Effects_of_Cadmium_Exposure_on_Syncytialization_in_Relation_to_Placental_Cell_Formation_and_Function.pdf

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Effects of Cadmium Exposure on Syncytialization in Relation to Placental Cell Formation and Function

Niharika Palakodety Chapel Hill, North Carolina

2020

A technical report submitted to the faculty at the University of North Carolina at Chapel Hill in partial fulfillment of the requirements for the Master of Science of Public Health Degree in the

Department of Environmental Sciences and Engineering in the Gillings School of Global Public Health

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2 Table of Contents

Abstract ... 3

List of Figures ... 4

List of Abbreviations ... 5

Introduction ... 6

Methods ... 10

Cell Culture and Cd Treatment. ... 10

Cytotoxicity Assay. ... 11

Protein Quantification and Human Chorionic Gonadotropin Secretion. ... 11

Statistical Analysis... 12

Results ... 13

hCG Production in Cd Exposed BeWo Cells ... 13

Figure 1 ... 13

hCG Secretion in Forskolin Treated Cytotrophoblasts. ... 13

Figure 2 ... 14

hCG Production in Cd-Forskolin Cotreated BeWo Cells. ... 14

Figure 3 ... 15

hCG Production in Cd-treated JEG-3 Cells. ... 15

Figure 4. ... 16

hCG Production in Cd-Forskolin Cotreated JEG-3 Cells ... 16

Figure 5. ... 17

Discussion... 18

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Abstract

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4 List of Figures

Figure 1. hCG secretion after CdCl2 treatment for 24 and 48 h in BeWo cells ... 13

Figure 2. hCG secretion after 24 and 48 h 100 μM treatment to F ... 14

Figure 3. hCG production after 24 and 48 h Cd-forskolin cotreatments in BeWo cells ... 15

Figure 4. hCG production in JEG-3 after exposure to CdCl2 ... 16

Figure 5. hCG production after Cd-forskolin cotreatments for 24 and 48 h in JEG-3 cells. ... 17

Supplemental Figure 1. BeWo cell % survivability after 48 h of CdCl2 treatment ... 23

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5 List of Abbreviations

cAMP – Cyclic Adenosine Monophosphate Cd - Cadmium

CdCl2 - Cadmium Chloride

cDNA - Complementary Deoxyribonucleic Acid

DNA - Deoxyribonucleic Acid

DOHaD - Developmental Origins of Health and Disease ELISA - Enzyme-linked Immunosorbent Assay

F - Forskolin

FBS - Fetal Bovine Serum

h - Hours

hCG - Human Chorionic Gonadotropin mRNA - Messenger Ribonucleic Acid

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Introduction

Cadmium (Cd) is a nonessential heavy metal used ubiquitously in the production of many consumer and industrial goods. In particular, it has been utilized in cigarettes, fertilizers, non-corrosive metal plating, and electronics (Chunhabundit, 2016). Because of its widespread use, Cd can readily enter the environment through natural and anthropogenic pathways, including

fertilizer application, smoking, sewage sludge, metal ore combustion, and electronic waste recycling (Chunhabundit, 2016). The primary source of non-occupational human exposure to Cd is through contaminated food (Satarug, Garrett, Sens, & Sens, 2010); the heavy metal has a high rate of soil-to-plant transfer, and can easily accumulate in food, such as shellfish, mushrooms, green leafy vegetables, and root crops (Chunhabundit, 2016; Jarup & Akesson, 2009).

Additionally, cigarette smoking is another major source of exposure (Adams et al., 2011; Rafati Rahimzadeh, Rafati Rahimzadeh, Kazemi, & Moghadamnia, 2017). Cd easily accumulates in the tobacco plant, and is subsequently released and inhaled during the burning of cigarettes (Adams et al., 2011). Exposure to Cd is a major public health issue because of its severe toxicity to the human body.

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focus in understanding how heavy metals, like Cd, affect pregnant women and developing fetuses. Cd exposure is associated with the increased risk for adverse pregnancy- and birth-related outcomes, such as preeclampsia, miscarriage, and fetal undernourishment (Edwards, Maxson, Miranda, & Fry, 2015). Furthermore, in utero exposure to Cd has been linked to

impaired growth and neurodevelopment (Gardner et al., 2013; Kippler et al., 2016; Kippler et al., 2012). Perturbations in the placenta may explain the underlying mechanisms that modulate these adverse health associations.

The placenta is a complex, temporary organ that is responsible for ensuring a safe and healthy pregnancy (Burton & Jauniaux, 2015). It plays a vital role in serving as a barrier between maternal and fetal blood by promoting the exchange of gases, nutrients, and waste (Gude,

Roberts, Kalionis, & King, 2004). The placenta consists of two portions: the fetal tissue derived from the chorionic sac and the maternal tissue derived from the endometrium. In between these two regions lies the intervillous space, which is lined with the placental membrane. During the initial stages of development, the placental membrane consists of four layers: (1) the

syncytiotrophoblast, an enlarged multinucleated cell, which is in direct contact with maternal blood, (2) cytotrophoblast cells, (3) connective villus tissue, and (4) the endothelial lining of the fetal capillaries. Maternal and fetal exchange occurs at the terminal ends of the intervillous space (Gude et al., 2004).

As the placenta develops, the mononucleated cytotrophoblast layer begins to differentiate into the syncytiotrophoblast, narrowing the distance between the maternal and fetal blood to promote efficient exchange (Gude et al., 2004). This differentiation process is known as syncytialization (Gupta, Malhotra, Malik, Verma, & Chaudhary, 2016). As the

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(hCG) hormone, which functions to maintain the corpus luteum, angiogenesis, and further trophoblast differentiation (Guibourdenche et al., 2010). The production of hCG induces cytotrophoblast invasion of the uterus to ensure proper embryo implantation and supply of nutrients and oxygen (Adams et al., 2011; Hiden, Bilban, Knofler, & Desoye, 2007; Movsas & Muthusamy, 2018).

hCG production serves as a marker for syncytialization and placental development. hCG secreted by cytotrophoblasts bind to the luteinizing hormone/choriogonadotropin receptor on adjacent trophoblasts, resulting in the formation of intracellular cyclic adenosine monophosphate (cAMP). Subsequently, cAMP binds to the regulatory subunit of protein kinase A (PKA), and thereby activates and releases the catalytic subunit of PKA. The release of the catalytic subunit phosphorylates transcription factors, such as the cAMP response element-binding protein (CREB) and chorion-specific transcription factor (GCMa). This upregulates the transcription of genes involved in the fusion of trophoblasts, including Syncytin1, Syncytin 2, GCM1,

Connexin43, CGA, and CGB (Gerbaud & Pidoux, 2015). Syncytialization plays an essential role

in ensuring a healthy pregnancy because it forms the syncytiotrophoblasts, which are in direct contact with the maternal blood; thus, its proper development enables the feto-maternal exchange needed for fetal growth (Gerbaud & Pidoux, 2015). Inadequate formation of the

syncytiotrophoblast has the potential to result in the onset of pregnancy diseases, including preeclampsia (Huppertz, Meiri, Gizurarson, Osol, & Sammar, 2013).

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maternal mortality globally (Huppertz et al., 2013; Rosen, Munoz, McElrath, Cantonwine, & Ferguson, 2018). An estimated 2-8% of the global population of pregnant women are diagnosed with the disease (Rosen et al., 2018). Current research suggests several potential explanations for the development of preeclampsia, including improper placentation or inadequate supply of maternal blood, inflammatory response to oxidative stress, and abnormal maternal response from pre-existing conditions (e.g. hypertension, obesity, and diabetes) (Redman & Sargent, 2005). Additionally, exposures to environmental contaminants, like Cd, have also been associated with preeclampsia (Adams et al., 2011; Laine et al., 2015; Rosen et al., 2018; F. Wang et al., 2018). In understanding how Cd perturbs biological mechanisms to result in preeclampsia, evidence suggests oxidative stress to impaired placental function, pro-inflammatory reactions, and perturbations of the glucocorticoid, or stress response, pathway may all contribute (Laine et al., 2015; F. Wang et al., 2018).

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Methods

Cell Culture and Cd Treatment. The JEG-3 and BeWo choriocarcinoma cell lines were

purchased from the American Type Culture Collection (Manassas, VA). JEG-3 cells were grown in Gibco Minimum Essential Medium with 10% fetal bovine serum (FBS), 1%

penicillin/streptomycin, and 1mM sodium pyruvate at 37 °C in 50% CO2. BeWo cells were grown in Kaighn’s modification of Ham’s F-12 Medium with 10% FBS and 1%

penicillin/streptomycin at 37 °C in 50% CO2. Cells were plated at 2 × 106 cells per 10 cm2 flask, and incubated under standard conditions until achieving 80-90% confluence. Cadmium Chloride (CdCl2) was resuspended in deionized water to create a stock concentration of 10 mM. To study the effects of Cd exposure in vitro, cells were seeded in a 12-well culture plate at

0.25 × 106 cells per well, and incubated for 24 hours (h) prior to treatment. On the day of treatment, the stock CdCl2 was added to the cell type’s corresponding media, and vortexed to create final concentrations of 0.044 μM, 1 μM, and 10 μM. Cells were incubated in the presence or absence of Cd for 24 or 48 h, and then harvested for messenger ribonucleic acid (mRNA) isolation. Treated and untreated cells were harvested in 350 μL of Buffer RLT, and placed in a QIAcube (Qiagen, Valencia, CA) for mRNA and DNA extraction using the AllPrep

DNA/RNA/miRNA Universal Kit (Qiagen, Valencia, CA). mRNA was further cleaned with the RNA Clean and Concentrator-5 kit (Zymo Research, Irvine, CA). mRNA was quantified using a Nanodrop 1000 spectrophotometer (Thermo Scientific, Waltham, MA). For protein harvests, cells were seeded in a six-well plate at 0.5 × 106 cells per well, and incubated for 24 h prior to

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μL RIPA buffer, supplemented with 1% phosphatase inhibitor and 1% protease inhibitor (Fisher Scientific, New Hampshire). Cell supernatant was also collected from protein and RNA samples. To address potential variation due differences in where the cells are in their life stage, forskolin (F) was added to the Cd treatments. F is a known cAMP inducer; thus, treatment in cells induce cells into syncytialization (Dubey, Malhotra, & Gupta, 2018; Omata, Ackerman, Vandre, & Robinson, 2013; R. Wang et al., 2014). By adding F to the Cd treatments, the cell cycles would sync, and move towards syncytialization together. Therefore, both cell lines were cotreated with 100 μM F and 0.044 μM, 1 μM, and 10 μM of CdCl2; they were treated for 24 or 48 h. The cells were harvested for mRNA, DNA, and protein with the same procedures previously described; supernatant was collected from the protein and mRNA samples.

Cytotoxicity Assay. To determine the cytotoxicity of the Cd treatments, JEG-3 and BeWo cells were seeded at 1.0 × 104 cells per well in 96-well culture plates, and incubated overnight for 24 h. The cells were treated as described above and incubated for 24 or 48 h. Resazurin dye, at a final concentration of 10 μg/L, was added to the culture, and then incubated for an additional two hours at 37 °C. During this time, viable cells convert resazurin to resorufin, the fluorescent product. After a two-hour incubation, fluorescence was measured at an excitation wavelength of 560 nm and an emission wavelength of 590 nm, according to the manufacturer’s protocol, with a Promega spectrophotometer microplate reader. Each well’s fluorescence value is in

correspondence with the number of viable cells in the population.

Protein Quantification and Human Chorionic Gonadotropin Secretion. Treated and untreated cells were harvested for protein in 150 μL RIPA buffer, supplemented with 1%

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protocol (Thermo Scientific, Waltham, MA). Supernatant from treated and untreated cells were measured for hCG concentrations using an hCG enzyme-linked immunosorbent assay (ELISA); the ELISA was followed according to manufacturer’s protocol (RayBiotech, Norcross, GA).

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Results

hCG Production in Cd Exposed BeWo Cells. To ensure that the Cd doses administered to the BeWo cells were not cytotoxic, a resazurin cytotoxicity assay was performed. The data

(Supplemental Figure 1) demonstrated that 0.044 μM CdCl2, 1 μM CdCl2, and 10 μM CdCl2 did not induce cytotoxicity in the BeWo cells. In order to understand whether the doses chosen impacted hCG secretion, supernatant from treated cells were collected, and associated protein concentrations were measured. hCG concentration was analyzed with an ELISA specific for hCGβ. Results showed that Cd did not significantly (p < 0.05) alter hCG secretion after 24 and 48 h of treatment (Figure 1).

Figure 1. hCG secretion after CdCl2 treatment for 24 and 48 h in BeWo cells. Values shown are means with SEM. Concentrations determined by the ELISAs (pg/mL) were adjusted to protein concentration (μg/mL) determined by the Pierce BCA assay. hCG production was not

significantly altered (p < 0.05) by Cd after 24 or 48 h.

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secretion in various cytotrophoblast cell lines, including BeWo and JEG-3 cells (Omata et al., 2013; R. Wang et al., 2014). For both cell types after 24 and 48 h exposure to F, there is a statistically significant (p < 0.05) increase in hCG secretion. Additionally, the BeWo cells secreted a greater concentration of hCG in comparison to the JEG-3 cells at both 24 and 48 h harvests.

Figure 2. hCG secretion after 24 and 48 h 100 μM treatment to F. Values shown are means with

SEM. hCG Concentrations (pg/mL) determined by ELISAs were adjusted for protein concentration (μg/mL). * indicates significance at p < 0.05; ** p < 0.05, *** p < 0.0005.

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analyzed using an hCG ELISA. After 24 h exposure to the Cd-forskolin cotreatments, there was no significant change in hCG secretion (Figure 3A). After 48 h, 10 μM Cd-Forskolin treatment significantly (P < 0.05) increased hCG production (Figure 3B).

Figure 3. hCG production after 24 and 48 h Cd-forskolin cotreatments in BeWo cells. The control lacks both F and CdCl2. F is the positive control. All Cd-forskolin cotreatments include 100 μM F followed by the indicated concentration of CdCl2. hCG concentrations (pg/mL) were determined by an hCGβ ELISA, and were adjusted by protein concentration (μg/mL) determined by the Pierce BCA assay. * indicates significance at p < 0.05 in comparison to F.

hCG Production in Cd-treated JEG-3 Cells. In order to ensure the selected Cd doses were not cytotoxic to JEG-3 cells, a resazurin cytotoxicity assay was conducted. The selected Cd doses (0.044 μM CdCl2, 1 μM CdCl2, and 10 μM CdCl2) were not cytotoxic to JEG-3 cells

(Supplemental Figure 2). To understand the effects of the non-cytotoxic Cd doses on the JEG-3 cells, supernatants from the cells were collected. Furthermore, CdCl2 exposure did not

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CdCl2-16

treated JEG-3 cells showed a significant decrease in hCG production at the highest dose of 10 μM CdCl2 (Figure 4B).

Figure 4. hCG production in JEG-3 after exposure to CdCl2. (A) The hCG concentrations derived from the ELISA were not adjusted to protein concentration. The JEG-3 cells were exposed to CdCl2 for 24 h, and showed a decrease in hCG secretion, although it was not significant. (B) The hCG concentrations (pg/mL) were adjusted to protein concentration (μg/mL). After exposure to CdCl2, there was a significant decrease in hCG secretion at the highest dose (10 μM). ** indicates significance at p < 0.05.

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Figure 5. hCG production after Cd-forskolin cotreatments for 24 and 48 h in JEG-3 cells. The hCG concentrations derived from the hCG ELISA (pg/mL) were adjusted for protein

concentration (μg/mL) determined by the Pierce BCA assay. The control lacks both F and CdCl2. F is the positive control. For each treatment, F concentration was 100 μM. The Cd doses (0.044 μM, 1 μM, and 10 μM CdCl2) varied as indicated.

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Discussion

Cd is a toxic metal that is widely used in consumer and industrial processes and products (Chunhabundit, 2016). Cd exposure is associated with kidney and bone damage, as well as various types of cancer (Godt et al., 2006; Il'yasova & Schwartz, 2005; Maruzeni et al., 2014; Nawrot et al., 2006; Rapisarda et al., 2018). Additionally, prenatal exposure to Cd has been linked with adverse pregnancy outcomes, such as increased risk for miscarriage and

preeclampsia (PMID: 25073434). There is a need to investigate the mechanisms that underly the relationship between prenatal Cd exposure and pregnancy-related outcomes, like preeclampsia. In the current literature, there is a gap in understanding how Cd exposure affects syncytialization, or the process by which mononucleated cytotrophoblasts differentiate into a multinucleated syncytiotrophoblast (Gupta et al., 2016). Syncytialization is a key process in the development of the placenta because it leads to the formation of syncytiotrophoblasts, which are in direct contact with the maternal blood. The syncytiotrophoblast allows for the exchange of nutrients, waste, and gases between the mother and fetus. During syncytialization, hCG hormone production increases to facilitate further placental development, thereby making it an ideal marker to measure (Hiden et al., 2007; Movsas & Muthusamy, 2018). There is evidence to suggest that inadequately formed syncytiotrophoblasts may play a role in the onset of pregnancy diseases, specifically preeclampsia (Huppertz et al., 2013). In this study, we aimed to investigate if non-cytotoxic doses of Cd could modulate syncytialization by measuring changes in hCG secretion.

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cotreatments. When cultured with or without forskolin, BeWo cell lines have the ability to form the syncytiotrophoblast, and produce higher concentrations of hCG, making them an appropriate model to study syncytialization(Bode et al., 2006; Drwal, Rak, & Gregoraszczuk, 2018). The results demonstrated that Cd-forskolin co-exposures in BeWos resulted in a significantly higher secretion of hCG after 48 h in comparison to forskolin alone. This suggests that forskolin and Cd together may have a synergistic effect on the BeWo trophoblasts, or the effects of Cd are

overpowered by the inducing effects of forskolin.

The second takeaway from the study is the effect of Cd on hCG production by JEG-3 cells. Because the JEG-3 cells are able to proliferative, migrate, and have endocrine functions, they can be used as first trimester model (Blanchon et al., 2002; Sokolov et al., 2015; Weiss et al., 2001). If JEG-3 cells are treated with forskolin, they will begin to secrete high concentrations of hCG, but they will not fuse to create the syncytiotrophoblast (Al-Nasiry, Spitz, Hanssens, Luyten, & Pijnenborg, 2006). When JEG-3 cells were exposed to Cd, they produced significantly less hCG at the highest dose (10 μM). In a normative pregnancy, hCG levels exponentially increase and peak during the first trimester, and then begin to decrease to steady state

concentration, where levels remain until the pregnancy reaches full term (Korevaar et al., 2015). Interestingly, deviations in hCG serum levels have a bidirectional association with increased risk for preeclampsia that is dependent upon the gestational age. On the one hand, a epidemiology study found that decreased hyperglycosylated-hCG, an isoform of hCG produced by extravillous cytotrophoblasts early in pregnancy, serum levels during the first trimester was linked to

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Ghosh, 2012; Heinonen, Ryynanen, Kirkinen, & Saarikoski, 1996). Based on the results of this study, JEG-3 and BeWo cells may be mimicking the placental environment at different

gestational stages; therefore, they have opposing reactions to the treatments.

Finally, the results demonstrated that the exposures had a differential response depending on the choriocarcinoma cell line used. Interestingly, Cd-forskolin treatment increased hCG secretion in BeWo cells significantly, but had no significant impact on JEG-3 cells. Conversely, Cd treatment decreased hCG secretion in JEG-3 cells significantly, but had no impact on BeWo cells. These results may be due to cellular differences between the JEG-3 and BeWo cells. JEG-3 cells are a representation of extravillous trophoblasts, while BeWo cells are more closely related to villous trophoblasts (Drwal et al., 2018). Furthermore, JEG-3 and BeWo cells differentially express approximately 2700 genes. In BeWo cells, Syndecan-1 (SDC1), metal regulatory transcription factor 1 (MTF1), Metallothionein 1E (MT1E), Metallothionein 1X (MT1X), Leptin Receptor (LEPR), Glycoprotein hormones alpha peptide (CGA), Chorionic gonadotropin beta polypeptide (CGB), placental growth hormone (GH2), Hydroxysteroid (17-beta)

dehydrogenase-1 (HSD17B1), and placental growth factor (PGF) are all upregulated; many of these genes play a

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(Al-Nasiry et al., 2006). cAMP induction in BeWo cells is associated with decreased cell

proliferation, but is associated with increased proliferation in JEG-3 cells (Al-Nasiry et al., 2006; Taylor, Newman, & Chen, 1991). The differential characteristics of the BeWos and JEG-3 cell lines may explain the varied response to Cd, especially in the presence of forskolin. When forskolin is present, BeWo cells may be progressing further into syncytialization compared to JEG-3 cells, which don’t necessarily follow the same pathway. With the Cd treatments and cotreatments, variation may be coming from differential functional characteristics based on where the cells are in their cell cycle.

While this study suggests that Cd exposure in placental choriocarcinoma cells lines, BeWo and JEG-3, may be impacting placental syncytialization, it is important to consider the limitations of the study. JEG-3 and BeWo cell lines are limited in their representation of the placenta. Because they are homogenous, their response lacks the complex interactions between various cell types present in the placenta and distal organ systems. Moreover, they may not reflect the interaction between the placenta and the fetus. Although hCG is a marker of

syncytialization, this study lacks data on the impact of Cd exposure on gene expression and cell morphology in relation to the development of the multinucleated syncytiotrophoblast due to the occurrence of the COVID-19 pandemic.

Future studies should aim to address the limitations of the investigation we conducted. The development of the syncytiotrophoblast is the essential end outcome of syncytialization. It will be important to explore how Cd exposure may be impacting cell morphology as

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known to play a role in syncytialization should be completed. Several known biomolecules of interest are syncytin-1, syncytin-2, glial cells missing transcription factor 1 (GCM1), human chorionic gonadotropin α-subunit (hCGα), and human chorionic gonadotropin β-subunit (hCGβ) (Azar et al., 2018; Lu et al., 2016; Malhotra, Suman, & Gupta, 2015). Furthermore, hCG

secretion might not be the only factor responsible for cell fusion. CREB phosphorylation and intracellular cyclic adenosine monophosphate (cAMP) concentrations are important mechanisms to consider (Gerbaud & Pidoux, 2015). To better understand how Cd exposure is affecting the hCG production, intracellular cAMP concentrations and the CREB phosphorylation should be measured. Finally, the results of this study should be replicated in animal and placental explant studies.

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Supplemental Figures

40 20 10 5 2.5 1 0.5 0

50 100 150

CdCl2Concentration (M)

% S u rv iv a l

Supplemental Figure 1. BeWo cell % survivability after 48 h of CdCl2 treatment. The red, dashed line indicates 100% survivability. All doses below 40 μM CdCl2 are not cytotoxic.

40 20 10 5 2.5 1.3 0.63 0.31 0

50 100 150

CdCl2 Concentration (M)

% S u rv iv a l 48 Hours 24 Hours

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