.jpgIMG-20130520-WA0005.jpg

( III ) ( IV )

( D )

Figure(5): Sperm morphology of control and treated groups showing : normal sperm shape (A),abnormal head (B), abnormal tail sperm (c), broken sperm and broken sperm(D) .

Discussion:

The primary goal of the present studies was to prospectively address the question ,Do Ca+2 channel blockers used as therapeutics against hypertension and epilepsy induce deleterious side effects on reproductive system and genome that could contribute to male sterility . This study was designed to fined answer for this question by utilizing exposure of male mice tonifedipine and ethosuximide at dosages comparable to those used for human therapy .

Oral treatment of nifedipine and ethosuximide induced high frequencies of numerical and structural chromosomal aberrations in bone morrow cells of treated mice ascompared to control group . Our result is in agreement with that of(1973)schmid who reported that chemicals in general produce aberrations in rodents.

Sperm are known to be vulnerable to oxidative damage during spermatogenesis and epididymal storage (2006)Agarwal et al.. Decreased in sperm count and motility were recorded , as resulting in peroxidation of sperm lipid membrane.In addition, decreased in anti- oxidant enzyme were reported to be linked with sperm apoptosis(. 2003 ) Agarwal et al.

The present study revealed that bothnifedipineandethosuximide caused high frequencies of sperm abnormalities . These findings are overall in line with(1997) katsoff et al.,( 2005)Robinson et al and (2009) Morakinyo et al..They regard to male sterility caused by Ca+2 channel blockers .Human sperms from patients treated bynifedipine fail to undergo acrosome reaction and fertilization. (2000)Son et al.

Several studies indicated that, both T- and L type Ca+2 channel blockers play an adverse role in normal spermatogenesis contributing to mal sterility (1997)katsoff& check , (. 2009) Ayodele et al, (2011) lee et al., ( 2011)Morakinyo et al.

Successful spermatogenesis and steroidogenesis are dependent on support from somatic cells in testis FSH and LH exerts their action on steroidogenesisby regulating intracellularCa+2 concentration through VDCCs in Sertoli cells and Leyding cells(2000)Rommerts et al.,& ( 2007) Costa et al. Thus any malformation of Ca+2 channels in somatic cells may result in failure of spermatogenesis and steroidogenesis

Inconclusions:Both T- and L- type Ca+2 channel blockers reduce fertility and induced mutaginicitythrough increasing sperm abnormalities and chromosomal aberrations level.

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2-(2006) Agarwal A , Sharma RK , Nallella KP , Thomas AJ Jr , Alvarez JG , Sikka SC: Reactive oxygen species as an independent marker of male factor infertility . FertilSteril .86 (4) 878- 85 .

3- (1993) Adeoya-Osiguwa&Fraser LR. A biphasic pattern of 45 Ca+2 uptake by mouse spermatozoa in vitro correlateswith changing functional potential . J Reprod Fertil.99(1):187-94.

4-(2011) Ayodele, O.;Morakinyo O. andBolanle, L .R; and Olufeyisipe, A .D ;:Calcium antagonists modulate oxidative stress and acrosomal reaction in rat spermatozoa. Archives of Medical Science 613-618 .

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6-(1464)Godfraind T . Antioxidant effect and therapeutic mode of action of calcium channel blockers in hypertension and atherosclerosis. Philos Trans R SocLond B Biol Sci.2005: Dec 29;360:2259-72 .

7- (1997) Goodwin , L . O ; Leeds , N. B ; Hurley , I . A ; Mandel , F . S ;Peryolizzi , R . G ; and Benoff , S . U . : Isolation and characterization of the primary structure of testis-specific L-type Calcium channel : implications for contraception . Molecur . Human Reproduction 5: 311-22 .

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calcium channels in male germ cells. Reproduction.123:203-15.

9-( 1997 ) Katsoff , D ; and check , J .H: A challenge to the concept that the use of calcium channel blockers causes reversible male infertility . Human reproduction 1480-1482.

10-(2011 ) Lee , J . H ;Ahn , H . J ; Lee , S .J ; Gye , M . C ; and Min , C . K : Effectof L- and T-type Ca channel blockers on spermatogenesis and steroidogenesis in the prepubertal mouse testis . J . Assist .Report .Genet . 28:23-30.

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12- ( 2009 ) Morakinyo , A . O ;Iranloye , B . O ; and Adegoke , O . A: Anti reproductive effect of calcium channel blockers on male rats . Reprod .Med .Biol . 8(3) : 97-102 .

13-( 2011 ) Morakinyo , A . O ;Iranloye , B . O ;Darmola , O . A ; and Adegoke , O . A: Antifertiliy effect of calcium channel blockers on male rats : association with oxidation stress . Advance in medical sciences . 95-105.

14-(1987) Park 2003 15- Preston , R . J ; Dean , B. D ; Galloway , S. ; Holden , H . ; Mcfec , A . F ; and shelly , M: Mammalian in vivo cytogenetic assays : analysis of chromosome aberration in bone marrow cells .mut .Res 189 :157-165 16-Robinson 2005

17-(2000) RommrtsFF,Lyng FM, vonLedebur E, Quinlan L,JoessGR,Warchol JB, et al: Calcium confusion –is the variability in calcum response by sertoil cells to specifusion hormones meaningfui or simply redunda? JEndocrinol.167:1-5.

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19-(2002) Son WY ,Han CT, Lee JH, Jung KY, Lee HM, Choo YK. :Dcvclopmcntalexprcssion Patterns of alpha1H T-typc ca2+ channels during spermatogenesis and organogenesis in mice. Dev Growth Differ.;44:181-90

20-(1992) Steele, G.L . , leung ,P.C: .Intragonadalsignalling mechanisms in the control of steroid hormone production . J Steroid Biochem Mo Biol.;41:515-22.

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22-(2000): Wennmuth G. , Westenbroek R.E. , Xu T, Hille B , Babcock DF.( :CaV2.2 and CaV2.3 (N- AND R-type ) Ca2+ channels in depolarization-evoked entry of Ca2+ into mouse sperm . J Biol Chem .(28):21210-7.

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التأثير السمي الوراثي لمثبطات الكالسيوم على ذكور

الفئران البيضاء

تلعب ايونات الكالسيوم دورا مهما وحيويا في العديد من الوظائف الحيوية مثل: التفاعلات الإنزيمية ,النشاط الكهربي للإفرازات الخلوية ,التخثر,ايض العظام العمليات الوظيفية للحيوانات المنوية 0 أدوية مثبطات الكالسيوم تمنع تقلص العضلات أثناء إزالة الاستقطاب بالإضافة إلى توسيع الأوعية الدموية عن طريق منع دخول ايونات الكالسيوم 0تستخدم هذه الأدوية في علاج العديد من الأمراض مثل ضغط الدم, الصداع النصفي والأزمات الصدرية 0نظرا لقلة المعلومات المتاحة عن تأثير هذه الأدوية على الخصوبة والجهاز الوراثي :تهدف هذه الدراسة إلى معرفة الآثار الجانبية التي تسببها هذه الأدوية على الجهاز الوراثي ومعدل الخصوبة في الذكور0 تم معالجة ذكور الفئران بجرعات 40 و100 مج / كجم من وزن الجسم من أدوية نيفيدبين وايثوسيكساميد عن طرق الفم لمدة 15 يوم لمعرفة 0تم إجراء تجارب لفحص نسبة التحورات الكروموسومية وتشوهات الحيوانات المنوية 0 أحدثت هذه المجموعة من الأدوية ارتفاع ملحوظ في نسبة التشوهات الكروموسومية والحيوانات المنوية مما يعطى دليل منطقي للتأثير الضار لهذه الأدوية على كل من الجهاز الوراثي ومعدل الخصوبة عند ذكور الفئران 0

التأثير السمي الوراثي لمثبطات الكالسيوم على ذكور

الفئران البيضاء

مقدم من الطالبات:

  • أسماء الحربي - هديل الحبيب
  • إيمان الحليس - بشائر الثقفي
  • سماهر القرشي - رؤيا الهوساوي
  • رهام العامري - أماني عجاج

باشراف:

د/ رشا علي عليوة

بحث مشروع تخرج مقدم لقسم الاحياء كلية العلوم التطبيقية 2013

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