Gestational Cd Exposure in the CD-1 Mouse Sex-Specifically Disrupts Essential Metal Ion Homeostasis

Abstract

In CD-1 mice, gestational-only exposure to cadmium (Cd) causes female-specific hepatic insulin resistance, metabolic disruption, and obesity. To evaluate whether sex differences in uptake and changes in essential metal concentrations contribute to metabolic outcomes, placental and liver Cd and essential metal concentrations were quantified in male and female offspring perinatally exposed to 500 ppb CdCl2. Exposure resulted in increased maternal liver Cd+2 concentrations (364 µg/kg) similar to concentrations found in non-occupationally exposed human liver. At gestational day (GD) 18, placental Cd and manganese concentrations were significantly increased in exposed males and females, and zinc was significantly decreased in females. Placental efficiency was significantly decreased in GD18-exposed males. Increases in hepatic Cd concentrations and a transient prenatal increase in zinc were observed in exposed female liver. Fetal and adult liver iron concentrations were decreased in both sexes, and decreases in hepatic zinc, iron, and manganese were observed in exposed females. Analysis of GD18 placental and liver metallothionein mRNA expression revealed significant Cd-induced upregulation of placental metallothionein in both sexes, and a significant decrease in fetal hepatic metallothionein in exposed females. In placenta, expression of metal ion transporters responsible for metal ion uptake was increased in exposed females. In liver of exposed adult female offspring, expression of the divalent cation importer (Slc39a14/Zip14) decreased, whereas expression of the primary exporter (Slc30a10/ZnT10) increased. These findings demonstrate that Cd can preferentially cross the female placenta, accumulate in the liver, and cause lifelong dysregulation of metal ion concentrations associated with metabolic disruption.

Keywords: epigenetic; essential metals; gestation; metallothionein; placenta.

Figure 1.

Effects of gestational CdCl₂ exposure on placental efficiency and tissue Cd levels. A, At GD18, placentas of female fetuses were unaffected by exposure to gestational CdCl₂, whereas male placenta weights were increased. B, Placental efficiency is shown as fetal weight divided by placental weight, where female offspring showed no effect of gestational CdCl₂ exposure and male placental efficiency is reduced. C, Cd concentration of tissues was quantified using ICP-MS. In the placenta of offspring exposed to CdCl₂ during gestation, both sexes showed accumulation with an average in females of 62 µg/kg and in males of 53 µg/kg. D, Offspring liver concentrations of Cd are shown plotted against age. E, Dam liver concentrations are shown for control dams and dams exposed to CdCl₂ at GD18 of their pregnancy. Placenta weight and efficiency: females: control, n = 17; CdCl₂, n = 14. Males: control, n = 20; CdCl₂, n = 22. Cd levels: GD18: females: control, n = 9; CdCl₂, n = 11. Males: control, n = 8; CdCl₂, n = 10. PND21: females: control, n = 4; CdCl₂, n = 4. PND42: females: control, n = 5; CdCl₂, n = 4. Males: control, n = 4, CdCl₂, n = 4. PND90: females: control, n = 6; CdCl₂, n = 4. Males: control, n = 4; CdCl₂, n = 6. PND120: females: control, n = 5, CdCl₂, n = 4. Males: control, n = 3; CdCl₂, n = 5. Dams: control, n = 3; CdCl₂, n = 3. Samples were collected from 3 litters per treatment. All values shown are mean ± SD. The level of statistical significance for differences between mean values of control and CdCl₂-exposed groups was determined by a two-way ANOVA (treatment, sex) with a Tukey’s post hoc test for all experiments and is indicated by * (p < .05). Litter was included as a covariate.

Figure 2.

Correlation matrix of metal concentration in offspring CD-1 mouse liver and placenta at GD18. A Pearson correlation matrix of liver and placental metal concentration is shown for females (A) and males (B). In females, hepatic Cd has a significant positive correlation with hepatic Zn and placental Cd, and a significant negative correlation with placental Zn. Hepatic Zn was positively correlated with placental Cd and negatively correlated with placental Zn. In males, hepatic Fe was negatively correlated with placental Mn and placental Cd was positively correlated with placental Mn. Liver: females: control, n = 9; CdCl₂, n = 11. Males: control, n = 8; CdCl₂, n = 10. Placenta: females: control, n = 10; CdCl₂, n = 11. Males: control, n = 10; CdCl₂, n = 12. All samples come from 3 unique litters per treatment.

Figure 3.

Effects of gestational exposure to CdCl₂ on essential metal concentration in offspring CD-1 mice liver. Liver concentration of the essential metals Zn (A, B), Fe (C, D), and Mn (E, F) is shown for offspring female (A, C, E) and male (B, D, F) livers isolated and flash frozen at GD18, PND21, PND42, PND90, and PND120. In females, hepatic Zn at GD18 was increased from 211 in controls to 266 µg/g in Cd-exposed offspring, at PND21 was decreased from 153 to 119 µg/g in Cd-exposed offspring, at PND42 was decreased from 141 to 116 µg/g in Cd-exposed offspring, at PND90 was decreased from 166 to 90 µg/g in Cd-exposed offspring, and at PND120 was decreased from 149 to 91 µg/g in Cd-exposed offspring. In females, hepatic Fe at GD18 was decreased from 291 to 271 µg/g in Cd-exposed offspring, at PND21 was unchanged, at PND42 was decreased from 284 to 186 µg/g in Cd-exposed offspring, at PND90 was decreased from 326 to 230 µg/g in Cd-exposed offspring, and at PND120 was decreased from 299 to 190 µg/g in Cd-exposed offspring. In females, hepatic Mn was unchanged at GD18, decreased from 4.7 to 3.9 µg/g in Cd-exposed offspring at PND21, decreased from 5.2 to 3.3 µg/g in Cd-exposed offspring at PND42, decreased from 4.8 to 3.7 µg/g in Cd-exposed offspring at PND90, and decreased from 4.6 to 3.5 µg/g in Cd-exposed offspring at PND120. In males, no changes were detected in essential metals at GD18 or PND42. In males at PND90 and PND120, the only changes noted in males were decreases in hepatic Fe from 324 to 256 µg/g in Cd-exposed offspring at PND90 and from 352 to 288 µg/g in Cd-exposed offspring at PND120. GD18 females: control, n = 9; CdCl₂, n = 11. Males: control, n = 8; CdCl₂, n = 10. PND21 females: control, n = 4; CdCl₂, n = 4. PND42 females: control, n = 5; CdCl₂, n = 4. Males: control, n = 4; CdCl₂, n = 4. PND90 females: control, n = 6; CdCl₂, n = 4. Males: control, n = 4; CdCl₂, n = 6. PND120 females: control, n = 5; CdCl₂, n = 4. Males: control, n = 4; CdCl₂, n = 5. The level of statistical significance for differences between mean values of control and CdCl₂-exposed groups was determined by a two-way ANOVA (treatment, sex) with a Tukey’s post hoc test for all experiments and is indicated by * (p < .05). All images show mean ± SD.

Figure 4.

Graphic representation of gestational Cd disruption ofplacental and hepatic metal concentration and alters mRNA expression of metal ion transporters. This graphic illustrates that dams were exposed to 500 ppb CdCl₂ in drinking water. Exposed dams accumulated ×850 more Cd than control dams by GD18. At GD18, ×15 more Cd was detected in the placenta and approximately ×8 more Cd in the liver of exposed female offspring than control female offspring. Gestational Cd exposure resulted in decreased placental Zn and increased placental Mn in exposed female offspring. Metallothionein mRNA expression was upregulated in exposed female placenta. The transporter responsible for uptake from maternal blood vessels into the placenta (ZIP14) and the transporters responsible for uptake from the placenta into the fetal cord blood (DMT1, ZnT2) were upregulated in the placentas of exposed female offspring relative to same-sex controls. In fetal livers from Cd-exposed animals, Cd and Zn concentrations were increased, whereas metallothionein levels were downregulated and remained downregulated into adulthood. The increased hepatic Zn was transient. Metal ion transporters responsible for influx of divalent metal cations into the liver were significantly downregulated in adulthood, whereas efflux transporters responsible for export into the bile were significantly upregulated. By adulthood, the hepatic Zn phenotypes reversed, with adult hepatic Zn decreases in exposed female offspring.