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Association of Ipomoea carnea and BCG reduces birth defects caused by cyclophosphamide in rats
Life Sciences 80 (2007) 430 – 435 www.elsevier.com/locate/lifescie
Association of Ipomoea carnea and BCG reduces birth defects caused by cyclophosphamide in rats Andréia Oliveira Latorre, Isis Machado Hueza, Silvana Lima Górniak ⁎ Department of Pathology, School of Veterinary Medicine, University of São Paulo, Brazil Received 28 March 2006; accepted 26 September 2006
Abstract Immunosuppressive drugs can induce the development of malformations in fetuses of mothers exposed to them, possibly affecting the placental function directly or by crossing the placenta to enter fetal circulation. However, activation of the maternal immune system with wellknown immunomodulator substances has been shown to produce a significant decrease in morphological defects caused by diverse teratogenic agents. All of these studies were performed on mice only, whereas the rat is the chosen species for developing teratological studies. Thus, the purpose of the present study was to investigate the possible protective effect of Bacillus Calmette Guérin (BCG) and/or the aqueous fraction (AF) of the plant Ipomoea carnea on the decrease of the teratogenic effect resulting from cyclophosphamide (CP), an antineoplastic and immunosuppressive drug, exposure in pregnant rats. It was verified that both BCG and/or AF attenuated the embryotoxic effects of CP in rats. All immune stimulated dams demonstrated an increase in placenta and fetus body weight. In conclusion, the present work showed that the rat is a good model for performing studies which aim for a clearer understanding of the mechanism by which maternal stimulation reduces malformations and how the association of I. carnea AF and BCG provided improved immunostimulation compared to BCG alone; however, additional studies are required to determine the specific mechanisms by which immune stimulant substances decrease malformation. © 2006 Elsevier Inc. All rights reserved. Keywords: Ipomoea carnea; BCG; Cyclophosphamide; Birth defects
Introduction Numerous chemical substances exist which can induce the development of malformations in fetuses of mothers exposed to them and several of these are drugs commonly ingested by patients chronically affected by diseases which require extensive therapy. Among these substances, immunosuppressive drugs are most frequently used for prophylaxis of allograft rejection in transplanted patients, in the treatment of aggressive lymphomas and autoimmune disease therapy (Matalon et al., 2004). These drugs may affect the placental function directly, or cross the placenta to enter fetal circulation, carrying a risk of fetal maldevelopment and malformations (Pacifici and Nottoli, 1995). However, for reasons not fully defined, activation of the maternal immune system with well-known immunomodulator substances, such as the synthetic copolymer pyran (Nomura ⁎ Corresponding author. Prof. Dr. Orlando Marques de Paiva, 87, CEP 05508900, São Paulo, Brazil. Tel.: +55 11 30917693; fax: +55 11 30917829. E-mail address: [email protected] (S.L. Górniak). 0024-3205/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2006.09.033
et al., 1990); granulocyte-macrophage colony stimulating factor – GM-CSF (Savion et al., 1999); attenuated Bacillus Calmette Guérin (BCG) (Holladay et al., 2000); interferon-gamma (IFNγ) and footpad injection with Freund's complete adjuvant (FCA) (Sharova et al., 2000) have been shown to produce a significant decrease in morphological defects caused by diverse teratogenic agents. Some results of these studies suggest that immunoregulatory cytokines secreted by activated maternal immune cells could mediate the protective effect against teratogenesis. Thus, studies conducted by Savion et al. (1999) showed the ability of granulocyte macrophage-colony stimulating factor (GM-CSF), as well as interleukin-2 (IL-2) and IL-3, to modulate the teratogenic activity induced by cyclophosphamide (CP). Moreover, Gorivodsky et al. (1999) showed that maternal immune stimulation reversed malformation and increased the expression of uteroplacental transforming growth factor β2 (TGF-β2) mRNA in mice treated with CP. Besides enhanced interleukin and growth factor levels in the mother, maternal immune stimulation also promoted diminished CP-induced brain and craniofacial
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anomalies in the fetal mice with decreased tumor necrosis factor-α (TNF-α) mRNA transcription (Ivnitsky et al., 1998). Therefore, it could be proposed that maternal immune stimulation may reverse teratogenic effects by increasing the synthesis and release of immune proteins with regulatory activity which could interact with the placenta and/or the fetus itself to relieve or partially relieve teratogenesis. These studies are important to provide pharmaceutical tools for women of childbearing age chronically exposed to potential teratogenic substances or even diabetic women who get pregnant and wish to carry to term. However, all of these studies were performed in mice only, whereas the rat is the chosen species for developing teratological studies (OECD, 1981). In addition, recently a group of immunotoxicology experts proposed the rat as the preferred model for developmental immunotoxicology (Holsapple et al., 2005). Thus, the purpose of the present study was to investigate the possible protective effect of BCG and/or the aqueous fraction (AF) of the plant Ipomoea carnea, which are known to activate macrophages (Holladay et al., 2000; Hueza et al., 2003, respectively) over the decrease of the teratogenic effect resulting from CP administration in pregnant rats.
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Immunostimulation The female rats were separated randomly into six groups: five experimental (ABC, AC, BC, A and B) and one control group (C). The treatment schedule is shown in Fig. 1. Briefly, rats from the ABC, AC and A groups were pretreated by gavage for 14 consecutive days with AF (3.0 g/kg of Ipomoea carnea dry leaves) 28 or 14 days prior to mating. Rats from the remaining groups (BC, B and C) were pretreated by gavage for the same period with filter water only. On day 14 before mating, BCG (10.0 mg/kg) was injected intraperitoneal (ip) in females from the ABC, BC and B groups and saline was injected ip in females from the other three groups (AC, A and C). Groups A and B were only used to show that the treatments AF and BCG did not promote teratological effects. Mating Mating was performed by placing one male into the cage of two females overnight. The day on which the females exhibited either sperm in the vaginal smear or a vaginal plug was designated as day 0 of pregnancy.
Material and methods
Cyclophosphamide-induced embryotoxicity
Plant material
Female rats were weighed on days 0, 11 and 21 of pregnancy. On day 11 of gestation, dams from the ABC, AC, BC and C groups were treated with a subcutaneous (sc) injection of CP (7.5 mg/kg) in the dorsal area of the neck (Gomes-Carneiro
I. carnea leaves were collected from plants cultivated at the Research Centre for Veterinary Toxicology (CEPTOX), University of São Paulo (USP), Pirassununga, Brazil, in May, 2001. A voucher specimen was deposited in the Herbarium of the Botanic Institute of São Paulo, Brazil (SP-360911) and its authenticity was confirmed by the taxonomist Rosangela Simão Bianchini of the same Institute. The AF resulting from extraction of I. carnea dry leaves was obtained according to previously described methods (Hueza et al., 2003). Reagents Bacillus Calmette Guérin-cepa Moreaux (BCG) was yielded by the Institute Butantã of São Paulo, Brazil and was suspended in a 0.9% NaCl solution at 0.1% concentration. CP (Genuxal®) was purchased from Asta Medica AG, Frankfurt, Germany and was dissolved in 0.9% NaCl solution. 10% neutral buffered formalin. Animals Male and nulliparous female Wistar rats aged about 70 days, bred in the Department of Pathology, School of Veterinary Medicine and Zootechny, were used. The animals received food and water ad libitum and were maintained under controlled conditions of temperature (22–25 °C), humidity (50–65%) and lighting (12/12 light/dark cycle). The experiments were carried out in accordance with the ethical principles for animal research adopted by the Bioethics Committee of the School of Veterinary Medicine and Zootechny, University of São Paulo.
Fig. 1. Treatment schedule: rats from the ABC, AC and A groups were pretreated for 14 consecutive days with AF (3.0 g/kg of I. carnea dry leaves) 28 or 14 days prior to mating. Rats from the remaining groups (BC, B and C) were pretreated for the same period with filter water only. On day 14 before mating, BCG (10.0 mg/kg) was injected ip in females from the ABC, BC and B groups and saline was injected ip in females from the other three groups (AC, A and C). On pregnancy day 11, CP (7.5 mg/kg) was injected sc in females from the ABC, AC, BC and C groups and saline was injected ip in females from the other groups (A and B).
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Table 1 Maternal weight (g) and cesarean section data of rats pretreated with AF (3.0 g/kg/day of Ipomoea carnea po) and/or BCG (10.0 mg/kg ip) 28 or 14 days before mating and subcutaneous injection of CP (7.5 mg/kg) or its vehicle alone (saline) on pregnancy day 11 Treatment 1
C
AC
ABC
BC
A
B
Pregnant females (N) Resorption of the whole litter(N)
8 2
9 1
6 0
7 0
10 0
8 0
Maternal weight (g)♦ Day 0 Day 11 Day 21 (minus utero wt)
231.9 ± 4.8 263.2 ± 6.1 260.4 ± 6.6
217.8 ± 3.3 250.6 ± 2.6 257.7 ± 3.5
228.4 ± 6.2 267.7 ± 9.1 264.8 ± 9.9
231.8 ± 4.7 265.9 ± 6.8 256.8 ± 7.2
229.8 ± 3.5 263.2 ± 3.3 267.1 ± 4.3
228.0 ± 4.3 265.8 ± 6.5 257.7 ± 5.8
Implantation sites• Per litter
100 (76.9–100)
100 (88.9–100)
100 (91.7–100)
100 (75–100)
100 (100–100)
100 (91.7–100)
Pre-implantation loss• Per litter
0 (0–23.1)
0 (0–11.1)
0 (0–8.8)
0 (0–25)
0 (0–0)
0 (0–8.3)
Post-implantation loss• Per litter
86.7 (58.3–92.8)
60 (25–90)
63.6 (21.4–78.6)
33.3 (8.3–69.2)⁎
8.3 (0–31.2)
4 (0–18.2)
1
A = aqueous fraction of Ipomoea carnea, B = Bacillus Calmette Guérin, C = cyclophosphamide. ♦Data are expressed as mean ± SEM and were analyzed by ANOVA followed by the Dunnett's test. •Percentages were analyzed by Kruskal–Wallis test followed by the Dunn's Multiple Comparisons Test. ⁎Differences are statistically significant (P b 0.05) between AC, ABC and BC groups compared to the C group.
et al., 2003). Rats from groups A and B were treated with an sc injection of saline in the same manner. Caesarian section and evaluation of the degree of severity of birth defects On day 21 of gestation all dams were anesthetized and killed by CO2 inhalation. The gravid uterus was weighed with its contents and the number of implantation sites, resorptions, living and dead fetuses was recorded. The living fetuses were
weighed, examined with a stereomicroscope for external visible anomalies such as cleft palate, exencephaly, craniofacial, limb, digits and tail defects, after death they were fixed in a formalin solution. The visible skull defects were scored for severity by using a grading scale as follows: 0 for absence of skull defects; 1 for very small opening on the skull; 2 for clearly defined opening on the skull; 3 for slight protrusion of the brain through a wider opening on the skull (mild exencephaly); 4 for pronounced protrusion of the brain outside the skull opening (severe exencephaly).
Table 2 Fetal weight (g) and data of fetuses from rats pretreated with AF (3.0 g/kg/day of Ipomoea carnea po) and/or BCG (10.0 mg/kg ip) 28 or 14 days before mating and subcutaneous injection of CP (7.5 mg/kg) or its vehicle alone (saline) on pregnancy day 11 Treatment1
C
AC
ABC
BC
A
B
Fetuses (litters+) examined
15 (6)
32 (8)
33 (6)
51 (7)
118 (10)
83 (8)
Resorptions Per implantation sites• Per litter♦
86.7 (58.3–92.9) 10.0 ± 1.0
60 (25–90) 6.2 ± 0.8*
63.6 (21.4–78.6) 7.6 ± 1.3
33.3 (8.3–69.2)* 4.7 ± 1.0**
8.3 (68.7–100) 1.6 ± 0.5
4.1 (0–18.18) 0.7 ± 0.3
Live fetuses Per implantation sites• Per litter♦
13.3 (7.1–41.7) 2.7 ± 0.9
40 (10–75) 4.0 ± 0.7
34.8 (21.4–78.6) 5.5 ± 1.2
66.7 (30.8–91.7)* 7.3 ± 1.1*
91.6 (68.7–100) 11.8 ± 0.5
95.8 (81.8–100) 10.4 ± 0.5
Fetal body weight (g)♦ Per litter Per group
3.00 ± 0.09 3.04 ± 0.08
3.67 ± 0.23 3.67 ± 0.11**
3.71 ± 0.32 3.61 ± 0.13*
3.47 ± 0.17 3.54 ± 0.08*
3.59 ± 0.11 3.59 ± 0.03
4.49 ± 0.30 4.58 ± 0.09
Placental weight (g)♦ Per litter Per group
0.28 ± 0.01 0.29 ± 0.01
0.37 ± 0.03 0.38 ± 0.01**
0.35 ± 0.03 0.39 ± 0.01**
0.37 ± 0.02 0.37 ± 0.01*
0.57 ± 0.02 0.57 ± 0.00
0.64 ± 0.02 0.64 ± 0.00
1
A = aqueous fraction of Ipomoea carnea, B = Bacillus Calmette Guérin, C = cyclophosphamide. Only litters with live fetuses. ♦Data are expressed as mean ± SEM and were analyzed by ANOVA followed by the Dunnett's test. •Percentages were analyzed by Kruskal–Wallis test followed by the Dunn's Multiple Comparisons Test. *Differences are statistically significant (P b 0.05), **(P b 0.01) between AC, ABC and BC groups compared to the C group.
+
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Table 3 Occurrence of external malformations in rat offspring pretreated with AF (3.0 g/kg/day of Ipomoea carnea po) and/or BCG (10.0 mg/kg ip) 28 or 14 days before mating and subcutaneous injection of CP (7.5 mg/kg) or its vehicle alone (saline) on pregnancy day 11 Treatment1
C +
Number of examined fetuses (litters )
15 (6)
% malformed fetuses per litter Skull closure deffects 0 (absence) 1 (slight) 2 (mild) 3 (severe) 4 (very severe) Polydactyly Oedema
0 (0–0) 0 (0–0) 45 (0–100) 30 (0–100) 0 (0–100) 0 (0–20) 80 (0–100)
AC 32 (8)
0 (0–50) 39.3 (0–100)⁎ 12.5 (0–75) 0 (0–80) 0 (0–100) 0 (0–40) 50 (16.7–100)
ABC 33 (6)
0 (0–9) 26.9 (0–85.7) 33.3 (14.3–100) 10 (0–33.3) 0 (0–33.3) 0 (0–0) 32.1 (0–100)
BC 51 (7)
25 (0–50)⁎ 9.1 (0–45.4) 28.6 (9.1–63.6) 18.2 (0–33.3) 0 (0–28.6) 16.7 (0–27.3) 18.2 (0–66.7)
A 118 (10)
100(100–100) 0 (0–0) 0 (0–0) 0 (0–0) 0 (0–0) 0 (0–0) 0 (0–0)
B 83 (8)
100 (100–100) 0 (0–0) 0 (0–0) 0 (0–0) 0 (0–0) 0 (0–0) 0 (0–0)
1
A = aqueous fraction of Ipomoea carnea, B = Bacillus Calmette Guérin, C = cyclophosphamide. Only litters with live fetuses. Percentages of malformed fetuses per litter (litter as statistical unit) were analyzed by Kruskal–Wallis test followed by the Dunn's Multiple Comparisons Test. ⁎Differences are statistically significant (P b 0.05) between AC, ABC and BC groups compared to the C group.
+
Statistical analysis Data were analyzed using only the C-group as control group against the ABC, BC and AC groups. The data was analyzed by one-way analysis of variance (ANOVA) followed by the Dunnett test for multiple comparisons and the data are expressed as mean ± S.E.M. Percentages data were compared by the Kruskall–Wallis test followed by Dunn's test and were expressed as median and range values. In all cases, differences were considered to be statistically significant at P b 0.05. Results Maternal weight and cesarean section data from rats pretreated with AF or BCG and submitted to CP treatment on day 11 of gestation No differences were observed in the weight gain of pregnant female rats pretreated with AF or BCG (i.e. A- and B-group, respectively), even when these pre-treatments were subsequently accompanied by CP injection on day 11 of gestation. Besides, no differences were observed in the percentages of the implantation sites and pre-implantation losses per litter between groups. In contrast, the percentage of post-implantation losses notably decreased in females from the BC-group (P b 0.05) when compared to that of C-group (Table 1).
placenta weight AC (P b 0.01), ABC (P b 0.01) and BC (P b 0.05) was verified when compared with dams from the control group. Occurrence of external malformations in fetuses from female rats pretreated with AF or BCG and submitted to CP treatment on day 11 of gestation No external malformation was noted in fetuses from dams pretreated with AF or BCG only (A- and B-group, respectively). In contrast, all fetuses from rats treated with CP alone, on pregnancy day 11, exhibited skull anomalies with severity scores varying between 2 and 4. Fetuses from the AC-group showed a concentration of skull anomalies between the scores 1 and 2, the same range of scores was observed in fetuses from the ABC-group; and fetuses from dams belonging to the BC-group showed skull anomaly scores concentrated between 0 and 2. Polydactylism was also observed among rat offspring exposed to CP on pregnancy day 11 (C-group), this malformation was observed, with greater frequency in fetuses from the BC-groups of dams, in contrast to fetuses of dams belonging to the ABCgroup, which showed no occurrence of polydactylism. Moreover, all the experimental groups pretreated with immunomodulator substances showed a decreased in the number of edematous fetuses over C-group (Table 3). Discussion
Analysis of fetuses from rats pretreated with AF or BCG and submitted to CP treatment on day 11 of gestation Fetal measurements are shown in Table 2, a remarkable increase in the number of live fetuses per litter was observed in the group of dams from the BC-group (P b 0.05) when compared with dams from the control group. Moreover, when the number of resorptions per litter was accessed, this was notably reduced in female rats from the AC-group (P b 0.05) and BC-group (P b 0.01). Additionally, when considering each individual group, an increase in fetal body weight AC (P b 0.01), ABC (P b 0.05) and BC (P b 0.05) and
CP is an antineoplastic and immunosuppressive drug which has been found to be teratogenic in rats, mice, chicks, rabbits, monkeys and humans (Matalon et al., 2004; Mirkes et al., 1984); on the other hand, many studies from independent laboratories show that maternal immune stimulation decreases fetal abnormalities caused by this substance; however, in all of these previous studies mice were used as the animal model; therefore, we extended this observation by verifying that materno-fetal immune interactions in rats also offer protection against maldevelopment induced by CP. In this regard, it should be pointed out that the rat is the chosen species for developmental
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(OECD, 1981) and immunotoxic studies (Holsapple et al., 2005), including developmental immunotoxicology evaluation (Ladics et al., 2005). Since the pioneer study, conducted by Nomura et al. (1990), showing that stimulation of peritoneal macrophages in pregnant mice reduced fetal malformations, many investigators have theorized regarding the mechanisms by which this protective effect occurs. Although the matter of how immune substrates and teratogenic metabolites interact at the cellular level is not yet clear, it is currently proposed that maternal immune stimulation may reduce chemically induced morphological lesions by an indirect effect via regulatory cytokine product of activated immune cells, in this respect Sharova et al. (2000) theorized that such soluble mediators restore the teratogen-altered expression of critical fetal genes. In the present study, it was verified that both BCG and/or AF attenuated the embryotoxic effects of CP in rats. In fact, BCG is known to promote macrophage activation, with the release of pro-inflammatory cytokines, such as IL-1, IL-6, TNF-α, GMCSF and other molecular components of inflammatory response and many studies have shown that most of these substances produce a reduction in chemically induced birth defects in mice (Holladay et al., 2002). Similarly, it is proposed that CP alters levels of cytokines in both the uteroplacental unit and the fetus, leading to an inappropriate apoptotic process which results in teratogenesis (Chernoff et al., 1989) which appears to be reversed by macrophage-release cytokines, such as GM-CSF (Savion et al., 1999). In relation to the AF of I. carnea, it is well recognized that the indolizidine alkaloid swainsonine, present in this plant, activates tissue macrophages (Das et al., 1995), releases IL-1 from these cells (Grzegorzewski et al., 1989) and enhances NK cell cytotoxicity (Bowlin et al., 1989) and for this reason has been considered as an antimetastatic agent (Roberts et al., 1998). In addition, a study conducted in this laboratory on rats, revealed that I. carnea extract promoted enhanced peritoneal macrophage activity (Hueza et al., 2003). Thus, it could be suggested that the reduction in CP-induced malformations verified in fetuses from both AC and ABCgroups was also due to maternal macrophage activation and, consequently, the production of cytokines. An unexpected observation in the present study was that BCG/CP-treated mothers showed a median of 16.7 fetuses with polydactylism, a frequency greater than that shown by fetuses from any other group, including CP-exposed fetuses (C-group). A similar effect was observed by Holladay et al. (2000), who verified that female mice stimulated with Freund's complete adjuvant and exposed to valproic acid (VA), a well-known chemical teratogen, produced fetuses displaying tail absence, a defect not typically seen in ICR mice exposed to VA. Collectively, these results clearly reveal that a teratogenic effect could be caused by maternal immune stimulation; however, at the present moment it is not possible to theorize the mechanisms by which this effect occurs. After a retrospective regarding all studies concerned with verifying the influence of immunostimulants on the suppression of developmental anomalies, we noted that many suppositions
were made regarding inflammatory cytokines in the placenta and fetus gene expression up-regulation (Sharova et al., 2003; Holladay et al., 2002), but until now no attention has been paid to the biotransformation mechanism of the teratological agents employed in these studies. In relation to CP, it is very well documented that this substance must be activated in the liver by microsomal cytochrome P-450 monooxygenases to achieve its teratogenic effects (Matalon et al., 2004; Mirkes et al., 1984). In fact, a study conducted by Gomes-Carneiro et al. (2003) showed the inhibition of CP-induced teratogenesis when pregnant rats were previously administered with β-ionone, an in vitro inhibitor of the isoenzyme CYP2B1. In addition it should be pointed out that other classic teratogens, such as valproic acid, urethane and polycyclic aromatic hydrocarbons, are also activated by liver microsomal enzymes (Parkinson, 2001). It is known that inflammatory cytokines (Crawford et al., 2004; Ke et al., 2001; Chen et al., 1995) and other inflammatory mediators, such as nitric oxide (Khatsenko, 1998; Carlson and Billings, 1996), can inhibit the efficacy of microsomal cytochrome P-450 enzymes, thus decreasing the biotransformation of the active metabolites of these drugs. Thus, it could be theorized that at least part of the improvement in fetal morphogenesis produced in all experiments using immunostimulants, such as BCG, interferons, FCA and other macrophage activators, is due to the impairment of the active metabolite production by these isoenzymes. Moreover, some teratogenic promoters; such as N-methyl-N-nitrosourea and streptozocin (nitrosourea compounds), substances that have been employed in numerous studies as teratogenesis inducers; produce their effect by DNA chain alkylation in the duplicating process (Prater et al., 2004). Moreover, some cytokines used to induce immune stimulation, such as interferon, can promote an increase in O6-alkylguanine-DNA alkyltransferase expression (Bertini et al., 1990) which is related to the repair of DNA alkylated by these nitrosourea compounds (Fang et al., 2005) that could lead to a diminished occurrence of DNA abnormalities and, consequently, a decrease in fetal maldevelopment. In conclusion, the present work showed that the rat is a good model for performing studies aimed at a clearer understanding of the mechanism by which maternal stimulation reduces malformations and how the association of I. carnea AF and BCG provided improved immunostimulation compared to BCG alone; however, additional studies are required to determine the specific role of immune stimulant substances on general metabolism and the possible influence of a non-immunological effect in decreasing malformation. Acknowledgements This research was supported by grants from Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), Brazil. References Bertini, R., Coccia, P., Pagani, P., Marinello, C., Salmona, M., D'Incalci, M., 1990. Interferon inducers increase O6-alkylguanine-DNA alkyltransferase in the rat liver. Carcinogenesis 11 (1), 181–183.
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Association of Ipomoea carnea and BCG reduces birth defects caused by cyclophosphamide in rats Andréia Oliveira Latorre, Isis Machado Hueza, Silvana Lima Górniak ⁎ Department of Pathology, School of Veterinary Medicine, University of São Paulo, Brazil Received 28 March 2006; accepted 26 September 2006
Abstract Immunosuppressive drugs can induce the development of malformations in fetuses of mothers exposed to them, possibly affecting the placental function directly or by crossing the placenta to enter fetal circulation. However, activation of the maternal immune system with wellknown immunomodulator substances has been shown to produce a significant decrease in morphological defects caused by diverse teratogenic agents. All of these studies were performed on mice only, whereas the rat is the chosen species for developing teratological studies. Thus, the purpose of the present study was to investigate the possible protective effect of Bacillus Calmette Guérin (BCG) and/or the aqueous fraction (AF) of the plant Ipomoea carnea on the decrease of the teratogenic effect resulting from cyclophosphamide (CP), an antineoplastic and immunosuppressive drug, exposure in pregnant rats. It was verified that both BCG and/or AF attenuated the embryotoxic effects of CP in rats. All immune stimulated dams demonstrated an increase in placenta and fetus body weight. In conclusion, the present work showed that the rat is a good model for performing studies which aim for a clearer understanding of the mechanism by which maternal stimulation reduces malformations and how the association of I. carnea AF and BCG provided improved immunostimulation compared to BCG alone; however, additional studies are required to determine the specific mechanisms by which immune stimulant substances decrease malformation. © 2006 Elsevier Inc. All rights reserved. Keywords: Ipomoea carnea; BCG; Cyclophosphamide; Birth defects
Introduction Numerous chemical substances exist which can induce the development of malformations in fetuses of mothers exposed to them and several of these are drugs commonly ingested by patients chronically affected by diseases which require extensive therapy. Among these substances, immunosuppressive drugs are most frequently used for prophylaxis of allograft rejection in transplanted patients, in the treatment of aggressive lymphomas and autoimmune disease therapy (Matalon et al., 2004). These drugs may affect the placental function directly, or cross the placenta to enter fetal circulation, carrying a risk of fetal maldevelopment and malformations (Pacifici and Nottoli, 1995). However, for reasons not fully defined, activation of the maternal immune system with well-known immunomodulator substances, such as the synthetic copolymer pyran (Nomura ⁎ Corresponding author. Prof. Dr. Orlando Marques de Paiva, 87, CEP 05508900, São Paulo, Brazil. Tel.: +55 11 30917693; fax: +55 11 30917829. E-mail address: [email protected] (S.L. Górniak). 0024-3205/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2006.09.033
et al., 1990); granulocyte-macrophage colony stimulating factor – GM-CSF (Savion et al., 1999); attenuated Bacillus Calmette Guérin (BCG) (Holladay et al., 2000); interferon-gamma (IFNγ) and footpad injection with Freund's complete adjuvant (FCA) (Sharova et al., 2000) have been shown to produce a significant decrease in morphological defects caused by diverse teratogenic agents. Some results of these studies suggest that immunoregulatory cytokines secreted by activated maternal immune cells could mediate the protective effect against teratogenesis. Thus, studies conducted by Savion et al. (1999) showed the ability of granulocyte macrophage-colony stimulating factor (GM-CSF), as well as interleukin-2 (IL-2) and IL-3, to modulate the teratogenic activity induced by cyclophosphamide (CP). Moreover, Gorivodsky et al. (1999) showed that maternal immune stimulation reversed malformation and increased the expression of uteroplacental transforming growth factor β2 (TGF-β2) mRNA in mice treated with CP. Besides enhanced interleukin and growth factor levels in the mother, maternal immune stimulation also promoted diminished CP-induced brain and craniofacial
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anomalies in the fetal mice with decreased tumor necrosis factor-α (TNF-α) mRNA transcription (Ivnitsky et al., 1998). Therefore, it could be proposed that maternal immune stimulation may reverse teratogenic effects by increasing the synthesis and release of immune proteins with regulatory activity which could interact with the placenta and/or the fetus itself to relieve or partially relieve teratogenesis. These studies are important to provide pharmaceutical tools for women of childbearing age chronically exposed to potential teratogenic substances or even diabetic women who get pregnant and wish to carry to term. However, all of these studies were performed in mice only, whereas the rat is the chosen species for developing teratological studies (OECD, 1981). In addition, recently a group of immunotoxicology experts proposed the rat as the preferred model for developmental immunotoxicology (Holsapple et al., 2005). Thus, the purpose of the present study was to investigate the possible protective effect of BCG and/or the aqueous fraction (AF) of the plant Ipomoea carnea, which are known to activate macrophages (Holladay et al., 2000; Hueza et al., 2003, respectively) over the decrease of the teratogenic effect resulting from CP administration in pregnant rats.
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Immunostimulation The female rats were separated randomly into six groups: five experimental (ABC, AC, BC, A and B) and one control group (C). The treatment schedule is shown in Fig. 1. Briefly, rats from the ABC, AC and A groups were pretreated by gavage for 14 consecutive days with AF (3.0 g/kg of Ipomoea carnea dry leaves) 28 or 14 days prior to mating. Rats from the remaining groups (BC, B and C) were pretreated by gavage for the same period with filter water only. On day 14 before mating, BCG (10.0 mg/kg) was injected intraperitoneal (ip) in females from the ABC, BC and B groups and saline was injected ip in females from the other three groups (AC, A and C). Groups A and B were only used to show that the treatments AF and BCG did not promote teratological effects. Mating Mating was performed by placing one male into the cage of two females overnight. The day on which the females exhibited either sperm in the vaginal smear or a vaginal plug was designated as day 0 of pregnancy.
Material and methods
Cyclophosphamide-induced embryotoxicity
Plant material
Female rats were weighed on days 0, 11 and 21 of pregnancy. On day 11 of gestation, dams from the ABC, AC, BC and C groups were treated with a subcutaneous (sc) injection of CP (7.5 mg/kg) in the dorsal area of the neck (Gomes-Carneiro
I. carnea leaves were collected from plants cultivated at the Research Centre for Veterinary Toxicology (CEPTOX), University of São Paulo (USP), Pirassununga, Brazil, in May, 2001. A voucher specimen was deposited in the Herbarium of the Botanic Institute of São Paulo, Brazil (SP-360911) and its authenticity was confirmed by the taxonomist Rosangela Simão Bianchini of the same Institute. The AF resulting from extraction of I. carnea dry leaves was obtained according to previously described methods (Hueza et al., 2003). Reagents Bacillus Calmette Guérin-cepa Moreaux (BCG) was yielded by the Institute Butantã of São Paulo, Brazil and was suspended in a 0.9% NaCl solution at 0.1% concentration. CP (Genuxal®) was purchased from Asta Medica AG, Frankfurt, Germany and was dissolved in 0.9% NaCl solution. 10% neutral buffered formalin. Animals Male and nulliparous female Wistar rats aged about 70 days, bred in the Department of Pathology, School of Veterinary Medicine and Zootechny, were used. The animals received food and water ad libitum and were maintained under controlled conditions of temperature (22–25 °C), humidity (50–65%) and lighting (12/12 light/dark cycle). The experiments were carried out in accordance with the ethical principles for animal research adopted by the Bioethics Committee of the School of Veterinary Medicine and Zootechny, University of São Paulo.
Fig. 1. Treatment schedule: rats from the ABC, AC and A groups were pretreated for 14 consecutive days with AF (3.0 g/kg of I. carnea dry leaves) 28 or 14 days prior to mating. Rats from the remaining groups (BC, B and C) were pretreated for the same period with filter water only. On day 14 before mating, BCG (10.0 mg/kg) was injected ip in females from the ABC, BC and B groups and saline was injected ip in females from the other three groups (AC, A and C). On pregnancy day 11, CP (7.5 mg/kg) was injected sc in females from the ABC, AC, BC and C groups and saline was injected ip in females from the other groups (A and B).
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Table 1 Maternal weight (g) and cesarean section data of rats pretreated with AF (3.0 g/kg/day of Ipomoea carnea po) and/or BCG (10.0 mg/kg ip) 28 or 14 days before mating and subcutaneous injection of CP (7.5 mg/kg) or its vehicle alone (saline) on pregnancy day 11 Treatment 1
C
AC
ABC
BC
A
B
Pregnant females (N) Resorption of the whole litter(N)
8 2
9 1
6 0
7 0
10 0
8 0
Maternal weight (g)♦ Day 0 Day 11 Day 21 (minus utero wt)
231.9 ± 4.8 263.2 ± 6.1 260.4 ± 6.6
217.8 ± 3.3 250.6 ± 2.6 257.7 ± 3.5
228.4 ± 6.2 267.7 ± 9.1 264.8 ± 9.9
231.8 ± 4.7 265.9 ± 6.8 256.8 ± 7.2
229.8 ± 3.5 263.2 ± 3.3 267.1 ± 4.3
228.0 ± 4.3 265.8 ± 6.5 257.7 ± 5.8
Implantation sites• Per litter
100 (76.9–100)
100 (88.9–100)
100 (91.7–100)
100 (75–100)
100 (100–100)
100 (91.7–100)
Pre-implantation loss• Per litter
0 (0–23.1)
0 (0–11.1)
0 (0–8.8)
0 (0–25)
0 (0–0)
0 (0–8.3)
Post-implantation loss• Per litter
86.7 (58.3–92.8)
60 (25–90)
63.6 (21.4–78.6)
33.3 (8.3–69.2)⁎
8.3 (0–31.2)
4 (0–18.2)
1
A = aqueous fraction of Ipomoea carnea, B = Bacillus Calmette Guérin, C = cyclophosphamide. ♦Data are expressed as mean ± SEM and were analyzed by ANOVA followed by the Dunnett's test. •Percentages were analyzed by Kruskal–Wallis test followed by the Dunn's Multiple Comparisons Test. ⁎Differences are statistically significant (P b 0.05) between AC, ABC and BC groups compared to the C group.
et al., 2003). Rats from groups A and B were treated with an sc injection of saline in the same manner. Caesarian section and evaluation of the degree of severity of birth defects On day 21 of gestation all dams were anesthetized and killed by CO2 inhalation. The gravid uterus was weighed with its contents and the number of implantation sites, resorptions, living and dead fetuses was recorded. The living fetuses were
weighed, examined with a stereomicroscope for external visible anomalies such as cleft palate, exencephaly, craniofacial, limb, digits and tail defects, after death they were fixed in a formalin solution. The visible skull defects were scored for severity by using a grading scale as follows: 0 for absence of skull defects; 1 for very small opening on the skull; 2 for clearly defined opening on the skull; 3 for slight protrusion of the brain through a wider opening on the skull (mild exencephaly); 4 for pronounced protrusion of the brain outside the skull opening (severe exencephaly).
Table 2 Fetal weight (g) and data of fetuses from rats pretreated with AF (3.0 g/kg/day of Ipomoea carnea po) and/or BCG (10.0 mg/kg ip) 28 or 14 days before mating and subcutaneous injection of CP (7.5 mg/kg) or its vehicle alone (saline) on pregnancy day 11 Treatment1
C
AC
ABC
BC
A
B
Fetuses (litters+) examined
15 (6)
32 (8)
33 (6)
51 (7)
118 (10)
83 (8)
Resorptions Per implantation sites• Per litter♦
86.7 (58.3–92.9) 10.0 ± 1.0
60 (25–90) 6.2 ± 0.8*
63.6 (21.4–78.6) 7.6 ± 1.3
33.3 (8.3–69.2)* 4.7 ± 1.0**
8.3 (68.7–100) 1.6 ± 0.5
4.1 (0–18.18) 0.7 ± 0.3
Live fetuses Per implantation sites• Per litter♦
13.3 (7.1–41.7) 2.7 ± 0.9
40 (10–75) 4.0 ± 0.7
34.8 (21.4–78.6) 5.5 ± 1.2
66.7 (30.8–91.7)* 7.3 ± 1.1*
91.6 (68.7–100) 11.8 ± 0.5
95.8 (81.8–100) 10.4 ± 0.5
Fetal body weight (g)♦ Per litter Per group
3.00 ± 0.09 3.04 ± 0.08
3.67 ± 0.23 3.67 ± 0.11**
3.71 ± 0.32 3.61 ± 0.13*
3.47 ± 0.17 3.54 ± 0.08*
3.59 ± 0.11 3.59 ± 0.03
4.49 ± 0.30 4.58 ± 0.09
Placental weight (g)♦ Per litter Per group
0.28 ± 0.01 0.29 ± 0.01
0.37 ± 0.03 0.38 ± 0.01**
0.35 ± 0.03 0.39 ± 0.01**
0.37 ± 0.02 0.37 ± 0.01*
0.57 ± 0.02 0.57 ± 0.00
0.64 ± 0.02 0.64 ± 0.00
1
A = aqueous fraction of Ipomoea carnea, B = Bacillus Calmette Guérin, C = cyclophosphamide. Only litters with live fetuses. ♦Data are expressed as mean ± SEM and were analyzed by ANOVA followed by the Dunnett's test. •Percentages were analyzed by Kruskal–Wallis test followed by the Dunn's Multiple Comparisons Test. *Differences are statistically significant (P b 0.05), **(P b 0.01) between AC, ABC and BC groups compared to the C group.
+
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Table 3 Occurrence of external malformations in rat offspring pretreated with AF (3.0 g/kg/day of Ipomoea carnea po) and/or BCG (10.0 mg/kg ip) 28 or 14 days before mating and subcutaneous injection of CP (7.5 mg/kg) or its vehicle alone (saline) on pregnancy day 11 Treatment1
C +
Number of examined fetuses (litters )
15 (6)
% malformed fetuses per litter Skull closure deffects 0 (absence) 1 (slight) 2 (mild) 3 (severe) 4 (very severe) Polydactyly Oedema
0 (0–0) 0 (0–0) 45 (0–100) 30 (0–100) 0 (0–100) 0 (0–20) 80 (0–100)
AC 32 (8)
0 (0–50) 39.3 (0–100)⁎ 12.5 (0–75) 0 (0–80) 0 (0–100) 0 (0–40) 50 (16.7–100)
ABC 33 (6)
0 (0–9) 26.9 (0–85.7) 33.3 (14.3–100) 10 (0–33.3) 0 (0–33.3) 0 (0–0) 32.1 (0–100)
BC 51 (7)
25 (0–50)⁎ 9.1 (0–45.4) 28.6 (9.1–63.6) 18.2 (0–33.3) 0 (0–28.6) 16.7 (0–27.3) 18.2 (0–66.7)
A 118 (10)
100(100–100) 0 (0–0) 0 (0–0) 0 (0–0) 0 (0–0) 0 (0–0) 0 (0–0)
B 83 (8)
100 (100–100) 0 (0–0) 0 (0–0) 0 (0–0) 0 (0–0) 0 (0–0) 0 (0–0)
1
A = aqueous fraction of Ipomoea carnea, B = Bacillus Calmette Guérin, C = cyclophosphamide. Only litters with live fetuses. Percentages of malformed fetuses per litter (litter as statistical unit) were analyzed by Kruskal–Wallis test followed by the Dunn's Multiple Comparisons Test. ⁎Differences are statistically significant (P b 0.05) between AC, ABC and BC groups compared to the C group.
+
Statistical analysis Data were analyzed using only the C-group as control group against the ABC, BC and AC groups. The data was analyzed by one-way analysis of variance (ANOVA) followed by the Dunnett test for multiple comparisons and the data are expressed as mean ± S.E.M. Percentages data were compared by the Kruskall–Wallis test followed by Dunn's test and were expressed as median and range values. In all cases, differences were considered to be statistically significant at P b 0.05. Results Maternal weight and cesarean section data from rats pretreated with AF or BCG and submitted to CP treatment on day 11 of gestation No differences were observed in the weight gain of pregnant female rats pretreated with AF or BCG (i.e. A- and B-group, respectively), even when these pre-treatments were subsequently accompanied by CP injection on day 11 of gestation. Besides, no differences were observed in the percentages of the implantation sites and pre-implantation losses per litter between groups. In contrast, the percentage of post-implantation losses notably decreased in females from the BC-group (P b 0.05) when compared to that of C-group (Table 1).
placenta weight AC (P b 0.01), ABC (P b 0.01) and BC (P b 0.05) was verified when compared with dams from the control group. Occurrence of external malformations in fetuses from female rats pretreated with AF or BCG and submitted to CP treatment on day 11 of gestation No external malformation was noted in fetuses from dams pretreated with AF or BCG only (A- and B-group, respectively). In contrast, all fetuses from rats treated with CP alone, on pregnancy day 11, exhibited skull anomalies with severity scores varying between 2 and 4. Fetuses from the AC-group showed a concentration of skull anomalies between the scores 1 and 2, the same range of scores was observed in fetuses from the ABC-group; and fetuses from dams belonging to the BC-group showed skull anomaly scores concentrated between 0 and 2. Polydactylism was also observed among rat offspring exposed to CP on pregnancy day 11 (C-group), this malformation was observed, with greater frequency in fetuses from the BC-groups of dams, in contrast to fetuses of dams belonging to the ABCgroup, which showed no occurrence of polydactylism. Moreover, all the experimental groups pretreated with immunomodulator substances showed a decreased in the number of edematous fetuses over C-group (Table 3). Discussion
Analysis of fetuses from rats pretreated with AF or BCG and submitted to CP treatment on day 11 of gestation Fetal measurements are shown in Table 2, a remarkable increase in the number of live fetuses per litter was observed in the group of dams from the BC-group (P b 0.05) when compared with dams from the control group. Moreover, when the number of resorptions per litter was accessed, this was notably reduced in female rats from the AC-group (P b 0.05) and BC-group (P b 0.01). Additionally, when considering each individual group, an increase in fetal body weight AC (P b 0.01), ABC (P b 0.05) and BC (P b 0.05) and
CP is an antineoplastic and immunosuppressive drug which has been found to be teratogenic in rats, mice, chicks, rabbits, monkeys and humans (Matalon et al., 2004; Mirkes et al., 1984); on the other hand, many studies from independent laboratories show that maternal immune stimulation decreases fetal abnormalities caused by this substance; however, in all of these previous studies mice were used as the animal model; therefore, we extended this observation by verifying that materno-fetal immune interactions in rats also offer protection against maldevelopment induced by CP. In this regard, it should be pointed out that the rat is the chosen species for developmental
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(OECD, 1981) and immunotoxic studies (Holsapple et al., 2005), including developmental immunotoxicology evaluation (Ladics et al., 2005). Since the pioneer study, conducted by Nomura et al. (1990), showing that stimulation of peritoneal macrophages in pregnant mice reduced fetal malformations, many investigators have theorized regarding the mechanisms by which this protective effect occurs. Although the matter of how immune substrates and teratogenic metabolites interact at the cellular level is not yet clear, it is currently proposed that maternal immune stimulation may reduce chemically induced morphological lesions by an indirect effect via regulatory cytokine product of activated immune cells, in this respect Sharova et al. (2000) theorized that such soluble mediators restore the teratogen-altered expression of critical fetal genes. In the present study, it was verified that both BCG and/or AF attenuated the embryotoxic effects of CP in rats. In fact, BCG is known to promote macrophage activation, with the release of pro-inflammatory cytokines, such as IL-1, IL-6, TNF-α, GMCSF and other molecular components of inflammatory response and many studies have shown that most of these substances produce a reduction in chemically induced birth defects in mice (Holladay et al., 2002). Similarly, it is proposed that CP alters levels of cytokines in both the uteroplacental unit and the fetus, leading to an inappropriate apoptotic process which results in teratogenesis (Chernoff et al., 1989) which appears to be reversed by macrophage-release cytokines, such as GM-CSF (Savion et al., 1999). In relation to the AF of I. carnea, it is well recognized that the indolizidine alkaloid swainsonine, present in this plant, activates tissue macrophages (Das et al., 1995), releases IL-1 from these cells (Grzegorzewski et al., 1989) and enhances NK cell cytotoxicity (Bowlin et al., 1989) and for this reason has been considered as an antimetastatic agent (Roberts et al., 1998). In addition, a study conducted in this laboratory on rats, revealed that I. carnea extract promoted enhanced peritoneal macrophage activity (Hueza et al., 2003). Thus, it could be suggested that the reduction in CP-induced malformations verified in fetuses from both AC and ABCgroups was also due to maternal macrophage activation and, consequently, the production of cytokines. An unexpected observation in the present study was that BCG/CP-treated mothers showed a median of 16.7 fetuses with polydactylism, a frequency greater than that shown by fetuses from any other group, including CP-exposed fetuses (C-group). A similar effect was observed by Holladay et al. (2000), who verified that female mice stimulated with Freund's complete adjuvant and exposed to valproic acid (VA), a well-known chemical teratogen, produced fetuses displaying tail absence, a defect not typically seen in ICR mice exposed to VA. Collectively, these results clearly reveal that a teratogenic effect could be caused by maternal immune stimulation; however, at the present moment it is not possible to theorize the mechanisms by which this effect occurs. After a retrospective regarding all studies concerned with verifying the influence of immunostimulants on the suppression of developmental anomalies, we noted that many suppositions
were made regarding inflammatory cytokines in the placenta and fetus gene expression up-regulation (Sharova et al., 2003; Holladay et al., 2002), but until now no attention has been paid to the biotransformation mechanism of the teratological agents employed in these studies. In relation to CP, it is very well documented that this substance must be activated in the liver by microsomal cytochrome P-450 monooxygenases to achieve its teratogenic effects (Matalon et al., 2004; Mirkes et al., 1984). In fact, a study conducted by Gomes-Carneiro et al. (2003) showed the inhibition of CP-induced teratogenesis when pregnant rats were previously administered with β-ionone, an in vitro inhibitor of the isoenzyme CYP2B1. In addition it should be pointed out that other classic teratogens, such as valproic acid, urethane and polycyclic aromatic hydrocarbons, are also activated by liver microsomal enzymes (Parkinson, 2001). It is known that inflammatory cytokines (Crawford et al., 2004; Ke et al., 2001; Chen et al., 1995) and other inflammatory mediators, such as nitric oxide (Khatsenko, 1998; Carlson and Billings, 1996), can inhibit the efficacy of microsomal cytochrome P-450 enzymes, thus decreasing the biotransformation of the active metabolites of these drugs. Thus, it could be theorized that at least part of the improvement in fetal morphogenesis produced in all experiments using immunostimulants, such as BCG, interferons, FCA and other macrophage activators, is due to the impairment of the active metabolite production by these isoenzymes. Moreover, some teratogenic promoters; such as N-methyl-N-nitrosourea and streptozocin (nitrosourea compounds), substances that have been employed in numerous studies as teratogenesis inducers; produce their effect by DNA chain alkylation in the duplicating process (Prater et al., 2004). Moreover, some cytokines used to induce immune stimulation, such as interferon, can promote an increase in O6-alkylguanine-DNA alkyltransferase expression (Bertini et al., 1990) which is related to the repair of DNA alkylated by these nitrosourea compounds (Fang et al., 2005) that could lead to a diminished occurrence of DNA abnormalities and, consequently, a decrease in fetal maldevelopment. In conclusion, the present work showed that the rat is a good model for performing studies aimed at a clearer understanding of the mechanism by which maternal stimulation reduces malformations and how the association of I. carnea AF and BCG provided improved immunostimulation compared to BCG alone; however, additional studies are required to determine the specific role of immune stimulant substances on general metabolism and the possible influence of a non-immunological effect in decreasing malformation. Acknowledgements This research was supported by grants from Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), Brazil. References Bertini, R., Coccia, P., Pagani, P., Marinello, C., Salmona, M., D'Incalci, M., 1990. Interferon inducers increase O6-alkylguanine-DNA alkyltransferase in the rat liver. Carcinogenesis 11 (1), 181–183.
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