Effect of Acyclovir on Rat Fetus Palate Mucosa

 
Marilena Chinali KOMESU
Luiz Guilherme BRENTEGANI
Reinaldo AZOUBEL
Ruberval Armando LOPES
Miguel Angel SALA
 
Departamento de Estomatologia, Faculdade de Odontologia de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP, Brasil

Braz Dent J (1995) 6(1): 17-23 ISSN 0103-6440

| Introduction | Material/Methods | Results | Discussion | Conclusions | References |


Five pregnant rats were treated during organogenesis with sc injections of acyclovir (50 mg/kg body weight) on days 9, 10 and 11 of pregnancy. The fetuses (N = 62) were evaluated on day 20 of gestation and presented decreased body weight as well as delayed differentiation of fetal rat palate epithelium, with increased nuclear volume, decreased cytoplasmic and cellular volumes, decreased epithelial and keratin thicknesses, and increased cellular numerical density.


Key words: Acyclovir, palate, rat fetus, stereology.


Introduction

Acyclovir has been shown to have teratogenic properties in rats and chicks producing gross malformations which vary according to the dose and the embryonic age at the time of injection (Stahlmann, 1988). Administration of acyclovir to rats during organogenesis resulted in anomalies of the skull (reduction of the crown-rump length, abnormal shape of the head, decreased width of the skull, resembling a beak-like visceral cranium, smaller or missing os timpanicum), anomalies of the vertebral column, missing tail, and protruding tongue (Stahlmann et al., 1988; Chahoud et al., 1988). The present study was undertaken to determine the alterations present in palatine mucosa induced by maternal injection of acyclovir, during the teratogenic period in rats.

Material and Methods

Wistar rats (N = 10) were kept under constant day/night cycle (12/12 h), relative humidity and at room temperature (23 + 2oC). They were fed Purina pellet feed and tap water ad libitum. One male was caged with two females overnight and females were examined the following morning for the presence of sperm in the vaginal smear. The day sperm were detected was designated as day one of gestation.

The commercially available preparation for intravenous application of acyclovir (sodium salt) was used (ZoviraxÒ; Wellcome Chemical Works, Dartford, Kent, England). Five animals were treated subcutaneously on days 9, 10 and 11 of gestation with 50 mg acyclovir/kg body weight. Control animals were treated similarly with saline.

The animals were sacrificed on day 20 of gestation, and the fetuses were removed. To examine for variations in pathology within and among litters, five complete litters of each group were fixed in a solution of 85 ml 80% ethanol, 10 ml formol, and 5 ml glacial acetic acid, and after processing, embedded in paraffin and serially sectioned at 6 mm. The sections were stained with hematoxylin and eosin.

Stereological analysis was performed with the curvilinear test system of Merz (1968). The following parameters were calculated after point counting (720 per animal) on palatine epithelium: nuclear, cytoplasmic and cellular volumes, nuclear/cytoplasm ratio, keratinized surface density, epithelial thickness, keratin thickness, free surface/basal surface ratio, and numerical density (described in detail by Ribeiro et al., 1990).

Comparison of the results for the experimental group and the controls was performed by the non-parametric Mann-Whitney test.


Results

The mean fetal body weight was 5.07 g for the control group and 3.83 for the acyclovir-treated group (P<0.05).

Histologically, the hard palate epithelium of experimental animals was thinner and less differentiated than in control fetuses. The epithelium was composed of smaller and more numerous cells, with larger nuclei. The keratin layer was thinner. The alterations in the soft palate epithelium were less evident. The mean values of stereological parameters of the epithelial cells from both hard and soft palates can be observed in Table 1.
 
 


 
 

The nuclear volume of the basal and spinous cells from either hard or soft palates was larger in the treated group. Statistical analysis showed a significant difference between the control and the treated fetuses only in the soft palate for basal cells, and significant in both palates for spinous cells. The cytoplasmic and cellular volumes for basal and spinous cells from hard palate were significantly smaller in the treated group. The mean values of the cytoplasmic and cellular volumes in cells from hard and soft palates were significantly smaller in treated fetuses.

Mean epithelial surface density was smaller in the control group than in the treated group. Statistical analysis showed the existence of a significant difference between the groups only in the hard palate. The hard and soft palate epithelia were significantly thinner in the treated fetuses than in the control ones. Keratin thickness was also decreased.

Table 1 shows that the values for the free surface/basal surface ratio were no different between groups. The number of cells by cubic millimeter of epithelium of hard palate was greater (P<0.05) in treated animals than in the controls, whereas in the soft palate the values were similar.


Discussion

Epithelial alterations were observed in hard and soft palates of fetuses whose mothers were injected with 50 mg acyclovir/kg body weight on days 9, 10 and 11 of pregnancy. These alterations were shown by stereology (larger basal and spinous nuclei and smaller cell volumes, thinner epithelial and keratin thicknesses, and greater numerical density). These findings demonstrate a delayed differentiation of the analyzed tissue in treated animals.

Acyclovir [9-(2-hydroxyethoxymethyl) guanine] is used as a specific virustatic agent for the treatment of infections with herpes-type viruses. With the aid of a specific virus-coded kinase, acyclovir is phosphorylated to the corresponding triphosphate (Furmann et al., 1981) and incorporated into the viral genome during replication. Inhibition of DNA synthesis (Furmann et al., 1979) by termination of DNA chain growth is assumed to be responsible for the virocidal effect observed.

All current drugs which inhibit viral DNA synthesis also affect the DNA of uninfected cells. Acyclovir shows an unusually high degree of selectivity: its conversion to the monophosphate is catalyzed with a high degree of specificity by the viral thymidine kinase. However, it is well known that at high concentrations an effect on the DNA metabolism of uninfected cells also occurs (Stahlmann et al., 1988). These findings explain the alterations observed in the epithelium in this study.

The dose of 3 x 50 mg acyclovir/kg daily, used in this research, exhibited no teratogenic effect. Acyclovir exhibited no teratogenic effect when administered to rats at a sc dose of 3 x 25 mg acyclovir/kg daily from day 6 to 15 of gestation (Moore et al., 1983). Eight doses of 50 mg acyclovir/kg during organogenesis (from early day 9 to day 11) induced no defects, but a single injection of 200 mg acyclovir/kg on day 10 produced severe head defects (Stahlmann et al., 1988). Information on the teratogenic potential of acyclovir without maternal influence comes from the rat whole-embryo in vitro studies (Klug et al., 1985).

The reduced fetal weight observed in this study was also present in the findings of Chahoud et al. (1988). Acyclovir reduces the placental weight in rats (Komesu et al., 1995), and small placentas have impaired circulation with reduced blood flow to the fetus and thereby impair nutrition, which results in fetuses with smaller body weight.


Conclusions

Prenatal exposure to acyclovir in rats results in developmental alterations characterized by a decrease in body weight as well as by the delayed differentiation of diverse organs and tissues, including the epithelium of hard and soft palates.

Acknowledgments

Research supported by CNPq (Proc. 300.535/90-2).

References

Chahoud I, Stahlmann R, Bochert G, Dillmann I, Neubert D: Gross-structural defects in rats after acyclovir application on day 10 of gestation. Arch Toxicol 62: 8-14, 1988

Furmann PA, St Clair MH, Fyfe JA, Rideout JL, Keller PM, Elion GB: Inhibition of Herpes simplex virus-induced DNA polymerase activity and viral DNA replication by 9-(2-hydroxyethoxymethyl) guanine and its triphosphate. J Virol 32: 72-77, 1979

Furmann PA, de Miranda P, St Clair MH, Elion GB: Metabolism of acyclovir in virus-infected and uninfected cells. Antimicrob Agents Chemother 20: 518-524, 1981

Klug S, Lewandowski C, Blankenburg G, Merker HJ, Neubert D: Effect of acyclovir on mammalian embryonic development in culture. Arch Toxicol 58: 89-96, 1985

Komesu MC, Brentegani LG, Lopes RA, Sala MA, Azoubel R, Garcia Lima E: Morfologia de fetos de ratas injetadas com aciclovir no 10o dia da gestação. Rev Reg Ciências 4: 151-156, 1995

Merz WA: Die Streckenmessung an gerichteten Strukturen im Mikroskop und ihre Anwendung zur Bestimmung von Oberflächen-Volumen-Relationen im Knochengewebe. Mikroskopie 22: 132-142, 1968

Moore Jr HJ, Szczech GM, Rodwell DE, Kapp Jr RW, de Miranda P, Tucker Jr WF: Preclinical toxicology studies with acyclovir: teratologic, reproductive and neonatal tests. Fund Appl Toxicol 3: 560-568, 1983

Ribeiro CAL, Maia Campos G, Melis MS, Sala MA, Lopes RA: Alteraciones morfológicas del epitelio lingual de fetos de rata provocadas por el ejercício materno. Arch Fac Med Zaragoza 30: 84-88, 1990

Stahlmann R, Klug S, Lewandowski C, Bochert G, Chahoud I, Rahm U, Merker H-J, Neubert D: Prenatal toxicity of acyclovir in rats. Arch Toxicol 61: 468-479, 1988


Correspondence:Prof. Dr. Ruberval A. Lopes, Departamento de Estomatologia (Patologia), Faculdade de Odontologia de Ribeirão Preto, USP, 14040-904, Ribeirão Preto, SP, Brasil.


Accepted October 9,1995
Electronic publication: March, 1996


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