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Efectos de bajas dosis de fenitoina en el plexo coroide de fetos de rata. Estudio cariometrico.

RESUMEN: El objetivo del presente trabajo fue caracterizar cariometricamente las alteraciones en el epitelio de los plexos corioides, provocadas por bajas dosis de fenitoina, cuando fue administrado en el 9[grados], 10[grados], 11[grados] dia de prenez, durante la organogenesis.

El estudio cariometrico demostro que los animales tratados con fenitoina, poseian nucleos mas pequenos y sin alteracion de forma. Los resultados obtenidos sugieren que la fenitoina actua en la embriogenesis provocando alteraciones del desarrollo en los plexos coroides.

PALABRAS CLAVE: 1. Cariometria; 2. Fenitoina; 3. Teratogenesis; 4. Plexo coroide. 5. Rata.

EFFECTS OF LOW-DOSE PHENYTOIN ADMINISTERED TO RATS FETUSES ON DEVELOPING CHOROID PLEXUS. KARIOMETRIC STUDY

SUMMARY: The aim of the present study was to determine, by kariometric parameters, the influence of phenytoin, as teratogen, on the epithelial layer of choroid plexus in rats fetuses, using single daily dose (75 mg Kg-1 bd.wt.), during gestation GD9 to GD 11, and lowest total dose used orally (225 mg Kg-1), to the best of our knowledge. In some of experimental studies in animals, the anticonvulsant drug phenytoin were administered in embriotoxic daily doses (100-1500 mg Kg-1 bd.wt.) increasing concentrations up to 20 times the human therapeutic plasma concentration. There is a significant lack of information regarding the embriotoxicity in pregnancy of these drug, phenytoin, in low doses.

Phenytoin was administered in a single daily dose (75 mg Kg-1 bd.wt.) to pregnanty rats in GD9 to GD11 days of gestation, during organogenesis. Histological sections were obtained for analysis of nuclear alterations in the cuboidal epithelium of choroid plexuses of lateral ventricles in the rat fetuses. Eleven kariometric parameters were measured in each of the nuclei. Statistical comparison were made with Mann-Whitney's test.

The nuclei of the phenytoin group showed significant reduction of the size parameters : longest axis, mean axis, nuclear volume, nuclear area, nuclear perimeter, ratio of the longest axis to the shortest axis, ratio of the nuclear volume to the nucleararea) but not in shortest axis and shape parameters (contour index, shape factor and eccentricity).

A distinctive pattern of nuclear abnormalities in choroid plexus ephitelium of rat fetuses is associated with the use of low dose of phenytoin during pregnancy. Variations in nuclear size might reflect fundamental nuclear alterations of significance during the process of embriogenesis and could represent teratogenic influence of phenytoin in rats. Even at low dosage and short period of use during gestation, phenytoin can induce embriotoxicity.

KEYWORDS: 1. Kariometry 2. Phenytoin 3. Teratogenesis 4. Choroid plexus 5. Rat.

INTRODUCTION

Phenytoin is a drug syntetized by Biltz, 1908 (Rall & Schleifer, 1991) which was introduced as anticonvulsant drug in 1938 (Merrit & Putnam, 1938). Its teratogenic potencial was recognized by some authors, including the Federal and Drug Administration (FDA) in United States. (Teratology Society Public Affairs Committee, 1994; Alvan et al., 1995; International League Against Epilepsy, 1993). Hanson & Smith (1975) also described the fetal hydantoin syndrome that includes: intra and extrauterine growth retardation, mental impairment, congenital heart lesions, facial dysmorphisms and limb anomalies. While all above are nonspecific malformations, distal digital and nail hypoplasias were considered malformations related to phenytoin (Holmes et al., 2001).

Kariometric parameters, such nuclear areas and volumes, nuclear perimeter, shape factor, contour index and others, have been used by some authors, especially, in carcinogenesis studies and morphometric studies about embriotoxicity of drugs, chemical and physical agents (Lopes-Paulo, 2002; Sala et al., 1994; Keep & Jones, 1990; Ferreira et al., 1967).

The aim of the present study was to determine, by kariometric parameters, the influence of phenytoin, as teratogen, on the epithelial layer of choroid plexus in rats fetuses, with lowest total dose used (225 mg Kg-l), to the best of our knowledge (Table I), using a single daily dose (75 mg Kg-1 bd.wt.) on days GD9 to GD11.

MATERIAL AND METHOD

Female Wistar rats (Rattus norvegicus) (N=10) (Kalter, 1974), mean weight 205g, were kept at a constant 12-hour-day/night cycle, with free access to a standard pellet diet (Purina) and tap water. Males were mated with females overnight and females examined the following morning for the presence of sperm in the vaginal smear. The day sperm was detected was considered to be day one of gestation (GD1).

Animals were treated orally (by gavage) with single daily dose (75 mg Kg-1 bd.wt.) of phenytoin (Epelin, Ache Laboratorios, Sao Paulo, Brasil) on days GD9 to GD11 of gestation. Control animals received the equivalent amount of saline solution. Rats were killed by anesthetic ether inhalation on GD20 and the fetuses removed. For the purposes of this study, five complete litters of each group were fixed in a solution of ethanol 80% (85 ml), formol (10 ml), and glacial acetic acid (5 ml). After processing, fetus heads were separated from bodies and embedded in paraffin. Histological serial sections were done in coronal plane, from eye level to 2 mm behind the eyes. The sections, 6 [micro]m thick, were stained with haematoxylin and eosin technique.

Five fetuses in each group were chosen at random. Under the microscope, were examined 50 nuclei in each one of the five fetuses in the group that received phenytoin (total = 250 nuclei) and examined 50 nuclei in each one of the five fetuses of the controlled group (total= 250 nuclei). Each nuclei was projected by optical microscope (H500 Hund Wetzlar) with a camera lucida (Leitz Wetzlar-Germany) at a magnification of x1000, and drawn with a soft pencil. On ellipsoid nuclei, two perpendicular axis were measured : longest axis and shortest axis. Then, the following nuclear parameters were calculated, according to the methods described by Sala et al.: mean diameter, ratio larger diameter to shorter diameter, perimeter, area, volume, ratio volume to area, coefficient of shape, contour index and eccentricity. Statistical comparison between the treated with phenytoin group and the control group were performed using the non-parametric U Mann-Whitney test. (Siegel, 1975a; Siegel, 1975b; Conover, 1999).

RESULTS

Mean fetal weight was 3,48 g for the control group and 2,78 g for the treated group (p< 0.01). No clinical difference was noted between the two groups.

Histopathological analysis revealed that the choroid plexus epithelium was thinner in the treated group fetuses, constituted by cells with picnotic nuclei with condensed material, thinner basal lamina and poor intersticial estroma inside the choroid vilosities. (Figs. 1a, b).

[FIGURE 1 OMITTED]

Karyometric study showed median values of each nuclear parameter determined in the two studied groups (250 nuclei in each group), as well as the statistical comparison are show in Table II.

Significant differences between the group that received phenytoin and the control group were noted in 7 of the 11 nuclear parameters studied. The longest axis of nuclei of epithelial cuboidal cells of the choroid plexuses, in animals treated with phenytoin, showed significant decrease in size, significant decrease of nuclear volume, area and perimeter and a reduction in mean axis, ratio of the longest axis to the shortest axis and of the ratio of nuclear volume to nuclear area.

There were no significant differences in the shortest axis, shape factor, contour index or eccentricity between the two studied groups. The results obtained demonstrated that there were no significant alterations in the parameters that assess the nuclear shape: contour index, shape factor and eccentricity (Fig.1a, b). On the other hand, the results showed that all parameters associated with nuclear size were significantly diminished (except the shortest axis) in the animals that were given phenytoin (Fig. 1a, b).

The present study allows us to claim that even the use of low dose of phenytoin during gestation could induce changes in nuclei of epithelial cuboidal cells of the choroid plexuses of lateral ventricles in rat fetuses.

DISCUSSION

The results obtained demonstrate that after prenatal exposure to phenytoin, despite the absence of significant alterations in nuclear shape, there were changes in nuclear size in the cuboidal epithelium of lateral ventricles in rat fetuses, reflected mainly through reduction in longest axis followed by reduction in other nuclear size parameters.

Other authors have used some or all the nuclear parameters studied in the present investigation, mainly to investigate neoplastic alterations, where the nuclear parameters of shape frequently registered more alterations than the nuclear parameters of size (Mandarim-de-Lacerda, 1999; Rich & Reade, 1996; Setala et al., 1997; Kronqvist et al., 1997; Kirillov & Iuschenko, 1996), kariometric evaluation of drug influence during pregnancy (Gomes Totti et al., 2001; Dechichi et al., 1997; Espiridiao et al., 1996).

Phenytoin continue to be widely used in the treatment of epilepsy. This drug have important shortcomings such asa highly variable and nonlinear pharmacokinetics, a narrow therapeutic index, suboptimal response rates, and a propensity to cause significant adverse effects and drug interactions. Its primary mechanism of action is modulation of the sustained repetitive firing of neurones by direct inhibition and blockage of voltage-gated sodium channels in the neuronal cell membrane, and by delay of cellular reactivation. The plasma protein binding of phenytoin is normally between 90% and 95%. The drug is rapidly distributed from the blood to the tissues and is almost completely metabolized in the liver. The therapeutic serum concentration of phenytoin is considered to be 10-20 [micro]g ml-1 and normally reaches the steady-state level within 1-2 weeks. The half-life of phenytoin is less than 20h in low doses, but is prolonged in high doses, newborn infants and elderly people (Iivanainen, 1998). Phenytoin is ah effective anticonvulsant, but high serum phenytoin concentrations may be associated with serious toxicity (Kozer et al., 2002). Phenytoin is eliminated almost entirely by hepatic oxidation. The principle enzymes responsible are cytochrome P450 (CYP)2C9 and CYP2C 19. CYP2C9 is saturated by therapeutic doses of phenytoin, and at steady state both enzymes are probably operant in most people (Bachmann & Belloto, 1999). The nonlinear pharmacokinetics of phenytoin make it a difficult drug for which to establish safe and effective administration regimens (Battino et al., 1995). Phenytoin's fetal adverse effect is related to its membrane stabilizing pharmacological properties (blockage of voltage-dependent ion channels). During a restricted sensitive period, this results in induction of concentration-dependent bradyarrhythmia in the embryo and episodes of hypoxia/reoxygenation (Azarbayjani & Danielsson, 1998).

The choroid plexus are vascular structures that lie in lateral ventricles of the brain. They are formed by a central arteriole surrounded by a simple cuboidal epithelium, that faces on one side to the central arteriole and on another side to the cavity of the ventricle. This specialized epithelium has a secretory function: active production of cerebrospinal fluid (CSF) and remains asa differentiated epithelium from primitive neural tube. In this study we considered the choroid plexus as the interface between blood and CSF, thus a structural blood-CSF barrier (Dohrmann, 1970; Cserr, 1971).

A teratogen is defined as any substance present during embrionic or fetal life capable of inducing abnormal postnatal structure or function (Dicke, 1989). However, animal teratology may not be a reliable predictor of human teratogenicity (Palmieri & Canger, 2002) and there is a significant lack of information regarding the teratogenic profile of these drug, phenytoin, in terapeutic concentrations (Ohmori et al., 1997).

In summary, the reduction of nuclear size in the fetuses exposed to phenytoin may reflect significant changes in nuclear material during the process of embriogenesis, induced by the presence of the phenytoin, originally, in low experimental used dosage. Therefore, the measurement of kariometric parameters can be used to assess teratological alterations in the cuboidal epithelium of choroid plexus in rats induced by teratogens and may be of human relevance.

Received: 28-09-2003

Accepted: 30-10-2003

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* M. J. Goes & ** R. Azoubel

* Department of Neurological Sciences, ** Department of Morphology Faculdade de Medicina de Sao Jose Rio Preto Sao Jose Rio Preto-SP, Brasil

Correspondence to:

Prof. Dr. Reinaldo Azoubel

Facultad de Medicina de Sao Jose do Rio Preto

Av. Brig. Faria Lima 5416

CEP 15090-000

Sao Jose do Rio Preto

Sao Paulo--BRASIL
Table I. Studies with phenytoin as embriotoxic agent, orally
administered.

Author                      Animal   Day dose *   Gestation day

Beekhuijzen et al. (2000)    rats       500       GD10
                             rats       1000      GD10
McCartney et al. (1999)      rats        50       GD7-GD18
Gelineau et al. (1999)       mice        60       GD6-GD18
Tsutsumi et al. (1998)       rats        50       GD7-GD18
                             rats       100       GD7-GD18
Tachibana et al. (1996)      rats       200       GD10-GD14
Abdel-Hamid et al. (1996)    mice        50       GD5-GD15

Author                      Animal    Total dose **

Beekhuijzen et al. (2000)    rats        500
                             rats        1000
McCartney et al. (1999)      rats        600
Gelineau et al. (1999)       mice        720
Tsutsumi et al. (1998)       rats        600
                             rats        1200
Tachibana et al. (1996)      rats        1000
Abdel-Hamid et al. (1996)    mice        550

* day dose: mg Kg-1 bd. wt.
** total dose: mg Kg-1 bd.wt

Table II. Nuclear parameters determined in control group and
phenytoin group.

Nuclear parameter                    Control   Phenytoin   Mann-Whitney
                                                              Test

Volume ([micro][m.sup.3])             192.68    145.89         0.028 **
Area ([micro][m.sup.2])                39.32     32.59         0.028 **
Perimeter ([micro]m)                   23.34     20.79         0.008 *
Longest axis ([micro]m)                 9.12      7.87         0.004 *
Shortest axis ([micro]m)                5.44      5.19         0.210
Mean axis ([micro]m)                    6.98      6.35         0.048 **
Ratio of longest to shortest axis       1.77      1.58         0.048 **
Ratio of volume to area ([micro]m)      4.66      4.23         0.048 **
Shape factor                            0.88      0.92         0.075
Contour index                           3.79      3.71         0.075
Eccentricity                            0.76      0.71         0.075

* p<0.01
** p<0.05
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Publication:International Journal of Morphology
Date:Dec 1, 2003
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