Effects of Low-Dose Drinking Water Arsenic on Mouse Fetal and Postnatal Growth and Development

© The Author(s), 2012. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in PLoS One 7 (2012): e38249, doi:10.1371/journal.pone.0038249.Arsenic (As) exposure is a significant worldwide environmental health concern. Chronic exposure via contaminated drinking water has been associated with an increased incidence of a number of diseases, including reproductive and developmental effects. The goal of this study was to identify adverse outcomes in a mouse model of early life exposure to low-dose drinking water As (10 ppb, current U.S. EPA Maximum Contaminant Level). C57B6/J pups were exposed to 10 ppb As, via the dam in her drinking water, either in utero and/or during the postnatal period. Birth outcomes, the growth of the F1 offspring, and health of the dams were assessed by a variety of measurements. Birth outcomes including litter weight, number of pups, and gestational length were unaffected. However, exposure during the in utero and postnatal period resulted in significant growth deficits in the offspring after birth, which was principally a result of decreased nutrients in the dam's breast milk. Cross-fostering of the pups reversed the growth deficit. Arsenic exposed dams displayed altered liver and breast milk triglyceride levels and serum profiles during pregnancy and lactation. The growth deficits in the F1 offspring resolved following separation from the dam and cessation of exposure in male mice, but did not resolve in female mice up to six weeks of age. Exposure to As at the current U.S. drinking water standard during critical windows of development induces a number of adverse health outcomes for both the dam and offspring. Such effects may contribute to the increased disease risks observed in human populations.This work was supported by National Institute of Environmental Health Sciences at the National Institutes of Health grants 1F32 ES019070 (CDK-H) and P42 ES007373 (BPJ, JWH, RIE and CDK-H, Dartmouth Superfund Research Program Project Grant, Project 2 and Pilot Project)

Effects of arsenic exposure on the distribution of cholesterol and triglycerides over serum lipoprotein fractions.

<p>Dams were fasted for 6 hours on PN day 21 and pooled serum of 4 As (IU&PN) treated dams (open triangles) and 3 controls (black circles) was used for lipoprotein profiling by FPLC. Fractions were assayed for TG (top panel) and total cholesterol levels (bottom panel).</p

Effects of arsenic exposure on nutritional composition of dam's breast milk.

<p>Breast milk was collected between day 10–12 PN. Breast milk was analyzed for (A.) total protein concentrations and (B.) total TG concentrations. Asterisks indicate statistical significance, ** p<0.01, *** p<0.001, One Way ANOVA compared to control. Error bars equal mean ± SEM (n = 8–9 mice per exposure).</p

Effects of gestational arsenic exposure on liver steatosis and gross organ changes in the dam.

<p>Dams were sacrificed at gestation day 15.5. (A, B.) Detection of liver steatosis at the gross level was observed in a significant percentage of the As exposed mice (C–F.) Histological hematoxylin and eosin staining in (C.,E.) control and (D., F.) As-exposed dams. Scale bars indicate magnification.</p

Effects of cross-fostering on postnatal growth of in utero arsenic-exposed pups.

<p>The average litter weight (grams) of offspring at weaning (day 21 PN) was assessed following fostering. Immediately following birth, all dams were placed on control drinking water through the weaning period. Groups were as follows: (A.) Control offspring remaining with biological mom through weaning/not fostered (n = 11 litters); (B.) 10 ppb IU As exposed mice remaining with biological mom/not fostered (n = 11 litters); (C.) Control offspring fostered by 10 ppb IU As mom (n = 5 litters); (D.) 10 ppb IU As offspring fostered by control mom (n = 5 litters); (E.) Control offspring fostered by a non-biological control mom (n = 3 litters); (F.) 10 ppb IU As offspring fostered by a non-biological 10 ppb IU As mom (n = 3 litters). Asterisks indicate statistical significance, * p<0.05, **** p<0.0001, One Way ANOVA compared to control mice remaining with biological mom (column A). Error bars represent mean ± SEM.</p

Experimental model of developmental arsenic exposure in C57BL/6 mice.

<p>Following the detection of cervical plugs, mated females were exposed to control water or 10 ppb As in drinking water through the gestational period. At the birth of their pups, dams in each exposure group were further divided into sub-groups receiving control water or 10 ppb As in drinking water through 30 days of age (n = 14–17 dams per exposure). Weaning from the dam took place at day 21 PN (or later if a pup did not reach the 7 gram weight cut-off). This resulted in 4 overall exposure groups: 1. Control (no As drinking water exposure; 2. Postnatal (PN, offspring receiving 10 ppb As from PN days 1–30); 3. In utero (IU, offspring receiving 10 ppb As from gestational day 1 through birth); 4. In utero & postnatal (IU&PN, offspring receiving 10 ppb As from gestational day 1 through day 30 PN) At day 30 PN, all offspring were placed on control drinking water and growth was assessed until 6 weeks PN.</p

Exposure to Arsenic during gestation and lactation affect total TG levels in the serum and liver.

<p>Asterisks indicate statistical significance,</p>*<p>p<0.05 and</p>**<p>p<0.01, compared to respective control. Values represent mean ± SEM. Two tailed student's t-test for gestational exposure; One Way ANOVA for post natal exposures.</p

Effects of in utero and postnatal arsenic exposure on growth of offspring.

<p>(A). The weight (grams) of offspring was monitored over the course of development and is shown across all four exposure groups at birth, day 10, 21, and day 28 PN. Male and female mice are included. Birth, n = 21–25; Day 10, n = 17–21; Day 21, n = 65–71; Day 28, n = 44–52. One Way ANOVA, compared to control for each respective time point (B.) Mouse weights separated by gender at day 42 PN across all four exposure groups; n = 49–54. Male mice in all exposure groups are represented with open shapes and female mice with closed shapes. One Way ANOVA for female mice, compared to unexposed female mice. Asterisks indicate statistical significance, * p<0.05, ** p<0.01*** p<0.001, ****p<0.0001. Error bars represent mean ± SEM.</p

Arsenic measurements^a in tissue samples from dams and pups.

a<p>Total As levels were measured by ICP-MS, as described in Methods (n = 3–6). Values represent mean ± SEM. Asterisks indicate statistical significance from respective control group in matching row.</p>*<p>p<0.05,</p>**<p>p<0.01,</p>***<p>p<0.001. Number sign indicates statistical significance from virgin female mouse exposed to 10 ppb As for 2 weeks. One Way ANOVA.</p