ToxSci Advance Access originally published online on June 14, 2007
Toxicological Sciences 2007 99(1):244-253; doi:10.1093/toxsci/kfm162
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Arsenic Exposure in Pregnant Mice Disrupts Placental Vasculogenesis and Causes Spontaneous Abortion
Department of Biomedical and Pharmaceutical Sciences and Center for Environmental Health Sciences, The University of Montana, Missoula, Montana 59812
1 To whom correspondence should be addressed at The University of Montana, Department of Biomedical and Pharmaceutical Sciences, SB 160, Missoula, MT 59812-1552. Fax: (406) 243-5228. E-mail: douglas.coffin{at}umontana.edu.
Received April 21, 2007; accepted June 11, 2007
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ABSTRACT |
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Arsenic is an abundant toxicant in ground water and soil around areas with extractive industries. Human epidemiological studies have shown that arsenic exposure is linked to developmental defects and miscarriage. The placenta is known to utilize vasculogenesis to develop its circulation. The hypothesis tested here states the following: arsenic exposure causes placental dysmorphogenesis and defective placental vasculogenesis resulting in placental insufficiency and subsequent spontaneous abortion. To test this hypothesis, pregnant mice were exposed to sodium arsenite (AsIII) through drinking water from conception through weanling stages. Neonatal assessment of birth rates, pup weights, and litter sizes in arsenic exposed and control mothers revealed that AsIII-exposed mothers had only 40% the fecundity of controls. Preterm analysis at E12.5 revealed a loss of fecundity at E12.5 from either 20 ppm or greater exposures to AsIII. There was no loss of fecundity at E7.5 suggesting that spontaneous abortion occurs during placentation. Histomorphometry on E12.5 placentae from arsenic-exposed mice revealed placental dysplasia especially in the vasculature. These results suggest that arsenic toxicity is causative for mammalian spontaneous abortion by virtue of aberrant placental vasculogenesis and placental insufficiency.
Key Words: arsenic; vasculogenesis; placenta; development; mouse.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
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The overall goal of this study was to determine whether arsenic exposure through drinking water reduces mammalian fecundity. Arsenic is a naturally occurring metalloid released into the environment by natural events and human activities. It is included in pesticides and wood preservatives, pigments, and glass. Arsenic occurs in both organic and inorganic forms, with two oxidation states of the latter as arsenite (AsIII) and arsenate (AsV), that speciate in the environment depending upon the level of dissolved oxygen. Arsenate can be converted to arsenite in vivo (Abernathy et al., 1999
) and either inorganic species can be methylated to monomethylarsonic acid and dimethylarsinic acid (Vahter and Concha, 2001). The main source of human exposure to arsenic is contaminated drinking water and some foods, such as rice, other grains, and fish (ACSH, 2002). High levels of arsenic contamination in drinking water from several hundred ppb to over 1 ppm have been observed in Argentina, Bangladesh, Chile, India, Mexico, Thailand, and Taiwan (ACSH, 2002). In the United States, Montana, New Mexico, Arizona, Nevada, Utah, Southern California, Idaho, and Nebraska, all have localities with 50–100 ppb of arsenic in their drinking water. These levels are higher than the currently approved 10 ppb maximum contaminant level (ACSH, 2002) and, therefore, represent potential environmental health risks. Consequently, we developed a mouse model to first determine whether exposure to high arsenic levels in drinking water reduced fecundity and then find the primary developmental dysfunction associated with arsenic toxicity.
Human epidemiological data suggest an association between arsenic exposure in drinking water and adverse reproductive outcomes (Ahmad et al., 2001). Correlations between relatively low levels of arsenic (0.8–2 ppb) and significant increases in spontaneous abortion have been reported (Aschengrau et al., 1989) with similar reports for fetal mortality (Hopenhayn-Rich et al., 2000) and stillbirth (Borzsonyi et al., 1992). Consistent with the data on in utero fatalities are data correlating human arsenic exposures with increases in preterm birth rates and reduced birth weights (Hopenhayn-Rich et al., 2000; Yang et al., 2003). The human epidemiologic studies are complicated and mostly correlative. Cumulatively, the abundance of independent human studies from different environments that affect all stages of pregnancy provides ample data to support a theory stating that arsenic exposure causes adverse pregnancy outcomes (i.e., spontaneous abortion, stillbirth, low birth weight, and preterm delivery) in humans.
The epidemiological data provide little or no insight for how arsenic exposure could adversely affect human pregnancies. Experimental animal studies support the arsenic/adverse pregnancy hypothesis by revealing that arsenic is a developmental toxicant (Holson et al., 2000; Hood, 1972; Hood et al., 1978). However, the intraperitoneal, intravenous, or gavage administration routes are dissimilar to the human exposures and fail to assess the direct toxicity of arsenic exposure in drinking water on fecundity of pregnant mice (Wang et al., 2006). There are no dose–response studies to determine the lowest concentration of AsIII that causes toxicity in developing mice. These studies are, therefore, a necessary prelude to developing a mouse model to study arsenic toxicity during mammalian development. Speculation on how arsenic exposure complicates pregnancy is based on data showing that arsenic is transferred to the developing embryo through the abundant placental vasculature. Inorganic arsenic of both oxidation states can easily cross the placenta as a low weight molecule and accumulate in the placenta (DeSesso et al., 1998; Lindgren et al., 1984) with levels of 34 µg/kg, almost fivefold higher than control women (7 µg/kg) (Concha et al., 1998a). Arsenic accumulation in the placenta has led to speculation that arsenic might have deleterious vascular effects, causing placental abnormalities or decreased blood flow, that then retard fetal growth (Hopenhayn et al., 2003).
Based on the current information, we exposed mice to increasing concentrations of arsenic and found that 20 ppm or greater exposures caused a significant decrease in fecundity. Histology and immunohistochemistry on the embryos and placentae suggest that placental insufficiency concomitant with defective vasculogenesis are the causes of arsenic mediated spontaneous abortion in mice.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
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Arsenic exposure.
Sodium arsenite (formula NaAsO2, molecular weight 129.91) was purchased from J. T. Baker (Phillipsburg, NJ), and dissolved in deionized distilled water (ddH2O) to yield 0 ppm (0µM), 10 ppm (133µM), 20 ppm (267µM), 37.5 ppm (0.50mM), 75 ppm (1mM), and 150 ppm (2.0mM) concentrations of drinking water for mice. The water bottles were changed twice weekly to assure maintenance of the desired AsIII oxidation state.
To generate mating stocks, FVB/NJ mice (7–10 weeks age) were bred from our colony. All mice were maintained under specific pathogen-free conditions in a Public Health Service, AAALAC facility. Experiments were conducted under a University of Montana IACUC approved animal care and use protocol where they received a diet of rodent chow (PMI, Brentwood, MO) and tap water ad libitum. In addition, they were kept on a 12:12-h light:dark cycle at an environmental temperature of 22 ± 1.5°C, with a minimum relative humidity of 40%. Matings consisted of two virgin females and one stud male placed on ddH2O. The females were examined each morning for the presence of a semen plug. Detection of a semen plug was designated embryonic day 0.5 (E0.5) of gestation. Plug-positive females were considered "pregnant" and immediately placed in separate cages and exposed to AsIII through drinking water on a continuous basis throughout the gestational and weaning stages. At least 10 pregnant females (n = 10) were exposed to each of the AsIII concentrations (0, 10, 20, 37.5, 75, 150 ppm). The control group (0 ppm, n > 8) was maintained under similar conditions but placed on ddH2O without AsIII.
Fecundity.
Each pregnant mouse was numbered and the number of newborn pups in her first litter was recorded. Pregnant (plug-positive) females that yielded no pups were considered a spontaneous abortion while "successful" pregnancies were considered those yielding any pups, regardless of the number. The mean pup weight per litter was recorded weekly (p = 7, 14, and 21 days) by weighing the entire litter together and then dividing the total litter weight by the number of pups. The mean litter size was obtained at each AsIII exposure concentration by finding the mean value for the number of pups per litter for all the litters born at each concentration. The birth rate was calculated by dividing the number of successful pregnancies by the total number of pregnant females. The spontaneous abortion rate was calculated by dividing the number of females that spontaneously aborted by the total number of pregnant females. Fecundity was defined as the product of the mean litter size and the birth rate. Normalized fecundity was calculated by dividing the experimental value by the control value and multiplying by 100. The female and her pups were sacrificed at the weanling stage. No pregnant females were reused regardless of whether they produced a litter.
Embryology and teratology.
At 7.5 and 12.5 days of gestation (E7.5 and E12.5), AsIII-treated and control plug-positive females were euthanized with carbon dioxide. The uterus was transferred into sterile phosphate-buffered saline (PBS) and the number of live embryos or plaque resorptions was counted. Live embryos were removed from their surrounding membranes, examined for gross defects, then the entire litter was collectively weighed. The mean embryo weight was calculated by dividing the total litter weight by the number of embryos in each litter. Embryos and placentae (or the decidua) were then stored in 4% formalin/PBS at room temperature for histochemistry.
Histology and anti-vonWillebrand factor immunohistochemistry.
Formalin-fixed placentae and embryos from E7.5 and E12.5 were embedded in paraffin, sectioned (7 µm), and mounted on microscope slides. A Varistain 24-4 automated tissue stainer (Shandon Corp., Pittsburgh, PA) was used for hematoxylin and eosin (H&E) staining of the placentae and embryos for light microscopy using a Nikon Eclipse E800. Photographs were taken with a digital camera (Nikon) and stored as .*tif files. Antisera against vonWillebrand's factor (VWF) (factor VIII related antigen) were used to label the placental endothelium in E12.5 tissue using published methods (Coffin et al., 1991). Briefly, the sections were deparaffinized, rehydrated into PBS, and then antigen retrieval step was conducted by boiling sections in 0.01M citrate pH 6 for 10 min and cooling in citrate for 10 min. This was followed by a PBS rinse and then a blocking step in 4% normal goat serum for 20 min. Anti-VWF (Chemicon, Temecula, CA) diluted 1:50 was incubated on the sections overnight at 4°C. Secondary antibody, a goat anti-rabbit IgG (Molecular Probes, Invitrogen, AlexaFluor 488) diluted 1:500 was applied for 1 h at room temperature. Following 3- to 5-min rinses in PBS, the slides were coverslipped using FluorSave (Calbiochem) for fluorescence microscopy.
BSL-B4 histochemistry.
After mounting the 4% formalin-PBS fixed placentae and embryos in frozen tissue matrix (VWR, Bristol, CT), 10-µm sections were cut on a Cryostat (Thermo-Shandon). Mouse embryos were cut longitudinally. To stain the endothelial cells in placenta and embryo, fixed tissues were soaked in CMPBS (PBS supplemented with 1mM CaCl2 and 1mM MgCl2) three times, 5 min each. The tissues were then permeabilized with 100% methanol for 20 min and rehydrated in ethanol series (95–70–50%) for 2 min each. After rinsing in CMPBS two times, biotinylated Bandeiraea simplicifolia lectin I isolectin B4 (BSL-B4) (Vector Laboratories, Burlingame, CA) that binds the (1,3)-galactoside moiety on endothelial cells was applied (1 µg/ml in CMPBS) for 30 min at room temperature. After a rinse with CMPBS, Texas Red avidin (546; Molecular Probes, Eugene, OR) was applied at 1:500 for 30 min. Following a final rinse in PBS, the slides were coverslipped with Fluorsave reagent (Calbiochem, San Diego, CA) and examined with a fluorescence microscope (Nikon Eclipse E800, Japan). The immunofluorescence staining was analyzed with a laser scanning cytometer (LSC; CompuCyte Corporation, Cambridge, MA). Each datum is an average of three repeated sections on each slide and five different embryo litters (one litter on one slide) in each group.
Statistical analysis.
Results are expressed as the mean ± standard deviation. Comparisons of means were analyzed by one-way analysis of variance (ANOVA) to determine intergroup differences. If the results of the ANOVA were significant (p < 0.05), Tukey's honestly significant difference (HSD), and Dunnett two-sided test were applied to the data to compare the treated group with control groups. Birth rates, fecundity, and prenatal viability were analyzed by one-way analysis of chi-square test to determine the confidence limits for determining AsIII treatment as the source of spontaneous abortion.
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RESULTS |
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Neonatal Dose–Response
To determine how arsenic exposure during pregnancy affected fecundity, a dose–response study was conducted where time-pregnant female mice were exposed to 0, 1, 10, 20, 37.5, 75, or 150 ppm sodium arsenite (AsIII) in their drinking water starting at E0.5 and continuing throughout the gestational and weaning stages. The following data were recorded at p = 0 (time of birth): number successful pregnancies, number of spontaneously aborted litters, litter size, and average pup weight per litter. From that data the birth rate, spontaneous abortion rate, and fecundity were calculated. A chi-square test was applied for statistical significance for expected spontaneous abortion frequencies and fecundity between AsIII-exposed mice versus controls. Average pup weight per litter was also measured at p = 1, 2, and 3 weeks.
Although there was a trend toward smaller litters, there was no significant difference in litter sizes (pups per litter) between the AsIII exposure groups at p = 0 for AsIII exposure concentrations of 1 ppm (7.9 ± 3.0), 10 ppm (7.4 ± 3.1), 20 ppm (6.9 ± 2.5), 37.5 ppm (5.6 ± 2.7), and 75 ppm (7.1 ± 1.7) compared to the dH2O treated controls (9 ± 4). In addition, there were no statistically significant differences in mean pup weights between the AsIII-treated groups and the control group at p = 0, p = 7 days, p = 14 days or p = 21 days (data not shown). Comparison of the number of litters born (p = 0) between the AsIII exposure and water (control) groups revealed strikingly increased spontaneous abortion (miscarriage) rates at AsIII exposures of 20, 37.5, and 75 ppm (Fig. 1). Note that there was no significant difference between the control group, the 1 ppm group and the 10 ppm group in delivery of neonates. However, the 20, 37.5, and 75 ppm groups were all significantly different from the control group (p < 0.01) regarding their spontaneous abortion rate at p = 0. All mothers in the group treated with 150 ppm AsIII died 2–4 weeks after exposure. The 150-ppm exposure level was used based on AsIII exposure cancer studies in the same strain of mice (Germolec et al., 1998). An essential difference was the use of high fat chow in the cancer studies that was not used here, plausibly explaining the high mortality at 150 ppm that occurred in this project.
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Birth rates were calculated as the inverse of miscarriage rates. Calculation of normalized fecundity (birth rate x litter size normalized to 100%; Fig. 1) was conducted to assess how AsIII exposure affected conception overall. Since fecundity and birth rate use different units both fecundity and birth rates were normalized for comparison and then the differences, due to litter size only, were calculated. AsIII exposures of 1 and 10 ppm resulted in 18% [(103.5 – 85)/100] and 23% [(115.9 – 85)/100] reductions, respectively, between normalized fecundity and normalized birth rates. The 29% [(67.1 – 47.8)/100] reduction at 20 ppm and the reduction of 46% [(52.5 – 28.2)/100] at 37.5 ppm were even more dramatic. This comparison shows that the combined effect of smaller litters and increased miscarriage was greater that either effect alone for AsIII mediated reductions in murine reproduction.
Arsenic Toxicity In Utero
Following the neonatal/p = 0 experiments, the effects of in utero AsIII exposure on development were examined at E7.5 and E12.5 stages. These stages were chosen based on our hypothesis stating that AsIII exposure is detrimental to vascular development. The most critical events for placentation and establishment of the embryonic circulatory system occur between the E7.5 and E12.5. The experimental design for the in utero exposure studies was based on the neonatal results. Plugged mice (E0.5) were exposed to AsIII concentrations of 0 (control), 10, 20, 37.5, and 75 ppm until the E12.5 stage when the mice were sacrificed. In the interest of minimizing the number of mice necessary to obtain meaningful data only 0 (control), 20, and 37.5 ppm levels were used for exposures until the E7.5 stage. Data for spontaneous abortion rates, viable litters, embryos weights, litter size, and fecundity were all obtained at the E7.5 and E12.5 stages. Note that compared to the neonatal studies, for the in utero studies mean embryo weight/litter replaced weekly pup weights and the number of viable embryos per litter replaced litter size.
Analysis of embryos at the E12.5 stage of development, exposed in utero to increasing concentrations of AsIII, generated results (Fig. 2) consistent with the neonatal experiments. There were no statistically significant differences in litter sizes (pups/litter) between the AsIII exposure groups at E12.5 for AsIII exposure concentrations of: 10 (8.9 ± 3.0), 20 (8.4 ± 2.8), and 37.5 ppm (6.8 ± 2.6) compared to the dH2O-treated controls (8.9 ± 2.9). Similarly, there were no statistically significant differences in mean embryo weight/litter between the AsIII-treated groups and the control group at E12.5 (data not shown). There was, however, a significant (p < 0.05) decrease in the proportion of E12.5 viable litters when mothers were exposed to either 20, 37.5, or 75 ppm AsIII through their drinking water (Fig. 2). There was no statistically significant difference between the control group and the 10-ppm exposure group (Fig. 2). At 75 ppm there was increased mortality and the mice were noticeably debilitated, possibly contributing to increased variance in the data. The mean values for the 75-ppm AsIII-treated group were still statistically significant different (p < 0.05) compared to controls but less than the 37.5-ppm group; closer to the means for the 20-ppm treatment group. Collectively, the E12.5 results are consistent with the neonatal results with both experiments showing a dramatic decrease in fecundity caused by greater than or equal to 20-ppm AsIII exposures (Fig. 2). However, in contrast to the neonatal data, the lower fecundity at E12.5 is primarily due to increased spontaneous abortions (Fig. 2) because factoring in litter sizes did not produce any significant change when comparing the normalized birth rates to normalized fecundity.
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Similar to the neonatal and E12.5 in utero experiments, exposures to AsIII in pregnant mice that were terminated at the E7.5 stage showed no statistically significant differences in litter sizes or mean embryo weights (data not shown). However, in contrast to results from the neonatal and E12.5 experiments, exposures terminated E7.5 showed no significant difference in the proportion of viable litters between the H2O control group (10/15, 67% viable) and the 37.5-ppm arsenic exposure group (8/14, 57% viable). Fecundity at E7.5 for exposures of 37.5 ppm AsIII was 95% of the water control group showing no significant effect. Comparison of the data for all three AsIII exposure experiments (neonatal, E12.5 and E7.5) showed a lower neonatal fecundity that resulted from embryo drop-out between E7.5 and E12.5. Moreover, these data show that there is no effect from AsIII exposure on prefertilization, fertilization, and preimplantation development, i.e., the effect occurs between E7.5 and E12.5 in postimplantation development. The principal factor causing lower fecundity during this developmental period was a substantial loss of embryo viability that was later manifest as higher spontaneous abortions for neonates (Fig. 1).
Placentation and Vasculature
Placentae and embryos were examined for morphological variance, teratogenesis, and vascular abnormalities. Gross examination of placentae from 37.5-ppm AsIII-exposed and control mice showed larger, darker placentae from the AsIII-exposed mice (Fig. 3). The larger, darker placentae from the AsIII-treated mice were indicative of dysplasia and vascular anomalies including venous/arterial anastomoses consistent with placental insufficiency. Further histological analysis was carried out with paraffin sections of those placentae that were stained with H&E and then used for microscopy to examine placental morphology (Fig. 4). Comparison of the AsIII-exposed placenta to the control showed an expanded decidua and trophoblast giant cell layer in the AsIII-exposed placenta (Fig. 4A). Furthermore, the labyrinth layer was smaller and the syncytiotrophoblast tissue was less differentiated revealing impaired morphogenesis in that tissue in particular (Fig. 4C). The surrounding blood vessels were fewer in the labyrinth layer (Fig. 4E) with hypertrophic endothelium in the poorly defined vasculature (Fig. 4G). Overall, the AsIII-exposed placentae appeared to be poorly developed featuring both undifferentiated and degenerating tissues.
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To specifically examine the vasculature, serial sections from the same samples used for H&E staining were stained with anti-VWF antibodies to highlight the placental vasculature (Fig. 5). Most notable was a greater density of blood vessels in the decidua and spongiotrophoblast resulting from AsIII exposure (Figs. 5A and 5C). The VWF staining was consistent with the H&E staining showing a relatively avascular, smaller, and poorly defined labyrinth layer in the AsIII-exposed placentae (Fig. 5E). The VWF data were consistent with an analysis of defective placental vasculogenesis resulting from AsIII toxicity.
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Although the H&E and the VWF staining were consistent, we wanted to confirm the VWF results with a second endothelial cell marker in a quantitative manner. Therefore, a quantitative analysis of placental vasculature was conducted using histochemical fluorescence with B. simplicifolia lectin I BSL-B4 (staining (1,3)-Gal of endothelial cells). BSL-B4 stained placentae from AsIII-exposed and control placentae were qualitatively and quantitatively analyzed with an LSC. Qualitative fluorescence microscopy (not shown) yielded results consistent with H&E and VWF staining, although baseline background was higher, BSL-B4 showed dramatic vascular dysmorphogenesis. Quantification of fluorescence showed a significant loss of BSL-B4–positive staining (p < 0.05) in placentae exposed to 37.5 ppm AsIII at E12.5 compared to the controls (Fig. 6). BSL-B4–stained E12.5 embryos analyzed in the same manner showed decreased BSL-B4 labeling from the control (not shown). This difference was, however, not statistically significant.
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DISCUSSION |
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The results show that AsIII exposure through drinking water in pregnant mice from E0.5 through weaning disrupts placental morphogenesis and lowers fecundity. The decrease in fecundity is most likely to due to placental insufficiency but detrimental effects on the embryo that could also contribute to spontaneous abortion are still a possibility. Drinking water exposure to AsIII did not cause a statistically significant difference in the litter size at any time point. Our study suggests that the primary effect of AsIII in pregnant mice was a dose-dependent increase in conceptus mortality (spontaneous abortion rate). Previous studies reported that embryonic malformations resulted from arsenic exposure by ip or iv routes in pregnant mice (Morrissey and Mottet, 1983
). In this study, we did not observe any obvious offspring malformations, which is consistent with the other results showing that oral administration of arsenic in rats by gavage only resulted in increased prenatal mortality (Stump et al., 1999). We did not observe any significant increase of the resorption plaques at E12.5 in 37.5-ppm AsIII-treated group compared to the control group suggesting that preterm deliveries that are eaten by the mother is also a possibility. To our knowledge, this is the first study associating arsenic exposure in drinking water with lower fecundity of pregnant mice.
Epidemiological data in humans showed higher miscarriage rates, premature deliveries, and lower birth rates associated with high ambient arsenic levels (Aschengrau et al., 1989). The primary routes of human arsenic exposure worldwide are dust and drinking water following extractive activities. By necessity, human studies are correlative without the ability to directly show cause and effect by experimentally administering arsenic under controlled conditions. Here we conducted those experiments in mice and the neonatal data convincingly show that AsIII exposures through drinking water do, indeed, cause a loss of fecundity, primarily through spontaneous abortion or spontaneous abortion from placental insufficiency. The lack of any significant change in birth weights, birth defects, or premature deliveries in this study, compared to humans, could be due to the lack of complicating factors missing in our controlled study. In the human populations complicating factors might include poor nutrition, other toxicants such as heavy metals, toxins such as organophosphates, alcohol use, or tobacco use. These are among several human activities known to cause pregnancy complications that were not included in this study. Inclusion of some or many of these factors in combination with arsenic exposures might have resulted in the birth defects, lower birth weights, and premature deliveries observed in humans.
The AsIII levels used here are also substantially higher than the environmental levels observed in the human studies. Our studies included a titration of increasing AsIII levels to determine the necessary exposure to achieve a statistically significant spontaneous abortion rate. Typically, the human exposure levels for "high arsenic" range from 20 ppb to 1 ppm (Borzsonyi et al., 1992; Hindmarsh and McCurdy, 1986; Tabacova et al., 1994; Willhite and Ferm, 1984). Our data showed that at least 20 ppm, x20–1000 higher than the ambient levels, was required to cause statistically significant spontaneous abortion. The species differences could result from two possibilities. First, the human environment has a number of complicating factors (discussed above) that were not included in our controlled, mouse exposure experiments. Those (human) environmental factors could result in lower arsenic exposure levels effectively causing spontaneous abortion. Alternatively, the many obvious physiological differences between mice and humans require higher arsenic levels in mice to produce the phenotype observed in humans (i.e., comparative arsenic toxicology). Rodents have substantial capacity to hypermethylate arsenic resulting in transition from a more toxic AsIII form to the methyl form and the methyl form is more easily excreted (Wu et al., 2004). These complications do not and should not preclude use of mice for arsenic toxicity studies in many fields including cancer research (Germolec et al., 1998; Vega et al., 2001), cardiovascular research (Bunderson et al., 2004), neuroscience (Machado et al., 1999; Willhite and Ferm, 1984; Wlodarczyk et al., 1996), and developmental biology (Concha et al., 1998b; Ferm, 1977; Machado et al., 1999; Tabacova et al., 1994; Willhite and Ferm, 1984; Wlodarczyk et al., 1996).
Vascular development in the postimplantation mouse embryo and placentation essentially begin at E6.5, resulting in a fully functional embryonic circulation by E12.5 (Coffin et al., 1991). Therefore, we suspected that AsIII damage to the embryonic vascular system should be occurring between those stages. Experimental testing of that hypothesis included assessment of fecundity by bracketing with the E7.5 and E12.5 stages. Statistical evaluation of the E7.5 data showed no difference between the AsIII-exposed mice and controls. Conversely, analysis of the E12.5 data and the neonatal data showed a statistically significant decrease in fecundity in AsIII-exposed mice. Therefore, the loss of litters is occurring at some point between those stages, a conclusion that is supported by the histomorphometry and VWF immunohistochemistry results. The data clearly show no increase in spontaneous abortion at E7.5. We now have preliminary data at E8.5 (not shown) showing no increase in spontaneous abortion or fecundity at that stage either. These results show that the AsIII-mediated spontaneous abortion occurs AFTER implantation and that the loss of fecundity from AsIII toxicity is NOT caused by effects on fertilization or preimplantation development.
Another interesting interpretation stems from results showing a difference between normalized birth rates and normalized fecundity at the neonatal stage. This difference (in neonates) occurred when litter size and birth rate factors were included in assessing fecundity suggesting that both smaller litters and lower birth rates contributed to the AsIII-mediated decline in fecundity. However, no such difference occurred at the E12.5 stage when comparing normalized viable litters (comparable to neonatal birth rates) and normalized fecundity. This suggests that the lower fecundity at the E12.5 was entirely due to increased spontaneous abortion. This difference suggests that the frequency of litters born declines between E12.5 and the neonatal stage and, furthermore, that loss of embryos in the later stages could be from premature birth or late term spontaneous abortion. Therefore, AsIII exposure in mice may indeed be causing a phenomenon (in mice) that is similar to preterm delivery in humans.
The lack of any morphological defects in the AsIII-exposed embryos while the placentae showed both gross and histological defects suggests that the AsIII-mediated loss of fecundity is due to placental insufficiency. These results are consistent with reports of cavernous damage to both endodermal and mesodermal tissues in arsenic treated mouse embryos, yolk sacs, and placentae (Li and Zhu, 1997). Furthermore, the vascular defects observed by gross appearance (Fig. 3) and in the VWF and BSL-B4 placental sections from AsIII-exposed mice (Figs. 4 and 5) support the conclusion that the placental insufficiency is directly related to a defect in placental vasculogenesis. These results are consistent with data from chicken chorioallantoic membrane (CAM) assays showing that low arsenic exposures (< 0.033µM or 2.5 ppb) stimulate angiogenesis while arsenic exposures over 1µM (75 ppb) inhibit angiogenesis (Soucy et al., 2003). There is, however, an important distinction to be made between placental vasculogenesis and the angiogenesis observed in the CAM and many other systems including tumor angiogenesis.
Classic studies using simple microscopy (Evans, 1909; Sabin, 1917, 1920) later expanded with immunohistochemistry (Coffin and Poole, 1988; Poole and Coffin, 1989) on avian embryos established that embryonic vascular development occurs through two fundamental modes termed "vasculogenesis" and "angiogenesis" with the distinction between the two based on morphogenesis (Risau et al., 1988). Vasculogenesis is the de novo formation of embryonic vascular anlagen to establish the first observed blood vessel rudiments in the embryo and extra-embryonic tissues. Angiogenesis then expands blood vessels from those anlagen by branching morphogenesis into avascular areas. The placental vasculature has been shown to develop principally through the vasculogenesis mode of blood vessels development (Huppertz and Peeters, 2005). This distinction, between placental vasculogenesis and other forms of angiogenesis, may explain differences in results whereby AsIII exposure has been shown to stimulate tumor angiogenesis (Soucy et al., 2003, 2005) while the results presented here clearly reveal defective placental vasculogenesis. In simplest terms, they are very different systems and different results from AsIII exposures should not be considered unusual.
Studies on arsenic toxicity provide an opportunity to study the differential regulatory pathways between angiogenesis and vasculogenesis. Published results have shown unique involvement of the circulating flk-1 vascular endothelial growth factor (VEGF) receptor-2 and flk-1–positive preendothelial cells in vasculogenesis but not angiogenesis (Tepper et al., 2005). Another unique role for the of the VEGF regulatory system in vasculogenesis is demonstrated by the hHex null mouse that includes defective vasculogenesis through elevated VEGFa in the phenotype (Hallaq et al., 2004). Obviously, the VEGF system is active in both angiogenesis and vasculogenesis, but our results and previously published data suggest that VEGF may be functioning in a different manner for each form of neovascularization. Studies on placental blood vessel development may help clarify the differences as we characterize the molecular mechanisms responsible for AsIII-mediated vascular dysplasia. Initial focus will be on the VEGF system because it has been shown to be altered by AsIII exposure (Duyndam et al., 2001; Soucy et al., 2005).
Overall, the previously published results, the E12.5 fecundity data and the histomorphometry conducted on E12.5 placentae are all consistent with a model where AsIII-related vascular toxicity causes placental insufficiency concomitant with aberrant vasculogenesis that results in spontaneous abortion. It is noteworthy that use of seminal plugs as in indicator of pregnancy probably leads to some increased variance in the data. Unfortunately, there are no reliable clinical pregnancy tests for mice and the only reliable means for accurate pregnancy testing is ultrasound (Visualsonics, Inc., Toronto, Canada). We did not have that procedure available at the time of this study. More refined arsenic exposures, developmental and biochemical studies are now required to determine how arsenic exposures alter endothelial cell biochemistry and physiology to cause placental endothelial dysfunction and subsequent spontaneous abortion. This includes an analysis of fecundity between the stages of E7.5 and E12.5 to examine the sources and mechanisms for placental dysplasia and at later stages including E15.5 and E18.5 to address the possibility of preterm delivery.
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FUNDING |
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National Institutes of Health and Centers for Disease Control grants (NCRR-1P20RR17670, NIEHS-R21ES01174902, RO1CCR822092).
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ACKNOWLEDGMENTS |
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We thank William McDonald for his technical assistance and Aaron Barchowsky for technical advice.
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REFERENCES |
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