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Prenatal phenobarbital exposure induced developmental changes in rat brain and muscle.

Introduction

Seizure incidence during the neonatal period is higher than any other period in the lifespan [1], yet there is little knowledge about this period in terms of the effect of seizures or of the drugs used in their treatment. The fact that several antiepileptic drugs (AEDs) [2] induce pronounced apoptotic neuronal death in specific regions of the immature brain prompts a search for AEDs that may be devoid of this action [3]. Phenobarbital (previously known as phenobarbitone in the UK) belongs to a group of medicines called barbiturates. It is used to treat epilepsy and works by stabilising electrical activity in the brain [4].

The brain and nerves are made up of many nerve cells that communicate with each other through electrical signals. These signals must be carefully regulated for the brain and nerves to function properly. When abnormally rapid and repetitive electrical signals are released in the brain, the brain becomes over-stimulated and normal function is disturbed. This can result in fits or seizures [5]. Neurotransmitters are chemicals that are stored in nerve cells and are involved in transmting messages between the nerve cells. GABA is a neurotransmitter that acts as a natural 'nerve-calming' agent. It helps keep the nerve activity in the brain in balance. Glutamate is a neurotransmitter that acts as a natural 'nerve-exciting' agent. It is released when electrical signals build up in nerve cells and subsequently excites more nerve cells. It is thought to play a key role in causing epileptic seizures [7].

Phenobarbital increases the activity of GABA and decreases the activity of glutamate in the brain. These actions help stabilise the electrical activity in the brain and prevent epileptic fits. Phenobarbital prevents epileptic fits by preventing the excessive electrical activity in the brain. It is thought to achieve this by affecting certain neurotransmitters in the brain [4].

The placenta offers no significant barrier to the passage of barbiturates to the foetus, Thus, these drugs become widely distributed in feotal tissues when consumed by dam during pregnancy [8] although the majority of children born to women with epilepsy are normal, they are at increased risk for malformations as well as for poor neuropsychological outcomes [9]. The risk of in utero exposure has to be measured against the risk of the underlying disease. Thus, understanding the magnitude and differential effects of AEDs on teratogenesis is important. Our studies are aimed at both of these issues. phenobarbital, induced substantial regionally specific cell death, In vitro study [10] has demonstrated that the secretion of testicular testestrone is in response to the stimulatory action of gonadotrophins. The release of these gonadotrophins from the adenohypophysis has been shown to be in response to stimulation of gland by hypothalamic luteinizing hormone releasing factor.

Since Phenobarbital diffuses to all parts of the central nervous system. It is possible that the neurons of the hypothalamus may be destroyed by this drug. Neuronal losses in the hypothalamus will dysrupt the hypothalamus-pituitary-testis regulatory axis which result in loss of or considerable reduction in the testosteron synthesis ability of testicular interstitial cells of Leydig. In the early 1940s, Albright and Reifenstein were among the first to refer to the antiosteoporotic and anabolic properties of androgens. Testosteron has an anabolic effect and stimulate the growth of muscle [11]. Several studies have confirmed the testosterone increase muscle mass [12]. Strength and endurance, other studies have reported even more specific effectsof testosterone in skeletal muscle [13]. The levator ani muscle, for example disappear during development of female rats, but can be maintained with testosterone administration.

The aim of this review is to address the question whether Phenobarbital can damage the brain cells and subsequent affect on the androgen and how androgens may affect muscle strength and provide protection for muscle growth.

Materials and Methods

Adult wistar rats of albino strain weighing 200 gms obtained from Animal house of the college of Urmia Veterinary school. Sixteen adult female rats and four adult male rats were used as breeding stock in this experiment. The rats were housed in standard cages, with 4 female and 1 male to a cage A commercially prepared diet and clean drinking water were provided adlibitum. The female rats were divided into control and treated groups. The treated groups divided into two subgroups, were given 3g of phenobarbital with their feed in group1 and 4g of phenobarbital per kg of diet in group 2 from day 12 to 18 of the pregnancy (vaginal plug = 1 day). This period was chosen based on earlier observation that the development of muscle and innervation of muscle fibers are completed on day 16th of gestation. From group 1 and group 2 females, a total of 24 male offsprings were randomly selected and equally allocated into 2 groups and constituted the prenatally phenobarbital exposed males. All the selected rats were maintained on commercially prepared diet. At 12 weeks of age the rats in all groups were sacrificed with CO2. The body weight of each rat was determined at 3 and 12. The Biceps brachii, Triceps brachii, Soleus, Tibialis, Gastronemius muscle were disected and weighed. The brains were collected from both the groups and were subjected to histological study after staining the paraffin sections by haematoxylin and eosin stains. Serum Testestron and estrogen was measured by a ELISA technique.

Statistical Analysis:

The data obtained for the muscle were subjected to analysis of variance using F-ratio and Duncan s New Multiple Range.

Results and Discussions

Hormonal Results:

The result of hormonal analysis is shown in Table 1. The normal range Testestron 5.4 and estrogen was 2,4ug/ml. The result of hormonal changes in different groups showed, marked suppression of serum Testosteron to less than 60% of baseline values was a consistent and highly significant predicator of androgen concentration (reduced to 2-7% that normal levels) and there was a correlation between the testosterone and estrogen deficiency.

Muscles:

The comparison of the absolute muscle weights of control and Phenobarbital exposed male rats showed significant differences between the groups. Comparison revealed that the absolute weights of soleus, tibialis cranialis and gastronemius muscles of the control males were significantly superior to those of the Phenobarbital exposed groups (P<0.01). There were no significant findings between the absolute weight of the two treatment groups (p>0.05). The absolute weight of triceps brachii muscle of control males was significantly superior to that of the Phenobarbital exposed (P< 0.05) (Table 2). In this study pair of groups were compared using student "t" test. The results showed that, tibialis cranialis, (p<0.05) gastrocnemius (p<0.01) of control males weighed heavier than those of Phenobarbital-exposed males but the other muscles were no significant (p>0.05) (Table 3). The absolute muscle weights of control male rat was heavier than those of male rat in groups 1 and 2, triceps brachii (p<0.05), tibialis cranialis (p<0.01) gastrocnemius(p<0.01). This study also demonstrated the muscle mass indices of muscles were significant except bicep brachii. Histological examination of the muscle showed muscle fiber degeneration and regeneration. Some muscle fibers were normal in size and dark pink. Others, which were degenerating, were smaller and lighter pink. They were sometimes vacuolated and had central nuclei rather than the normal peripheral nuclei. Small fibers with a bluish tinge and enlarged nuclei were regenerating fibers. Some of the smaller fibers were cut into smaller pieces (fig.5).

Brain:

Phenobarbital caused widespread apoptotic neurodegeneration in the brains of rats (Fig. 1). The microscopic examination of brain revealed various degree of neuron degeneration and necrosis to hemorrhages in different parts of the brain in phenobarbital exposed rats( Fig. 2).The area of necrosis in some area of the brain was large enough to see as vacuole(Fig. 4) included foci of hemorrhages accompanied with severe vasogenic and cytotoxic edema (Fig. 3), rarefaction and necrosis was observed in the cortical and medullary areas of brain hemisphere. The deteriorated regions were surrounded by gliosis were predominantly located in the white matter of the medullary layer more on the white mater (Fig. 1). Hemorrhages were visible either within the necrotic areas or were restricted to areas surrounding the cystic necrosis (fig. 3).

[FIGURE 1 OMITTED]

[FIGURE 2 OMITTED]

[FIGURE 3 OMITTED]

[FIGURE 4 OMITTED]

[FIGURE 5 OMITTED]

Discussion:

Althogh There are a few reports about antiepileptic drug induced congenital malformations [1,14] but none of these authors reported brain and muscle anomalies induced by phenobarbital. Here we report that Phenobarbital one of the major AEDs cause sensitive neurons to undergo apoptotic death in the developing rat forebrain. These findings apply to compounds that block voltage-gated sodium channels, enhance GABA ergic inhibition, or block glutamatemediated excitation. Neurotoxicity of AEDs is age-dependent and is associated with impairment of neurotrophin--mediated, survivalpromoting signals in the brain [7]. The combination of AEDs with different modes of action results in a substantially higher apoptotic response compared with monotherapy.

The primary pathological lesion that was observed was congestion and edema in the brain. It may be possible that via [Na.sup.+] channels, it affects the osmolarity of blood vessels, which interacts with various factors responsible at the time of growth and development of the affected fetuses. This activity explains the mechanism of cellular toxicity of treated rat brains. Programmed cell death (apoptosis), in contrast to necrosis, is characterized by uniform internucleosomal DNA fragmentation, nuclear shrinkage, chromatin compaction as well as by cytoplasmatic condensation, and disintegration [15-16].

Numerous studies have provided evidence of an association between antiepileptic drugs (AEDs) and muscle in persons treated with. As well, numerous biochemical abnormalities have been described including hypocalcemia, hypophosphatemia, reduced levels of biologically active vitamin D metabolites, hyperparathyroidism [17]. However few studies have evaluated the affect of Phenobarbital on androgen and muscle in rat.

Serum concentrations of testosteron and estrogen during the postoperative period was much lower than basal line. All of the serum hormonal concentrations were significantly affected by the operation. In this retrospective analysis, we demonstrated a significant association between lower serum level of androgen and brain damage in male rats.

Reduction in muscle mass of males prenatally exposed to Phenobarbital was reported by [18]. The smaller muscle mass of the phenobarbital-exposed group as observed from the analysis of the soleus muscle was due to a smaller number of muscle fibres being present than in the control group, since the muscle fibre sizes were similar in both groups. This indicated that prenatal administration of phenobarbital inhibits normal hyperplasia of muscle fibres. This reduction may be attributed to loss of influence of testosterone on muscles. The reduction in the muscle mass may have resulted hypothalamic neuronal losses in the prenatally--phenobarbital exposed mice. The histopathological study of the brain showed neuronal losses and vacuolation in brain tissues, [19] reported neuron necrosis in different region of the brain in mice. It was assumed that the prenatal exposure of rat to Phenobarbital may have resulted the destruction of neurons at hypothalamic levels, hence disrupting the hypothalamus--pituitary testis axis regulatory mechanism. Impairment in the production of the releasing factor by the hypothalamus, may adversely affect the testosterone--synthesizing ability of the the interstisial cells of Leydig whose function has been shown to be dependent on the stimulation by pituitary interstitial cell stimulating hormone [20]. Thus it is concluded that the clinicians should very carefully justify and prescribe the therapy of Phenobarbital especially to pregnant women.

References

[1.] Morrei, M.J., 1996. The new antiepileptic drugs and women: efficacy, reproductive health, pregnancy and fetal outcome. Epilepsia, 37(34): 2013-44.

[2.] Giselon, L.G., C.R. Curtin, L.D. Kramer, 1994. The steady state (SS) pharmacokinetics (PK) of phenytoin (Dilantin) and Topiramate (Topamax) in epileptic patients on monotherapy and during combination therapy Epilepsia, 35: 54.

[3.] Hauck, L. and D. Quinn, Minor anomalies accompanying prenatal exposure to topiramate. Epilepsia, 56: 115.

[4.] Kwan, P., M.J. Brodie, 2004. Phenobarbital for the treatment of epilepsy in the 21st century, a critical review. Epilepsi, 45(9): 1141-1149.

[5.] Kim, J.S., A. Kondratyev, Y. Tomita, K. Gale, 2007. Neurodevelopmental impact of antiepileptic drugs and seizures in the immature brain. Epilepsia, 19-28.

[6.] Wu, W.N., J.B. Hubner, A.J. Streeter, 1994. Evaluation of absorption, excretion, pharmacokinetics and metabolism of the anticonvulsant, topiramate in healthy men. Pharm. Res., 11: 336.

[7.] Udensi, M., C. Emeruwa and N. Daniel, 2001. Retardation of muscle growth in prenatally Phenobarbital exposed male mice: Evidence for disruption of Hypothalamus-Pituitary--Testis regulatory Axis. Philipp. J. Vet. Med., 38: 85-91.

[8.] Booth, D., D.J. Evans, 2004. Aniconvulsant for neonate. Cochrane Database of Systematic Reviews (3). Retrieved on 2006-09-06.

[9.] Warren, G.B., M.D. Houslay, J.C. Metcalfe & N.J.M. Birdsall, 1975. Nature (London), 255: 684-687.

[10.] Urban, R.J., 1999. Effects of testosterone and growth hormone on muscle function. J. Lab. Clin. Med., 136: 7-10.

[11.] Hikim, I.S., J. Artaza, L. Woodhouse, 2002. Testosterone-induced increase in muscle size in healthy young men is associated with muscle fiber hypertrophy. Am. J. Physiol. Endocrinol., 283: 154-164.

[12.] Nnodim, J.O., 1999. Quantitative study of the effects of denervation and castration on the levator ani muscle of the rat. Anat Rec., 255: 324-333.

[13.] Glauser, T.A., 1999. Topiramate. Epilepsia, 40: 71-80.

[14.] Ketter, T.A., 1999. Metabolism and excretion of mood stabilizers and newer anticonvulsan Cell Mol. Neurobiol., 19(511): 53.

[15.] Bredesen, D.E., 1995. Neural apoptosis. Ann Neurol., 38: 839-851.

[16.] Alison, M., 2007. Aniepileptic drugs and bone disease. Clinical review in Bone and Mineral

[17.] Metabolism, 2: 159-165.

[18.] Ihemelandu, E.C., 1993. Effect of maternal Phenobarbital consumption on muscle development in mice. ACTA Anatomica, 148: 22-26.

[19.] Yanai, J. and Bergman, 1981. Neuronal deficits in mice after neonatal exposure to Phenobarbital. A mental neurology experi., 199: 208-73l.

[20.] Swerdloff, R.S., P.C. Walsh and W.P. Odell, 1972. Control of leutenizing and follicle stimulating hormo Riggs BC, Khosla S, Melton LJ. 2002 Sex steroids and the construction and conservation of the adult skeleton. Endocr Rev., 23: 279-302. nes secretion in the male.Steroids, 20: 13-22.

(1) Ali Asghar Tehrani, (2) Maryam Faramarzpr, (3) Navid Hosseini Mansoub and (4) Afsaneh Azhari

(1) Department of Pathobiology. Faculty of Veterinary Medicine, Urmia University, Iran.

(2) Department, of Biology, Faculty of Science, University of Payame Noor, Esfehan, Iran.

(3) Islamic Azad University, Maragheh Branch, Maragheh, Iran.

(4) Department of Pharmacology, Faculty of Medicine, Marmara University, Turkey.

Corresponding Author

Navid Hosseini Mansoub, Islamic Azad University, Maragheh Branch, Maragheh, Iran.

E-mail: n.h.mansoub@gmail.com
Table 1. Comparison of hormonal changes in two groups.

Hormonal Assay   Treatments

                 T1-Control                T2

Testosterone     4.60 [+ or -] 0.499 (a)   3.60 [+ or -] 0.168 (b)
  (ug/ml)
17-B Estradiol   2.70 [+ or -] 0.213 (a)   233 [+ or -] 0.122 (b)
  (ug/ml)

Hormonal Assay   Treatments

                 T3- Test              Significance

Testosterone     2.80 [+ or -] 0.168   **
  (ug/ml)
17-B Estradiol   2.3 [+ or -] 0.422
  (ug/ml)

(ab): Values in the same row and variable with no common
superscript differ significantly. * : P<0.05, : P<0.01, NS: Not
Significant. (1) Values are means of nine observations per
treatment and mean [+ or -] S.E giveu for each measurement. (2)
T1 = control males, T2 = group I, T3 = group II

Table 2. Comparison Of Body Weights (G) Of Three Groups Of Male
Rats Of Phenobarbital-Exposed And Control, In Age Of 3 And 12
Week

Groups                               Age

               3 Weeks                  12 Weeks

T1 Control     58.5 [+ or -] 0.29 (a)   196.25 [+ or -] 0.05 (a)
T2- PhB        44.8 [+ or -] 0.22 (b)   166.53 [+ or -] 0.04 (b)
T3             54.1 [+ or -] 0.22 (b)   160.18 [+ or -] 0.05 (b)
SEM            0.245                    0.044
Significance   *                        **

(ab): Values in the same row and variable with no common
superscript differ significantly . * : P<0.05, **: P<0.01, NS:
Not Significant. (1) Values are means of six observations per
treatment and their pooled SEM. (2) T1 = control males, T2 =
PhBgroup 2,T3 = Phbgroup 3

Table 3. Comparison of muscle weights (mg) of three groups of
male rats of control, phenobarbital-exposed, in age of 12 week.

Groups               Treatments

                     T1-Control                T2-phB

Biceps brachii       0.189 [+ or -] 0.78 (a)   0.206 [+ or -] 0.84 (a)
Triceps brachii      0.468 [+ or -] 0.06 (a)   0.244 [+ or -] 0.05 (b)
Soleus               0.115 [+ or -] 0.02 (a)   0.025 [+ or -] 0.01 (b)
Tibialis cranialis   0.433 [+ or -] 0.02 (a)   0.246 [+ or -] 0.04 (b)
Gastrocnemius        0.624 [+ or -] 0.31 (a)   0.341 [+ or -] 0.34 (b)

Groups               Treatments

                     T3                        SEM     Significance

Biceps brachii       0.080 [+ or -] 0.81 (a)   0.724   NS
Triceps brachii      0.212 [+ or -] 0.09 (b)   0.070   *
Soleus               0.028 [+ or -] 0.01 (b)   0.010   **
Tibialis cranialis   0.232 [+ or -] 0.02 (b)   0.028   **
Gastrocnemius        0.322 [+ or -] 0.32 (b)   0.322   **

(ab): Values in the same row and variable with no common
superscript differ significantly . * : P<0.05, **: P<0.01, NS:
Not Significant. (1) Values are means of six observations per
treatment and their pooled SEM. Mean [+ or -] S.E giveu for each
measurement. (2) T1 = control males, T2 = PhB, phenobarbital
group 1., T3 = Phb group2
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Title Annotation:Original Article
Author:Tehrani, Ali Asghar; Faramarzpr, Maryam; Mansoub, Navid Hosseini; Azhari, Afsaneh
Publication:Advances in Environmental Biology
Article Type:Report
Geographic Code:7IRAN
Date:Aug 1, 2011
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