Application of Mouse Embryonic Stem Cell Test to Detect Gender

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Application of mouse embryonic stem cell test to detect gender-specific effect of chemicals: a supplementary tool for embryotoxicity prediction Wei Cheng, Ren Zhou, Fan Liang, Hongying Wei, YAN FENG, and Yan Wang Chem. Res. Toxicol., Just Accepted Manuscript • DOI: 10.1021/acs.chemrestox.6b00197 • Publication Date (Web): 21 Jul 2016 Downloaded from http://pubs.acs.org on July 26, 2016

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Chemical Research in Toxicology

Application of mouse embryonic stem cell test to detect genderspecific effect of chemicals: a supplementary tool for embryotoxicity prediction Wei Cheng†, Ren Zhou†, Fan Liang†, Hongying Wei †‡, Yan Feng†, Yan Wang *,†,‡,§ †

College of Public Health, School of Medicine, Shanghai Jiaotong University, Shanghai, P.R.

China, 200025 ‡

Hongqiao International Institute of Medicine, School of Medicine, Shanghai Jiaotong

University, Shanghai, P.R. China, 200336 §

Shanghai Ninth People’s Hospital affiliated to Shanghai Jiaotong University School of

Medicine, Shanghai, P.R. China, 200011 *Corresponding Author: Dr. Yan Wang e-mail: [email protected] Telephone: +86 21 63846590-776353 Fax number: +86 21 63842157 Postal address: Room328, Building No.7, No.500 Quxi Road, Shanghai, China, 200011.

Keywords: Gender-specific effect; Alternative test; Mouse embryonic stem cell test; Embryotoxicity; Molecular endpoints; Hazard identification;

ACS Paragon Plus Environment

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Table of Content Graphic


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Abstract:

Gender effect is an inherent property of chemicals, characterized by variations caused by chemical-biology interaction. It is widely existed, but the shortage of appropriate model restricts the study on gender-specific effect. The embryonic stem cell test (EST) has been utilized as an alternative test for developmental toxicity. Despite its numerous improvements, mouse embryonic stem cells with an XX karyotype have not been used in the EST, which restricts the ability of the EST to identify gender-specific effect during high-throughput-screening (HTS) of chemicals to date. To address this, embryonic stem cell (ESC) SP3 line with an XX karyotype was used to establish a “female” model as a complement to EST. Here we proposed a “doubleobjects in unison” (DOU)-EST, which consisted of male ESC and female ESC; a seven-day EST protocol was utilized, and the gender-specific effect of chemicals was determined and discriminated; the replacement of myosin heavy chain (MHC) with myosin light chain (MLC) provided a suitable molecular biomarker in the DOU-EST. New linear discriminant functions were given in the purpose of distinguishing chemicals into three classes, namely no genderspecific effect, male-susceptive and female-susceptive. For fifteen chemicals in training set, the concordances of prediction result as no gender effect, male susceptive and female susceptive were 86.67%, 86.67% and 93.33% respectively, the sensitivities were 66.67%, 83.33% and 83.33% respectively, and the specificities were 91.67%, 88.89% and 100% respectively; the total accuracy of DOU-EST was 86.67%. For three chemicals in test set, one was incorrectively predicted. The possible reason for misclassification may due to the absence of hormone environment in vitro. Leave-one-out cross validation (LOOCV) indicated a mean error rate of 18.34%. Taken together, these data suggested a good performance of the proposed DOU-EST.


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Emerging chemicals with undiscovered gender-specific effect are anticipated to be screened with the DOU-EST.

Text: Introduction Experimental animals are widely used to assess toxicity by endpoints for hazard identification, and investigation of adverse effects. However, according to the Registration Authorization and Evaluation of Chemicals (REACH) projection, the quantity of experimental animals used is enormous, and the majority of animals are subjected to experiments involving carcinogenicity, mutagenicity and reproduction related toxicity; of those, approximately 64% were sacrificed in order to perform developmental and reproductive toxicity tests (DART).1 In advocating animal welfare, the 3R’s principle (refinement, reduction and replacement) was outlined and alternative tests to DART using in vitro methods were developed, for both economical and ethical reasons.2 Embryonic stem cells (ESC) utilize the advantages of pluripotency, self-renewal ability and tissue-specific differentiation potential, which can mimic organogenesis in vitro, and allows accurate determination of chemically induced adverse effects during embryo development.3 Embryonic stem cell test (EST), which is established based on the cardiac differentiation of ESC, functions as a prediction model for embryotoxicity. Based on the test-set of chemicals, the parameters for embryotoxicity prediction includes 50% inhibition of cellular proliferation (IC50) on 3T3 fibroblasts and ESC, together with 50% inhibition of differentiation (ID50) of ESC into cardiomyocytes.4 Up to date, ESC derived neural cells,5, 6 osteoblasts,7, 8 and vascular endothelial cells,9-11 were developed to predict embryotoxicity following exposure to xenobiotics. In the validated EST,


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microscopy observation of contractility in ESC-derived cardiomyocytes was selected as the endpoint for ID50 calculation.4 In order to improve ID50 determination, utilization of quantitative biological technique provided reliability and convenience, whereby fluorescence activated cell sorting (FACS) was used to obtain ID50 values by employing antibodies against sarcomeric myosin heavy chain protein (MHC); further, a 96 well plate-based ESC assay was developed to achieves the high-throughput screening for EST.12,13 At regulatory level, some in vivo test required a matched number of male and female animals, which was a tiny but ignored point for development of alternative test. Despite various improvements to EST, it is still unable to investigate the gender difference since that no ESC line with XX karyotype was used in mouse EST. It is gradually acceptable that “cells have a sex”. Independent from pharmacokinetics and hormone environment, at cellular level, sex difference exists. Many investigations have proved that cells from females revealed better performance in adaption to environmental stress and metabolic disturbances, finally surviving better than those from male.14, 15 Differences existed in apotosis and autophagy, as well as in gender-related or cell-death-related cellular pathway.16-18 Deeper studies in these fields will provide more information to understand the mechanisms behind the gender-specific effect; currently, at least, the fact that chemicals induced genderspecific effect should be envisaged. As an inherent property of chemical-biology interaction, gender-specific response to stressor in cells should be taken advantage of to monitor the risk of chemicals in blooming. The shortage of appropriate model places restriction on the study of gender-specific effect, therefore, it is of great importance to establish a developmental toxicity prediction system


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applying cells of both genders, which may benefit the screening of chemicals with genderspecific effect. Therefore, a SP3 ESC with XX karyotype was utilized to establish a “female” model to supplement the current EST, in addition to R1 ESC with XY karyotype as a “male” model derived from the same strain. It was hypothesized that in the EST, the female ESC would response equally to the developmental adverse effect induced by chemicals without gender effect, as male ESC did; when male ESC and female ESC were exposed to chemicals with gender-specific susceptibility, variations in response were expected to be observed. To test this hypothesis, a set of fifteen chemicals was selected according to previous studies, which included three chemicals with gender effect reported, six chemicals with male susceptibility and six chemicals with female susceptibility. Following exposure to chemicals in the training set and test set, the IC50 and ID50 values of different chemicals in R1 ESC and SP3 ESC models were determined applying a seven-day protocol of EST; comparison and discrimination were performed accordingly. Materials and methods Culture and differentiation of ESCs ESC line R1 and SP3, both derived from 129 mouse strain, were used in current study between passages 25 and 30. R1 ESC (ATCC:SCRC-1011) was commercially purchased, and SP3 ESC was a gift from Professor Ying Jin from the Key Laboratory of Stem Cell Biology, Shanghai Institutes of Biological Sciences of Chinese Academy of Sciences. Pluripotency of ESCs were maintained by cultivating cells on mitomycin C inactivated feeder layer of C57BL/6 mouse strain derived mouse embryonic fibroblasts. ESC medium consisted of KnockOut Dulbecco’s modified Eagle’s medium (KO DMEM, Gibco, USA), 15% fetal calf serum (Gibco, USA), 0.1


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mM beta-mercaptoethanol (Gibco, USA), 2 mM L-glutamine (Gibco, USA), 0.1 mM nonessential amino acids (Gibco, USA) and 1000 U/ml leukemia inhibitory factor (LIF, Millipore, USA). Then, ESCs were digested into single cells with a 0.25% Trypsin/EDTA, the feeder was discarded, and the LIF were removed. Afterward, 800 cells of R1 ESC and SP3 ESC were cultured in 20 µl hanging drops to generate embryoid bodies in DMEM medium with 20% fetal calf serum (Gibco, USA), namely the cardiac differentiation medium (CDM), one petri dish (Alpha, China) per concentration of each test chemical for 3 days; then, EBs were cultivated in suspension as one petri dish per concentration of each test chemical for 2 days. On day 5, 60 EBs of both R1 and SP3 were plated into each well of 96-well plate (Corning, USA), respectively; 10 wells per concentration of each test chemical. On day 7 of differentiation, EB-derived cardiomyocytes were harvested for further study. In addition, ten-day cultivation was performed to compare the positive beating rate of R1 ESC and SP3 ESC underwent cardiac differentiation. As described previously, on day 5, 30 EBs of R1 ESC and SP3 ESC were plated into each well of 96-well plate (Corning, USA) respectively. Contracting cardiomyocytes were determined microscopically on day 10 (Supporting information 1 and 2), the number of positive beating was expressed as fraction of total 30 EBs, and data were recorded as positive beating rate. Karyotype analysis of ESC Two ESC lines were seeded onto a slider with a total number of 1 x 106, and were incubated with 10 ml fresh medium for 24 h. Colcemid (Sigma, USA) was added to a final concentration of 50 ng/ml for 2 h. The slider was then transferred to a petri dish containing hypotonic solution (0.56% w/v KCl) for 5 min at 37°C. An ice-cold fixative (methanol: acetic acid = 3:1, v/v) was


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dripped onto the slider, then the hypotonic solution was removed and new cold fixative was added for 20 min. The slides were left to air dry. Chromosomes were C-banded, and DAPI staining was performed to confirm the karyotypic characteristics. Alkaline phosphatase (AKP) staining R1 and SP3 ESC were cultured as described above on gelatin-coated culture dishes without induced differentiation, and NIH 3T3 were cultivated as negative control for AKP activity determination. Culture medium was removed and cells were rinsed with phosphate-buffered saline (PBS; pH 7.4) three times. AKP solution was prepared in advance with Tris-HCl (pH 8.2) and was added into culture dishes, after incubation at 37°C in the dark for 20 mins; cells were rinsed with PBS and were with 4% paraformaldehyde for 2 mins according to manufacturer’s instruction. Observation was carried out under a light microscope. DNA extraction and sex-specific gene expression determination R1 and SP3 ESC were cultured as described above on gelatin-coated culture dishes without induced differentiation. Qiagen FlexiGene DNA Kit (Qiangen, Germany) was used for genomic DNA extraction. The PCR amplification procedures were set as followed: 95°C, 5 mins for 1 cycle; 95°C, 15s, 56°C, 30s and 72°C, 30s for 31 cycles; 72°C, 5 mins for 1 cycle and 4°C for preservation. The primers were commercially synthesized by Shanghai Sunny Biotechnology Co., Ltd. Primers and amplification sizes of PCR products were listed in table S1. RNA extraction and quantitative real-time PCR analysis ESC derived cardiomyocytes were harvested with Trizol regent (Life technology, USA) from day 5 to day 10, without chemicals exposure. Total RNA was extracted according to the


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manufacturer’s protocol. The concentration of total RNA was quantified by the absorbance at 260 nm using a Nanodrop spectrophotometer (Thermo, USA). Reverse transcription was performed with Prime Script® RT reagent Kit (Takara, Japan), and the cDNA was prepared for real-time PCR. According to the manufacturer’s protocol, real-time PCR was performed in triplicate in a 10 µl volume system with SYBR® Premix Ex TaqTM II RT-PCR Kit (Takara, Japan) on a ABI 7900HT Fast Real-Time PCR System (Applied Biosystems Inc., USA). Primers and amplification sizes of PCR products were shown in table S1. Relative gene expressions of Myh6 and Myl4 were calculated by the -2△△Ct method against internal reference gene of glyceraldehydes-3-phosphate dehydrogenase (GAPDH). The quantitative PCR amplification procedures were set as followed: 95°C, 30s for 1 cycle; 95°C, 5s and 60°C, 30s for 40 cycles for PCR reaction. Additional 95°C, 15s and 60°C, 1min and 95°C, 15s were set for dissociation curve acquisition. Cytotoxicity assay Alamar blue reagent (Invitrogen, USA) was used to determine cellular viability and the IC50 of each chemical was determined based on this assay. Then 500 cells of R1 and SP3 ESC were seeded in each well of a 96-well tissue culture plate respectively; then cells were exposed to varying concentration of test chemicals (summarized in Table 1), ethanol and PBS were used as solvent for test chemicals. Final concentration of solvent in medium was less than 0.5%.19 Medium with different concentrations of chemicals were changed every second day; on day 7 of cultivation, the cytotoxicity of each chemical was determined using TriStar LB941 Multimode microplate reader (Berthold, Germany). As described previously, culture medium was removed, and cells were rinsed with PBS; alamar blue solution in fresh medium were prepared in advance in fresh medium, then added into each well of 96-well plate.20 After 2 h incubation, alamar blue


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fluorescence was measured according to manufacturer’s instruction. This experiment was preformed three times independently, with five replicates each time. According to EST protocol, IC50 value was calculated. Additionally, the cytotoxicity of test chemicals on NIH 3T3 cells were determined as described above. One chemical in each group was randomly selected and was assigned into test set; the rest chemicals were assigned into training set (table 1). Flow cytomery Cells were cultivated as described above; cells exposed to test chemicals were dissociated on day 7 with 4 U/ml DNase I in 0.25% Trypsin-EDTA, intracellular staining for flow cytometry analysis was performed according to Seiler’s protocol.4 Briefly, cells were fixed with 2% paraformaldehyde in PBS (pH 7.4), permeabilized with 0.2% saponin in PBS; blocking solution consisted of 10% goat serum, 1% BSA and 0.2% saponin was used to block nonspecific reaction. The immunostaining was performed as the cells were incubated at 4°C with 1:400 diluted primary antibody of mouse anti-MHC (Cat#: ab50967, Abcam, USA) or 1:200 diluted primary antibody of mouse anti-myosin light chain (MLC) (Cat#: MA1-24940, Thermo, USA) in blocking solution for 2 h. Then, cells were rinsed with PBS for three times, cells were incubated for 30 min at 4°C with 1:1000 diluted Alexa Fluor® 565 labeled goat anti-mouse IgG (Thermo, USA). The cells were washed three times and re-suspended in 500 µl of PBS. Untreated cells that were lacking of primary antibody were used as control. Flow cytometry analysis was performed on a BD AccuriTM C6 instrument (Becton Dickinson, Germany) using the default software, 15000 events were determined in each plot by using a 585 nm filter in the FL-3


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channel. Differentiation was determined by comparing the fluorescence intensity of chemicaltreated cells against untreated cells obtained from solvent control. According to the FACS-EST protocol,10-20% of the chemical-untreated cells population (gated) should be MHC positive.4 Isotype control group was set to exclude the nonspecific antibody binding. R1 ESC and SP3 ESC were exposed to varying concentrations of test chemicals during spontaneous cardiac differentiation, the proportion of MLC at each dosage of each chemical was determined as described; ID50 values of test chemicals were calculated and recorded. According to the EST protocol, the embryotoxicity prediction of test chemicals were performed in R1 ESC and SP3 ESC, and prediction results were listed in table S2. Statistical analysis The experiment was performed three times independently. Data were shown as the mean ±SE. There were four endpoints obtained in all: IC50 ESC and ID50 for R1 ESC and SP3 ESC respectively. Prior to chemical exposure, positive beating rate over 80% for ESC differentiation were set as the acceptance criteria. As mentioned above, IC50 and ID50 values were calculated accordingly.4 One-way ANOVA was performed to compare the difference of IC50 and ID50 values between R1 ESC and SP3 ESC. In order to analyze the variances between endpoints of R1 ESC and SP3 ESC, namely the “gender effect of chemicals” being proposed in current study, general linear model (GLM) was applied. To fit the normal distribution, IC50 and ID50 values were logarithmic-transferred and were set as dependent variables. Test chemicals and cells were set as term factor, in combination with their interactions (test chemicals × cells); if the effect of test chemicals on the IC50 or ID50 values affected by cells, interaction was considered. The contribution of the term factors was


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expressed by significance values; a significance p value smaller than 0.05 were considered to be significant. In order to calculate the practical significance of each term, partial eta squared statistics were executed. Larger values of partial eta squared suggested greater variation of the effect divided by the sum of variations of the effects and the distance of variations to error; the maximum variation was 1. Meanwhile, pairwise scatter plots were drafted and the determination coefficients (R2) were calculated to reveal the correlations between endpoints values. An R2 value of over 0.8 indicated a well-correlation. SPSS software version 19 for Windows (SPSS Inc., Chicago, USA) was used for statistical analysis. As illustrated in previous study, the prediction model (PM) of EST was established to distinguish the three classes of embryotoxicity in vitro.21 Fisher’s classification functions allowed the assignment of chemical into one of the three embryotoxicity classes.4, 22 Similarly, in current study, in addition to the embryotoxicity, there were three classes of test chemicals: chemicals with no-gender effect, with male susceptibility and with female susceptibility. In order to testify if chemicals with different gender susceptibility could be classified, two canonical discriminant functions were applied to visualized test chemicals in a canonical plot. The coefficients of the two canonical discriminant functions were calculated in the R statistical computing environment (R Development Core Team, 2009) with lda function of the MASS package.23 The classification results of the prediction models were validated by self recognition of the model; the robustness of the two canonical discriminant functions was verified with leave-one-out-cross-validation (LOOCV). The decision boundaries in the canonical plot were and drafted as described previously.23 Finally, the proposed PM was re-expressed in terms of three Fisher’s classification functions for further application.


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Result Pluripotency identification of R1 ESC and SP3 ESC R1 ESC and SP3 ESC were cultivated on mitomycin C-inactivated feeder layer of C57BL/6 mouse strain derived mouse embryonic fibroblasts. Followed by a 48-hour culture, morphology observation was carried out by light microscopy. As shown in Fig.1A and B, small island-like clones with higher diopter than feeder layer represented good proliferation status of R1 ESC and SP3 ESC respectively. The normal karyotypic rate of both ESCs in current study were exceeding 50% (Fig.1C and D) In Fig.1E and F, clones of R1 ESC and SP3 ESC in dark blue revealed positive activity of AKP; whereas 3T3 fibroblasts as the negative control had no colored staining (Fig.1G). Clones with positive staining of Nanog and Ssea-1 were observed in R1 ESC and SP3 ESC respectively, as shown in Fig.1H and I; whereas NIH 3T3 cells were set as negative control, and negative staining of Nanog and Ssea-1 were seen in Fig.1J. The expression of pluripotent markers Oct4 and Ssea-1 in R1 ESC, SP3 ESC and NIH 3T3 fibroblasts were determined. As displayed in Fig.1K, Ssea-1 was highly expressed in R1 ESC and SP3 ESC respectively, together with high expression of Oct4; whereas 3T3 cells as the negative control had no expression of both markers. X chromosome specific gene Zfx and Y chromosome specific gene Sry were selected to identify the gender of each ESC. As demonstrated in Fig.1L, R1 ESC was determined to be a “male”, while SP3 ESC turned out to be a “female”. Time-sequenced expressions of cardiac differentiation-related genes


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Time-sequenced expressions of cardiac differentiation-related genes were determined from day 5 on. Differentiated cells were harvested on each day in order to compare endpoints proposed for ID50 determination. As shown in Fig.2A, the expressions of Myh6 and Myl4 in both ESC lines were drastically increased with time. However, significant difference in the expression of Myh6 between R1 ESC and SP3 ESC were observed on each day, with exception of that on day 8. In contrast, significant difference in the expression of Myl4 between R1 ESC and SP3 ESC was observed on day 10 only. the positive beating rate acquired by microscopy counts of the 30 EBs revealed similar contractile ability in these two ESC-derived cardiomyocytes on day 10 (Fig.2B), which suggested that the SP3 ESC model were qualified for chemicals testing, as R1 ESC did. Quantification of cardiac marker protein expression determined intracellularly by flow cytometry was performed on day 7. Control was analyzed and gate was set accordingly (Fig.2C, right panel). As shown in Fig.2C, 20.6% R1 ESC-derived cardiomyocytes was positive-stained with MLC, and 20.1% SP3 ESC-derived cardiomyocytes was positive-stained with MLC on day 7 (left panel). In terms of MHC, 19.5% R1 ESC-derived cardiomyocytes was positive-stained, however only 13.7% was positive-stained on day 7 (middle panel). The expression of MHC in both R1 ESC and SP3 ESC were between 10-20%, which matched the criteria indicated in the FACS-EST protocol. However, the proportion of MHC-positive cells varied significantly between R1 ESC and SP3 ESC. In contrast, the proportion of MLC-positive cells in both R1 ESC and SP3 ESC were close to each other, which were about 20%. The data suggested that MHC may not be an ideal biomarker for ID50 values determination, since the heterogeneity may incorrectly reflect effects that chemicals with different gender effects produced in both R1 ESC and SP3 ESC; instead, MLC was proposed to replace the MHC as biomarker for ID50 values determination.


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Concentration-dependent responses in cell proliferation and cardiac differentiation The dose-response curves of test chemicals represented their cytotoxicities on undifferentiated R1 ESC and SP3 ESC (IC50), as well as their inhibitions on cardiomyocyte differentiation (ID50) respectively, as shown in Fig.3 to Fig.5; IC50 and ID50 values were listed in table 2 and table 3. In addition, the IC50 data acquired in NIH 3T3 cells were represented in table S2. For chemicals without gender effect, significant differences between IC50 SP3 ESC and IC50 R1 ESC were observed in FU. For chemicals with male susceptibility, significant differences were observed in all chemicals of training set; the IC50 SP3 ESC values of these mentioned chemicals were larger than those of IC50 R1 ESC values. For chemicals with female susceptibility, significant differences were observed in BPA, CF, OB and DH, the IC50 R1 ESC values of CF and OB were larger than their values obtained in SP3 ESC model, while the IC50 SP3 ESC values of BPA and DH were larger than their values obtained in R1 ESC model. No significant difference was observed in IC50 values of both R1 ESC and SP3 ESC in other chemicals (table 2 and Fig.6A). In terms of ID50 values, no significant difference between R1 ESC and SP3 ESC was observed among chemicals without gender effect. However, significant differences between R1 ESC and SP3 ESC were observed in all chemicals with male susceptibility and with female susceptibility. The ID50 values acquired in R1 ESC of chemicals with male susceptibility were all smaller than that in SP3 ESC; in contrast, the ID50 values acquired in SP3 ESC with female susceptibility were all smaller than that in R1 ESC (table 3 and Fig.6B). Meanwhile, the concentration-dependent reductions in cellular viability and differentiation potential of chemicals in training set could indicate the differences between IC50 and ID50 values


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of R1 ESC and SP3 ESC as well (Fig.3 to Fig.5). In addition, dose-response curves of chemicals in test set were shown in Fig.8. Embryotoxicity of test chemicals were predicted according to EST protocol. As listed in table S2, prediction results of most or chemicals were coincident in male EST and female EST. However, different prediction results occurred in VZ and DX; in female EST, they were lower evaluated, as compared to that in male EST. Correlation analysis between endpoints of R1 ESC and SP3 ESC The pairwise scatter plots between IC50 and ID50 values of R1 ESC and SP3 ESC were drawn to represent the inter-gender deviation. Regular scatter plots were made to visualize the correlation between the endpoints, where x axis represented IC50 and ID50 values of SP3 ESC, and y axis represented IC50 and ID50 values of R1 ESC respectively (Fig.6). A strong correlation between two endpoints would result in a systematic pattern, with R2 values of 0.843 and 0.955 for the endpoints of IC50 and ID50. Several plots diverged from the straight line, higher points represented a greater tolerance when cells exposed to test chemicals, and vice versa for lower points. Variability evaluation between IC50 and ID50 values of R1 ESC and SP3 ESC GLM univariate analysis was applied to evaluate the difference between IC50 and ID50 values of R1 ESC and SP3 ESC. As shown in table 4, due to the variety of gender susceptibility of these test chemicals, in addition to their different teratogenic potential, the test chemicals would have a strong impact on both IC50 and ID50; the partial eta squared values of IC50 and ID50 were 0.997 and 0.996 respectively, which indicated that the two endpoints were able to characterize the


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variations of test chemicals. Significant difference between R1 ESC and SP3 ESC was observed based on IC50 and ID50 values, where the partial eta squared values were 0.636 and 0.405 respectively, which outlined the difference between the two ESC lines. A significant difference in the interaction of chemical and cells was observed based on IC50 and ID50 values, where the partial eta squared values were 0.807 and 0.910 respectively. Linear discriminant analysis and prediction model The PM expressed in the form of the Fisher’s functions in EST was used to classify chemicals into three classes of embryotoxicity. Application of the EST PM to current data resulted in different prediction results, as summarized in table S2. In order to classify test chemicals into different gender susceptibility, two canonical discriminant functions LD1 and LD2 were established. According to the result of linear discriminant analysis, functions LD1 and LD2 were expressed as followed: LD1= 2.374 × log10IC50 R1 – 1.864 × log10ID50 SP3 + 1.044 × (IC50 R1 ÷ IC50 SP3) + 0.788 × (ID50 R1 ÷ ID50 SP3) LD2= -0.706 × log10IC50 R1 + 1.067 × log10ID50 SP3 + 0.759 × (IC50 R1 ÷ IC50 SP3) - 0.199 × (ID50 R1 ÷ ID50 SP3) By utilizing LD1 and LD2, chemicals were divided into three groups: no gender effect, male susceptibility and female susceptibility; the decision boundaries were drawn to discriminate the three groups (Fig.7). The Wilks’ Lambda values were 0.124 and 0.699 for LD1 through LD2 and LD2, with significance p values of II and I > III, the prediction result would be no gender-specific; if II > I and II > III, the prediction result would be male-susceptive; if III > I and III > II, the prediction result would be female-susceptive. Accordingly, chemicals in training set were predicted (Table 5), and the accuracy of prediction result was calculated (Table 6), which suggested a good performance of DOU-EST. Regarding chemicals in test set, PG and IN were classified as no gender-specific, and BPS was classified as female-susceptive.


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Discussion The EST was developed as an alternative test for developmental toxicity in vivo, in order to reduce the use of animals by providing creditable judgment on the teratogenic potential of xenobiotics. According to the classification criteria, based on the IC50 and ID50 values, chemicals are categorized as non-embryotoxic, weak embrotoxic, and strong embryotoxic.4,

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Gender

differences during embryogenesis were less compared in the perinatal period. Many epidemiological investigations have indicated there are differences between male and female embryos, as well as between human and rodent embryos during embryogenesis.24, 25 However, female model has not yet been developed in EST, which may restrict discovery of genderspecific effects in chemicals. In order to resolve this, we utilized the SP3 ESC with a karyotype of XX to establish a model for teratogenicity prediction in vitro, as well as to supplement upon the currently validated EST method. To ensure that SP3 ESC is able to undergo developmental toxicity screening, karyotypic analysis and pluripotency examination were performed, compared to R1 ESC. As represented in Fig.1, the positive staining of AKP activity, pluripotent markers Nanog and Ssea-1, and the high expression of pluripotent markers Oct4 and Ssea-1 indicate the high level maintenance of pluripotency in both R1 ESC and SP3 ESC. The karyological analysis results of R1 ESC and SP3 ESC in current study showed a more than 50% normal karyotype in both ESC cells, which were similar to what had been reported.26, 27 By detecting the sex chromosome specific genes Zfx and Sry, R1 ESC was determined to be XY, while SP3 ESC was found to be XX, representing the “male” and “female” respectively. Spontaneous cardiac differentiation could be monitored by contractile observation microscopically on day 10, the positive beating rates between R1 ESC and SP3 ESC were similar (Fig.2B). Even though the mRNA level of Myh6 and Myl4, which


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encodes MHC and embryo-specific MLC respectively, were different significantly on day 10, it didn’t affect the phenotype of contractile in ESC-derived cardiomyocytes. Myosin is a crucial structural protein in cardiac muscle, whereby both MHC and MLC proteins are required for heart morphogenesis.28 Myh6, which encodes α-MHC, and, is extensively homologous across species, and is predominantly expressed in the atrial region during embryonic cardiogenesis, which extends towards the ventricular chambers following birth; it relates to shortening velocity, energy consumption and oxygen consumption in heart, and therefore is selected as molecular endpoint in EST instead of contractile observation.29 In current study, however, the different expression of Myh6 in R1 ESC- and SP3 ESC-derived cardiomyocytes on day 7 was significant (Fig.2A and 2C). This heterogeneity, in a way of producing inconsistent starting line, may incorrectly reflect the effects that chemicals with different gender effects induced on both ESC lines. Alternatively, based on the contractile property of ESC-derived cardiomyocytes, embryo-specific MLC was proposed as candidate molecular endpoints. It functions as maintaining the integrity of myosin and to modulate force generation by decreasing myosin neck region compliance and promoting strong cross-bridge formation by enhancing myosin attachment to actin, which helps to accelerate shortening velocity and isometric tension production.30 Abnormal expression of embryo-specific MLC (encoded by Myl4) has been identified to associate with congenital heart disease (CHD) and cardiomyopathies, such as Tetralogy of Fallot, hypertrophy of the left ventricle and heart failure.28, 31-34 On day 7, the expression of MLC in SP3 ESC was similar to that in R1 ESC (Fig.2A and 2C), suggesting the feasibility of applying MLC as molecular biomarker to detect potential gender effect of chemicals.


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Gender-specific effect is an inherent property of chemicals, characterized by variations caused by chemical-biology interaction. Generally, differences in pharmacokinetics and hormone environment account for the gender-specific effect of drug clinically and in vivo; however, both of the two factors are hardly mimicked in vitro. Interestedly, despite of these differences, behind the phenotype of contractile of cardiomyocytes, variations in disruption of endogenous hormone, sensitivity of ion channels and alteration in cellular Ca2+ handling following xenobiotics exposure in cellular level make the discrimination of chemicals induced gender-specific effect possible in male and female cells.35, 36 Accordingly, chemicals in training set with different gender susceptibility were selected. GE, RA, OT, VZ, DX, BPA, LC and CF disrupt endogenous hormones, such as sexual hormones or thyroid hormones, which are required for cardiac contractile; these endogenous hormones may be secreted during the spontaneous cardiac differentiation. BPA and OB produced different alteration patterns of Ca2+ controlling in female, compared with in male. Difference in sensitivity of Na+-K+-ATPase resulted in different responses to GE and DG in male and female. Large amount of studies indicated that, compared to male, female tolerated better to AS, and thereby is subject to aspirin resistance, although the conclusion is controversial and the mechanism has not been fully elucidated.37-39 Even though no systemic study on female susceptibility has been performed, a great number of clinical cases revealed a higher incidence of some adverse drug reactions in female cardiovascular system, which provided an evidence of potential female susceptibility. When R1 ESC and SP3 ESC exposed to chemicals in the training set during cardiac differentiation, difference in a hypothetical distance between IC50 and ID50 was expected to be recognized to identify the gender effect in chemicals with gender susceptibility, i.e. |IC50-


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ID50|R1≠ |IC50-ID50|SP3; in contrast, when cells exposed to chemicals without gender susceptibility, it turned out to be |IC50-ID50|R1= |IC50-ID50|SP3. Preliminary hint was acquired in table 4, where significant differences were observed in biological response to chemicals in “male” and “female” ESC models, as well as in interaction between chemicals and cells. With the assistance of three Fisher’s linear discriminant functions, three values of one chemical were obtained respectively, which reflected different probabilities of the group the chemical belonged to. Take RA as example, the values of three functions were -11.475, -2.772 and -31.307 respectively, represented the probabilities of RA subordinated to no gender effect, male-susceptive and female-susceptive; among them, the second value was larger than the first value and the third value, indicating a higher probability of RA was male-susceptive, rather than female-susceptive or no gender-specific. Generally, the classical EST was established focused on great differences in ID50 values of chemicals against the IC50 values acquired in ESC and 3T3 fibroblasts. Unlike the teratogenicity potential of chemicals, the difference in gender-specific effect was much smaller than the teratogenicity potential in real world; however, by utilizing discriminant function, successfully, the gender-specific effect of chemicals could be predicted by DOU-EST, with an overall concordance greater than 85% (Table 6). Still, in training set, FU and DG were incorrectively predicted, as well as IN in the test set. Doubtfully, few study indicated that FU, a widely used drug for treating cancers, maybe more toxic in female patients with metastatic colorectal cancer only, but biological mechanism has not been elucidated up to date.40, 41 And in current study, FU was predicted as strong-embryotoxicity but male-susceptive, due to a better tolerance in SP3 ESC, as revealed by a significant higher IC50 value. DG was a cardiotonic steroid, since it could inhibit the Na+-K+-ATPase and execute


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cardiotonic and vasotonic effect via controlling the Na+/Ca2+ exchange; in this process, estrogen played a crucial role; so do OB.42 However, an in vitro system being lack of the hormonal environment was restricted to reproduce this biological function. Unlike OB, the distance of IC50 and ID50 values between R1 and SP3 may be not enough to be recognized by the PM of DOUEST. In terms of IN, it functioned as a prostaglandin inhibitor, resulting in the de-activation of MLC kinase and muscular contraction through decreasing intracellular calcium influx and its release from the sarcoplasmic reticulum in vivo.43 Similar to the case of DG, being short of prostaglandin in vitro caused misclassification of IN, even thought significant difference in IC50 and ID50 values of R1 and SP3 ESC were observed. Despite of the drawbacks, enlargement in number of chemicals for testing will help to improve the DOU-EST model in the future. An emerging concept of developmental origins of health and disease (DOHaD) emphasize in the early-life exposure and connection to later-life disease; as one of risk factors, environmental chemicals exposure contribute to the different susceptibility, incidence and severity to some diseases with remarkable gender-specific effect.44, 45 The DOU-EST will benefit the screening and warning of chemicals with gender-specific effect, in addition to alert of their embryotoxicity. Also, disease-related changes in MLC expression induced by xenobiotics exposure can be monitored, which may further contribute to understand the mechanism of DOHaD partially.46,47 In conclusion, in current study, we established the first “female” EST; we proposed a so-called DOU-EST consisted of male ESC and female ESC; a seven-day EST protocol was utilized, and the gender-specific effect of chemicals was determined and discriminated; the replacement of MHC with MLC provided a suitable molecular biomarker in the DOU-EST. New linear


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discriminant functions were given in the purpose of distinguishing chemicals into three classes, namely no gender-specific effect, male-susceptive and female-susceptive. The linear discriminant functions were acceptable to predict the gender susceptibility; further studies are expected to perform inter-laboratory tests by using more chemicals to improve the DOU-EST. Emerging chemicals with undiscovered gender-specific effect are anticipated to be screened and predicted.

Conflict of interest The authors declare that there are no conflicts of interest.

Funding Sources This work was supported by the National Natural Science Foundation of China (21577091) and Shanghai Science & Technology Development Foundation (12140901000). Acknowledgement Many appreciations to Professor Ying Jin from the Key Laboratory of Stem Cell Biology, Shanghai Institutes of Biological Sciences of Chinese Academy of Sciences, for SP3 ESC line supporting. Supporting Information Videos of contractile ESC-derived cardiomyocytes; primer sequence; table of IC50 values of chemicals in NIH3T3 cells for embryotoxicity prediction. This material is available free of charge via the Internet at http://pubs.acs.org.


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Abbreviations Abbr. AKP AN AS BPA BPS CDM CF CHD DART DG DH DOHaD DOU-EST DX ESC EST FACS FU GAPDH GE GLM HTS HU IN LC LIF LOOCV MHC MLC Myh6 Myl4 OB OT PG PM RA REACH Sry VZ Zfx

Full term Alkaline phosphatase 6-aminonicotinamide Aspirin Bisphenol A Bisphenol S cardiac differentiation medium Caffein congenital heart disease developmental and reproductive toxicity tests Digoxin Dihenhydramine developmental origins of health and disease “double-objects in unison”-embryonic stem cell test Dexamethasone embryonic stem cell embryonic stem cell test fluorescence activated cell sorting 5-Fluorouracil Glyceraldehydes-3-phosphate dehydrogenase Genistein General linear model High-throughput-screening Hydroxyurea Indomethacin Lithium chloride Leukemia inhibitory factor Leave-one-out cross validation Myosin heavy chain Myosin light chain Myosin heavy chain 6 Myosin light chain 4 Ouabain Ochratoxin A Penicillin G Prediction model Retinoic acid Registration Authorization and Evaluation of Chemicals Sex determining region of Chr Y Vinclozolin Zinc finger protein, X-linked


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Ochratoxin A: developmental and reproductive toxicity-an overview. Birth Defects Res. B Dev. Reprod. Toxicol. 98, 493-502. (62) Ueta, E., Kodama, M., Sumino, Y., Kurome, M., Ohta, K., Katagiri, R., and Naruse, I. (2010) Gender-dependent differences in the incidence of ochratoxin Ainduced neural tube defects in the Pdn/Pdn mouse. Congenital Anomalies 50, 29-39. (63) Bisenius, E. S., Veeramachaneni, D. N., Sammonds, G. E., and Tobet, S. (2006) Sex differences and the development of the rabbit brain: effects of vinclozolin. Biol. Reprod. 75, 469-476. (64) Walker, D. M., and Gore, A. C. (2011) Transgenerational neuroendocrine disruption of reproduction. Nature reviews. Endocrinology 7, 197-207. (65) Rivkees, S. A., and Stephenson, K. (2009) Low-dose dexamethasone therapy from infancy of virilizing congenital adrenal hyperplasia. Int. J. Pediatr. Endocrinol. 2009, 274682. (66) Rivkees, S. A., and Crawford, J. D. (2000) Dexamethasone treatment of virilizing congenital adrenal hyperplasia: the ability to achieve normal growth. Pediatrics 106, 767-773. (67) Seltenrich, N. (2015) Comparing BPS and BPA: cardiovascular effects in female rat hearts. Environ. Health Perspect. 123, A157. (68) Posnack, N. G., Brooks, D., Chandra, A., Jaimes, R., Sarvazyan, N., and Kay, M. (2015) Physiological response of cardiac tissue to bisphenol A: alterations in ventricular pressure and contractility. Am. J. Physiol.: Heart Circ. Physiol. 309, H267275. (69) Gao, X., Ma, J., Chen, Y., and Wang, H. S. (2015) Rapid responses and mechanism of action for low-dose bisphenol S on ex vivo rat hearts and isolated myocytes: evidence of female-specific proarrhythmic effects. Environ. Health Perspect. 123, 571-578. (70) Baptista, T., Murzi, E., Hernandez, L., Burguera, J. L., and Burguera, M. (1991) Mechanism of the sex-dependent effect of lithium on body weight in rats. Pharmacol., Biochem. Behav. 38, 533-537. (71) Allagui, M. S., Hfaiedh, N., Vincent, C., Guermazi, F., Murat, J. C., Croute, F., and El Feki, A. (2006) Changes in growth rate and thyroid- and sex-hormones blood levels in rats under sub-chronic lithium treatment. Hum. Exp. Toxicol. 25, 243-250. (72) Luo, H., Deng, Z., Liu, L., Shen, L., Kou, H., He, Z., Ping, J., Xu, D., Ma, L., Chen, L., and Wang, H. (2014) Prenatal caffeine ingestion induces transgenerational neuroendocrine metabolic programming alteration in second generation rats. Toxicol. Appl. Pharmacol. 274, 383-392. (73) Li, J., Luo, H., Wu, Y., He, Z., Zhang, L., Guo, Y., Ma, L., Magdalou, J., Chen, L., and Wang, H. (2015) Gender-specific increase in susceptibility to metabolic syndrome of offspring rats after prenatal caffeine exposure with post-weaning high-fat diet. Toxicol. Appl. Pharmacol. 284, 345-353. (74) Palacios, J., Marusic, E. T., Lopez, N. C., Gonzalez, M., and Michea, L. (2004) Estradiol-induced expression of N(+)-K(+)-ATPase catalytic isoforms in rat arteries: gender differences in activity mediated by nitric oxide donors. Am. J. Physiol.: Heart Circ. Physiol. 286, H1793-1800. (75) Tsai, W. C., Chen, Y. C., Kao, Y. H., Lu, Y. Y., Chen, S. A., and Chen, Y. J. (2013) Distinctive sodium and calcium regulation associated with sex differences in atrial


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electrophysiology of rabbits. Int. J. Cardiol. 168, 4658-4666. (76) Rathore, S. S., Wang, Y., and Krumholz, H. M. (2002) Sex-based differences in the effect of digoxin for the treatment of heart failure. N. Engl. J. Med. 347, 1403-1411. (77) Alemanni, M., Rocchetti, M., Re, D., and Zaza, A. (2011) Role and mechanism of subcellular Ca2+ distribution in the action of two inotropic agents with different toxicity. J. Mol. Cell. Cardiol. 50, 910-918. (78) Sype, J. W., and Khan, I. A. (2005) Prolonged QT interval with markedly abnormal ventricular repolarization in diphenhydramine overdose. Int. J. Cardiol. 99, 333-335. (79) Ramachandran, K., and Sirop, P. (2008) Rare complications of diphenhydramine toxicity. Conn. Med. 72, 79-82.


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Table 1 Summary of test chemicals Mechanism of action

Species

Group

Ref.

-

disruption in mitosis

H, R, D

Training

48, 49

No

-

disruption in DNA Synthesis

H, M

Training

50, 51

329-89-5

No

-

disruption in glucose metabolism

H, R, M

Training

48, 52, 53

PG

69-57-8

No

-

antibiotics

H, M

Test

54

GE

446-72-0

Yes

male > female

disruption in endogenous hormone; difference in sensitivity of Na+-K+-ATPase

M, G

Training

55, 56

R

Training

57, 58

disruption in hormone/neuroendocrine

H, M

Training

38, 59

H

Test

60

H, M

Training

61, 62

H, M, Rb

Training

63, 64

H, M

Training

65, 66

R

Training

67, 68

R

Test

69

R

Training

70, 71

R

Training

72, 73

R

Training

74, 75

M, R, Rb

Training

Test chemical

Abbr.

Hydroxyurea

HU

127-07-1

No

5-Fluorouracil

FU

51-21-8

6-aminonicotinamide

AN

Penicillin G Genistein

all-trans-retinoic acid

RA

CAS

Gender-specific effect

302-79-4

Yes

Susceptibility

male > female

Aspirin

AS

50-78-2

Yes

male > female

lower tolerence of cytotoxicity in male

Indomethacin

IN

53-86-1

Yes

male > female

disruption in endogenous hormone; alteration in myocyte Ca2+ handling

Ochratoxin A

OT

303-47-9

Yes

male > female

Vinclozolin

VZ

50471-44-8

Yes

male > female

Dexamethasone

DX

50-02-2

Yes

male > female

Bisphenol A

BPA

80-05-7

Yes

female > male

Bisphenol S

BPS

80-09-1

Yes

female > male

Lithium chloride

LC

7447-41-8

Yes

female > male

Caffein

CF

58-08-2

Yes

female > male

Ouabain

OB

630-60-4

Yes

female > male

disruption in endogenous hormone

disruption in endogenous hormone disruption in endogenous hormone

disruption in endogenous hormone; alteration in myocyte Ca2+ handling disruption in endogenous hormone; alteration in myocyte Ca2+ handling disruption in endogenous hormone; alteration in myocyte Ca2+ handling disruption in endogenous hormone disruption in endogenous hormone

Digoxin

DG

20830-75-5

Yes

female > male

alteration in myocyte Ca2+ handling

76, 77


32

Page 33 of 48 Dihenhydramine

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48

Chemical Research in Toxicology DH

147-24-0

Yes

female > male difference in sensitivity of Na+-K+-ATPase; difference in histamine H1 receptor agonism

H

Training

78, 79

Note: H, human; M, mouse; R, rat; Rb, rabbit; G, guinea pig; D, dog.


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Page 34 of 48

Table 2 IC50 values of chemicals Test chemical Susceptibility

no gender effect

male > female

female > male

IC50 R1 ESC (µg/ml) mean

SE

IC50 SP3 ESC (µg/ml) mean

SE

HU

1.785

0.757

1.542

0.479

FU

0.228

0.016

0.370

0.096

AN

0.018

0.004

0.019

0.003

PG

1019.055

14.126

1024.766

17.023

GE

4.981

0.024

10.502

0.133

**

RA

0.007

0.001

0.011

0.002

**

AS

251.021

18.891

475.999

14.071

**

IN

86.550

4.774

133.852

9.064

**

OT

12.838

0.061

23.085

1.645

**

VZ

1.315

0.032

2.355

0.063

**

DX

45.129

7.375

185.046

9.126

**

BPA

21.350

1.779

25.020

1.374

**

BPS

26.171

2.890

24.166

1.974

LC

26.963

1.967

25.890

1.556

CF

185.800

8.197

170.900

4.969

**

OB

0.227

0.050

0.126

0.039

**

DG

0.407

0.039

0.375

0.103

DH

24.949

1.896

40.055

2.936

**

**

* p < 0.05, compared to R1 ESC; ** p < 0.01, compared to R1 ESC.

Table 3 ID50 values of chemicals Test chemical Susceptibility

no gender effect

ID50 R1 ESC (µg/ml) mean

SE

ID50 SP3 ESC (µg/ml) mean

SE

HU

1.139

0.071

1.187

0.121

FU

0.742

0.070

0.748

0.084

AN

0.003

0.0004

0.003

0.0006

PG

1027.055

33.551

1016.729

29.751


34

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male > female

female > male

GE

1.451

0.399

1.859

0.257

**

RA

0.001

0.0003

0.066

0.028

**

AS

97.949

4.729

340.786

11.304

**

IN

2.182

0.559

18.537

2.139

**

OT

10.065

1.588

18.260

1.617

**

VZ

0.381

0.078

0.627

0.081

**

DX

19.016

1.443

81.626

4.725

**

BPA

9.708

0.047

5.762

0.032

**

BPS

12.931

1.589

9.407

0.110

**

LC

5.075

0.037

4.108

0.044

**

CF

179.500

8.386

128.412

9.081

**

OB

1.241

0.104

0.103

0.093

**

DG

0.752

0.239

0.486

0.105

**

DH

10.741

2.092

1.092

0.155

**

* p < 0.05, compared to R1 ESC; ** p < 0.01, compared to R1 ESC.

Table 4 GLM univariate procedure result of chemicals in training set Endpoint IC50

Factor Chemical

F

Significance p value

Partial Eta Squared

14

1458.938

< 0.0001

***

0.997

1

104.853

< 0.0001

***

0.636

Chemical * Cell

14

17.871

< 0.0001

***

0.807

Error

60

-

Chemical

14

987.819

< 0.0001

***

0.996

1

40.867

< 0.0001

***

0.405

Chemical * Cell

14

43.444

< 0.0001

***

0.910

Error

60

Cell

ID50

df

Cell

-

-

-

-

-

* p

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