Environmental Pollution
Exposure to tris(1,3-dichloro-2-propyl) phosphate (TDCPP) induces vascular toxicity through Nrf2-VEGF pathway in zebrafish and human umbilical vascular endothelial cells
Xiali Zhong, Jiahuang Qiu, Jianmeng Kang, Xiumei Xing, Xiongjie Shi, Yanhong Wei
To appear in: Environmental Pollution
TDCPP exerts its vascular toxicity by suppressing Nrf2 and the downstream VEGF pathway in vascular endothelial cells, which accounts for the impaired angiogenesis in vascular development.
Exposure to tris(1,3-dichloro-2-propyl) phosphate (TDCPP) induces vascular toxicity through Nrf2-VEGF pathway in zebrafish and human umbilical vascular endothelial cells
Xiali Zhong a, Jiahuang Qiu a, Jianmeng Kang a, Xiumei Xing a, Xiongjie Shi b,
Yanhong Wei a Guangdong Provincial Key Laboratory of Food, Nutrition and Health, Department of Toxicology, School of Public Health, Sun Yat-sen University, Guangzhou 510080,
China
b Hubei Key Laboratory of Cell Homeostasis, College of Life Sciences, the Institute for Advanced Studies, Wuhan University, Wuhan 430072, China
* Corresponding author
Mailing address: Department of Toxicology, School of Public Health, Sun Yat-sen University, No.74 Zhongshan Rd. 2, Guangzhou 510080, China.
Phone: +86 20 87331122
E-mail: [email protected]
1 Abstract
2 The growing production and extensive use of organophosphate flame retardants
3 (OPFRs) have led to an increase in their environmental distribution and human
4 exposure. Developmental toxicity is a major concern of OPFRs’ adverse health effects.
5 However, the impact of OPFRs exposure on vascular development and the toxicity
6 pathway for developmental defects are poorly understood. In this study, we
7 investigated the effects of exposure to tris(1,3-dichloro-2-propyl) phosphate (TDCPP),
8 a frequently detected OPFR, on early vascular development, and the possible role of
9 nuclear factor erythroid 2-related factor (Nrf2)-dependent angiogenic pathway in
10 TDCPP’s vascular toxicity. TDCPP exposure at 300 and 500 µg/L impeded the growth
11 of intersegmental vessels (ISV), a type of microvessels, as early as 30 hpf.
12 Consistently, a similar pattern of decreased extension and remodeling of common
13 cardinal vein (CCV), a typical macrovessel, was observed in zebrafish at 48 hpf and
14 72 hpf. Developing vasculature in zebrafish was more sensitive than general
15 developmental parameters to TDCPP exposure. The expression of genes related to
16 VEGF signaling pathway dose-dependently decreased in TDCPP-treated larvae. In in
17 vitro experiments using human umbilical vascular endothelial cells (HUVECs), the
18 increased cell proliferation induced by VEGF was suppressed by TDCPP exposure in
19 a dose-dependent fashion. In addition, we found a repression of Nrf2 expression and
20 activity in TDCPP-treated larvae and HUVECs. Strikingly, the application of
21 CDDO-Im, a potent Nrf2 activator, enhanced VEGF and protected against defective
22 vascular development in zebrafish. Our results reveal that vascular impairment is a
23 sensitive index for early exposure to TDCPP, which could be considered in the
24 environmental risk assessment of OPFRs. The identification of Nrf2-mediating VEGF
25 pathway provides new insight into the adverse outcome pathway (AOP) of OPFRs.
26
27 Capsule of main finding: TDCPP induces vascular toxicity via the suppression of
28 Nrf2-dependent VEGF pathway
29
30 Keywords: Tris(1,3-dichloro-2-propyl) phosphate (TDCPP); Developmental toxicity;
31 Vascular toxicity; Vascular endothelial growth factor (VEGF); Nuclear factor
32 erythroid 2-related factor (Nrf2); Adverse outcome pathway
33
34 Introduction
35 Flame retardants (FRs) are manufactured chemicals that are applied to various
36 materials to prevent fire or diminish the chance of ignition. Over the past several
37 decades, FRs have been widely used in a wide range of commercial products,
38 including electronics, vehicles, furniture, upholstery, textiles, and toys (Cooper et al.,
39 2016). Most are additive to the base materials (without chemical bonds), which allows
40 for the easy release of FRs from products. Consequently, they are ubiquitously
41 distributed in environmental and biological samples (Deng et al., 2018, LeBel and
42 Williams, 1983). In recent years, the production and application of organophosphate
43 flame retardants dramatically increased to serve as the substitutes for PBDEs (Deng et
44 al., 2018, Dodson et al., 2012, Stapleton et al., 2008). A good amount of OPFRs have
45 been detected in human serum (Li et al., 2017). Of OPFRs’ adverse health effects,
46 impact on development is the primary concern. Evidence from epidemiological
47 studies has revealed an association between exposure to OPFRs and developmental
48 problems including lower birth weights, compromised IQ, anxiety, and hyperactivity
49 (Castorina et al., 2017, Hendriks et al., 2015). In addition, developmental defects
50 caused by OPFRs exposure have also been demonstrated in a variety of animal
51 experiments (Dishaw et al., 2014, Oliveri et al., 2015, Oliveri et al., 2018).
52 Tris(1,3-dichloro-2-propyl) phosphate (TDCPP) is one of the most common
53 OPFRs in baby products (Hoffman et al., 2015, Stapleton et al., 2011). Infants have
54 greater exposure to TDCPP than adults, which is reflected by the high concentration
55 of TDCPP metabolites detected in their urine (Butt et al., 2014, Hoffman et al., 2015) .
56 In light of its presence in house dust, daily OPFRs intake, primarily TDCPP, among
57 toddlers was estimated at ~20 times higher than that of adults (Tan et al., 2017).
58 Experimental and epidemiological studies have revealed that developmental exposure
59 to TDCPP leads to gastrulation defects, aberrant germ-layer formation (Dasgupta et
60 al., 2018), hyperactivity (Moser et al., 2015). However, the mechanisms of
61 developmental defects resulting from TDCPP exposure are poorly understood.
62 The formation of circulation system is one of the earliest events in
63 embryogenesis, and proper blood supply is fundamental to cellular differentiation and
64 proliferation (Gore et al., 2012, Walls et al., 2008). Disruption of vascular
65 development has been directly correlated with miscarriages, birth defects, maternal
66 placental complications, and neurodevelopmental problems (Kleinstreuer et al., 2011).
67 As the absorption, distribution, and excretion of exogenous chemicals (xenobiotics)
68 relies on the circulatory system, blood vessels are prone to contact with xenobiotics
69 upon developmental exposure. Thus, the blood vasculature is probably an important
70 target of their developmental toxicity (Helker et al., 2013). Given role of blood vessel
71 growth in the early stages of embryogenesis, the impacts of xenobiotics on vascular
72 development could be good candidates for the identification of biomarkers for their
73 early exposure and effects. Despite the central role of blood vessels in developmental
74 toxicity, the adverse effects on vascular growth by developmental exposure to TDCPP
75 have been under-emphasized in previous studies.
76 Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) is a key transcription factor
77 responsible for the defense response to environmental toxic insults such as hazardous
78 chemicals. It works by regulating the expression of genes related to redox balance
79 (antioxidants) and detoxification (Zhao et al., 2015, Wang et al., 2012, Wei et al.,
80 2016). Apart from cytoprotection, our recent studies have found that Nrf2 plays a
81 critical role in vascular growth via a context-dependent mechanism. When tissue is
82 injured, Nrf2 is activated in the ischemic tissue and promotes vascular regeneration
83 through the regulation of NADPH oxidase-2 (Wei et al., 2016) and semaphorin
84 6A-mediating pathway (Wei et al., 2015). In addition to injury, Nrf2 has been shown
85 to regulate physiological vascular development in an endothelial autonomous fashion
86 via VEGF-Dll4-Notch signaling (Wei et al., 2015). Taken together, the uncovered
87 Nrf2 functions suggest the potential role of Nrf2 in modulating vascular growth upon
88 chemical exposure.
89 The zebrafish is a powerful model for developmental studies, as its rapid
90 organogenesis is readily observed in the live and transparent embryos outside the
91 body (Childs et al., 2002, Hogan and Schulte-Merker, 2017). Vascular development in
92 zebrafish embryos is accomplished within several days, faster than is the case in
93 rodents (Gore et al., 2012, Walls et al., 2008). In addition, the zebrafish model is a
94 valuable, widely used resource in the study of environmental chemicals’
95 developmental toxicities (Hill et al., 2005, Padilla et al., 2012). In this study, we
96 investigate the effect of exposure to TDCPP on the vascular development of zebrafish,
97 and the possible role of Nrf2-dependent angiogenic pathways in TDCPP’s vascular
98 toxicity. Using both in vivo and in vitro approaches, we found that exposure to
99 TDCPP inhibited developmental vascular growth and suppressed VEGF signaling via
100 Nrf2-dependent pathway. Our findings provide new insight into the adverse outcome
101 pathway (AOP) of developmental exposure to OPFRs. They also help identify
102 potential biomarkers for early exposure and effects.
103
104
Materials and methods
105 2.1 Chemicals and reagents
106 Tirs(1,3-dichloro-2-propyl) phosphate (TDCPP) and dimethyl sulfoxide (DMSO)
107 were obtained from sigma (St. Louis, USA). TDCPP was dissolved in DMSO as stock
108 solution and stored at -20℃. CDDO-imidazolide (CODD-Im) was purchased from
109 MedChemExpress (Monmouth Junction, USA). 1-phenyl-2-thiourea (PTU) and
110 tricaine were obtained from Sigma (St. Louis, USA).
111
112 2.2 Zebrafish and chemical exposure
113 Wild-type AB and Tg (kdrl:eGFP) transgenic zebrafish embryos were obtained
114 from the Core Lab for Medical Science at Sun Yat-sen University. Maintenance and
115 exposure were performed according to previous report (Xing et al., 2018). Embryos
116 were collected and randomly distributed into glass dishes containing 10 mL of
117 TDCPP exposure solution at various concentrations (0, 50, 300 and 500 µg/L) at
118 about 2 hours post-fertilization (hpf). The dose range was designed according to the
119 previous studies (Wang et al., 2013) and our preliminary experiment. For the
120 CDDO-Im experiment, embryos were treated with 25 nM CDDO-Im for 2 hours at 2
121 hpf, prior to the addition of TDCPP. Zebrafish embryos and larvae were maintained at
122 26±1 ℃ with a 14/10 h light/dark cycle, and the exposure solution was changed every
123 day. After 24 hpf, PTU was applied to suppress the growth of melanin.
124 124
125 125
126 2.3 Assessment of general developmental toxicity
127 Cumulative mortality, malformation rate, and hatching rate were recorded as
128 preciously described (Xing et al., 2018). Cumulative mortality was calculated as
129 percentage of the total number of dead individuals from 2 to 30 hpf or 72 hpf
130 following TDCPP exposure. Malformations including pericardial edema, yolk sac
131 edema and tail abnormality were recorded at 30 hpf. Pericardial edema, yolk sac
132 edema, trunk curvature, tail abnormality, and craniofacial malformation were recorded
133 at 72 hpf.
134 134
135 2.4 Intersegmental vessels (ISV) measurement
136 Vasculatures were observed and measured, as previously described (Xing et al.,
137 2018). Tg (kdrl:eGFP) transgenic zebrafish embryos were collected at 30 hpf. After
138 removing the chorion with a needle, the embryos were anesthetized in a freshly
139 prepared solution containing 0.2 mg/ml tricaine. They were then embedded on their
140 sides in a petri dish containing 1.0 % low melting agarose. Images were captured
141 under a confocal microscope with a 10× objective at 488 nm (Leica, Germany). The
142 total number of ISV and completed ISV were counted for each larva to calculate the
143 percentage of completed ISV.
144
145 2.5 Common cardinal vein (CCV) measurement
146 Tg (kdrl:eGFP) transgenic zebrafish embryos were collected at 48 hpf or 72 hpf.
147 The following steps for zebrafish mounting and capture of images were performed as
148 mentioned above for ISV measurement. The CCV of each larva was outlined and the
149 area measured using Adobe Photoshop.
150
151
2.6 Measurement of superoxide dismutase (SOD) activity, catalase (CAT) activity
152 and GSH levels in zebrafish
153 Zebrafish larvae were collected at 72 hpf and homogenized in either 0.9% NaCl
154 (for SOD and GSH assay), or RIPA buffer (CAT assay). SOD activity, CAT activity
155 and GSH levels were examined according the manufacturer’s instructions (SOD
156 activity assay kit A001, GSH assay kit A006, Nanjing Jiancheng Bioengineering
157 Institute, China; catalase activity assay kit, S0051, Beyotime, China). Results were
158 normalized by protein concentrations.
159
160
2.7 Cell culture
161 Primary human venous endothelial cells (HUVECs) were obtained from Cell
162 System (USA). HUVECs were cultured in endothelial growth medium-2 (EGM-2,
163 Lonza, USA) and maintained at 37 ℃ with 5% CO2, as previously described (Ge et al.,
164 2014).
165 2.8 HUVEC viability and proliferation assay
166 HUVECs were seeded and cultured in a 96-well plate. When the cells reached
167 80-90% confluence, 10-200 µM of TDCPP were added. TDCPP doses for cell culture
168 study were applied according to the previous study (Xiang et al., 2017a, Ta et al.,
169 2014). Cell viability was measured with a Calcein-AM assay kit (Trevigen, USA)
170 after 24 hours. In VEGF-induced proliferation experiment, HUVECs were seeded in a
171 96-well plate. When cells were at 60-70% confluence, media were replaced with
172 endothelial basal medium-2 (EBM-2, lonza, USA), EBM-2+25 ng/mL VEGFA, or
173 EBM-2+25 ng/mL VEGFA+TDCPP. After 24 hours, cell proliferation was assessed
174 using a Calcein-AM assay kit (Trevigen, USA) according to the manufacturer’s
175 instructions.
176 176
177 2.9 Immunofluorescence staining
178 HUVECs were plated on the coverslip and maintained in EGM-2 medium until
179 the cells reached 80-90% confluence. The cells were treated with TDCPP for 24 hours,
180 then fixed with 4% paraformaldehyde, washed three times in PBS and blocked for 1
181 hour in blocking solution (10% goat serum, 1% BSA, 0.3% Triton X-100 in PBS) at
182 room temperature. Cells were then incubated with primary antibody NRF2 （1:100，
183 Cell Signaling Technology，USA）overnight at 4 °C. Goat anti-rabbit secondary
184 antibody (Alexa Fluor 594, 1:700, Thermo Fisher, USA) was incubated for 1 hour at
185 room temperature. Nuclei were stained with DAPI for 5 min, and mount on slides
186 with coverslips. Images were obtained with confocal microscope (Zeiss, Germany).
187 187
188 2.10 Quantitative real-time PCR
189 Zebrafish larvae were collected at 72 hpf and HUVEC cells were obtained 24
190 hours after TDCPP exposure. Total RNA was extracted with Trizol reagent
191 (Invitrogen, USA). Total RNA was reverse-transcribed to cDNA using a Quantscript
192 RT Kit (Thermo Fisher, USA). Real-time PCR was performed using an SYBR Green
193 PCR Master Mix reagent kit (TOYOBO, China) and along with zebrafish specific-
194 and human based- primers (designed by Primer-blast). Data were analyzed with the
195 comparative threshold cycle (CT) method as means of relative quantitation, and were
196 normalized to an endogenous reference beta-2 microglobulin (B2m).
197 197
198 2.11 Statistical analysis
199 Data are presented as mean± standard error of mean. Statistical analyses were
200 conducted with one way ANOVA to compare the difference among groups. A
201 comparison between the two groups was performed with a Newman-Keuls Multiple
202 Comparison test. P<0.05 was considered statistically significant.
203 203
204 3. Results and Discussion
205 3.1 The growths of ISV and CCV are sensitive to TDCPP exposure
206 ISV and CCV are typical microvessels and macrovessel in zebrafish, respectively.
207 To investigate the effect of TDCPP on microvasculature, we observed ISV growth in
208 zebrafish embryos (Figure 1A). ISV sprouting starts at the 24 somite stage (about 22
209 hpf) from the dorsal aorta. At ~30 hpf, ISV growth is finished and the dorsal
210 longitudinal anastomotic vessel (DLAV) grows to connect with the adjacent ISVs
211 (Figure 1B) (Tobia et al., 2015). Thus, ISV images were captured at 30 hpf, and the
212 completed ISVs (Helker et al., 2013) were calculated. Compared with the control
213 group, zebrafish embryos exposed to 300 µg/L showed a dramatic decrease in the
214 percentage of completed ISV. A more pronounced reduction was exhibited in the 500
215 µg/L TDCPP treatment group (Figure 1C and D). We also assessed cumulative
216 mortality and malformations including yolk sac edema, pericardial edema, and tail
217 abnormality at 30 hpf. Significant increase only exhibited in zebrafish embryos
218 treated with 500 µg/L TDCPP (Figure 1E and F).
219 CCV, a typical macrovessel in zebrafish, is located in the anterior trunk of the
220 embryo, where it collects venous blood and transports it to the heart (Helker et al.,
221 2013). The endothelial cells of the CCV emerge from the junction of the anterior and
222 posterior cardinal veins at ~24 hpf, and gradually migrate ventrally and reach the
223 ventral margin of the yolk sac at ~56 hpf. After CCV area peaks, the CCV area
224 reduces as it is remodeled into a tube at ~74 hpf (Figure 2A) (Bello et al., 2004). To
225 investigate whether the extension and remodeling of CCVs were affected by TDCPP
226 exposure, CCV images were obtained at 48 hpf and 72 hpf. The CCV area in
227 TDCPP-treated embryos at concentrations of 300 or 500 µg/L was less than that of the
228 control group (Figure 2B and C). A similar pattern was observed in the counting of
229 endothelial cells (Figure S1). At 72 hpf, CCV area in zebrafish larvae exposed to
230 TDCPP increased in a dose-dependent fashion (Figure 2D and E). This indicated a
231 delayed CCV remodeling caused by TDCPP exposure. Similar to the results at 30 hpf,
232 TDCPP exposure at 500 µg/L significantly increased cumulative mortality (Figure 2F)
233 and malformations (including yolk sac edema, pericardial edema, trunk curvature and
234 tail abnormality) (Figure 2G) at 72 hpf. In addition, hatching rate at 72 hpf slightly
235 decreased in TDCPP-exposed group at 500 µg/L (Figure 2H). As compared with
236 control group, no significant difference of cumulative mortality, malformations or
237 hatching rate was observed in other groups.
238 In addition, heart rates (including both atrium and ventricle beat) slightly
239 decreased in 300 µg/L and 500 µg/L groups, whereas the atrium to ventricle beat
240 ratios were 1:1 in all groups, indicating that TDCPP exposure had mild effect on heart
241 function and did not lead to atrioventricular block (Figure S1). The alteration was not
242 as remarkable as the vascular phenotype. Although heart and blood vessels belong to
243 cardiovascular system and share many characteristics in common, blood vessels in
244 zebrafish seem to be more sensitive to TDCPP exposure.
245 Collectively, our results indicate that TDCPP exposure impairs the growths of
246 both microvessels and macrovessels in zebrafish. Developing vasculature is more
247 sensitive to TDCPP exposure than the conventional developmental parameters in the
248 early life stage. Thus, the effect on vascular development in zebrafish could be
249 applied to the assessment of developmental toxicity of OPFRs and the screening of
250 biomarkers for early exposure. Consistent with our study, Xiang et al. have reported
251 that the flame retardant TBPH and its metabolites could inhibit HUVEC growth and
252 induce cell cycle arrest and apoptosis (Xiang et al., 2017b). Similar to TDCPP,
253 BDE-47, a predominant brominated flame retardant, was found to impede the growth
254 of ISV and CCV in zebrafish in our previous study (Xing et al., 2018). However, the
255 range of toxic BDE-47 doses for vascular growth was from 30 to 300 µg/L, lower
256 than that of TDCPP (300-500 µg/L) in this study. These results indicate that TDCPP,
257 as an alternative FR, was less toxic to vascular development than BDE-47. In the
258 present study, we emphasized the morphological changes of vasculature. To better
259 understand the vascular toxicity of TDCPP, the functional alterations of blood vessels
260 should be investigated in the further study.
261 Dishaw et al. showed that TDCPP levels were about 10 pmol/fish on 1 dpf and 1
262 pmol/l fish on 5 dpf in zebrafish exposed to 1 µM (430.9 µg/L) TDCPP (Dishaw et al.,
263 2014). Given the similar levels of TDCPP we used (50-500 µg/L), we may speculate
264 the comparable concentrations of TDCPP in zebrafish (about 1-10 pmol/fish) in our
265 experiment. TDCPP concentrations of 50-500 µg/L are relatively higher than the
266 reported environmentally relevant concentrations. Tan et al reported that the
267 concentrations of TDCPP ranged from 0.42 to10.19 µg/g in South China house dust
268 (Tan et al., 2017), and 61.2 µg/g dust in Brazilian city (Cristale et al., 2018). TDCPP
269 ranging from 520 to 4000 ng/L was found in the wastewater (Greaves and Letcher,
270 2017). In zebrafish, vascular growth starts at about 20 hpf and vascular development
271 proceeds until about 1 month post fertilization (Gore et al., 2012). The objective of
272 the study is to investigate the vascular toxicity of TDCPP and identify the sensitive
273 biomarkers for its early exposure. Thus, the early stage of vascular development (from
274 30 hpf to 72 hpf) was emphasized in our study. Whether long-term exposure to
275 TDCPP at environmental related concentrations during vascular development (for
276 example, exposure for 1 month) may impair vascular growth is necessary to be
277 considered in the further study.
278 278
279 3.2 TDCPP exposure suppressed VEGF pathway in zebrafish larvae and
280 HUVECs
281 VEGF pathway governing vascular sprouting, proliferation and EC migration, is
282 the primary pathway for the regulation of developmental angiogenesis (Li and Harris,
283 2009). VEGFA, a predominant isoform of VEGF, is directly induced by HIF1a and
284 binds to VEGFR1 and VEGFR2. This mediates the extension of vascular networks
285 and promotes angiogenesis (Pitulescu et al., 2017). To investigate the potential
286 mechanism by which TDCPP exposure impairs vascular development, mRNA levels
287 of the essential genes in VEGF pathway were assessed by real-time PCR. The mRNA
288 levels of Vegfa, Vegfr1, Vegfr2 and Vegfa inducer Hifa dose-dependently decreased
289 following exposure to TDCPP in zebrafish larvae (Figure 3A). To further confirm the
290 effect of TDCPP exposure on VEGF pathway, we examined the VEGF signaling in
291 HUVECs following exposure to TDCPP for 24 hours. TDCPP at concentrations of 10
292 µM and 100 µM were applied as the cell survival was not affected by treatment with
293 10-100 µM of TDCPP (Figure S3). In line with in vivo results, TDCPP exposure
294 markedly reduced the mRNA level of VEGF at 100 µM in HUVECs (Figure S4).
295 Moreover, we examined cell proliferation in HUVECs following treatment with both
296 VEGF and TDCPP for 24 hours. The results showed that VEGFA-induced cell
297 proliferation was dose-dependently suppressed by TDCPP exposure (Figure 3B). The
298 results suggest that TDCPP exposure inhibits VEGF pathway. This correlates with the
299 defective vascular development in zebrafish as shown in figure 1 and 2.
300 VEGF pathway is sensitive to xenobiotic toxicants in developmental process.
301 Consistent with our results, administration of O-(chloroacetyl-carbamoyl) fumagillol
302 (TNP-470) significantly decreased VEGF expression and VEGFR2 phosphorylation
303 in the ocular vasculature in rodents at early stages of pregnancy (Joussen et al., 2001).
304 Therefore, the results implicate that TDCPP inhibits vascular growth by suppressing
305 VEGF signaling. Wang et al., reported that exposure to 300-600 µg/L TDCPP
306 disrupted the thyroid endocrine system at 144 hpf (Wang et al., 2013). In our study,
307 vascular toxicity could be detected as early as 30 hpf following exposure to 300 or
308 500 µg/L TDCPP. These results showed that TDCPP-induced vascular toxicity may
309 occur earlier than endocrine disruption. Therefore, the results suggested that the onset
310 of vascular damage is likely independent of VEGF signaling. Whether endocrine
311 system is involved in the progression of vascular toxicity needs to be further
312 investigated.
313 313
314 3.3 TDCPP exposure inhibited Nrf2 function in zebrafish larvae and HUVECs
315 Our previous study demonstrated that Nrf2 plays an important role in the
316 regulation of developmental angiogenesis (Wei et al., 2013). To investigate whether
317 Nrf2 is involved in the effect of TDCPP on vascular growth, we then assessed the
318 Nrf2 expression and activity in zebrafish larvae and HUVECs. The results showed
319 that the mRNA levels of the Nrf2 and its target genes Sod1, Sod2, Gclm and Txn in
320 zebrafish were reduced at 72 hpf following exposure to TDCPP in a dose-dependent
321 fashion (Figure 4A), suggesting that TDCPP exposure suppresses Nrf2 expression and
322 transcriptional activity during vascular development. We also assessed the Nrf2
323 expression and activity in cultured HUVECs. Immunofluorescence staining showed a
324 dose-dependent reduction in NRF2 protein (red) in cells treated with 10 or 100 µM
325 TDCPP for 24 hours (Figure 5A and 5B). Likewise, the mRNA levels of Nrf2, Sod1,
326 Sod2, Gclm, and Txn in HUVECs dose-dependently decreased in TDCPP-treated cells
327 (Figure 5C). Taken together, these results suggest the inhibition of Nrf2 function by
328 TDCPP exposure. This correlates with the reduced VEGF pathway and defective
329 vascular development as shown above.
330 Nrf2 is a stress-response transcription factor frequently activated by chemical
331 exposure (Abolaji et al., 2014). . The activation of Nrf2 induces an adaptive response,
332 acting as a powerful intracellular mechanism for coping with unfavorable
333 environments (Gaisina et al., 2018). However in this study, Nrf2 was activated in
334 neither zebrafish in early life stage nor cultured HUVECs following TDCPP exposure.
335 Conversely, we observed the repression of Nrf2 expression and activity due to TDCPP
336 treatment. Upon different stimuli in specific subcellular compartments, there are three
337 E3 ubiquitin ligase complexes responsible for the ubiquitylation and degradation of
338 NRF2. NRF2 binds to the CUL3-RBX1-KEAP1 complex to respond to
339 electrophilic/oxidative stress in the cytosol. The SCF/β-TrCP complex, which can be
340 either nuclear or cytosolic, is more sensitive to metabolic changes and is regulated by
341 GSK3-β. During ER stress, HRD1 localizes to the endoplasmic reticulum and
342 ubquitylates NRF2 (Dodson et al., 2018, Harder et al., 2015, Liu et al., 2018). . In the
343 present study, we speculate that TDCPP exposure may affect KEAP1 complex activity
344 and that the ubiquitylation of Nrf2 suppresses Nrf2 activity. Further studies are
345 needed to investigate the regulatory mechanism of Nrf2 by TDCPP exposure in our
346 future studies.
347 Nrf2 plays a key role in the maintenance of redox homeostasis through
348 regulation of antioxidants. In our study, TDCPP exposure at 300 or 500 µg/L led to a
349 reduction in GSH level (Figure 4B) and SOD activity (Figure S2) in zebrafish larvae.
350 A decrease in GSH is frequently attributed to the elevated ROS and reduced activities
351 of antioxidants including SOD. Thus, we speculated that a perturbed redox balance
352 accounted for the impairment of vascular growth caused by TDCPP. The
353 down-regulation of a variety of antioxidants was observed in both zebrafish and
354 cultured HUVECs following TDCPP exposure (Figure 4A and 5B). Previous studies
355 have shown that OPFRs interrupt the antioxidant enzyme system to cause oxidative
356 stress both in vitro and in vivo (Chen et al., 2015a, Chen et al., 2015b). The
357 endogenous antioxidant defense system maintains redox homeostasis and is essential
358 for angiogenesis. Disruption of endogenous antioxidants hinders angiogenesis (Jin et
359 al., 2016) and application of antioxidants obliterates pathological angiogenesis
360 (Radomska-Lesniewska et al., 2017) or rescue angiogenesis defects (Jin et al., 2016).
361 Therefore, our results suggest that the impaired vascular growth due to TDCPP
362 exposure is correlated with disturbance of redox homeostasis.
363 363
364 3.4 Activation of Nrf2 by CDDO-Im abrogated the impairment of vascular
365 development due to TDCPP exposure
366 We next sought to confirm that Nrf2-depedent VEGF pathway is crucial for the
367 vascular toxicity of TDCPP upon developmental exposure. Since TDCPP exposure
368 suppressed Nrf2 function in vascular development, we aimed to determine whether
369 boosting Nrf2 activity can counteract TDCPP’s impact on vascular growth. CDDO-Im
370 is a potent Nrf2 activator and has been shown to enhance Nrf2 function and induce its
371 target gene expression in our previous studies (Wei et al., 2015). Zebrafish embryos
372 were pretreated with CDDO-Im 2 hours prior to TDCPP exposure. The mRNA levels
373 of the Nrf2 and its target genes Txn, Sod1, Sod2 soared in CDDO-Im treated zebrafish
374 (> 2 fold, Figure 6A). In addition, CDDO-Im also induced Vegfa expression (> 3 fold,
375 Figure 6A). We further investigated whether CDDO-Im treatment could protect
376 zebrafish from vascular toxicity in development. We found that CDDO-IM treatment
377 alleviated the reduction in the percentage of completed ISV by 300 or 500 µg/L
378 TDCPP (Figure 6B and D). Delayed CCV remodeling in zebrafish exposed to 300 or
379 500 µg/L TDCPP was abrogated by CDDO-IM treatment (Figure 6C and D). These
380 results indicate that the activation of Nrf2 induces VEGF signaling and protects
381 against TDCPP’s vascular toxicity in development. This strongly supports that the
382 defective vascular growth caused by TDCPP exposure is mediated by Nrf2-dependent
383 VEGF pathway.
384 Nrf2 has been shown to regulate angiogenesis dependent on VEGF signaling in
385 previous studies. Kuang et al. have reported that the knockdown of Nrf2 significantly
386 decreases VEGF levels, and activation of Nrf2 up-regulates VEGF expression with a
387 concomitant increase in cell migration and vascular tube formation (Kuang et al.,
388 2013). Our previous studies have shown that Nrf2 regulates physiological vascular
389 development in an endothelial autonomous fashion via VEGF-Dll4-Notch signaling
390 (Wei et al., 2013). In contrast to vascular development, Nrf2 is activated in the
391 ischemic tissue and promotes vascular regeneration through the regulation of NADPH
392 oxidase-2 and VEGF-HIF1a-Sema6A pathway in tissue injury (Wei et al., 2016, Wei
393 et al., 2015). In addition to physiological and pathological condition, the present study
394 has demonstrated that the suppression of Nrf2-dependent VEGF pathway was
395 responsible for the impairment of vascular growth due to TDCPP exposure and thus
396 might be regarded as a crucial toxicity pathway in the adverse effect of TDCPP on
397 development.
398 398
399 4. Conclusion
400 To our best knowledge, this study is the first time to report that TDCPP could
401 impede the growth of vasculature during early developmental stage in zebrafish. We
402 further demonstrated that TDCPP dose-dependently suppressed the Nrf2-mediated
403 VEGF pathway in both zebrafish and cultured vascular endothelial cells. Due to the
404 growing production and increased contents of OPFRs in both the environment and
405 biota, identifying powerful biomarkers for early exposure and delineating the toxicity
406 pathways for the characterization of AOP are urgently needed. In accordance with our
407 results, vascular impairment is a sensitive index for early exposure, which should be
408 considered in the environmental risk assessment of OPFRs. In addition, the
409 identification of Nrf2-dependent VEGF pathway by TDCPP exposure provides further
410 insight into the AOP of OPFRs.
411 411
412 Acknowledgements
413 We thank Drs. Jiayin Dai (Chinese Academy of Sciences) and Wen Chen (Sun
414 Yat-sen University) for helpful suggestions and technical assistance. This work was
415 supported by the National Science Foundation of China (21777199) and National Key
416 R & D program (2017YFC1600205).
417
418 Declarations of interest: none
419
420 Abbreviations
421 Tris(1,3-dichloro-2-propyl) phosphate (TDCPP); intersegmental vessel (ISV);
422 common cardinal vein (CCV); nuclear factor erythroid 2-related factor (Nrf2);
423 1[2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oy] imidazolide (CDDO-IM); vascular
424 endothelial growth factor (VEGF); beta-2 microglobulin (B2m); dimethyl sulfoxide
425 (DMSO); glutamate-cysteine ligase, modifier (Gclm); thioredoxin (Txn); Kelch
426 domain of Kelch-like ECH-associated protein (keap1).
427 427
428 References
429 Abolaji, A. O., Kamdem, J. P., Lugokenski, T. H., Nascimento, T. K., Waczuk, E. P.,
430 Farombi, E. O., Loreto, E. L. & Rocha, J. B. (2014) Involvement of oxidative
431 stress in 4-vinylcyclohexene-induced toxicity in Drosophila melanogaster. Free
432 Radic Biol Med, 71, 99-108.
433 Bello, S. M., Heideman, W. & Peterson, R. E. (2004)
434 2,3,7,8-Tetrachlorodibenzo-p-dioxin inhibits regression of the common cardinal
435 vein in developing zebrafish. Toxicol Sci, 78 (2), 258-266.
436 Butt, C. M., Congleton, J., Hoffman, K., Fang, M. & Stapleton, H. M. (2014)
437 Metabolites of organophosphate flame retardants and 2-ethylhexyl
438 tetrabromobenzoate in urine from paired mothers and toddlers. Environ Sci
439 Technol, 48 (17), 10432-10438.
440 Castorina, R., Bradman, A., Stapleton, H. M., Butt, C., Avery, D., Harley, K. G.,
441 Gunier, R. B., Holland, N. & Eskenazi, B. (2017) Current-use flame retardants:
442 Maternal exposure and neurodevelopment in children of the CHAMACOS cohort.
443 Chemosphere, 189, 574-580.
444 Chen, G., Jin, Y., Wu, Y., Liu, L. & Fu, Z. (2015a) Exposure of male mice to two
445 kinds of organophosphate flame retardants (OPFRs) induced oxidative stress and
446 endocrine disruption. Environ Toxicol Pharmacol, 40 (1), 310-318.
447 Chen, G., Zhang, S., Jin, Y., Wu, Y., Liu, L., Qian, H. & Fu, Z. (2015b) TPP and
448 TCEP induce oxidative stress and alter steroidogenesis in TM3 Leydig cells.
449 Reprod Toxicol, 57, 100-110.
450 Childs, S., Chen, J. N., Garrity, D. M. & Fishman, M. C. (2002) Patterning of
451 angiogenesis in the zebrafish embryo. Development, 129 (4), 973-982.
452 Cooper, E. M., Kroeger, G., Davis, K., Clark, C. R., Ferguson, P. L. & Stapleton, H.
453 M. (2016) Results from Screening Polyurethane Foam Based Consumer Products
454 for Flame Retardant Chemicals: Assessing Impacts on the Change in the
455 Furniture Flammability Standards. Environ Sci Technol, 50 (19), 10653-10660.
456 Cristale, J., Aragao Bele, T. G., Lacorte, S. & Rodrigues de Marchi, M. R. (2018)
457 Occurrence and human exposure to brominated and organophosphorus flame
458 retardants via indoor dust in a Brazilian city. Environ Pollut, 237, 695-703.
459 Dasgupta, S., Cheng, V., Vliet, S. M. F., Mitchell, C. A. & Volz, D. C. (2018)
460 Tris(1,3-dichloro-2-propyl) Phosphate Exposure During the Early-Blastula Stage
461 Alters the Normal Trajectory of Zebrafish Embryogenesis. Environ Sci Technol,
462 52 (18), 10820-10828.
463 Deng, W. J., Li, N., Wu, R., Richard, W. K. S., Wang, Z. & Ho, W. (2018) Phosphorus
464 flame retardants and Bisphenol A in indoor dust and PM2.5 in kindergartens and
465 primary schools in Hong Kong. Environ Pollut, 235, 365-371.
466 Dishaw, L. V., Hunter, D. L., Padnos, B., Padilla, S. & Stapleton, H. M. (2014)
467 Developmental exposure to organophosphate flame retardants elicits overt
468 toxicity and alters behavior in early life stage zebrafish (Danio rerio). Toxicol Sci,
469 142 (2), 445-454.
470 Dodson, M., de la Vega, M. R., Cholanians, A. B., Schmidlin, C. J., Chapman, E. &
471 Zhang, D. D. (2018) Modulating NRF2 in Disease: Timing Is Everything. Annu
472 Rev Pharmacol Toxicol. doi: 10.1146/annurev-pharmtox-010818-021856
473 Dodson, R. E., Perovich, L. J., Covaci, A., Van den Eede, N., Ionas, A. C., Dirtu, A. C.,
474 Brody, J. G. & Rudel, R. A. (2012) After the PBDE phase-out: a broad suite of
475 flame retardants in repeat house dust samples from California. Environ Sci
476 Technol, 46 (24), 13056-13066.
477 Gaisina, I. N., Lee, S. H., Kaidery, N. A., Ben Aissa, M., Ahuja, M., Smirnova, N. N.,
478 Wakade, S., Gaisin, A., Bourassa, M. W., Ratan, R. R., Nikulin, S. V., Poloznikov,
479 A. A., Thomas, B., Thatcher, G. R. J. & Gazaryan, I. G. (2018) Activation of Nrf2
480 and Hypoxic Adaptive Response Contribute to Neuroprotection Elicited by
481 Phenylhydroxamic Acid Selective HDAC6 Inhibitors. ACS Chem Neurosci, 9 (5),
482 894-900.
483 Ge, L., Trujillo, G., Miller, E. J. & Kew, R. R. (2014) Circulating complexes of the
484 vitamin D binding protein with G-actin induce lung inflammation by targeting
485 endothelial cells. Immunobiology, 219 (3), 198-207.
486 Gore, A. V., Monzo, K., Cha, Y. R., Pan, W. & Weinstein, B. M. (2012) Vascular
487 development in the zebrafish. Cold Spring Harb Perspect Med, 2 (5), a006684.
488 Greaves, A. K. & Letcher, R. J. (2017) A Review of Organophosphate Esters in the
489 Environment from Biological Effects to Distribution and Fate. Bull Environ
490 Contam Toxicol, 98 (1), 2-7.
491 Harder, B., Jiang, T., Wu, T., Tao, S., Rojo de la Vega, M., Tian, W., Chapman, E. &
492 Zhang, D. D. (2015) Molecular mechanisms of Nrf2 regulation and how these
493 influence chemical modulation for disease intervention. Biochem Soc Trans, 43
494 (4), 680-686.
495 Helker, C. S., Schuermann, A., Karpanen, T., Zeuschner, D., Belting, H. G., Affolter,
496 M., Schulte-Merker, S. & Herzog, W. (2013) The zebrafish common cardinal
497 veins develop by a novel mechanism: lumen ensheathment. Development, 140
498 (13), 2776-2786.
499 Hendriks, H. S., Koolen, L. A., Dingemans, M. M., Viberg, H., Lee, I., Leonards, P. E.,
500 Ramakers, G. M. & Westerink, R. H. (2015) Effects of neonatal exposure to the
501 flame retardant tetrabromobisphenol-A, aluminum diethylphosphinate or zinc
502 stannate on long-term potentiation and synaptic protein levels in mice. Arch
503 Toxicol, 89 (12), 2345-2354.
504 Hill, A. J., Teraoka, H., Heideman, W. & Peterson, R. E. (2005) Zebrafish as a model
505 vertebrate for investigating chemical toxicity. Toxicol Sci, 86 (1), 6-19.
506 Hoffman, K., Butt, C. M., Chen, A., Limkakeng, A. T., Jr. & Stapleton, H. M. (2015)
507 High Exposure to Organophosphate Flame Retardants in Infants: Associations
508 with Baby Products. Environ Sci Technol, 49 (24), 14554-14559.
509 Hogan, B. M. & Schulte-Merker, S. (2017) How to Plumb a Pisces: Understanding
510 Vascular Development and Disease Using Zebrafish Embryos. Dev Cell, 42 (6),
511 567-583.
512 Jin, P., Li, T., Li, X., Shen, X. & Zhao, Y. (2016) Suppression of oxidative stress in
513 endothelial progenitor cells promotes angiogenesis and improves cardiac function
514 following myocardial infarction in diabetic mice. Exp Ther Med, 11 (6),
515 2163-2170.
516 Joussen, A. M., Beecken, W. D., Moromizato, Y., Schwartz, A., Kirchhof, B. &
517 Poulaki, V. (2001) Inhibition of inflammatory corneal angiogenesis by TNP-470.
518 Invest Ophthalmol Vis Sci, 42 (11), 2510-2516.
519 Kleinstreuer, N. C., Judson, R. S., Reif, D. M., Sipes, N. S., Singh, A. V., Chandler, K.
520 J., Dewoskin, R., Dix, D. J., Kavlock, R. J. & Knudsen, T. B. (2011)
521 Environmental impact on vascular development predicted by high-throughput
522 screening. Environ Health Perspect, 119 (11), 1596-1603.
523 Kuang, L., Feng, J., He, G. & Jing, T. (2013) Knockdown of Nrf2 inhibits the
524 angiogenesis of rat cardiac micro-vascular endothelial cells under hypoxic
525 conditions. Int J Biol Sci, 9 (7), 656-665.
526 LeBel, G. L. & Williams, D. T. (1983) Determination of organic phosphate triesters in
527 human adipose tissue. J Assoc Off Anal Chem, 66 (3), 691-699.
528 Li, J. L. & Harris, A. L. (2009) Crosstalk of VEGF and Notch pathways in tumour
529 angiogenesis: therapeutic implications. Front Biosci (Landmark Ed), 14,
530 3094-3110.
531 Li, P., Jin, J., Wang, Y., Hu, J., Xu, M., Sun, Y. & Ma, Y. (2017) Concentrations of
532 organophosphorus, polybromobenzene, and polybrominated diphenyl ether flame
533 retardants in human serum, and relationships between concentrations and donor
534 ages. Chemosphere, 171, 654-660.
535 Liu, D., Xue, J., Liu, Y., Gu, H., Wei, X., Ma, W., Luo, W., Ma, L., Jia, S., Dong, N.,
536 Huang, J., Wang, Y. & Yuan, Z. (2018) Inhibition of NRF2 signaling and
537 increased reactive oxygen species during embryogenesis in a rat model of retinoic
538 acid-induced neural tube defects. Neurotoxicology, 69, 84-92.
539 Moser, V. C., Phillips, P. M., Hedge, J. M. & McDaniel, K. L. (2015)
540 Neurotoxicological and thyroid evaluations of rats developmentally exposed to
541 tris(1,3-dichloro-2-propyl)phosphate (TDCIPP) and tris(2-chloro-2-ethyl)
542 phosphate (TCEP). Neurotoxicol Teratol, 52 (Pt B), 236-247.
543 Oliveri, A. N., Bailey, J. M. & Levin, E. D. (2015) Developmental exposure to
544 organophosphate flame retardants causes behavioral effects in larval and adult
545 zebrafish. Neurotoxicol Teratol, 52 (Pt B), 220-227.
546 Oliveri, A. N., Ortiz, E. & Levin, E. D. (2018) Developmental exposure to an
547 organophosphate flame retardant alters later behavioral responses to dopamine
548 antagonism in zebrafish larvae. Neurotoxicol Teratol, 67, 25-30.
549 Padilla, S., Corum, D., Padnos, B., Hunter, D. L., Beam, A., Houck, K. A., Sipes, N.,
550 Kleinstreuer, N., Knudsen, T., Dix, D. J. & Reif, D. M. (2012) Zebrafish
551 developmental screening of the ToxCast Phase I chemical library. Reprod Toxicol,
552 33 (2), 174-187.
553 Pitulescu, M. E., Schmidt, I., Giaimo, B. D., Antoine, T., Berkenfeld, F., Ferrante, F.,
554 Park, H., Ehling, M., Biljes, D., Rocha, S. F., Langen, U. H., Stehling, M.,
555 Nagasawa, T., Ferrara, N., Borggrefe, T. & Adams, R. H. (2017) Dll4 and Notch
556 signalling couples sprouting angiogenesis and artery formation. Nat Cell Biol, 19
557 (8), 915-927.
558 Radomska-Lesniewska, D. M., Balan, B. J. & Skopinski, P. (2017) Angiogenesis
559 modulation by exogenous antioxidants. Cent Eur J Immunol, 42 (4), 370-376.
560 Stapleton, H. M., Allen, J. G., Kelly, S. M., Konstantinov, A., Klosterhaus, S., Watkins,
561 D., McClean, M. D. & Webster, T. F. (2008) Alternate and new brominated flame
562 retardants detected in U.S. house dust. Environ Sci Technol, 42 (18), 6910-6916.
563 Stapleton, H. M., Klosterhaus, S., Keller, A., Ferguson, P. L., van Bergen, S., Cooper,
564 E., Webster, T. F. & Blum, A. (2011) Identification of flame retardants in
565 polyurethane foam collected from baby products. Environ Sci Technol, 45 (12),
566 5323-5331.
567 Ta, N., Li, C., Fang, Y., Liu, H., Lin, B., Jin, H., Tian, L., Zhang, H., Zhang, W. & Xi,
568 Z. (2014) Toxicity of TDCPP and TCEP on PC12 cell: changes in CAMKII,
569 GAP43, tubulin and NF-H gene and protein levels. Toxicol Lett, 227 (3),
570 164-171.
571 Tan, H., Peng, C., Guo, Y., Wang, X., Wu, Y. & Chen, D. (2017) Organophosphate
572 Flame Retardants in House Dust from South China and Related Human Exposure
573 Risks. Bull Environ Contam Toxicol, 99 (3), 344-349.
574 Tobia, C., Gariano, G., Guerra, J. & Presta, M. (2015) Zebrafish embryo
575 intersegmental vessels: a tool for investigating sprouting angiogenesis. Methods
576 Mol Biol, 1214, 173-184.
577 Walls, J. R., Coultas, L., Rossant, J. & Henkelman, R. M. (2008) Three-dimensional
578 analysis of vascular development in the mouse embryo. PLoS One, 3 (8), e2853.
579 Wang, L., Kou, M. C., Weng, C. Y., Hu, L. W., Wang, Y. J. & Wu, M. J. (2012)
580 Arsenic modulates heme oxygenase-1, interleukin-6, and vascular endothelial
581 growth factor expression in endothelial cells: roles of ROS, NF-kappaB, and
582 MAPK pathways. Arch Toxicol, 86 (6), 879-896.
583 Wang, Q., Liang, K., Liu, J., Yang, L., Guo, Y., Liu, C. & Zhou, B. (2013) Exposure
584 of zebrafish embryos/larvae to TDCPP alters concentrations of thyroid hormones
585 and transcriptions of genes involved in the hypothalamic-pituitary-thyroid axis.
586 Aquat Toxicol, 126, 207-213
587 Wei, Y., Gong, J., Thimmulappa, R. K., Kosmider, B., Biswal, S. & Duh, E. J. (2013)
588 Nrf2 acts cell-autonomously in endothelium to regulate tip cell formation and
589 vascular branching. Proc Natl Acad Sci U S A, 110 (41), E3910-3918.
590 Wei, Y., Gong, J., Xu, Z. & Duh, E. J. (2016) Nrf2 promotes reparative angiogenesis
591 through regulation of NADPH oxidase-2 in oxygen-induced retinopathy. Free
592 Radic Biol Med, 99, 234-243.
593 Wei, Y., Gong, J., Xu, Z., Thimmulappa, R. K., Mitchell, K. L., Welsbie, D. S., Biswal,
594 S. & Duh, E. J. (2015) Nrf2 in ischemic neurons promotes retinal vascular
595 regeneration through regulation of semaphorin 6A. Proc Natl Acad Sci U S A, 112
596 (50), E6927-6936.
597 Xiang, P., Liu, R. Y., Sun, H. J., Yang, Y. W., Cui, X. Y. & Ma, L. Q. (2017a) Effects
598 of novel brominated flame retardant TBPH and its metabolite TBMEHP on
599 human vascular endothelial cells: Implication for human health risks. Environ Res,
600 156, 834-842.
601 Xiang, P., Liu, R. Y., Li, C., Gao, P., Cui, X. Y. & Ma, L. Q. (2017b) Effects of
602 organophosphorus flame retardant TDCPP on normal human corneal epithelial
603 cells: Implications for human health. Environ Pollut, 230, 22-30.
604 Xing, X., Kang, J., Qiu, J., Zhong, X., Shi, X., Zhou, B. & Wei, Y. (2018) Waterborne
605 exposure to low concentrations of BDE-47 impedes early vascular development
606 in zebrafish embryos/larvae. Aquat Toxicol, 203, 19-27.
607 Zhao, M., Chen, J., Zhu, P., Fujino, M., Takahara, T., Toyama, S., Tomita, A., Zhao, L.,
608 Yang, Z., Hei, M., Zhong, L., Zhuang, J., Kimura, S. & Li, X. (2015)
609 Dihydroquercetin (DHQ) ameliorated concanavalin A-induced mouse
610 experimental fulminant hepatitis and enhanced HO-1 expression through
611 MAPK/Nrf2 antioxidant pathway in RAW cells. Int Immunopharmacol, 28 (2),
612 938-944.
613 Figure legends
614 Figure 1. TDCPP exposure inhibited ISV growth in zebrafish embryos. (A) ISV in
615 Zebrafish (outlined in yellow). (B) Schematic of ISV growth. ISV sprouts from the
616 DA. At ~30 hpf, ISV growth is finished and the DLAV grows dorsally to connect with
617 the adjacent ISVs. (C) Representative images of ISV at 30 hpf following exposure to
618 TDCPP. Completed ISVs are denoted by arrows. (D) The percentage of completed
619 ISV at 30 hpf. (E) Cumulative mortality from ~2 hpf to 30 hpf. (F) Percentage of
620 zebrafish embryos displaying malformations (including yolk sac edema, pericardial
621 edema, and tail abnormality) at 30 hpf. ISV: intersegmental vessel; DA: dorsal aorta;
622 DLAV: dorsal longitudinal anastomotic vessel. Scale bars in A: 200 µm; B: 20 µm; C:
623 100 µm. Data are presented as mean ± SEM. n = 10-15 embryos (in A-D) or 5 dishes
624 (in E and F, each dish contains 40 embryos before treatment with TDCPP). * p<0.05,
625 ** p<0.01 and *** p<0.001 as compared with the control group.
626 626
627 Figure 2. TDCPP exposure impeded the extension and remodeling of CCV in
628 zebrafish embryos and larvae. (A) Schematic of CCV in zebrafish. The vascular
629 endothelial cells (*) of CCV begin to emerge from the junction of the anterior and
630 posterior cardinal veins on both sides of the embryo at ~24 hpf. The CCV then
631 extends ventrally from the junction as a sheet of vascular endothelial cells until
632 reaching the ventral margin of the yolk sac at ~56 hpf. The arrow indicates the
633 direction of CCV extension. (B) Representative images of CCV at 48 hpf following
634 exposure to TDCPP. (C) CCV area at 48 hpf. (D) Representative images of CCV at 72
635 hpf following exposure to TDCPP. CCV growth involves a remodeling process from
636 48 to 74 hpf and within the process CCV transforms from a sheet of endothelial cells
637 into a tube. (E) CCV area at 72 hpf. (F) Cumulative mortality from ~2 hpf to 72 hpf.
638 (G) Percentage of zebrafish embryos displaying malformations (including yolk sac
639 edema, pericardial edema, trunk curvature and tail abnormality) at 72 hpf. (H)
640 Hatching rate at 72 hpf. Scale bars in A: 200 µm, 5µm (insert); B and E: 100 µm.
641 Data are presented as mean ± SEM. n = 15-20 embryos or larvae (in A-E) or 5 dishes
642 (in F-H, each dish contains 40 embryos before treatment with TDCPP). * p<0.05, **
643 p<0.01 and *** p<0.001 as compared with the control group.
644 644
645 Figure 3. TDCPP exposure suppressed VEGF pathway in zebrafish larvae and
646 HUVECs. (A) The mRNA levels of Vegfa, Vegfr1, Vegfr2 and Hif1a in zebrafish
647 larvae at 72 hpf following exposure to TDCPP. (B) VEGFA-induced cell proliferation
648 was dose-dependently suppressed by TDCPP exposure. HUVECs were treated with
649 25 ng/ml VEGFA and 10-200 µM TDCPP for 24 hours. Data are presented as mean ±
650 SEM. n = 8 pooled samples (10 larvae in each sample) or 3 independent experiments
651 in cell culture experiment. * p<0.05, ** p<0.01 and *** p<0.001 a as compared with
652 the control group (A) or the group treated with VEGF only (B).
653 653
654 Figure 4. TDCPP exposure inhibited Nrf2 function and decreased GSH content in
655 zebrafish larvae. (A) The mRNA levels of Nrf2, Sod1, Sod2, Gclm, and Txn at 72 hpf.
656 (B) GSH level at 72 hpf. Data are presented as mean ± SEM. n = 5. * p<0.05 and **
657 p<0.01 as compared with the control group.
658 658
659 Figure 5. TDCPP exposure inhibited Nrf2 expression and activity in HUVECs. (A)
660 Representative images of HUVECs subjected to immunofluorescence staining of
661 NRF2 (red). Nuclei were stained with DAPI. Scale bar: 50 µm. (B) Quantification of
662 NRF2 immunofluorescence in (A). The result shows a dose-dependent reduction in
663 NRF2 protein in HUVECs treated with 10 or 100 µM TDCPP for 24 hours. n=8
664 random fields (40 cells) per group. (C) mRNA levels of Nrf2, Sod1, Sod2, Gclm, and
665 Txn in HUVECs treated with 10 or 100 µM TDCPP for 24 hours. Scale bar: 10 µm. n
666 = 3 independent experiments. Data are presented as mean ± SEM. * p<0.05, **
667 p<0.01 and *** p<0.001 as compared with the control group.
668
669 Figure 6. Activation of Nrf2 by CDDO-Im abrogated the impairment of vascular
670 development due to TDCPP exposure. (A) The mRNA expression level of Nrf2, Nrf2
671 target genes, and Vegfa in zebrafish treated with 25 nM CDDO-IM. n = 8 pooled
672 samples (10 larvae in each sample) (B) The percentage of completed ISV following
673 exposure to CDDO-Im and TDCPP at 30 hpf. (C) CCV area following CDDO-Im
674 treatment at 72 hpf. (D) Upper panels: representative images of ISV at 30 hpf
675 following exposure to CDDO-IM and TDCPP; Botton panels: representative images
676 of CCV at 72 hpf following exposure to CDDO-IM and TDCPP. Scale bar: 100 µm.
677 Data are presented as mean ± SEM. n = 15-20 embryos or larvae. * p<0.05, ** p<0.01
678 and ***p<0.001 as compared with the control group.
Highlights
TDCPP exposure impeded the growth of microvessels (ISV), as early as 30 hpf, and delayed the extension or remodeling of macrovessels (CCV) at 48 hpf and 72 hpf in zebrafish.
Developing vasculature in zebrafish was more sensitive than CDDO-Im general developmental parameters to TDCPP exposure.
TDCPP exposure inhibited Nrf2-depedent VEGF signaling pathway in zebrafish and cultured human umbilical vascular endothelial cells.