139(1), 220–233 2014 doi: 10.1093/toxsci/kfu024 Advance Access publication February 4, 2014
TOXICOLOGICAL SCIENCES
Testicular Effects Following In Utero Exposure to the Antivirals Acyclovir and Ganciclovir in Rats Fab´ıola Nihi,* Davyson Moreira,† Ana Carolina Santos Lourenc¸o,‡ Caroline Gomes,‡ Samanta Luiza Araujo,‡ Renata Mercer Zaia,* Natalia Botelho Trevisani,* Leonardo de Athayde Pinto,* Daniele Dietrich Moura-Costa,§ Rosana Nogueira de Morais,* Francisco Jos´e Roma Paumgartten,† and Anderson Joel Martino-Andrade*,1
1 To
whom correspondence should be addressed at Departamento de Fisiologia, Universidade Federal do Paran´a, Rua Francisco H. dos Santos S/N, P.O. Box 19031, Curitiba 81531-980, Brazil. Fax: +55 41 3361 1716. E-mail: [email protected]. Received October 16, 2013; accepted January 29, 2014
In utero exposure to the antivirals acyclovir and ganciclovir has been reported to induce gross structural defects in rat offspring. The present study investigated the effects of maternal antiviral treatment on gestation day 10 on reproductive and nonreproductive organs in male rat offspring with a particular focus on the testes. Vehicle and two doses of acyclovir and ganciclovir, 75 and 300 mg/kg, were administered to rat dams. The total doses were fractioned into three subcutaneous applications (3 × distilled water, 3 × 25 mg/kg, and 3 × 100 mg/kg) that were administered on gestation day 10 at 8:00 a.m., 1:00 p.m., and 6:00 p.m. The antiviral concentrations were measured in the serum of the dams 1 h after the last administration. Exposure to 300 mg/kg ganciclovir induced germ cell deficiency in both fetal and adult testes, an effect that was not seen in any other treatment group. Adult rats exposed in utero to this high ganciclovir dose exhibited Sertoli cellonly tubules intermingled with seminiferous tubules that displayed a normal size and normal cell counts, alterations that resemble focal Sertoli cell-only syndrome in humans. The serum concentrations of ganciclovir were markedly higher than those of acyclovir, particularly at the high dose tested. However, although 300 mg/kg acyclovir did not induce germ cell deficiency, other specific effects were seen in exposed animals, including incomplete eye opening and reduced thymus weight. Key words: acyclovir; ganciclovir; testis; rats; gestation.
Acyclovir and ganciclovir are synthetic acyclic guanosine analogues used as antivirals. Acyclovir is used against infections caused by herpes simplex virus, but it also has activity against the varicella zoster and Epstein Barr viruses. Ganciclovir is mainly used to treat cytomegalovirus, particularly in immunocompromised patients (Scholar and Pratt, 2000). These drugs exhibit high specificity for infected cells because they depend on the activity of the viral thymidine kinase to be converted into monophosphate derivatives, which are further metabolized into active triphosphate compounds by host cells.
The triphosphate compounds are competitive inhibitors of viral DNA polymerases, and the incorporation of these analogues into viral DNA can slow or halt replication (Haynes et al., 1996; Scholar and Pratt, 2000). Toxicological studies have shown that repeated-dose treatment with high acyclovir doses is nephrotoxic because of the poor water solubility and crystallization of this compound in renal tubules in rats (Stahlmann et al., 1988). However, maternal treatment with high doses fractioned into three subcutaneous administrations on gestation day 10 has allowed the detection of the teratogenic effects of acyclovir and ganciclovir without causing nephrotoxicity or other secondary adverse effects (Chahoud et al., 1988; Klug et al., 1991; Stahlmann et al., 1988). Additionally, acyclovir and ganciclovir induced gross structural defects in a whole-embryo culture system (Klug et al., 1991; Stahlmann et al., 1988). Neyts et al. (1995) demonstrated that the subcutaneous administration of 100 mg/kg ganciclovir for 14 days in adult mice induced severe germ cell depletion when the testes were analyzed 4 weeks after final dosing. Faqi et al. (1997) showed similar results in rats that were subcutaneously administered 100 mg/kg ganciclovir three times at 4-h intervals for one day or received a single daily dose of 60 mg/kg injected for five consecutive days. Nevertheless, the reproductive effects of acyclovir and ganciclovir following in utero exposure have not been extensively investigated. An abstract published by Hartmann et al. (1991) indicated that maternal ganciclovir administration (3 × 100 mg/kg) on gestation day 10 caused testicular atrophy in adult male offspring. However, a full description of the testicular effects was not provided. In mice and rats, testicular differentiation occurs between gestation days 10 and 13 as a result of multiple morphological and cellular events that are triggered by the expression of the sex-determining region Y (SRY) gene and several other genetic influences (Ross and Capel, 2005). However, germ cells arise
C The Author 2014. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For permissions, please email: [email protected]
Downloaded from http://toxsci.oxfordjournals.org/ at FU BerlinFB Humanmedizin on May 14, 2015
*Departamento de Fisiologia, Universidade Federal do Paran´a, Centro Polit´ecnico, Curitiba 81531-980, Brazil; †Laborat´orio de Toxicologia Ambiental, Escola Nacional de Sa´ude P´ublica, Fundac¸a˜ o Oswaldo Cruz, Rio de Janeiro 21040-900, Brazil; ‡Departamento de Farmacologia, Universidade Federal do Paran´a, Centro Polit´ecnico, Curitiba 81531-980, Brazil; and §Departamento de Biologia Celular, Universidade Federal do Paran´a, Centro Polit´ecnico, Curitiba 81531-980, Brazil
ANTIVIRALS AND TESTICULAR DEVELOPMENT
MATERIALS AND METHODS
Animals Female Wistar rats were obtained from the animal facility of the Universidade Federal do Paran´a. The animals were maintained under controlled temperature (21 ± 1◦ C) and luminosity (12 h/12 h light/dark cycle). Standard pellet food (Nuvilab, Colombo, Brazil) and tap water were available ad libitum. Three nongravid female rats were mated with one male for 3 h, and the day of sperm detection in the vaginal smear was considered gestation day 0 (Chahoud and Kwasigrocch, 1997). All of the experimental procedures were approved by the Committee on Animal Research and Ethics of the Universidade Federal do Paran´a (consent no. 513), which is in accordance with national and international animal welfare guidelines. Drugs and Treatments Acyclovir (Eurofarma, S˜ao Paulo, Brazil) and ganciclovir (Novafarma, An´apolis, Brazil) were available pharmaceutical preparations intended for intravenous application in humans. The content of the vials was diluted with distilled water to yield appropriate concentrations for an administration volume of 1 ml/kg. The gravid females were randomly assigned to five experimental groups. The dams were treated on gestation day 10 with the following three subcutaneous applications (8:00 a.m., 1:00 p.m., and 6:00 p.m.) of distilled water or antivirals: control (3 × distilled water), 75 mg/kg acyclovir (3 × 25 mg/kg), 300 mg/kg acyclovir (3 × 100 mg/kg), 75 mg/kg ganciclovir (3 × 25 mg/kg), and 300 mg/kg ganciclovir (3 × 100 mg/kg). A total of three experiments were conducted using the same exposure protocol described above. In experiment 1, the dams (n = 9–11 per group) were euthanized on gestation day 20 to evaluate the early effects on male fetuses. In experiment 2, the dams (n = 10–13 per group) were allowed to deliver, and the male offspring was observed until postnatal day 91 (±1 day) to
evaluate the possible late effects on the testes and other organs. In experiment 3, the dams (n = 3–7 per group) were euthanized 1 h after the last subcutaneous administration, and blood was collected to quantify antivirals in maternal serum. Table 1 contains the detailed information on the number of animals and litters in each experiment. In all cases, the litter was used as the statistical unit. Experiment 1: Effects of Acyclovir and Ganciclovir on Rat Fetuses The body weight of the rat dams was monitored throughout pregnancy. On gestation day 20, the dams were euthanized by decapitation followed by excision of the uterus and removal of the fetuses. The number, sex, and body weight of the fetuses were recorded. The absolute and relative weights of the liver, kidneys, thymus, and spleen of the dams were also recorded. The relative weights were calculated as the following: organ weight/body weight × 100. The anogenital distance (AGD) was recorded for male and female fetuses using a digital caliper (Carrera Precision, Guangdong, China). The AGD of each animal was divided by the cube root of body weight to avoid errors caused by differences in body size (Gallavan et al., 1999). Fetal testicular histology. Following AGD measurements, the fetuses were euthanized by decapitation, and the testis of one rat per litter was submitted to histological processing whenever possible. Not all litters could be investigated because some testes were lost during manipulation or histological processing. The right testis was immersed and fixed in Bouin’s fixative (75% saturated picric acid, 20% formalin, and 5% acetic acid) for 1 h and maintained in 70% ethanol until embedment in paraffin. Testicular sections (5 m) were obtained, stained with hematoxylin and eosin, and microscopically analyzed to determine the seminiferous cord diameter and number of gonocytes per cord. In each testis, the diameters of seminiferous cords were measured in 20 randomly selected circular cross sections whenever possible. The measurements were made with UTHSCSA ImageTool 3.0 software (UTHSCSA, San Antonio, TX) using images acquired on a Zeiss Axiophot microscope (Oberkochen, Germany) coupled with an imaging system at ×200 magnification (ASI, Vista, CA). The number of gonocytes per cord was determined by counting the nuclei of these cells at ×400 magnification (Zeiss Axiophot microscope) in 15–20 circular cross sections chosen at random for each testis. The crude count was corrected by section thickness and the mean nuclear diameter of gonocytes according to Abercrombie’s formula (Abercrombie, 1946): count = crude count × (section thickness/[section thickness + nuclear diameter]). The diameters of 20 randomly selected gonocyte nuclei per testis were measured using UTHSCSA ImageTool 3.0 software. The calculated power was 0.99, considering five experimental groups and the cell counts obtained by the analysis of 15–20 circular cross sections of seminiferous cords, with ␣ = 0.05, mean standard deviation of 0.5,
Downloaded from http://toxsci.oxfordjournals.org/ at FU BerlinFB Humanmedizin on May 14, 2015
outside the urogenital ridge prior to testis or ovary formation. Once they reach the developing gonad, around gestation day 10, they are referred to as gonocytes (Culty, 2009). Several aspects of gonadal differentiation are still unknown, and the effects induced by chemicals during critical windows of development may be helpful to understand normal and abnormal testis differentiation and function. In the present study, we investigated the early and late reproductive effects of two doses of acyclovir and ganciclovir in male rats following maternal exposure on gestation day 10, which corresponds to the initial stage of gonadal differentiation in rodents. In humans, acyclovir and ganciclovir are used by oral and intravenous routes with short administration intervals because these compounds have very short halflives. Pregnant rats were treated subcutaneously three times on gestation day 10 in order to achieve serum concentrations similar to those seen in humans.
221
222
NIHI ET AL.
TABLE 1 Number of Animals and Litters in the Three Experiments Conducted Parameter
Control
Acyclovir (mg/kg) 75
300
75
300
10 10
10 10
11 11
9 8
8
6–7
8–9
7–8
11 (88.9, 100, 100) (91.6, 100, 100) 11
10 (87.5, 100, 100) (66.7, 100, 100) 8
12 (100, 100, 100) (100, 100, 100) 12
13 (100, 100, 100) (93.2, 100, 100) 11
32
24
36
32
22
16
23
22
11
8
11
10
—
—
—
5
7
7
3
4
a Animals from different litters. Not all litters could be investigated, because some testes were lost during manipulation or histological processing. b Live birth index = (number of live offspring/number of delivered offspring) × 100; data indicate first quartile (Q1), median and third quartile (Q3).
c Weaning index = (number of live offspring at weaning/number of live offspring at birth) × 100; data indicate first quartile (Q1), median and third quartile (Q3). No significant differences were observed for birth (p = 0.177) and weaning (p = 0.066) indices (Kruskal-Wallis). d n = 1–3 rats per litter. In all cases the litters were used as the statistical units.
and total sample size of 35 (n = 6–8 litters per group). Power was calculated with the software G*Power version 3.1.3 (University of Dusseldorf, Germany). Experiment 2: Postnatal Effects of Acyclovir and Ganciclovir The dams were treated on gestation day 10, and their body weights were monitored throughout pregnancy and lactation. The body weight of the pups was monitored every 7 days from birth to postnatal day 21 (weaning). The dams and female pups were euthanized on lactation day 22, whereas the male pups continued to be evaluated. The age at preputial separation was monitored daily in all male rats by manual retraction of the prepuce starting on postnatal day 38. From postnatal day 22 onward, the body weight of male offspring was recorded every 7 days until postnatal day 91 ± 1 (adulthood). At this age, 1–3 males per litter were euthanized by decapitation to collect blood and organs. Organ weights. The absolute and relative weights of the following reproductive organs and tissues were recorded: testes, epididymis, ventral prostate, seminal vesicle (with coagulating glands), and levator ani/bulbocavernosus muscle (LABC). The entire epididymis was carefully removed, dissected free of fat, and weighed. The vas deferens was cut at its junction with the
cauda epididymis. The ventral prostate was weighed without the prostatic capsule, and the seminal vesicle was weighed without its contents. The absolute and relative weights of the liver, kidneys, thymus, and spleen were also recorded. The relative weights were calculated as the following: organ weight/body weight × 100. Number of homogenization-resistant spermatids per testis. The left testes were used to determine the number of homogenization-resistant spermatids, which correspond to spermatids in stages 17–19 of development. After removal of the tunica albuginea, each testis was minced and homogenized for 1 min in 10 ml of 0.9% NaCl that contained 0.5% Triton X-100 at medium speed in a Potter S Tissuemizer (Sartorius, Goettingen, Germany). The homogenate was diluted 10 times in 0.9% NaCl for the microscopic counting of homogenization-resistant spermatids in a B¨urker hemocytometer chamber (Brand GmbH, Wertheim, Germany). Testicular histology. The testis of one rat per litter was submitted to histological processing, whenever possible. The right testes were immersed and fixed in Bouin’s fixative for approximately 2 h. The capsule of each testis was cut at the poles to
Downloaded from http://toxsci.oxfordjournals.org/ at FU BerlinFB Humanmedizin on May 14, 2015
Experiment 1—fetal endpoints Number of pregnant rats 9 Number of dams with viable male 8 fetuses Number of animals analyzed for fetal 6–7 testicular histologya Experiment 2—adult endpoints Number of pregnant rats 10 Live birth index% (Q1, median, Q3)b (90.5, 100, 100) Weaning index% (Q1, median, Q3)c (100, 100, 100) Number of litters with viable adult 9 male offspring Number of adult male rats 25 investigated for organ weightsd Number of males with counts of 17 homogenization-resistant spermatidsd Number of rats investigated for adult 9 testicular histologyd Testicular cell counts (round 5 spermatids and Sertoli cells) Experiment 3—concentrations of antivirals in maternal serum Number of pregnant rats with blood — samples
Ganciclovir (mg/kg)
ANTIVIRALS AND TESTICULAR DEVELOPMENT
Experiment 3: Concentrations of Acyclovir and Ganciclovir in Maternal Serum
Testicular cell counts. Testicular sections from the control and 300 mg/kg ganciclovir groups were further analyzed to determine the number of round spermatids and Sertoli cells per tubular cross section at stages VII to VIII of the seminiferous epithelium cycle. For each animal, round spermatids nuclei and Sertoli cell nucleoli were counted in 10 round (i.e., not germcell-deficient) seminiferous tubule cross sections at stages VII to VIII of the cycle (n = 5 rats per group). The crude count was corrected by section thickness and the mean nuclear or nucleolar diameters according to Abercrombie’s formula (Abercrombie, 1946): count = crude count × (section thickness/[section thickness + nuclear or nucleolar diameter]). The diameters of 10–20 randomly selected spermatid nuclei or Sertoli cell nucleoli per testis were measured using UTHSCSA ImageTool 3.0 software.
The rat dams were treated with three subcutaneous applications (8:00 a.m., 1:00 p.m., and 6:00 p.m.) of distilled water or antivirals on day 10 of gestation. One hour after the final administration (i.e., at 7:00 p.m.), the dams were anesthetized with ketamine (75 mg/kg) and xylazine (1.5 mg/kg). After opening the abdominal cavity, blood was collected from the posterior vena cava with a 23-gauge needle attached to a 1-ml syringe. Blood samples were centrifuged for 15 min at 1000 × g, and the resulting serum was stored at −20◦ C until analysis. The serum concentrations of acyclovir and ganciclovir were determined by high-performance liquid chromatography (HPLC) based on the methods described by Boulieu et al. (1997). Acyclovir (>99% purity) and ganciclovir (>99% purity) standards were purchased from Sigma (Schnelldorf, Germany), and HPLC-grade acetonitrile was obtained from Tedia (Rio de Janeiro, Brazil). Potassium dihydrogenphosphate (KH2 PO4 ) was supplied by Merck (Darmstadt, Germany). The analyses were performed on a reverse-phase LiChrospher-100 RP-18 Merck column (inner diameter, 250 mm × 4.6 mm; 5 m particle size; Darmstadt, Germany) equipped with a Merck guard column (inner diameter, 4.0 mm; length, 1 cm) packed with the same packing material and placed before the inlet of the analytical column. For the quantification of ganciclovir, the mobile phase consisted of a mixture of 10mM KH2 PO4 (pH 3.4) and acetonitrile at a ratio of 98:2 (vol/vol). For acyclovir, the mobile phase consisted of a mixture of 20mM KH2 PO4 (pH 3.2) and acetonitrile at a ratio of 96:4 (vol/vol). For both analyses, the flow rate was set at 1.5 ml/min. Detection was performed at 254 nm, and the mobile phase was previously filtered through a 0.45 mm filter (Millipore, Bedford, MA) and degassed. For sample preparation, a 100 l aliquot of serum was subjected to precipitation with 50 l of a 25% zinc sulfate solution in water (wt/vol). After mixing, the sample was centrifuged at 15,000 × g for 15 min. Afterward, 20 l of the supernatant was automatically injected into the column. Additional details of the methods, including the calibration curve, limits of detection and quantification, precision, and accuracy, are described in the supplementary data.
Serum testosterone. Blood was collected by decapitation and clotted at room temperature. After centrifugation, the serum samples were transferred to clean tubes and kept at −20◦ C. Serum testosterone was quantified using an enzyme immunoassay (Munro et al., 1991). The antibody against testosterone (R1556/7) and the respective horseradish peroxidase conjugate were obtained from Coralie Munro at the University of California, Davis (Davis, CA). Serial dilutions of pooled serum rat samples produced displacement curves parallel to those of standard curves. The intra- and interassay coefficients of variation were
TOXICOLOGICAL SCIENCES
Testicular Effects Following In Utero Exposure to the Antivirals Acyclovir and Ganciclovir in Rats Fab´ıola Nihi,* Davyson Moreira,† Ana Carolina Santos Lourenc¸o,‡ Caroline Gomes,‡ Samanta Luiza Araujo,‡ Renata Mercer Zaia,* Natalia Botelho Trevisani,* Leonardo de Athayde Pinto,* Daniele Dietrich Moura-Costa,§ Rosana Nogueira de Morais,* Francisco Jos´e Roma Paumgartten,† and Anderson Joel Martino-Andrade*,1
1 To
whom correspondence should be addressed at Departamento de Fisiologia, Universidade Federal do Paran´a, Rua Francisco H. dos Santos S/N, P.O. Box 19031, Curitiba 81531-980, Brazil. Fax: +55 41 3361 1716. E-mail: [email protected]. Received October 16, 2013; accepted January 29, 2014
In utero exposure to the antivirals acyclovir and ganciclovir has been reported to induce gross structural defects in rat offspring. The present study investigated the effects of maternal antiviral treatment on gestation day 10 on reproductive and nonreproductive organs in male rat offspring with a particular focus on the testes. Vehicle and two doses of acyclovir and ganciclovir, 75 and 300 mg/kg, were administered to rat dams. The total doses were fractioned into three subcutaneous applications (3 × distilled water, 3 × 25 mg/kg, and 3 × 100 mg/kg) that were administered on gestation day 10 at 8:00 a.m., 1:00 p.m., and 6:00 p.m. The antiviral concentrations were measured in the serum of the dams 1 h after the last administration. Exposure to 300 mg/kg ganciclovir induced germ cell deficiency in both fetal and adult testes, an effect that was not seen in any other treatment group. Adult rats exposed in utero to this high ganciclovir dose exhibited Sertoli cellonly tubules intermingled with seminiferous tubules that displayed a normal size and normal cell counts, alterations that resemble focal Sertoli cell-only syndrome in humans. The serum concentrations of ganciclovir were markedly higher than those of acyclovir, particularly at the high dose tested. However, although 300 mg/kg acyclovir did not induce germ cell deficiency, other specific effects were seen in exposed animals, including incomplete eye opening and reduced thymus weight. Key words: acyclovir; ganciclovir; testis; rats; gestation.
Acyclovir and ganciclovir are synthetic acyclic guanosine analogues used as antivirals. Acyclovir is used against infections caused by herpes simplex virus, but it also has activity against the varicella zoster and Epstein Barr viruses. Ganciclovir is mainly used to treat cytomegalovirus, particularly in immunocompromised patients (Scholar and Pratt, 2000). These drugs exhibit high specificity for infected cells because they depend on the activity of the viral thymidine kinase to be converted into monophosphate derivatives, which are further metabolized into active triphosphate compounds by host cells.
The triphosphate compounds are competitive inhibitors of viral DNA polymerases, and the incorporation of these analogues into viral DNA can slow or halt replication (Haynes et al., 1996; Scholar and Pratt, 2000). Toxicological studies have shown that repeated-dose treatment with high acyclovir doses is nephrotoxic because of the poor water solubility and crystallization of this compound in renal tubules in rats (Stahlmann et al., 1988). However, maternal treatment with high doses fractioned into three subcutaneous administrations on gestation day 10 has allowed the detection of the teratogenic effects of acyclovir and ganciclovir without causing nephrotoxicity or other secondary adverse effects (Chahoud et al., 1988; Klug et al., 1991; Stahlmann et al., 1988). Additionally, acyclovir and ganciclovir induced gross structural defects in a whole-embryo culture system (Klug et al., 1991; Stahlmann et al., 1988). Neyts et al. (1995) demonstrated that the subcutaneous administration of 100 mg/kg ganciclovir for 14 days in adult mice induced severe germ cell depletion when the testes were analyzed 4 weeks after final dosing. Faqi et al. (1997) showed similar results in rats that were subcutaneously administered 100 mg/kg ganciclovir three times at 4-h intervals for one day or received a single daily dose of 60 mg/kg injected for five consecutive days. Nevertheless, the reproductive effects of acyclovir and ganciclovir following in utero exposure have not been extensively investigated. An abstract published by Hartmann et al. (1991) indicated that maternal ganciclovir administration (3 × 100 mg/kg) on gestation day 10 caused testicular atrophy in adult male offspring. However, a full description of the testicular effects was not provided. In mice and rats, testicular differentiation occurs between gestation days 10 and 13 as a result of multiple morphological and cellular events that are triggered by the expression of the sex-determining region Y (SRY) gene and several other genetic influences (Ross and Capel, 2005). However, germ cells arise
C The Author 2014. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For permissions, please email: [email protected]
Downloaded from http://toxsci.oxfordjournals.org/ at FU BerlinFB Humanmedizin on May 14, 2015
*Departamento de Fisiologia, Universidade Federal do Paran´a, Centro Polit´ecnico, Curitiba 81531-980, Brazil; †Laborat´orio de Toxicologia Ambiental, Escola Nacional de Sa´ude P´ublica, Fundac¸a˜ o Oswaldo Cruz, Rio de Janeiro 21040-900, Brazil; ‡Departamento de Farmacologia, Universidade Federal do Paran´a, Centro Polit´ecnico, Curitiba 81531-980, Brazil; and §Departamento de Biologia Celular, Universidade Federal do Paran´a, Centro Polit´ecnico, Curitiba 81531-980, Brazil
ANTIVIRALS AND TESTICULAR DEVELOPMENT
MATERIALS AND METHODS
Animals Female Wistar rats were obtained from the animal facility of the Universidade Federal do Paran´a. The animals were maintained under controlled temperature (21 ± 1◦ C) and luminosity (12 h/12 h light/dark cycle). Standard pellet food (Nuvilab, Colombo, Brazil) and tap water were available ad libitum. Three nongravid female rats were mated with one male for 3 h, and the day of sperm detection in the vaginal smear was considered gestation day 0 (Chahoud and Kwasigrocch, 1997). All of the experimental procedures were approved by the Committee on Animal Research and Ethics of the Universidade Federal do Paran´a (consent no. 513), which is in accordance with national and international animal welfare guidelines. Drugs and Treatments Acyclovir (Eurofarma, S˜ao Paulo, Brazil) and ganciclovir (Novafarma, An´apolis, Brazil) were available pharmaceutical preparations intended for intravenous application in humans. The content of the vials was diluted with distilled water to yield appropriate concentrations for an administration volume of 1 ml/kg. The gravid females were randomly assigned to five experimental groups. The dams were treated on gestation day 10 with the following three subcutaneous applications (8:00 a.m., 1:00 p.m., and 6:00 p.m.) of distilled water or antivirals: control (3 × distilled water), 75 mg/kg acyclovir (3 × 25 mg/kg), 300 mg/kg acyclovir (3 × 100 mg/kg), 75 mg/kg ganciclovir (3 × 25 mg/kg), and 300 mg/kg ganciclovir (3 × 100 mg/kg). A total of three experiments were conducted using the same exposure protocol described above. In experiment 1, the dams (n = 9–11 per group) were euthanized on gestation day 20 to evaluate the early effects on male fetuses. In experiment 2, the dams (n = 10–13 per group) were allowed to deliver, and the male offspring was observed until postnatal day 91 (±1 day) to
evaluate the possible late effects on the testes and other organs. In experiment 3, the dams (n = 3–7 per group) were euthanized 1 h after the last subcutaneous administration, and blood was collected to quantify antivirals in maternal serum. Table 1 contains the detailed information on the number of animals and litters in each experiment. In all cases, the litter was used as the statistical unit. Experiment 1: Effects of Acyclovir and Ganciclovir on Rat Fetuses The body weight of the rat dams was monitored throughout pregnancy. On gestation day 20, the dams were euthanized by decapitation followed by excision of the uterus and removal of the fetuses. The number, sex, and body weight of the fetuses were recorded. The absolute and relative weights of the liver, kidneys, thymus, and spleen of the dams were also recorded. The relative weights were calculated as the following: organ weight/body weight × 100. The anogenital distance (AGD) was recorded for male and female fetuses using a digital caliper (Carrera Precision, Guangdong, China). The AGD of each animal was divided by the cube root of body weight to avoid errors caused by differences in body size (Gallavan et al., 1999). Fetal testicular histology. Following AGD measurements, the fetuses were euthanized by decapitation, and the testis of one rat per litter was submitted to histological processing whenever possible. Not all litters could be investigated because some testes were lost during manipulation or histological processing. The right testis was immersed and fixed in Bouin’s fixative (75% saturated picric acid, 20% formalin, and 5% acetic acid) for 1 h and maintained in 70% ethanol until embedment in paraffin. Testicular sections (5 m) were obtained, stained with hematoxylin and eosin, and microscopically analyzed to determine the seminiferous cord diameter and number of gonocytes per cord. In each testis, the diameters of seminiferous cords were measured in 20 randomly selected circular cross sections whenever possible. The measurements were made with UTHSCSA ImageTool 3.0 software (UTHSCSA, San Antonio, TX) using images acquired on a Zeiss Axiophot microscope (Oberkochen, Germany) coupled with an imaging system at ×200 magnification (ASI, Vista, CA). The number of gonocytes per cord was determined by counting the nuclei of these cells at ×400 magnification (Zeiss Axiophot microscope) in 15–20 circular cross sections chosen at random for each testis. The crude count was corrected by section thickness and the mean nuclear diameter of gonocytes according to Abercrombie’s formula (Abercrombie, 1946): count = crude count × (section thickness/[section thickness + nuclear diameter]). The diameters of 20 randomly selected gonocyte nuclei per testis were measured using UTHSCSA ImageTool 3.0 software. The calculated power was 0.99, considering five experimental groups and the cell counts obtained by the analysis of 15–20 circular cross sections of seminiferous cords, with ␣ = 0.05, mean standard deviation of 0.5,
Downloaded from http://toxsci.oxfordjournals.org/ at FU BerlinFB Humanmedizin on May 14, 2015
outside the urogenital ridge prior to testis or ovary formation. Once they reach the developing gonad, around gestation day 10, they are referred to as gonocytes (Culty, 2009). Several aspects of gonadal differentiation are still unknown, and the effects induced by chemicals during critical windows of development may be helpful to understand normal and abnormal testis differentiation and function. In the present study, we investigated the early and late reproductive effects of two doses of acyclovir and ganciclovir in male rats following maternal exposure on gestation day 10, which corresponds to the initial stage of gonadal differentiation in rodents. In humans, acyclovir and ganciclovir are used by oral and intravenous routes with short administration intervals because these compounds have very short halflives. Pregnant rats were treated subcutaneously three times on gestation day 10 in order to achieve serum concentrations similar to those seen in humans.
221
222
NIHI ET AL.
TABLE 1 Number of Animals and Litters in the Three Experiments Conducted Parameter
Control
Acyclovir (mg/kg) 75
300
75
300
10 10
10 10
11 11
9 8
8
6–7
8–9
7–8
11 (88.9, 100, 100) (91.6, 100, 100) 11
10 (87.5, 100, 100) (66.7, 100, 100) 8
12 (100, 100, 100) (100, 100, 100) 12
13 (100, 100, 100) (93.2, 100, 100) 11
32
24
36
32
22
16
23
22
11
8
11
10
—
—
—
5
7
7
3
4
a Animals from different litters. Not all litters could be investigated, because some testes were lost during manipulation or histological processing. b Live birth index = (number of live offspring/number of delivered offspring) × 100; data indicate first quartile (Q1), median and third quartile (Q3).
c Weaning index = (number of live offspring at weaning/number of live offspring at birth) × 100; data indicate first quartile (Q1), median and third quartile (Q3). No significant differences were observed for birth (p = 0.177) and weaning (p = 0.066) indices (Kruskal-Wallis). d n = 1–3 rats per litter. In all cases the litters were used as the statistical units.
and total sample size of 35 (n = 6–8 litters per group). Power was calculated with the software G*Power version 3.1.3 (University of Dusseldorf, Germany). Experiment 2: Postnatal Effects of Acyclovir and Ganciclovir The dams were treated on gestation day 10, and their body weights were monitored throughout pregnancy and lactation. The body weight of the pups was monitored every 7 days from birth to postnatal day 21 (weaning). The dams and female pups were euthanized on lactation day 22, whereas the male pups continued to be evaluated. The age at preputial separation was monitored daily in all male rats by manual retraction of the prepuce starting on postnatal day 38. From postnatal day 22 onward, the body weight of male offspring was recorded every 7 days until postnatal day 91 ± 1 (adulthood). At this age, 1–3 males per litter were euthanized by decapitation to collect blood and organs. Organ weights. The absolute and relative weights of the following reproductive organs and tissues were recorded: testes, epididymis, ventral prostate, seminal vesicle (with coagulating glands), and levator ani/bulbocavernosus muscle (LABC). The entire epididymis was carefully removed, dissected free of fat, and weighed. The vas deferens was cut at its junction with the
cauda epididymis. The ventral prostate was weighed without the prostatic capsule, and the seminal vesicle was weighed without its contents. The absolute and relative weights of the liver, kidneys, thymus, and spleen were also recorded. The relative weights were calculated as the following: organ weight/body weight × 100. Number of homogenization-resistant spermatids per testis. The left testes were used to determine the number of homogenization-resistant spermatids, which correspond to spermatids in stages 17–19 of development. After removal of the tunica albuginea, each testis was minced and homogenized for 1 min in 10 ml of 0.9% NaCl that contained 0.5% Triton X-100 at medium speed in a Potter S Tissuemizer (Sartorius, Goettingen, Germany). The homogenate was diluted 10 times in 0.9% NaCl for the microscopic counting of homogenization-resistant spermatids in a B¨urker hemocytometer chamber (Brand GmbH, Wertheim, Germany). Testicular histology. The testis of one rat per litter was submitted to histological processing, whenever possible. The right testes were immersed and fixed in Bouin’s fixative for approximately 2 h. The capsule of each testis was cut at the poles to
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Experiment 1—fetal endpoints Number of pregnant rats 9 Number of dams with viable male 8 fetuses Number of animals analyzed for fetal 6–7 testicular histologya Experiment 2—adult endpoints Number of pregnant rats 10 Live birth index% (Q1, median, Q3)b (90.5, 100, 100) Weaning index% (Q1, median, Q3)c (100, 100, 100) Number of litters with viable adult 9 male offspring Number of adult male rats 25 investigated for organ weightsd Number of males with counts of 17 homogenization-resistant spermatidsd Number of rats investigated for adult 9 testicular histologyd Testicular cell counts (round 5 spermatids and Sertoli cells) Experiment 3—concentrations of antivirals in maternal serum Number of pregnant rats with blood — samples
Ganciclovir (mg/kg)
ANTIVIRALS AND TESTICULAR DEVELOPMENT
Experiment 3: Concentrations of Acyclovir and Ganciclovir in Maternal Serum
Testicular cell counts. Testicular sections from the control and 300 mg/kg ganciclovir groups were further analyzed to determine the number of round spermatids and Sertoli cells per tubular cross section at stages VII to VIII of the seminiferous epithelium cycle. For each animal, round spermatids nuclei and Sertoli cell nucleoli were counted in 10 round (i.e., not germcell-deficient) seminiferous tubule cross sections at stages VII to VIII of the cycle (n = 5 rats per group). The crude count was corrected by section thickness and the mean nuclear or nucleolar diameters according to Abercrombie’s formula (Abercrombie, 1946): count = crude count × (section thickness/[section thickness + nuclear or nucleolar diameter]). The diameters of 10–20 randomly selected spermatid nuclei or Sertoli cell nucleoli per testis were measured using UTHSCSA ImageTool 3.0 software.
The rat dams were treated with three subcutaneous applications (8:00 a.m., 1:00 p.m., and 6:00 p.m.) of distilled water or antivirals on day 10 of gestation. One hour after the final administration (i.e., at 7:00 p.m.), the dams were anesthetized with ketamine (75 mg/kg) and xylazine (1.5 mg/kg). After opening the abdominal cavity, blood was collected from the posterior vena cava with a 23-gauge needle attached to a 1-ml syringe. Blood samples were centrifuged for 15 min at 1000 × g, and the resulting serum was stored at −20◦ C until analysis. The serum concentrations of acyclovir and ganciclovir were determined by high-performance liquid chromatography (HPLC) based on the methods described by Boulieu et al. (1997). Acyclovir (>99% purity) and ganciclovir (>99% purity) standards were purchased from Sigma (Schnelldorf, Germany), and HPLC-grade acetonitrile was obtained from Tedia (Rio de Janeiro, Brazil). Potassium dihydrogenphosphate (KH2 PO4 ) was supplied by Merck (Darmstadt, Germany). The analyses were performed on a reverse-phase LiChrospher-100 RP-18 Merck column (inner diameter, 250 mm × 4.6 mm; 5 m particle size; Darmstadt, Germany) equipped with a Merck guard column (inner diameter, 4.0 mm; length, 1 cm) packed with the same packing material and placed before the inlet of the analytical column. For the quantification of ganciclovir, the mobile phase consisted of a mixture of 10mM KH2 PO4 (pH 3.4) and acetonitrile at a ratio of 98:2 (vol/vol). For acyclovir, the mobile phase consisted of a mixture of 20mM KH2 PO4 (pH 3.2) and acetonitrile at a ratio of 96:4 (vol/vol). For both analyses, the flow rate was set at 1.5 ml/min. Detection was performed at 254 nm, and the mobile phase was previously filtered through a 0.45 mm filter (Millipore, Bedford, MA) and degassed. For sample preparation, a 100 l aliquot of serum was subjected to precipitation with 50 l of a 25% zinc sulfate solution in water (wt/vol). After mixing, the sample was centrifuged at 15,000 × g for 15 min. Afterward, 20 l of the supernatant was automatically injected into the column. Additional details of the methods, including the calibration curve, limits of detection and quantification, precision, and accuracy, are described in the supplementary data.
Serum testosterone. Blood was collected by decapitation and clotted at room temperature. After centrifugation, the serum samples were transferred to clean tubes and kept at −20◦ C. Serum testosterone was quantified using an enzyme immunoassay (Munro et al., 1991). The antibody against testosterone (R1556/7) and the respective horseradish peroxidase conjugate were obtained from Coralie Munro at the University of California, Davis (Davis, CA). Serial dilutions of pooled serum rat samples produced displacement curves parallel to those of standard curves. The intra- and interassay coefficients of variation were
