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International Journal of Morphology

On-line version ISSN 0717-9502

Int. J. Morphol. vol.21 no.4 Temuco  2003

http://dx.doi.org/10.4067/S0717-95022003000400007 

Int. J. Morphol., 21(4):299-302, 2003.

EFFECTS OF PYRAZINAMIDE ON PREGNANT ALBINO RATS

EFECTOS DE LA PYRAZINAMIDA SOBRE LA RATA PREÑADA

*Marisa Pascale Quintino; *Manuel de Jesus Simões; *Mary Uchiyama Nakamura;
**Ricardo Martins Oliveira-Filho; ***Silvia Espiridião & *Luiz Kulay Júnior


QUINTINO, M. P.; SIMÕES, M. J.; NAKAMURA, M. U.; OLIVEIRA-FILHO, R. M.; ESPIRIDIÃO, S. & KULAY, Jr. L. Effects of pyrazinamide on pregnant albino rats. Int. J. Morphol., 21(4):299-302, 2003.

SUMMARY: Although being used for years in the treatment of tuberculosis, no data are available in the literature on the safety of pyrazinamide during pregnancy. Accordingly, we aimed to make a first approach to this problem by evaluating the effects of this drug administered during the entire pregnancy of albino rats. Fourty female, EPM-1 Wistar albino rats of about 250 g b.w. were used. Upon conception (day zero of pregnancy) the animals were randomly divided in 4 groups of 10 rats each and labeled as follows. Controls (C), animals treated with the drug vehicle (destilled water); experimental groups (E1, E2 and E3), animals treated with 35, 105 or 315 mg/kg b.w. pyrazinamide by gavage (oral route) once daily up to the term (20th day of pregnancy). Drug or vehicle volume was always 0.5 ml. Body weight gain was followed up every week. At term, upon sacrifice (in excess of anesthesia) and histerectomy, the following parameters have been studied: number of implantations and reabsorptions; intrauterine deaths; number of living foetuses and of placentae; weights of concepts and of placentae; major foetal malformations; maternal mortality index. No significant effects of pyrazinamide on rat pregnancy have been observed regarding the maternal body weight gain, the weights of concepts, the number of implantations and reabsorptions and the weights of placentae and foetuses. Also, no deleterious effects have been noticed regarding major foetal malformations, intrauterine deaths or maternal mortality. With the highest pyrazinamide dosis employed (315 mg/kg b.w.), however, a significant lowered uterine weight was recorded. Although otherwise safe, a high-dose regimen of pyrazinamide during rat pregnancy can induce a slight yet significant reduction of uterine weight.

KEY WORDS: 1. Pyrazinamide; 2. Toxicology; 3. Rat; 4. Pregnancy.


INTRODUCTION

Tuberculosis (TB) and other mycobacterial diseases follow the history of humanity since ancient man started eating bovine meat and milk contaminated with Mycobacterium bovis-like bacilli such as its close-related M. tuberculosis. Initially it was a disease of lower mammals, and the etiologic agent probably preceded the development of man on earth. It became an uncommon endemic disease in man about the time man began to settle in villages and develop agriculture (Bates & Stead, 1993).

In some countries, estimated new case rates are as high as 400 per 100.000 per year. In general, poverty and tuberculosis go hand in hand. In countries where human immunodeficiency virus infection is endemic, TB is commonly the single most important cause of morbidity and mortality in AIDS patients. A reasonable estimate of the magnitude of TB in the world is that 1/3 of the population is infected with M. tuberculosis, that there are 30 million cases of active TB in the world, that 10 million new cases occur annually, and that 3 million people die of TB each year. TB probably causes 6% of all deaths worldwide (Daniel, 1994).

After the initial entry of tubercle bacilli into the lungs or another site in a previously uninfected individual, primary tuberculous lesions almost always heal; most of those destined to develop TB do so during adolescence or young adulthood that is to say, during the reproductive years. The tuberculous disease is more common in young adult women, and this fact calls the attention for the use of antimycobacterial agents during pregnancy.

The history of the binomial tuberculosis/pregnancy has had three distinct phases. The first one, at the end of the 19th century and hold by the German School stated that pregnancy was in order because it collapsed the tuberculous cavities in lungs (Anderson, 1994). In the second phase, according to Young (1936), it was said that the TB woman, if a virgin, should not get married; if married, should not become pregnant; if pregnant, should abort and, if a mother, should not breast-feed. Currently, it is believed that no reciprocal influences exist between TB and pregnancy since a proper treatment is established (Silva, 1980).

With regard to the several protocols recommended, some concerns during gestation refer to ethionamide and streptomycin due to their foetal toxicities (Quagliato Jr., 2000). Other drugs such as ethambutol, isoniazid, rifampin and pyrazinamide are commonly used in protocol treatments during pregnancy (Uehara & Santoro, 2001). In relation to pyrazinamide, it was recently showed an association of the drug with hypospadia (Czeizel et al., 2001), but there is no reference to experimental animals or controlled studies in humans (Uehara & Santoro; Briggs et al., 2002). In order to investigate the chronic effects of pyrazinamide on gestation, in this paper we administered this drug daily during the entire pregnancy period of albino rats in a search for its maternal and/or foetal toxic effects.

MATERIAL AND METHOD

Adult, virgin EPM-1 Wistar rats weighing ca. 200 g under routine laboratory care with Purina rat chow and water ad libitum, were mated in the proportion of 2 females for every male, during 2 h. Pregnancy was verified according to Hamilton & Wolfe (1938) by the finding of spermatozoa in vagina; this was considered the day zero of pregnancy. Forty pregnant rats were then randomly divided into 4 groups and treated from the 1st up to the 20th day of gestation as follows: C, control group, treated with drug vehicle (0.5 ml destilled water by gavage); P1, P2 and P3, groups of animals treated respectively with 35, 105 or 315 mg/kg b.w. of pyrazinamide by gavage, once a day, starting at the day 0 until the 20th day of gestation. Body weight gain was monitored by weighing the animals at days 0, 7th, 10th and 20th of pregnancy.

At term (20th day) the animals were sacrificed by deep ether anesthesia. Upon laparotomy and uterine horns opening, the sites of implantations and reabsorptions were recorded; the living foetuses and their placentae were carefully removed, loosely passed onto filter paper to remove excess liquid and weighed to the nearest of 0.1 mg. The foetuses were examined for major malformations under a stereo microscope.

Data were analyzed by one-way analysis of variance and the Kruskal-Wallis multiple comparisons tests (Sokal & Rohlf, 1969; Winer, 1971). A 2.01 version GraphPad Instat® software was employed for calculations.

RESULTS

Figure 1 shows the linear regressions of body weight increase of pregnant rats throughout the experimental period. Similarly to what occurs in normal rats, it should be noticed that all groups reached their fastest weight gain in the last third of pregnancy. No significant differences were recorded among the groups treated with pyrazinamide with regard to the control group.


Fig. 1. Body weight gain of pregnant rats (groups of 10 animals each) treated with vehicle (destilled water) or pyrazinamide once a day by gavage during the entire period of pregnancy (P1, 35 mg/kg; P2, 105 mg/kg; P3, 315 mg/kg). Drug or vehicle volume given was always 0.5 ml. No significant differences were detected among the groups.

With respect to the gestation products, we observed (Table I) that the treatment with pyrazinamide did not affect the foetal weight, but significantly reduced the mean placental weight in the group treated with the highest dose (group P3). On the other hand, no differences were seen among the groups in relation to the number of implantations, of placentae or of living foetuses at term. Similarly, there were no deaths in utero or shortly after uterine opening.

Table I. Effects of pyrazinamide on rat pregnancy.1
 


Control P1 P2 P3
Nº of implantations2 11 ± 2 10 ± 3  8 ± 3 10 ± 4
Foetal weight (mg ) 4.44 ± 0.67 4.11 ± 0.21 3.96 ± 0.18 3.59 ± 0.84
Placental weight (mg)  0.62 ± 0.09 0.61 ± 0.11 0.66 ± 0.16 0.48 ± 0.08*

1Groups of 10 animals were treated with vehicle (destilled water) or pyrazinamide once a day by gavage during the entire period of pregnancy (P1, 35 mg/kg; P2, 105 mg/kg; P3, 315 mg/kg). Drug or vehicle volume given was always 0.5 ml.
2These data stand for the number of implantations, of living foetuses and of placentae.
*P<0.05 in relation to the control group.

DISCUSSION

By evaluating the DNA content in rat tissues, Winick (1968) identified a common sequence of cell growth in all organs, namely hyperplasia > hyperplasia/hypertrophy > hypertrophy, and then maturity follows. In this regard, two types of interferences can alter the process (Winick et al., 1972): intrinsic causes can be due mainly to chromosome alterations and result in normal placentae with multiple foetal malformations; among the extrinsic causes there are maternal infections, inadequate nutrient input and drug use, which can cause placental weight reduction without foetal major malformation (Vemeersch, 1980).

Our results show a reduction of placental weight in the group treated with the highest dosis of pyrazinamide but no fetal malformations were recorded, a finding which presumably correlates with a deficit of nutritional input.

In fact, pyrazinamide can produce, among other severe side effects, nausea and anorexia (Girling, 1982) and was administered to the animals during the hyperplasic period of placental development (Kulay Jr., 1968). This time-coincidence could be responsible for the deleterious effect of pyrazinamide observed on placental weight in group P3.

It is noticeable that this drug, although capable of producing an anorexigenic effect due to its use in rats at a dosis 9-fold higher than the therapeutic dosis recommended for humans, might not produce a critical reduction of nutrient intake. This is so because if there were a reduction higher than 25% of the caloric needs, it should be produced a significant impairment of the maternal body gain, followed by reduction of the mean foetal weights and foetal deaths (Chow & Lee, 1964).


QUINTINO, M. P.; SIMÕES, M. J.; NAKAMURA, M. U.; OLIVEIRA-FILHO, R. M.; ESPIRIDIÃO, S. & KULAY, Jr. L. Efectos de la pyrazinamida sobre la rata preñada. Int. J. Morphol., 21(4):299-302, 2003.

RESUMEN: La pyrazinamida es un fármaco muy utilizado para el tratamiento de la tuberculosis, pero no hay datos en la literatura respecto a sus potenciales riesgos para la preñez. Debido a esto, en este trabajo tratamos ratas preñadas durante toda la gestación (desde el día 0 hasta el día 20) con 3 dosis de pyrazinamida (35, 105 o 315 mg/kg, una vez al día) para una primera observación sobre los efectos de la pyrazinamida durante la preñez. Las ratas control recibieron el vehículo de la droga (agua destilada). Los resultados mostraron que no hubo alteraciones significativas en cuanto a la evolución del peso corporal de las ratsa madres, al peso de los fetos, al número de implantaciones y reabsorciones, ni tampoco en cuanto a los pesos de placentas y fetos. Además, no se observaron malformaciones fetales mayores, muertes intrauterinas o aumento de la mortalidad materna. El único efecto significativo observado con la dosis de 315 mg/kg de pyrazinamida, fue una reducción del peso uterino.

PALABRAS CLAVE: 1. Pyrazinamida; 2. Toxicología; 3. Rata hembra; 4. Preñez


REFERENCES

Anderson, G. Tuberculosis in pregnancy. Perinatology, 21:328-97, 1994.         [ Links ]

Bates, J. H.; Steab & W. W. The history of tuberculosis as a global epidemic. Med. Clin. North Am., 77:6-12, 1993.         [ Links ]

Briggs, G.G.; Freeman, R. & Yaffe, S.J. Drugs in pregnancy and lactation. 6th ed. Lippincott Williams & Wilkins, Philadelphia, 2002.         [ Links ]

Chow, B.F. & Lee, C. T. Effect of dietary restriction of pregnant rats on body weight gain of offsprings. J. Nutr., 82:10-8, 1964.         [ Links ]

Czeizel, A.E.; Rockenbauer, M. & Olsen, J. A population-based case-control study on the safety oral anti-tuberculosis drug treatment in pregnancy. Int. J. Tuberc. Lung. Dis., 5:564-8, 2001.         [ Links ]

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Hamilton, J.B.; Wolfe, J. M. Effect of male hormone substance upon birth and prenatal development in rat. Anat. Rec., 70:433-40, 1938.         [ Links ]

Kulay Jr, L. Histochemical study (polisaccharides, lipids and proteins) of rat's placenta during the functional period. Acta Anat., 70:607-22, 1968.         [ Links ]

Quagliato Jr., R. Pneumopatias na gestação. In: Neme B: Obstetrícia Básica, 2th ed. Sarvier, São Paulo, 2000.         [ Links ]

Silva, L.C.C. Tuberculose pulmonar e gravidez. Rev. AMRIGS, 24:39-45, 1980.         [ Links ]

Sokal, R. R. & Rohlf, F. J. Biometry. W.H. Freeman, San Francisco, 1969, p. 776.         [ Links ]

Uehara, C. & Santoro, I. L. Tuberculose pulmonar e pleural. In: Borges, D.R & Rothschild, H.A. - Atualização Terapêutica, 20th ed., Artes Médicas, São Paulo, 2001.         [ Links ]

Vemeersch, J. Nutrição materna e o resultado da gestação. In: Worthington, B.S.; Vemeersch, J. & Williams, S.R. - Nutrição na gravidez e na lactação. Interamericana, Rio de Janeiro, 1980.         [ Links ]

Winer, B. J. Statistical Principles in Experimental Design. McGrow-Hill, New York, 1971.         [ Links ]

Winick, M. Nutrition and cell growth. Nutr. Rev., 26:195-201, 1968.         [ Links ]

Winick, M.; Brasel, J.A. & Rosso, P. Nutrition and cell growth. In: Winick, M. - Nutrition and development. John Wiley & Sons, New York, 1972.         [ Links ]

Young, S. Pulmonary tuberculosis and pregnancy. Brit. Med, 2:749-59, 1936.         [ Links ]

Correspondence to:
Prof. Dr. Luiz Kulay Júnior
Rua Dr. Haberbeck Brandão, 68/71
CEP: 04027-040
São Paulo
BRAZIL

Received : 09-06-2003
Accepted: 10-10-2003
 


* Federal University of São Paulo School of Medicine, São Paulo (SP), Brazil.

** University of São Paulo Institute of Biomedical Sciences, São Paulo (SP), Brazil.

*** Department of Obstretrics and Ginaecology - Faculty of Medicine, Fundação ABC, São Paulo (SP), Brazil.

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