Toxicological effects of cadmium during pregnancy in ... - Springer Link

Toxicological Effects of Cadmium during Pregnancy in Wistar Albino Rats Jonah Sydney Aprioku1, Barikpoar Ebenezer2 & Maxwell Azubuike Ijomah3 1

Department of Pharmacology, Faculty of Basic Medical Sciences, University of Port Harcourt, Port Harcourt, Nigeria 2 Department of Clinical Pharmacy and Management, Faculty of Pharmaceutical Sciences, University of Port Harcourt, Port Harcourt, Nigeria 3 Department of Mathematics and Statistics, Faculty of Science, University of Port Harcourt, Port Harcourt, Nigeria Correspondence and requests for materials should be addressed to J.S. Aprioku ([email protected]) Received 25 October 2013 / Received in revised form 24 February 2014 Accepted 26 February 2014 DOI 10.1007/s13530-014-0183-z ©The Korean Society of Environmental Risk Assessment and Health Science and Springer 2014

Abstract Cadmium is a heavy metal and widespread environmental toxicant. This study investigated the effects of prenatal Cd exposure on fetal growth and limb development in rats. Pregnant rats were given 0, 4 or 8 mg/kg/day (equivalent to � 0, 30 or 60 ppm) of cadmium as CdSO4 in their drinking water from concep⁄0.001) tion to gestation day 20. Cd significantly (p⁄ and dose-dependently inhibited maternal weight gain and caused abortion of pregnancy. In addition, Cd ⁄0.001) decreased fetal body weight, significantly (p⁄ forelimb and hindlimb bone lengths, compared to controls. These effects were sex-dependent, greater in the female offspring. Furthermore, there were reductions in the weights, and alterations in the histology of maternal placenta, ovary and liver of Cd-exposed rats. The results indicate that cadmium will cause abortion of pregnancy and sex-dependent impairment of fetal growth and limb development, which may be consequent upon alterations in ovarian and placental functions. Keywords: Cadmium, Dimorphism, Gestation, Limb anomalies, Placenta

Introduction Cadmium (Cd) is a heavy metal and an important

inorganic toxicant widely distributed in the environment. The potential for human exposure to Cd has generally increased with the increasing industrial use of this metal1. Tobacco smoke is the most important source of environmental exposure to Cd for smokers, whereas the major source of exposure for the general population is via Cd-contaminated food and water2,3. Other pathways of Cd exposure in humans include incineration of municipal waste materials, and combustion of fossil fuels. However, the most significant contemporary source of occupational exposure results from the production of nickel-cadmium batteries, while zinc smelting, pigment plants, and soldering activities are other sources4. Importantly, the metal has been shown to be a major product of petroleum refining activities5, which increases the level of its toxicity in regions involved with oil production such as the southern part of Nigeria. Cadmium accumulates in soft tissues and has a long half-life of about 15-30 years in human beings6, primarily because of its low rate of excretion from the body. The metal is highly potent and deleterious to many tissues and organs in both experimental animals and humans, and has been a focus of numerous studies. The liver is the primary target in acute Cd toxicity, whereas in chronic Cd-induced toxicity, the kidney is the primary organ mostly affected2. Cadmium exposure has also been shown to cause carcinogenesis and mutagenesis7,8. Cd has also been linked to a wide spectrum of deleterious effects on reproductive tissues9,10. In the testis, changes due to disruption of the bloodtestis barrier and oxidative stress have been noted, with onset of widespread necrosis at higher dosage exposures6. In the female, Cd has been shown to alter ovarian function with associated failure in ovulation and impairment of implantation11,12. Exposure of experimental animals to oral or parenteral Cd has also been reported to cause a wide range of abnormalities in the embryo, including craniofacial, neurological, cardiovascular, gastrointestinal and genitourinary anomalies13. Single intravenous administrations of Cd (1.25-2 mg/kg) at different gestational days (GDs) have been shown to cause different forms of fetal malformations, ranging from hydrocephalus, facial abnormalities, anophthakmia, hepatic/placental cell necrosis and limb defects14-16. Cd has also been reported to cause variable effects (either enhancing or inhibiting) on pro-

Effects of Maternal Cadmium Exposure on Pregnancy

Results and Discussion Results Effects of Cadmium on Maternal Body Weight and Pregnancy

Average body weights of pregnant rats in control group increased, p⁄0.001 during pregnancy, but there was no significant (p¤0.05) change in body weights of rats that received Cd-4, 8 mg/kg (Figure 1). Analysis of maternal body weights across time in the control group showed that maternal weights of rats on days 5, 10, 15 and 20 were significantly p⁄0.001 higher compared to their weight at conception, i.e., day zero of pregnancy (Figure 1). Maternal weights on days 10, 15 and 20 were also significantly (p⁄0.001) higher when compared to their weight on day 5. Comparing to body weight on day 10, those on days 15 and 20 were significantly (p⁄0.01, p⁄0.001) higher. The weight at day 20 was also significantly (p⁄0.05) higher than that of day 15 (Figure 1). In addition, all 6 rats in the control group had fetuses in their uteri; while 1 out of 6 in 4 mg/kg exposed rats and none in 8 mg/kg exposed rats had fetuses in their uteri (Table 1). In addition, the average number of litters delivered in control =0.034) than that of rats was higher (6.50±0.50, p= the 4 mg/kg Cd-exposed rats, 4.00±0.10 (Figure 2). The fetuses of control rats were all alive, healthy and active, while one of the four fetuses of 4 mg/kg Cdexposed rats was a stillbirth, and the others were less

350 300

*#‡a

*#α *#

Body weight (g)

250 * 200 150 100 Control 4 mg/kg 8 mg/kg

50 0

Day 0

Day 5

Day 10

Day 15

Day 20

Day of gestation

Figure 1. Effect of oral maternal Cd (4, 8 mg/kg) exposure from GD0-GD20 on maternal body weight in Wistar albino =8 animals per rats. Data are expressed as mean±SEM, n= group. Statistical differences between the groups were evaluated by one-way ANOVA, followed by Newman-Keuls Multiple Comparison Test. *Significant at p⁄0.001 (Day 1 vs Days 5, 10, 15 and 20), #Significant at p⁄0.001 (Day 5 vs Days 10, 15 and 20), αSignificant at p⁄0.01 (Day 10 vs Day 15), ‡ Significant at p⁄0.001 (Day 10 vs Day 20), aSignificant at p⁄0.05 (Day 15 vs Day 20).

Average number of fetuses delivered per animal

gesterone biosynthesis depending on the dose6. Furthermore, Mikolic et al.17 and Piasek et al.18 have reported alterations in maternal and fetal distribution and concentrations of essential trace elements (growth factors) following maternal exposure of Cd (3, 5 mg/kg per day) from GD0 to term. However, most of these studies did not evaluate the fetal toxicity that will occur following exposure of Cd from onset to delivery stage of pregnancy. The present work investigates the effects of prenatal oral exposure of cadmium-4, 8 mg/kg/day (equivalent � 30, 60 ppm per day) from GD0-GD20 on fetal growth and limb development in Wistar albino rats. The study further aims to evaluate if such effects are gender related.

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8 7 6 5 *

4 3 2 1 0

Control

4 mg/kg

Figure 2. Number of fetuses in utero of pregnant Wistar albino rats following oral administration of Cd from GD0GD20. Data are expressed as mean±SEM. Statistical differences between the groups were evaluated by unpaired Student’s t-test. *Represents value significantly different from control value at p⁄0.05. Note: Pregnancies of 8 mg/kg Cd exposed animals were aborted.

Table 1. Effects of oral maternal Cd exposure from GD0-GD20 on pregnancy outcome in Wistar albino rats. Dose

No. of rats having fetus in utero

Average no. of fetuses delivered per animal

No. of stillbirths

Physical appearance of fetuses delivered

Control 4 mg/kg 8 mg/kg

6 1 Nil

6.5 4 -

1 -

Normal and active Inactive, pale in color and in distress -

Note: Pregnancies of 8 mg/kg Cd exposed animals were aborted.

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Toxicol. Environ. Health. Sci. Vol. 6(1), 16-24, 2014

6

0.8

A

0.6

4

Bone length (mm)

Fetal body weight (g)

A

3 2

Control 4 mg/kg Cd

0.7

* *

5

1

* *

0.5 0.4

*

0.3 0.2 0.1

0 Control

4 mg/kg

0 Humerus

6

B

0.7

5 * 4 3 2 1

Radius

B

0.5

Metacarpus

Male Female

0.6 Bone length (mm)

Fetal body weight (g)

*

*

0.4

* *

0.3 0.2

0 Male

Female

Figure 3. A. Effect of oral maternal exposure of 4 mg/kg Cd from GD0-GD20 on fetal body weight in Wistar albino rats. B. Comparison of Cd-induced fetal body weights between =8 animals per sexes. Data are expressed as mean±SEM, n= group. Statistical differences between the groups were evaluated by unpaired Student’s t-test. *Represents value significantly different from control value at p⁄0.05, **Represents value significantly different from control value at p⁄0.0001. Note: Pregnancies of 8 mg/kg Cd exposed animals were aborted.

0.1 0 Humerus

Radius

Metacarpus

Figure 4. A. Lengths of fetal forelimb bones following oral maternal exposure of 4 mg/kg Cd from GD0-GD20. B. Comparison of Cd-induced fetal forelimb bone lengths between sexes. Data are expressed as mean±SEM. Statistical differences between the groups were evaluated by unpaired Student’s t-test. *Represents values significantly different from control value at p⁄0.05, **Represents value significantly different from control values at p⁄0.01. Note: Pregnancies of 8 mg/kg Cd exposed animals were aborted.

active, pale in color and in distress as shown by the presence of meconium-stained liquor amnii (Table 1). Effects of Cadmium on Fetal Body Weight and Limb Development

Furthermore, average body weight of fetuses of 4 mg/kg Cd-exposed rats was significantly (p⁄0.0001) lower, compared to control: 4.40±0.20 g vs 5.51± 0.05 g (Figure 3A). When analyzed by sex, female’s =0.0206) lower than male’s weight was significantly (p= (Figure 3B). Furthermore, average fetal forelimb bone lengths of 4 mg/kg Cd-exposed rats were significantly reduced compared to controls: humerus, 0.47±0.05 =0.0045; radius, 0.43±0.05 mm vs 0.71±0.03 mm, p= =0.0185; metacarpus, 0.29 mm vs 0.61±0.04 mm, p= =0.0202 (Figure 4A). ±0.02 mm vs 0.38±0.02 mm, p= Fetal hindlimb bone lengths of 4 mg/kg Cd-exposed rats were also significantly reduced compared to con-

= trols: femur, 0.51±0.07 mm vs 0.68±0.02 mm, p= 0.0306; tibia, 0.40±0.07 mm vs 0.77±0.04 mm, p⁄ 0.0008; metatarsus, 0.40±0.06 mm vs 0.71±0.01 mm, =0.0006 (Figure 5A). These effects were observed p= to be more pronounced in the females, p⁄0.01, than in the males, p⁄0.05 (Figure 4B and 5B), similar to the result on birth weight. Effects of Cadmium on Maternal Organ Weights and Histology

There was a significant (p⁄0.05) decrease in placental weight of pregnant rats that received 4 mg/kg Cd compared to control, while no placenta was found in group that received 8 mg/kg Cd (Table 2). Histopathological analysis of placenta of 4 mg/kg Cd-exposed mothers showed congestion of blood vessels and mild


degenerative changes in trophoblastic cells (Figure 6A, 6B and 6C). Furthermore, while uterine weights did not change, the weights of ovary in Cd-treated pregnant rats were lower, p⁄0.05 than control animals (Table 2). Histology of uteri of Cd-exposed rats were normal (Figure 7A, 7B and 7C), but histopathological 0.9 0.8

A

Control 4 mg/kg Cd

Bone length (mm)

0.7 *

0.6

* * *

0.5

* * *

0.4 0.3 0.2 0.1 0 Femur

0.8

Tibia

B

Metatarsus

Male Female

0.7

Bone length (mm)

0.6 0.5

* * * *

0.4 0.3 0.2 0.1 0 Femur

Tibia

Metatarsus

Figure 5. A. Lengths of fetal hindlimb bones following oral maternal exposure of 4 mg/kg Cd from GD0-GD20. B. Comparison of Cd-induced fetal hindlimb bone lengths between sexes. Data are expressed as mean±SEM. Statistical differences between the groups were evaluated by unpaired Student’s t test. *Represents value significantly different from control value at p⁄0.05, **Represents values significantly different from control value at p⁄0.01, ***Represents values significantly different from control value at p⁄0.001. Note: Pregnancies of 8 mg/kg Cd exposed animals were aborted.

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analysis of their ovarian tissues revealed follicular degeneration in 4 mg/kg Cd-exposed rats, and marked degeneration of follicles and hemorrhage in 8 mg/kg Cd-exposed rats (Figure 8A, 8B and 8C). In addition, liver weights in Cd-treated pregnant rats were significantly (p⁄0.05) lower compared to controls (Table 2). Histology of livers of 4 mg/kg Cd-exposed rats were observed to have inflammations around the portal tract with congestion of vessels and periportal necrosis; while hydropic degeneration and fatty changes with intraparenchymal and portal tract inflammations were observed in the hepatic tissues of 8 mg/kg Cdexposed rats (Figure 9A, 9B and 9C).

Discussion A number of studies have demonstrated that Cd causes alteration in male and female reproductive functions9,10,15. The metal has also been shown to adversely affect pregnancy, but such effects have been observed to be largely dependent on the dose, animal specie, route and gestational age of exposure4,16,19. In the present study, we hypothesize that exposure of Cd from conception through end of pregnancy will impact more severe fetal effects, and these effects may be gender related. Adequate weight gain during pregnancy is vital for the wellbeing of mother and baby20. In this study, Cd (4, 8 mg/kg, equivalent to � 30, 60 ppm per day) prevented maternal weight gain, which suggests that the metal may adversely affect normal growth of embryo and pregnancy outcome in the rat. This observation, which is consistent with that of Castilo et al.21, was highly correlated with the inability of Cd exposed rats to sustain pregnancies. Importantly, all pregnancies were aborted at 8 mg/kg; while about 80% were aborted at 4 mg/kg. This suggests that exposure to Cd levels ›4 mg/kg/day during pregnancy may have serious consequences on pregnancy outcome in animals. Suboptimal weight gain during pregnancy is known to be a major risk factor for the delivery of infants with low birth weights22. In our study, prenatal exposure of Cd decreased fetal body weights in a sex-dependent manner, females’ weight lower than males’, indicating that female rats may be more prone to Cd-induced develop-

Table 2. Effects of maternal Cd exposure on weights of maternal placenta, uterus, ovary and liver in Wistar albino rats. Dose Control 4 mg/kg 8 mg/kg

Weight (g) Placenta

Uterus

Ovary

Liver

1.10±0.09 0.64±0.05* -

0.27±0.01 0.32±0.04 0.31±0.05

0.19±0.02 0.10±0.02* 0.08±0.03*

7.57±0.45 7.32±0.44 5.64±0.50*

=8 animals per group. Data are expressed as mean±SEM, n= Statistical differences between the groups were evaluated by ANOVA. *Significant at p⁄0.05

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A

A

B

B

C

C

Figure 6. Photomicrographs showing histology of placenta following oral maternal exposure of 4 mg/kg Cd from GD0GD20 in Wistar albino rats (400×). A. Control, showing normal histology of the placenta with normal trophoblastic tissues (arrow heads). B. 4 mg/kg Cd, showing congestion of blood vessels and mild degenerative changes in trophoblastic cells (arrows). C. 8 mg/kg Cd, showing numerous vascular spaces in placenta (arrows).

Figure 7. Photomicrographs showing histology of uterus following oral maternal exposure of 4 mg/kg Cd from GD0GD20 in Wistar albino rats (400×). A. Control, showing normal uterine tissue with normal proliferative endometrium (arrow heads). B. 4 mg/kg Cd, showing normal uterine tissue with normal endometrium (arrows). C. 8 mg/kg Cd, showing normal uterine tissue with normal endometrium (arrows).


A

A

B

B

C

Figure 8. Photomicrographs showing histology of ovary following oral maternal exposure of 4 mg/kg Cd from GD0GD20 in Wistar albino rats (400×). A. Control, showing normal ovarian tissue with numerous ovarian follicles (arrow heads), ovarian stroma (up arrow). B. 4 mg/kg Cd, showing degenerating follicles in the ovary (arrows). C. 8 mg/kg Cd, showing markedly degenerating ovarian follicles and hemorrhage (arrows).

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C

Figure 9. Photomicrographs showing histology of liver following oral maternal exposure of 4 mg/kg Cd from GD0-GD20 in Wistar albino rats (400×). A. Control, showing normal liver histology; hepatocytes (arrow head), central vein (up arrow). B. 4 mg/kg Cd, showing mild inflammation around the portal tract (left arrow) with congestion of vessels and periportal necrosis (down arrow). C. 8 mg/kg Cd, showing hydropic degeneration/fatty changes in hepatic tissue (left arrows), as well as intraparenchymal and portal tract inflammation (arrow head).

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mental abnormalities. Previous works have shown fetal weight reductions by Cd18, but sexual dimorphism of this effect has not been reported. Exposure of experimental animals to oral or parenteral Cd has been reported to cause a range of abnormalities in the embryo of experimental animals13. However, reports on Cd-induced fetal toxicity are variable and dependent on the dose, route of exposure, animal strain, and gestational age4,16,19. Additionally, data on Cd-induced fetal limb defects at the dose range and gestational age (GD) of exposure used in this study has not been reported in the rat prior to this study. Our study showed that Cd (4 mg/kg) caused significant reductions in all forelimb and hindlimb bone lengths. When examined individually, sexual dimorphism of this effect was also observed, female more affected than the male fetuses, similar to the result obtained in fetal body weights. This gives evidence that female offspring of Cd-exposed mothers may be at greater risks of developmental toxicity. Normal ovarian and placental functions are critical for maintenance of pregnancy23,24. Examination of the ovarian tissue revealed that Cd decreased weight of maternal ovary dose-dependently, and also caused varying degrees of follicular degenerations and hemorrhage. Normal ovarian function is necessary for development of the embryo and maintenance of pregnancy23,25. This observation suggests that Cd may affect development of the embryo probably by causing resorption and death of the embryo which may partly account for the abortion of pregnancies. We also observed that Cd altered placental morphology in our study. With the multifunctional role of the placenta including, oxygen and carbon dioxide exchange, transfer of metabolic waste and hormonal regulation of the fetus24, this may affect normal fetal nutrition and other metabolic processes with consequent impairment of fetal growth. Cadmium-induced congestion of placental blood vessels and degeneration of trophoblastic cells observed in this study could cause death of the rat embryo by interrupting uteroplacental blood flow and this may contribute to resorption of fetuses. Also, damage to maternal placenta may cause impairment of vital transport systems like the placental Zn2+ transport26, which is critical for organogenesis and embryonic development. This may be responsible for the Cd-induced reduction in fetal body weights and abnormal forelimb and hindlimb developments observed in our results. At the higher dose (8 mg/kg), Cd caused more pronounced effect on pregnancy as evidenced in the higher incidence of loss of pregnancy (100%) and absence of placenta. The absence of placenta may be due to inhibition of placental development or degeneration of placental tissue due to ovar-

ian dysfunction by cadmium. Furthermore, Cd decreased maternal liver weight and caused dose-dependent inflammatory, degenerative and necrotic changes in different hepatic cells. This is consistent with previous reports of the liver being a target organ of cadmium toxicity27,28.

Conclusions In the present study, we report that oral Cd exposure (4, 8 mg/kg) to pregnant rats, from conception to term, affects sustenance of pregnancy, and weight and limb development of the offspring. Over the dose range, route, and gestational age of exposure used in this study, Cd possesses the potential of causing dose- and sex-dependent reductions in fetal body weights and limb bone lengths. Risk assessment of environmental impact by cadmium and other heavy metal toxicants may need to include considerations of potential developmental disruptions and probably their later life consequences.

Materials and Methods Cadmium sulphate salt (Riedel-de Haenag, France) was obtained from the Department of Pharmacology, University of Port Harcourt. Other chemicals were all of reagent standards.

Animals Non pregnant female albino rats weighing between 170-190 g and male albino rats weighing between 200220 g were obtained from the animal house of the University of Port Harcourt, Port Harcourt, Nigeria. The animals were maintained on standard laboratory chow and water ad libitum with a 12 h light-dark cycle. The experimental protocol followed the Guide for Care and Use of Laboratory Animals29, and was approved by our local Committee for Ethics in Animal Experimentation at the College of Health Sciences, University of Port Harcourt, Port Harcourt, Nigeria. Experimental Design The animals were divided into different groups and allowed to mate freely. Mating was confirmed by the presence of a vaginal copulation plug (day 0 of gestation) when they were randomly distributed into three =8). Each group was given 0, 4 or 8 mg/kg/ groups (n= day (equivalent to � 0, 30 or 60 ppm per day) in their drinking water from GD0 to GD20. Fetuses were delivered by caesarean section at the end of treatments under ketamine/diazepam anaesthesia, 75/5 mg/kg ip30.


The birth weight and gross appearance of offspring were registered. The fetuses were later sacrificed by putting them in a CO2 chamber and then dissected and the lengths of their forelimb and hindlimb bones were measured. Maternal uterus, ovary, placenta and liver were removed and weighed. Thereafter, the tissues were routinely processed and stained with hematoxylin and eosin (H&E) and examined with light microscope (Nikon Eclipse E400). The images were photographed with an Olympus Model BX51 microscope at magnifications of 400x.

Statistical Analysis Data were expressed as means±SEM. Statistical differences between the groups were evaluated by paired Student’s t-test and ANOVA. Comparisons between groups were made by the Newman-Keuls or Bonferroni’s Multiple Comparison Test to compare maternal weights across time. The level of significance was set at p⁄0.05 and all statistical analyses were performed using GraphPad prism 5 software package.

Acknowledgements This work was carried out in collaboration by all authors. Aprioku J.S. designed the study, managed the literature searches, wrote the protocol and wrote the first draft of the manuscript. Ijoma M.A. performed the statistical analysis. Aprioku J.S. and B. Ebenezer managed the analyses of the study, and read and approved the final manuscript. The authors are grateful to the laboratory staff of the Department of Pharmacology, University of Port Harcourt for their technical assistance.

Conflict of Interest Statement The authors declare that there is no conflict of interest.

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