Prednisone reduces ketoconazole-induced skeletal ... - Springer Link

Arch Toxicol (2009) 83:863–871 DOI 10.1007/s00204-009-0439-6

R E P R O D U CT IV E T O X I C O L O G Y

Prednisone reduces ketoconazole-induced skeletal defects in rat fetuses Vanessa Cristiane Santana Amaral · Guilhermino Pereira Nunes Jr

Received: 15 January 2009 / Accepted: 12 May 2009 / Published online: 29 May 2009 © Springer-Verlag 2009

Abstract Ketoconazole (KT) is a broad-spectrum antifungal agent whose pharmacological activity is based on the capability to interfere with steroid biosynthesis through an interaction with fungal cytochrome P-450 enzymes and thereby avoiding the formation of fungal walls. As the inhibition of fungal cytochrome P-450 by KT is not speciWc, the mammalian cytochrome P-450 species, which play an important role in the biosynthesis of steroidogenesis, are also aVected. The reproductive and developmental toxicity of KT have been assessed. This antimycotic agent has been reported as embryotoxic and teratogenic when administered in high doses (80 mg/kg) to pregnant rats. The mechanisms by which KT exert teratogenic eVects remains to be elucidated. When considering the potential inhibitory eVect of KT on mammalian steroid biosynthesis as a possible responsible for the skeletal anomalies induced by this drug, this study aimed at determining whether steroid maternal supplementation may prevent the skeletal anomalies induced by KT. To test this hypothesis, maternal supplementation with prednisone (PRED) (0.1, 0.2 or 0.4 mg/kg) and 80 mg/kg of KT were administered to pregnant Wistar rats (n = 10) during organogenesis period. On gestational day 21, the dams were euthanized and examined for standard parameters of reproductive outcome. In summary, the

V. C. S. Amaral (&) · G. P. Nunes Jr Departamento de Ciências Fisiológicas, Instituto de Ciências Biológicas, ICB II, Universidade Federal de Goiás., Campus II, Samambaia, Goiânia, GO CEP 74001-970, Brazil e-mail: [email protected] V. C. S. Amaral Unidade de Ciências Exatas e Tecnológicas, Universidade Estadual de Goiás, Anápolis, GO 75001-970, Brazil

results showed that PRED supplementation therapy may cause reductions in the incidence of KT-induced cranial and appendicular skeletal anomalies as well as cleft palate in the rat, being these results more consistent with 0.4 mg/ kg of this drug. These results suggest an important role for glucocorticoids in KT-induced teratogenesis Keywords Embryotoxicity · Ketoconazole · Maternal toxicity · Prednisone · Skeletal anomalies

Introduction Since the late 1970s ketoconazole (KT) has been used as a broad-spectrum oral antifungal agent in the treatment of superWcial and systemic mycoses (Sheehan et al. 1999; Moudgal and Sobel 2003). A few human controlled epidemiological studies of structural birth defects in infants born to mothers who were treated with KT during pregnancy have been reported (Friedman and Polifka 1996; Briggs et al. 1998; Kazy et al. 2005). However, some studies have reported that KT is both teratogenic and embryotoxic in animals at doses ranging from 10 to 80 mg/kg (Buttar et al. 1989; Briggs et al. 1998; King et al. 1998; Sobel 2000). Syndactyly and oligodactyly have been reported in rat fetuses after maternal administration of 80 mg/kg (10 times the maximum recommended human dose) of KT (Briggs et al. 1998; Amaral and Nunes 2008). The antifungal activity of KT is mediated via the inhibition of the syntheses of ergosterol, a component of the fungal cell membrane (Vanden Bossche 1985; Rodriguez and Acosta 1995). This inhibition occurs through an interaction with a P450-dependent enzyme, 14 -demethylase (Como and Dismukes 1994; Zarn et al. 2003). In addition to its antifungal activity, KT is also known to act on the P450

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enzymes of the mammalian steroidogenesis system such as P450scc (cholesterol side-chain cleavage enzyme) (A.H.F.S. 1998), P450c17 (17-hydroxylase and C17–20 lyase) (Engelhardt et al. 1991) and P450c11 (11-hydroxylase) (Iranmanesh et al. 1987). As a result, endocrine abnormalities such as gynecomastia, oligoespermia, decreased libido, impotence, irregular menses and less frequently adrenal insuYciency have been described as side eVects in humans who received 400 mg/day of KT (Van Cauteren et al. 1989, 1990; Vanden Bossche 1992; Como and Dismukes 1994). The mechanism of teratogenesis for KT remains to be elucidated. Some studies have reported the inhibitory eVect on steroid biosynthesis (Loose et al. 1983; Sonino 1987; Vanden Bossche 1992; Como and Dismukes 1994). Thus, this work aimed at investigating whether the inhibition of maternal steroid synthesis induced by KT during the organogenesis could contribute to fetal anomalies. For this purpose, a prednisone supplementation therapy was implemented concomitantly with the administration of KT during organogenesis. This glucocorticoid was selected because according to FDA classiWcation, prednisone (P) belongs to category B, which states that animal studies do not indicated a risk to the fetus and there have been no controlled studies of pregnant women (Briggs et al. 1998).

Materials and methods Subjects were female Wistar rats (Federal University of Goiás/UFG, GO, Brazil), weighing 180–200 g. They were maintained in polyethylene cages and used in this experiment only after a 15-day acclimatization period. They were kept under a controlled temperature of 22 § 2°C and a normal 12-h light cycle. The procedures involving the use of experimental animals in this study were approved by the Ethics Committee for Animals Use of Federal University of Goiás. Fifty nulliparous females were housed overnight with males of the same stock in the ratio of one male to one female. Copulation was ascertained daily by vaginal smear or copulation plug. The day on which spermatozoa were found in a smear of vaginal contents or a copulation plug was observed in situ was designated as gestational day zero (GD 0). The pregnant rats were housed individually. Body weight changes, food and water consumption were recorded from GD 0 to 21. The rectal temperature was taken on days 0, 7, 15 and 21. Animals were observed daily for survival and any reactions to treatment. The study was composed of Wve groups of randomly chosen pregnant rats each, assigned to three treatment groups, i.e., KTP1 (ketoconazole 80 mg/kg + prednisone 0.1 mg/kg), KTP2 (ketoconazole 80 mg/kg + prednisone

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0.2 mg/kg), KTP4 (ketoconazole 80 mg/kg + prednisone 0.4 mg/kg) and two control groups, i.e., KT (ketoconazole 80 mg/kg) and SAL (isotonic saline 4 mL/kg). Ketoconazole and saline were administered orally by daily gavage, whereas prednisone was administered subcutaneously in a single dose from GD 6 to 15 (organogenesis period). The drugs were dissolved in physiological saline (NaCl 0.9%). On GD 21, the females were euthanized and number of corpora lutea in each ovary were recorded. The fetuses were removed from the uterus and the number of live and dead fetuses and macroscopic abnormalities were recorded. The fetuses and the placentas were weighed individually. Early and late resorptions and the number of implantation sites was recorded. Resorption was classiWed “early” when only placental tissue was visible and “late” when placenta as well as embryonic tissue was visible. Fetuses were cleared with KOH and stained with alizarin red (Manson et al. 1982) and examined with a stereodissection microscope. Skeletal anomalies were analyzed according to the method of Manson et al. (1982). The number of ossiWed metacarpals, metatarsals, distal and proximal phalanges was obtained by taking the sum of bones present in both paws in each of the fetuses. The 14th thoracic ribs were evaluated according to Kimmel and Wilson (1973) and separated into groups as follows: rudimentary ribs if less than half the length of the 13th thoracic rib and supernumerary if half or greater than half the length of the preceding 13th rib. Both maternal data and the variables of reproductive outcome were analyzed using ANOVA followed by Tukey– Kramer’s multiple comparison test. Skeletal anomalies were analyzed by Fisher’s exact test and chi-square test. Statistical signiWcance was assumed at P < 0.05.

Results Maternal eVects The results of maternal toxicity are shown in Table 1. Clinical signs of toxicity during treatment were observed only in KT group. Diarrhea was noted in the great majority of dams of KT group, and vaginal bleeding was noted in two dams only in the same group. The mean food consumption by KTP1 and KTP2 groups during organogenesis was signiWcantly decreased when compared with the SAL group. On the other hand, increased food consumption by the KTP4 was signiWcant when compared with KTP2 during the same period. Nevertheless, no statistically signiWcant decrease in the maternal body weight gain was observed in the KTP1, KTP2 and KTP4 groups during organogenesis. The mean food consumption by KTP2 group was signiWcantly decreased when compared with SAL and KTP4

Arch Toxicol (2009) 83:863–871

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Table 1 EVect of prednisone supplementation during organogenesis on ketoconazole-induced maternal toxicity in the rat Parameter

Number of dams/group

Groups SAL saline 4 mL/kg

KT ketoconazole 80 mg/kg

KTP1 ketoconazole 80 mg/kg + prednisone 0.1 mg/kg



10

10

10

10

10

Weight gain (g) Days 0–5

17.4 § 1.5

18.1 § 2.1

17.4 § 1.9

14.7 § 1.8

16.0 § 2.0

6–15

24.9 § 1.7

16.1 § 3.8

13.2 § 5.3

15.3 § 2.1

24.3 § 4.3

16–21

45.9 § 4.6

34.5 § 8.2

33.9 § 8.8

24.1 § 8.1

53.2 § 3.0 ‡

0–5

16.7 § 0.4

16.8 § 0.6

16.8 § 0.4

15.9 § 0.3

15.8 § 0.7

6–15

18.1 § 0.7

15.5 § 0.7

14.2 § 0.5*

13.8 § 0.4*

16.9 § 0.9‡

16–21

21.3 § 0.9

19.9 § 1.2

17.3 § 0.5

16.6 § 1.1*

21.4 § 1.2‡

0–5

36.6 § 1.3

39.5 § 1.7

32.9 § 1.4**

37.4 § 1.1

32.8 § 1.7**

6–15

43.6 § 3.1

41.4 § 1.7

34.6 § 1.9

39.8 § 2.0

37.4 § 2.0

16–21

48.5 § 3.5

50.2 § 3.2

42.8 § 1.9

46.7 § 2.8

49.3 § 2.3

37.3 § 0.1

37.5 § 0.1

38.0 § 0.1*

37.7 § 0.1

37.7 § 0.1

Food intake (g) Days

Water intake (mL) Days

Rectal temperature (°C)a All values are mean § SE a

Mean rectal temperature on days 0, 7, 14 e 21 of gestation * P < 0.05 when compared with SAL group ** P < 0.05 when compared with KT group ‡ P < 0.05 when compared with KTP2 group (ANOVA + Tukey–Kramer’s multiple comparison test)

groups from 15 to 21 days of gestation. During the same period, an increase in the mean of maternal body weight gain was observed in KTP4 group when compared with KTP2 group. In addition, a decrease in water intake during implantation was observed in KTP1 and KTP4 when compared with KT group. No statistically signiWcant diVerence in mean rectal temperature was noted in the SAL, KT, KTP2 and KTP4 groups. However, an increase in the mean rectal temperature was noted in the KTP1 group when compared with SAL group.

the KTP4 when compared with KT, but not when compared with SAL group. A signiWcant decrease in the mean of fetal body weights per litter was noted only in the KTP1 when compared with SAL group.

Reproductive outcome

Axial skeletal anomalies

Reproductive performance data are summarized in Table 2. A statistically signiWcant increase both in the mean percentages of postimplantation loss and in the number of both early and late postimplantation resorptions was observed in KT group. Similarly, a signiWcant increase both in the mean percentages of postimplantation loss and in the number of early postimplantation resorptions was noted in KTP1 and KTP2 groups when compared with SAL group. On the other hand, a signiWcant decrease in the number of both early and late postimplantation resorptions was observed in

Cranial skeletal anomalies data are shown in Table 3. There was an increase in the percentage of fetuses with frontal, parietal and interparietal incomplete ossiWcation of KT group when compared with SAL group. In addition, there was an increase in the incidence of fetuses with frontal incomplete ossiWcation of KTP1 and KTP2 groups when compared with SAL group. In contrast, no anomaly of that kind was found in the fetuses of KTP4 group. Also, the incidence of that anomaly seemed to be reduced in a dosedependent manner in the KTP1, KTP2 and KTP4 groups

External fetal anomalies There were no anomalies in any of the fetuses in the SAL, KTP1, KTP2 and KTP4 groups. However, syndactyly was found in three fetuses from the same litter of KT group.

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Arch Toxicol (2009) 83:863–871

Table 2 EVect of prednisone supplementation during organogenesis on ketoconazole-induced fetal toxicity in the rat Parameters


Number of corpora luteac





12.1 § 0.8

12.3 § 0.4

11.5 § 0.5

13.1 § 0.8

11.7 § 0.5

Number of implantation sitesc

7.5 § 1.1

11.1 § 0.8

9.3 § 0.8

9.8 § 1.1

8.6 § 0.9

Number of preimplantation resorptionsc

4.6 § 1.1

1.2 § 0.6

2.2 § 0.7

3.3 § 0.9

3.1 § 1.0

Preimplantation loss (%)a,c

36.6 § 9.1

9.8 § 5.4

19.1 § 5.9

26.4 § 7.5

25.6 § 8.2

Postimplantation loss (%)b,c

3.2 § 2.3

51.9 § 8.6*

53.3 § 12.3*

50.1 § 10.8*

20.1 § 9.5

Number of postimplantation resorptionsc

0.3 § 0.2

5.9 § 1.1*

4.5 § 1.0*

4.7 § 1.4*

Number of early resorptionsc

0.3 § 0.2

Number of late resorptionsc Total number of fetusesc

0 7.2 § 1.0

c

5.0 § 1.2*

4.2 § 1.1*

4.1 § 1.2*

1.1 § 0.4**

0.9 § 0.3*

0.3 § 0.1

0.6 § 0.3

0**

5.2 § 0.9

4.8 § 1.2

5.1 § 1.1

7.6 § 1.0 0.2 § 0.1

0.1 § 0.1

0.2 § 0.1

0.5 § 0.2

7.2 § 1.0

5.1 § 1.0

4.6 § 1.2

4.6 § 1.2

7.4 § 0.9

Placental weight (g)c

0.63 § 0.02

0.98 § 0.06

0.74 § 0.13

0.90 § 0.11

0.73 § 0.09

Fetal weight (g)c

5.02 § 0.18

4.14 § 0.22

3.27 § 0.61*

3.35 § 0.48

4.36 § 0.51

Number of dead fetuses Number of live fetusesc

0

1.0 § 0.4**

All values are mean § SE * P < 0.05 when compared with SAL group ** P < 0.05 when compared with KT group a Preimplantation loss (%) = [(no. of corpora lutea – no. of implantation sites)/no. of corpora lutea] £ 100 b Postimplantation loss (%) = [(no. of implantation sites – no. of live fetuses)/no. of implantation sites] £ 100 c The litter was used as a statistical unit for calculation of maternal and fetal values, thus these values represent means of litter means within each group

Table 3 EVect of prednisone supplementation during organogenesis on ketoconazole-induced craniofacial anomalies in rat fetuses Anomaly

Groups SAL saline KT ketoconazole 4 mL/kg 80 mg/kg

KTP1 ketoconazole KTP2 ketoconazole 80 mg/kg + prednisone 80 mg/kg + prednisone 0.1 mg/kg 0.2 mg/kg

Total number of fetuses

72

51

47

47

Frontal incomplete ossiWcation

0 (0)

7 (13.73)*

9 (19.15)*

6 (12.77)*

Parietal incomplete ossiWcation

0 (0)

21 (41.18)*

,

37 (78.72)* ** ,

75 0 (0)**,9,‡ ,

7 (9.33)*,**,9,‡

,

23 (30.67)*,**,9,‡

29 (61.70)* **

Interparietal incomplete ossiWcation

0 (0)

6 (11.76)*

Supraoccipital incomplete ossiWcation

1 (1.39)

0 (0)

5 (10.64)*,**

1 (2.13)

0 (0)9

Absent left tympanic ring

0 (0)

3 (5.88)

3 (6.38)

2 (4.26)

0 (0)

0 (0)

0 (0)

8 (17.02)*,**,9

8 (10.67)*,**,9

Absent tympanic ring (both sides)

0 (0)

Cleft palate

0 (0)

0 (0) 31 (60.78)*

35 (74.47)* **


3 (6.38) 32 (68.09)*

29 (61.70)* **

Values represent number of fetuses aVected. Numbers in parentheses represent the percentage of fetuses aVected * P < 0.05 when compared with SAL group ** P < 0.05 when compared with KT group 9 P < 0.05 when compared with KTP1 group ‡ P < 0.05 when compared with KTP2 group (2 followed by Fisher’s exact test)

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Arch Toxicol (2009) 83:863–871

867

when compared with KT group. An increase in the incidence of parietal incomplete ossiWcation in the fetuses of KTP1 and KTP2 groups was observed when compared with KT and SAL groups. The KTP4 group, in turn, showed a signiWcant decrease in the incidence of that anomaly when compared with KT, KTP1 and KTP2 groups. Concerning the incidence of interparietal incomplete ossiWcation, a signiWcant increase was found in KTP1, KTP2 and KTP4 groups when compared with KT group, while a decrease was found in KTP4 group when compared with KTP1 and KTP2 groups. An increase in the incidence of supraoccipital incomplete ossiWcation was found only in the fetuses of KTP1 group when compared with SAL and KT groups.

SigniWcant prevalence of cleft palate in KT, KTP1, KTP2 and KTP4 was observed when compared with SAL group. In addition, the number of fetuses with this anomaly was signiWcantly increased in KT and KTP1 groups when compared with KTP2 and KTP4, revealing a dose relationship pattern for the development of such anomaly. Skeletal anomalies in the trunk are described in Table 4. A decreased number of ossiWed sternebrae in several fetuses of KT group was found when compared with the fetuses of the SAL group. More than half of the fetuses (62.75%) in the KT group showed a complete ossiWcation of six sternebrae, whereas 31.37% of the fetuses in the same group had only Wve ossiWed sternebrae on the 21st gestational day.

Table 4 EVect of prednisone supplementation during organogenesis on ketoconazole-induced anomalies of axial skeleton in rat fetuses Anomaly






72

51

47

47

75

Six ossiWed sternebrae

61 (84.72)

32 (62.75)*

19 (40.42)*,**

25 (53.20)*

55 (73.33)9,‡

Five ossiWed sternebrae

9 (12.50)

16 (31.37)*

21 (44.68)*

15 (31.91)*

14 (18.67)9 2 (2.67)

Total number of fetuses Sternum

Four ossiWed sternebrae

2 (2.78)

3 (5.88)

6 (12.77)

3 (6.38)

Three ossiWed sternebrae

0 (0)

0 (0)

0 (0)

1 (2.13)

3 (4.00)

Two ossiWed sternebrae

0 (0)

0 (0)

1 (2.13)

0 (0)

1 (1.33)

Absent sternebrae

0 (0)

0 (0)

0 (0)

3 (6.38)

0 (0)

5 (6.94)

39 (76.47)*

17 (36.17)*,**

Supernumerary 14th ribs Left side

,

14 (29.79)*,** ,

23 (30.67)*,**

Right side

6 (8.33)

33 (64.71)*

14 (29.79)* **

12 (25.53)* **

15 (20.00)**

Wavy ribs

1 (1.39)

0 (0)

8 (17.02)*,**

0 (0)9

1 (1.33)9

0 (0)

19 (37.25)*

16 (34.04)*

4 (8.51)*,**,9

Rudimentary 7th ribs Left side

,9

3 (4.00)**,9

0 (0)

19 (37.25)*

15 (31.91)*

4 (8.51)* **

3 (4.00)**,9

Left side

32 (44.44)

11 (21.57)*

30 (63.83)*,**

33 (70.21)*,**

52 (69.33)*,**

Right side

26 (36.11)

17 (33.33)

33 (70.21)*,**

35 (74.47)*,**

59 (78.67)*,**

Left side

0 (0)

6 (11.76)*

14 (29.76)*,**

6 (12.77)*

1 (1.33)**,9,‡

Right side

0 (0)

7 (13.73)*

13 (27.66)*

5 (10.64)*

1 (1.33)**,9,‡

Bipartite cervical centra

0 (0)

0 (0)

1 (2.13)

1 (2.13)

0 (0)

Right side

,

Rudimentary 14th ribs

Rudimentary 15th ribs

Bipartite thoracic centra

0 (0)

0 (0)

3 (6.38)

,

8 (17.02)* **

21 (28.00)*,**,9

Bipartite lombar centra

0 (0)

0 (0)

0 (0)

3 (6.38)

3 (4.00)

Presence of three cervical vertebra

0 (0)

0 (0)

0 (0)

6 (12.77)*,**,9

0 (0)‡

Absent cervical vertebra

0 (0)

0 (0)

0 (0)

6 (12.77)*,**,9

0 (0)‡


123

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Arch Toxicol (2009) 83:863–871

Comparative analysis of groups KTP1, KTP2 and KTP4 has shown that the prevalence of six sternebrae bones apparently improves in a dose-dependent way in such groups. In the KTP4 group, 73.33% of the fetuses have shown six sternebrae bones, whereas in groups KTP1 and KTP2 it was observed that 40.42 and 53.20% of the fetuses, respectively, had developed the same number of bones. Regarding the development of supernumerary ribs, it was observed that the KT group has shown a signiWcant increase in prevailing supernumerary 14th ribs and rudimentary 7th and 15th ribs when compared with the SAL group. Fetuses of groups KTP1, KTP2, and KTP4 have shown a decrease in the prevalence of supernumerary 14th ribs and an increase in the prevalence of rudimentary 14th ribs when compared with the KT group (Table 4). In both cases, the prevalence of these ribs was not dose dependent. A dose dependent decrease in the prevalence of rudimentary 7th ribs was also observed in groups KTP1, KTP2 and KTP4, when compared with the KT group. The prevalence of rudimentary 15th ribs was lower in the KTP4 group only when compared with the KT, KTP1 and KTP2 groups.

An increase in the prevalence of bipartite thoracic centra was also observed in groups KTP2 and KTP4 when compared with the SAL and KT groups and this increase was even more signiWcant in group KTP4. The KTP2 group has also shown an increase in the alterations of vertebral bodies when compared with the other groups. About 12.77% of the fetuses in this group have shown only three ossiWed vertebral centra, whereas another 12.77% of the fetuses did not even show such vertebral centra. Besides that the KTP1 group has shown a signiWcant increase in the prevalence of wavy ribs when compared with the SAL and KT groups. Appendicular skeletal anomalies The anomalies of the forelimbs are summarized in Table 5. Fetuses of the KT group have shown a lesser number of ossiWed metacarpals than the fetuses from the SAL group. Only 21.57% of the fetuses in the KT group had eight ossiWed metacarpals, whereas 93.05% of the fetuses in the SAL group had this same number of ossiWed metacarpals.

Table 5 EVect of prednisone supplementation during organogenesis on ketoconazole-induced anomalies of forelimb skeleton in rat fetuses Anomaly






72

51

47

47

75

67 (93.05)

11 (21.57)*

29 (61.70)*,**

26 (55.32)*,**

58 (77.33)*,**,‡

7

4 (5.56)

0 (0)

0 (0)

0 (0)

2 (2.67)

6

1 (1.39)

31 (60.78)*

17 (36.17)*,**

20 (42.55)*

15 (20.00)*,**,‡

5

0 (0)

1 (1.96)

0 (0)

0 (0)

0 (0)

4

0 (0)

3 (5.88)

1 (2.13)

1 (2.13)

0 (0)

2

0 (0)

5 (9.81)*

0 (0)

0 (0)

0 (0)**


Number of ossiWed metacarpals 8

Number of ossiWed phalanges 8

10 (13.89)

0 (0)*

0 (0)*

0 (0)*

12 (16.00)**,9,‡

7

2 (2.78)

0 (0)

0 (0)

0 (0)

0 (0)

6

15 (20.83)

0 (0)*

0 (0)*

1 (2.13)*

6 (8.00)

4

22 (30.56)

5 (9.81)*

24 (51.06)*,**

19 (40.42)**

27 (36.00)**

3

5 (6.94)

1 (1.96)

0 (0)

2 (4.26)

2 (2.67)

2

3 (4.17)

4 (7.84)

4 (8.51)

8 (17.02)

11 (14.66)

1

1 (1.39)

1 (1.96)

1 (2.13)

1 (2.13)

3 (4.00)

0 (zero)

14 (19.44)

40 (78.43)*

18 (38.30)*,**

16 (34.04)**

14(18.67)**,9


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Arch Toxicol (2009) 83:863–871

More than half of the fetuses in the KTP1, KTP2 and KTP4 groups have shown a signiWcant dose dependent increase in the number of ossiWed metacarpals when compared with the KT group. Eight metacarpals were ossiWed in 29 (61.70%) of the fetuses in the KTP1 group, 26 (55.32%) of the KTP2 group and 58 (77.33%) of the KTP4 group. The other fetuses in these groups had six and/or four ossiWed metacarpals. As to the proximal phalanges, it was observed that several fetuses of the KT group have shown a decrease in the ossiWcation of such bones when compared with the SAL group, and 78.43% of the fetuses in the KT group and only 19.44% of the fetuses in the SAL group have not shown such bones. As opposed to previously described results, groups KTP1, KTP2 and KTP4 have shown a signiWcant increase in the number of ossiWed proximal phalanges when compared with the KT group. Such results were even more signiWcant in the KTP4 group as it has shown statistically similar values to the SAL group. No anomalies of the humerus, radius and ulna were observed in the experimental groups. Skeletal anomalies of the forelimbs are shown in Table 6. The KT group has shown a lesser prevalence of ossiWed metatarsals and distal phalanges when compared with the SAL group. However, KTP1, KTP2 and KTP4 groups have shown a dose-dependent tendency towards an increase in the total number of ossiWed metatarsals when compared with the KT group. A better performance was achieved by the KTP4 group, which has proven to be statistically similar to the SAL group. The analysis of groups KTP1, KTP2 and KTP4 revealed that no signiWcant diVerences were found either in the number of ossiWed distal phalanges among these three groups or when compared with the KT group, thus demonstrating a high prevalence of fetuses lacking distal phalanges. Other skeletal anomalies such as shortened Wbula, pubis and femur, or lack of Wbula, which were found in the fetuses of the KT group, were also found in the fetuses of groups KTP1, KTP2 and KTP4. Except for the results obtained in the KTP1 and KTP2 groups relating to the size reduction of the Wbula, which have shown similarity to the number of aVected fetuses in comparison with the KT group, the other results have shown a signiWcant statistical decrease in the above-described anomalies in groups KTP1, KTP2 and KTP4 when compared with the KT group.

Discussion The results of this study show that only KT group has signs of maternal toxicity (diarrhea, vaginal bleeding and maternal death). Although signiWcant diVerences had been

869

observed in food and water consumption in the animals of groups KTP1 and KTP2, such diVerences have not hindered maternal weight gains. Only the animals from the KTP4 group have shown an increase in weight gain from GD 15 to 21, accompanied by an increase in food consumption during the same period, which does not suggest any toxicity to the treatment, but rather an eVect of prednisone in the mother’s metabolism. In addition, the animals from the KTP1 group have also shown a signiWcant increase in their mean rectal temperature when compared with the animals from the SAL group. This result, however, has not been considered to be relevant, as it is found to be within normal temperature limits for rats (37.5–38.5°C). Animals from the KT, KTP1 and KTP2 groups have shown typical signs of embryotoxicity in terms of a signiWcant increase in the percentages of postimplantation loss, in the number of postimplantation resorptions and in the fetal growth restriction when compared with the animals of the SAL group. On the other hand, the animals from the KTP4 group have shown a decrease in the number of both early and late postimplantation resorptions, and an increase in mean fetal weight when compared with the animals from the KT group, which suggests that the treatment with prednisone 0.4 mg/kg apparently improves animal reproductive performance. SigniWcant increase in postimplantation losses and in the reduction of fetal weight due to the use of ketoconazole which were observed in our present study corroborate results previously (Buttar et al. 1989; Cummings et al. 1997), which suggests that such drug may negatively aVect rat gestation and fetal development. In general, the fetuses from the KT group have also shown a higher prevalence of skeletal anomalies when compared with the fetuses from the SAL group. However, the fetuses of groups KTP1, KTP2 and KTP4 have shown a signiWcant improvement in the prevalence of several skeletal anomalies induced by ketoconazole. Decreases were observed in the prevalence of the cleft palate, in the number of both supernumerary and rudimentary ribs (except for the rudimentary 14th ribs), as well as in the size and/or absence of hind limb bones in the fetuses of these three groups when compared with the KT group. Interesting to note that the improvement in the prevalence of some of these anomalies has apparently occurred in a dose-dependent way, whereas this relation has not been maintained for the other anomalies. In addition, the fetuses of these three groups have revealed an increase in the prevalence of the ossiWed sternebrae, metacarpals, metatarsals and forelimbs, which suggests a more complete skeletal development of these fetuses. The enhanced rates of ossiWcation of sternebrae, metacarpals and metatarsals of prednisone supplemental group possibly indicate the growth promoting eVects of this drug. These results suggest that the treatment with prednisone

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870

Arch Toxicol (2009) 83:863–871

Table 6 EVect of prednisone supplementation during organogenesis on ketoconazole-induced anomalies of hind limb skeleton in rat fetuses Anomaly







72

51

47

47

75

10 (19.61)*

29 (61.70)**

22 (46.80)*,**

59 (78.67)**,‡

Number of ossiWed metatarsals 10

55 (76.39)

9

4 (5.56)

1 (1.96)

3 (6.38)

1 (2.13)

1 (1.33)

8

13 (18.05)

16 (31.37)

11 (23.40)

15 (31.91)

15 (20.00)

7

0 (0)

0 (0)

0 (0)

2 (4.26)

0 (0)

6

0 (0)

11 (21.57)*

2 (4.26)**

5 (10.64)*

0 (0)**,‡

5

0 (0)

2 (3.92)

0 (0)

0 (0)

0 (0)

4

0 (0)

10 (19.61)*

0 (0)**

2 (4.26)**

0 (0)**

3

0 (0)

0 (0)

2 (4.26)

0 (0)

0 (0)

0 (zero)

0 (0)

1 (1.96)

0 (0)

0 (0)

0 (0)

Number of ossiWed phalanges 8

11 (15.28)

0 (0)*

0 (0)*

0 (0)*

0 (0)*

7

2 (2.78)

0 (0)

0 (0)

0 (0)

0 (0)

6

7 (9.72)

0 (0)*

0 (0)*

0 (0)*

1 (1.33)*

5

2 (2.78)

0 (0)

0 (0)

0 (0)

0 (0)

4

2 (2.78)

0 (0)

0 (0)

0 (0)

0 (0)

3

1 (1.39)

0 (0)

0 (0)

0 (0)

0 (0)

2

0 (0)

0 (0)

0 (0)

0 (0)

2 (2.67)

1

0 (0)

1 (1.96)

1 (2.13)

0 (0)

1 (1.33)

0 (zero)

47 (65.27)

50 (98.04)*

46 (97.87)*

47 (100)*

71 (94.67)*

Short Wbula

0 (0)

3 (5.88)

8 (17.02)*

5 (10.64)*

1 (1.33)9,‡

Absent Wbula

0 (0)

15 (29.41)*

1 (2.13)**

1 (2.13)**

0 (0)**

Short femur

0 (0)

7 (13.73)*

0 (0)**

0 (0)**

0 (0)**

Absent femur

0 (0)

1 (1.96)

0 (0)

0 (0)

0 (0)

Short tibia

0 (0)

4 (7.84)*

0 (0)

0 (0)

0 (0)

Absent tibia

0 (0)

3 (5.88)

0 (0)

0 (0)

0 (0)

Short pubis

0 (0)

11 (21.57)*

1 (2.13)**

0 (0)**

4 (5.33)**

Absent pubis

0 (0)

6 (11.76)*

1 (2.13)

1 (2.13)

3 (4.00)

Values represent number of fetuses aVected Numbers in parentheses represent the percentage of fetuses aVected * P < 0.05 when compared with SAL group ** P < 0.05 when compared with KT group 9 ‡

P < 0.05 when compared with KTP1 group P < 0.05 when compared with KTP2 group (2 followed by Fisher’s exact test)

apparently oVers the fetus a certain protection, as it reduces the prevalence of several fetal anomalies induced by ketoconazole. On the other hand, there has been an increase in the prevalence of anomalies located in the skull (incomplete ossiWcation), in the vertebrae and in the hind limbs of the fetuses from groups KTP1, KTP2 and KTP4, which were induced by ketoconazole. The increase in the prevalence of these anomalies in groups KTP1, KTP2 and KTP4 may

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stem from a possible eVect of glucocorticoid in the metabolism of fetal bones, or may represent—in the case of hind limbs and other bipartite vertebra centra—variations in skeletal development, rather than an anomaly in itself (Kimmel and Wilson 1973). The mechanisms by which KT produces teratogenic eVects have not yet been elucidated, but evidence points to a biochemical and/or molecular involvement of this drug in the genesis of skeletal anomalies. The biochemical

Arch Toxicol (2009) 83:863–871

mechanism is apparently related to the lack of speciWcity of KT in inhibiting only the fungal enzymes, which results in the inhibition of cytochrome P450 enzymes that are also part of the steroidogenesis process in mammals (Iranmanesh et al. 1987; Engelhardt et al. 1991; A.H.F.S. 1998). Although promoting this enzyme inhibition, KT may reduce serum levels of maternal steroids and cause maternal–fetal toxicities (MacDonald 1978; Cummings et al. 1997). The hypothesis that steroids are involved in the teratogenicity of this drug has been evidenced in our study, considering that after steroid replacement with prednisone we have noted a signiWcant decrease in the prevalence of anomalies induced by ketoconazole. Therefore, it is suggested that deWciency in glucocorticoid synthesis induced by ketoconazole during organogenesis—the period in which treatment with this drug was conducted—may result in skeletal anomalies, thus suggesting an important role for glucocorticoids in ketoconazoleinduced teratogenesis. However, further investigation will be necessary to verify this hypothesis. Acknowledgments The authors are grateful to Fundação de Apoio à Pesquisa—FUNAPE for supporting this work. The authors also thank Janssen Cilag Pharmaceutica (São Paulo-Brazil) for generously providing ketoconazole for this study.

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