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Environmental Pollution 268 (2021) 115809

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Environmental Pollution

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Review

Xenopus in revealing developmental toxicity and modeling humandiseases*

Juanmei Gao a, b, Wanhua Shen a, *

a Zhejiang Key Laboratory of Organ Development and Regeneration, College of Life and Environmental Sciences, Hangzhou Normal University, Hangzhou,Zhejiang, 311121, Chinab College of Life and Sciences, Zhejiang University, Hangzhou, Zhejiang, 310058, China

a r t i c l e i n f o

Article history:Received 14 June 2020Received in revised form1 October 2020Accepted 9 October 2020Available online 13 October 2020

Keywords:XenopusToxicityToxicantOocyteEmbryo developmentDisease model

* This paper has been recommended for acceptanc* Corresponding author.

E-mail address: shen@hznu.edu.cn (W. Shen).

https://doi.org/10.1016/j.envpol.2020.1158090269-7491/© 2020 Elsevier Ltd. All rights reserved.

a b s t r a c t

The Xenopus model offers many advantages for investigation of the molecular, cellular, and behavioralmechanisms underlying embryo development. Moreover, Xenopus oocytes and embryos have beenextensively used to study developmental toxicity and human diseases in response to various environ-mental chemicals. This review first summarizes recent advances in using Xenopus as a vertebrate modelto study distinct types of tissue/organ development following exposure to environmental toxicants,chemical reagents, and pharmaceutical drugs. Then, the successful use of Xenopus as a model for dis-eases, including fetal alcohol spectrum disorders, autism, epilepsy, and cardiovascular disease, isreviewed. The potential application of Xenopus in genetic and chemical screening to protect againstembryo deficits induced by chemical toxicants and related diseases is also discussed.

© 2020 Elsevier Ltd. All rights reserved.

1. Introduction

The South African clawed frog has been used as one of the mainvertebrate animal models since their introduction as a model or-ganism in the 1950s (Beck and Slack, 2001; Tadjuidje and Heasman,2010). The Xenopus model is a time- and cost-efficient model forin vivo studying embryonic development and environmentaltoxicity. The diploid Xenopus tropicalis (X. tropicalis) and the allo-tetraploid Xenopus laevis (X. laevis) have two distinct geneticbackgrounds and are widely used in developmental, biomedical,and cancer research (Buryskova et al., 2006; Davis et al., 1981;Hardwick and Philpott, 2015; Yao et al., 2017; Zhu et al., 2017, 2018).In addition, frogs can produce massive numbers of eggs that arefertilized into tadpoles in vitro in amatter of days during all seasons(Showell and Conlon, 2009). Both embryos and adults live in thewater and are independent of maternal influence, which makes iteasy for them to absorb exogenous hormones or other chemicalcompounds (Beck and Slack, 2001). Most importantly, Xenopus as atetrapod is evolutionarily closer to higher vertebrates than other

e by Dr. Sarah Harmon.

aquatic models in terms of organ development, physiology, andgene expression (Nenni et al., 2019). Other evolutionary similaritiesto mammals are also observed at the level of organ structure andfunction, such as lungs (Galdiero et al., 2017), heart (Blum and Ott,2018), kidneys (Desgrange and Cereghini, 2015), lymphatics (Nyet al., 2006) and immune system (Wheeler and Brandli, 2009)(Fig. 1). Therefore, Xenopus has become an important vertebrateanimal model for answering developmental questions, toxic effects,and human disease mechanisms.

Many aspects of Xenopus organs can be used to investigate themechanism of organogenesis and embryogenesis (Hoppler andConlon, 2019). In its ease use of manipulation and molecularintervention, studies in Xenopus tissues/organs have made a sig-nificant contribution to current understanding of embryo devel-opment, including the tectal brain, eyes, heart and kidneys(Frontera et al., 2016a; Tomlinson et al., 2005). The visual retino-tectal system has been extensively used to study neurogenesis,visual circuit function and synaptic plasticity in vivo (Akerman andCline, 2006; Faulkner et al., 2015; He et al., 2018; Lee et al., 2010;McKeown et al., 2013; Pratt and Aizenman, 2007; Shen et al., 2011,2014; Truszkowski et al., 2016). Free swimming and visually guidedavoidance behaviors can be analyzed to measure visual-motorfunction, which helps in the understanding of sensory processing

Fig. 1. Schematic diagram of evolutionarily conserved tissues and organs used for toxicological studies in zebrafish, Xenopus, and mouse. (A) Various tissues and organs are used fordevelopmental and toxicological studies in zebrafish, Xenopus, and mouse, such as heart, kidney, and craniofacial. The size of each organ is not proportionally scaled. A, atrium; V:ventricle; RA, right atrium; LA, left atrium; RV: right ventricle; LV: left ventricle.

J. Gao and W. Shen Environmental Pollution 268 (2021) 115809

in an intact neural circuit (He et al., 2018; Pratt and Aizenman,2007; Shen et al., 2011). The development of Xenopus pronephrickidney is similar to the amniotes metanephros, which is used toexamine nephron formation and model human kidney diseases(Cizelsky et al., 2014; Desgrange and Cereghini, 2015; Hwang et al.,2019) (Fig. 1). Amphibian metamorphosis is utilized in the researchof thyroid hormone-mediated cell apoptosis and stem cell devel-opment during vertebrate developmental transitions (Buchholz,2017; Das et al., 2002). Unlike mammals, Xenopus tadpoles havehigh regenerative potential for structures including the spinal cord(Aztekin et al., 2019; Borodinsky, 2017; Edwards-Faret et al., 2017;Munoz et al., 2015), heart (Liao et al., 2017; Marshall et al., 2019),epithelium (Frontera et al., 2016b; Kim et al., 2020), and limbs(Herrera-Rincon et al., 2018; Yokoyama et al., 2011), making Xen-opus an attractive model to uncover the mechanisms governingregeneration (Borodinsky, 2017; Phipps et al., 2020).

Exposure to environmental toxicants can cause short-termreversible or long-term irreversible damage to various tissues andorgans, leading to malformations and diseases (Fig. 2). Morerecently, several disease models, such as those for fetal alcoholspectrum disorders (FASDs), epilepsy, autism, and cardiovasculardiseases, have been successfully developed in Xenopus (Sater andMoody, 2017). Toxicant-induced developmental deficits and func-tional alterations in embryos can be characterized through a varietyof elegant techniques to gain insight into the etiology and

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pathophysiology of human diseases. In addition, Xenopus has beensuccessfully used to identify chemically responsive factors andcharacterize the disease mechanisms of pathogenic human vari-ants. Hence, Xenopus model provides an integrative approach forunderstanding the development of organs and the mechanisms ofhuman diseases. In this review, we summarized the recent progressin using Xenopus as an animal model for developmental studies,toxicological investigations, and disease modeling, with a descrip-tion of advances in methodological applications and drugscreenings.

2. Advantages of using Xenopus as an in vivo model fortoxicological studies

The Xenopus system has been traditionally used as a develop-mental model to investigate neurogenesis, embryogenesis, organ-ogenesis, and metamorphosis (Tadjuidje and Heasman, 2010). Asamphibian species have both aquatic larval phase and adultterrestrial phase, these organisms are sensitive to water and soilpollutants, which makes them to be a unique model for monitoringenvironmental risks with low experimental costs. The Xenopus eggis a huge cell that is easily manipulated. The Xenopus oocyteexpression system is utilized to determine the properties of ionchannels and neurotransmitter transporters in single cells withoutinterfering with the complexities of brain tissues. Cell extracts have

Fig. 2. Schematic diagram of several malformations used for testing chemical toxicity in Xenopus. Representative Xenopus stages at 32, 45 and 60 are used for studying chemical-induced developmental deficits. (A) Representative defects are shown as loss of eyes, abnormal embryogenesis, and embryo malformations at the developing stage 32. (B)Representative defects are shown as pigment aggregation, pigment loss, misfolded gut, abnormal eye, heart enlargement, decreased spine length, and tail flexure at stage 45. (C)Representative defects are shown as pigment aggregation, dispersed melanosomes, increased limb length, limb deformities, abnormal metamorphosis, and growth inhibition atstage 60. The size of embryos is not proportionally scaled.

J. Gao and W. Shen Environmental Pollution 268 (2021) 115809

been used to examine the fundamental processes of developmentalbiology, genome maintenance mechanisms and chemical genetics(Cross and Powers, 2009; Hoogenboom et al., 2017; Maia et al.,2017). Other tissues in Xenopus embryos have been widely usedto study embryogenesis, toxicology and developmental diseasesusing optic tectum (Pratt and Khakhalin, 2013), intestine (Ikuzawaet al., 2006), kidneys (Hwang et al., 2019), heart (Duncan andKhokha, 2016) and eyes (Imaoka et al., 2007; Luehders et al.,2015). Combining with methodological advances, such as in vivoelectrophysiological recordings (Hiramoto and Cline, 2014;Kutsarova et al., 2016; Shen et al., 2011; Tao and Poo, 2005),genomic microarray chips (Fu et al., 2017; Huyck et al., 2015; Zhuet al., 2017), RNA deep sequencing (Paranjpe et al., 2013), electrontransfer dissociation (ETD) (McGivern et al., 2009), and in vivobioorthogonal noncanonical amino acid tagging (BONCAT) (Liuet al., 2018; Shen et al., 2014), the Xenopus system has become avaluable vertebrate model system to decipher the mechanism ofchemical toxicants-induced defects and screen potential pharma-ceutical drugs to treat the damage (Liao et al., 2020). However, thedependence on specific antibodies can also be a limitation, while itis time consuming and expensive to generate antibodies to detectXenopus proteins. The collection of large tissue samplings would bealso labor consuming.

2.1. Xenopus oocytes and egg extracts

Unfertilized Xenopus oocytes removed from female frogs havebeen broadly used to investigate endocytosis mechanisms, ionchannels, and cytological processes (Maia et al., 2017). The lowendogenous expression of ion channels in oocytes makes themamenable to expressing exogenous channel proteins or disease-related proteins and performing electrophysiological recordingsto study ion transport and channel physiology (Schmidt et al.,2009). The physiological properties of ion channels expressed inoocytes, such as NMDAR (Cruz et al., 2000), AMPAR (Chang et al.,2016), GABAR (Storustovu and Ebert, 2006), and nAChRs (van

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Kleef et al., 2008), provide convenient tools to characterize the ef-fects of active compounds or environmental pollutants, such astoluene (Cruz et al., 1998) and ototoxic solvents (van Kleef et al.,2008) on corresponding receptors. As an in vitro model, Xenopusoocytes will continue to provide an efficient system in identifyingnovel drug targets within hundreds of potential candidates andfurther understanding the mechanism of human diseases (Zenget al., 2020).

Xenopus egg extracts are a cell-free, biologically active cyto-plasm that mimics the cellular physiological environment andcontains all cytoplasmic proteins, organelles, and other essentialcomponents. The Xenopus oocyte extract system stands out as apowerful tool in determining signaling pathway, studying genomemaintenance mechanisms (Hardwick and Philpott, 2015;Hodskinson et al., 2020; Hoogenboom et al., 2017; Hyde et al.,2016). Due to its low cost and efficient nature, the system hasbeenwidely used in screening potential activators or inhibitors andrevealing drug toxicology effects within a large number of com-pounds (Maia et al., 2017; Thorne et al., 2011). Thesemethodologiesallow researchers to build systematic tools to study chemical effectson cell signaling and screen potential anti-toxic drugs.

2.2. Xenopus embryos

Most tissues and organs of Xenopus have been extensively usedto study embryo development and toxicological studies from theembryonic to metamorphic stages (Taft et al., 2018; Zhang et al.,2020) (Fig. 1). The retinotectal visual system is a powerful modelfor examining neurotransmission in neural circuit developmentand function in response to different environmental factors(Igarashi et al., 2015; James et al., 2015; Liao et al., 2020). The visualbehavior of Xenopus tadpoles is currently used to determine visual-motor functions and neurotoxicity induced by chemical toxicants(Dong and Aizenman, 2012; Gao et al., 2016a; Shen et al., 2014).Pigment cells (melanophores in amphibians; melanocytes inmammals) are aggregated in light and dispersed in dark in the wild

J. Gao and W. Shen Environmental Pollution 268 (2021) 115809

type Xenopuswithout the influence of time of day. A complex seriesof chemical and physiological mechanisms regulates melanophorepigment distribution, making these skin cells a valuable tool tostudy pigment cell development, assess chemical toxicity andperform drug screening, as an in vivo model (Dahora et al., 2020;Iuga et al., 2009; Tomlinson et al., 2009; Wheeler and Brandli,2009). In contrast to wide type Xenopus, the transparent nature ofAlbino Xenopus tadpole skin is well suited for live tracking of neuralmorphology, cardiovascular dynamics, heart development, lym-phangiogenesis, and angiogenesis over hours to dayswith exposureto chemical toxicants (Duncan and Khokha, 2016; Hoppler andConlon, 2019; Liao et al., 2020; Ny et al., 2006).

While zebrafish shares many advantages with X. laevis in addi-tion to its well-developed genetic manipulation strategies, theirgenome may not perform same function as mammals (Lasser et al.,2019). Zebrafish genome is evolutionarily distant to humans andmake it difficult in identifying human orthologs. Moreover,X. tropicalis has a true diploid genome with substantial sharedsynteny with the human genome, which is feasible to create per-manent transgenic lines for genetic manipulations. In addition,Xenopus shares greater similarities in tissues and organs withhigher vertebrates, such as heart, kidneys, vascular, lungs andlymphatic system (Ny et al., 2006) (Fig.1). Additionally, Xenopus hasa three-chambered heart, while zebrafish has a two-chamberedheart with one atrium and one ventricle, which cannot fullymodel four-chambered human heart abnormalities (Blum and Ott,2018; Duncan and Khokha, 2016) (Fig. 1). Also, Xenopus tadpoleswill develop lungs and limbs, which are absent from zebrafish.Importantly, amphibian metamorphosis physiologically resemblesmammalian postembryonic development (Wheeler and Brandli,2009). These advantages make Xenopus a unique model to studythe impact of environmental factors on its development frommolecular and cellular to circuit and behavioral levels (Hasebe et al.,2017; Thompson and Cline, 2016; Tomlinson et al., 2005).

3. Toxicological studies using Xenopus

Accumulating evidence has shown that Xenopus embryos aresensitive to environmental chemicals, which makes Xenopus aunique model animal for cytotoxicity research (Wheeler andBrandli, 2009). Multiple tissues and organs in Xenopus have beenused to examine the developmental effects of chemical pollutants,hormones, pesticides, and other environmental factors (Table 1).Xenopus has become a good model to build links between pheno-types of malformations and changes of gene expression due toestablished molecular and cellular technologies. The frog embryoteratogenesis assay in Xenopus (FETAX) has been used for manyyears to test mortality and malformations in response to variousenvironmental factors (Fort and Mathis, 2018; Newman andDumont, 1983). Multidisciplinary techniques such as electrophysi-ology, calcium imaging, click chemistry labeling, cryo-electronmicroscopy and time-lapse imaging have been used extensivelyto study the effects of chemical toxicity on tadpole development(Bestman and Cline, 2020; Ciarleglio et al., 2015; Glasgow et al.,2019; Hasebe et al., 2017; Khakhalin et al., 2014; Liu and Haas,2011; Shen et al., 2014). Proteomic analysis using the iTRAQ tech-nique in the gonads or transcriptome-wide profile in the testis ofX. laevis has been used to examine global protein changes or m (6)amethylome profiles in response to atrazine (Chen et al., 2015).Combined with swimming behavior analysis, Xenopus can be usedas an important animal model for biosafety assessment and po-tential drug screening at an early stage of clinical trials (Maia et al.,2017; Tomlinson et al., 2005; Zhang et al., 2014), especially forscreening early event targets in cell differentiation and organo-genesis (Hwang et al., 2019). Therefore, the successful use of

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advanced techniques in Xenopus is amenable to facilitate theresearch on developmental toxicity and the feasibility of screeningpotential drugs (Schmitt et al., 2014).

3.1. Chemical pollutants

Xenopus is a commonly used vertebrate model in the study ofthe effects of toxic compounds on embryo development and neuralcircuit function during development (Wang et al., 2018). Numerouschemical pollutants, such as benzene, methylmercury, cadmium,zinc and polychlorinated biphenyls, have been found to causeneurodevelopmental toxicity in Xenopus (Huyck et al., 2015;Mouchet et al., 2015; Rice, 2008) (Table 1).

The benzene, toluene, ethylbenzene and xylene aromatic hy-drocarbons, collectively called BTEX, are emitted from oil refineriesand gas stations and are known to cause eye inflammation, head-ache, asthma and severe diseases such as leukemia and multiplemyeloma (McKenzie et al., 2012). The developing Xenopus retino-tectal system has been used to assess chemical toxicity and screenpotential pharmaceutical drugs, which would provide us an effi-cient and convenient opportunity to treat pollutants-induced def-icits and diseases. Studies have found that para-xylene (PX) cancause developmental delay and neural apoptosis in the developingXenopus tectum through methylation and acetylation of histones(Gao et al., 2016a). Further studies have shown that PX exposureinduces deficits in neuronal structure and visual stimulation-induced excitatory compound synaptic currents without alteringneurotransmitter release probability (Liao et al., 2020). The toxiceffects of ethylbenzene and xylene are also revealed by inhibitingNMDA-induced currents (Cruz et al., 2000) and acetylcholine-induced ionic currents (van Kleef et al., 2008) in Xenopus oocytesexpressing NMDAR or a9a10 nAChR. Interestingly, the coapplica-tion of D-glucuronolactone (GA) with PX rescues all deficitsinduced by PX exposure (Gao et al., 2016a; Liao et al., 2020). Theseresults demonstrate that PX is toxic to brain development andplasticity and that GA may serve as a promising candidate to treatPX-induced defects in the developing brain.

The FETAX assay has been extensively used to assess teratogenicand toxic effects of chemical pollutants. Four aromatic ami-nesdacridine, aniline, pyridine, and quinoline have been revealedas potential environmental hazards following their exposure toX. laevis embryos (Davis et al., 1981). Polycyclic aromatic hydro-carbons (PAHs) and their derivatives have been shown to induceembryo malformations and oxidative stress at sublethal concen-trations (Buryskova et al., 2006). Alcohol ethoxylate (AE) andalcohol ethoxy sulfate (AES) cause teratogenic and toxic effects inXenopus embryos and tadpoles, in particular to the epithelia and gillcartilage (Cardellini and Ometto, 2001). Most importantly, re-searchers have used the Xenopus system to compare the toxicseverity score and find less toxic plasticizer. For example, theyexamined the developmental toxicity of citrate esters and indicatedthat triethyl 2-acetylcitrate (ATEC) could be considered an alter-native to the phthalate plasticizer of dibutyl-phthalate (DBP),which has higher toxicity in amphibian embryos (Xu and Gye,2018).

The phenotype of dispersion and aggregation of skin pigment isa good marker for monitoring chemical toxicity in water ecosys-tems. The first study to assess chemical toxicity in drinking waterwas performed in cultured Xenopus melanophores (Iuga et al.,2009). They found that some chemicals, including ammonia,arsenic, copper, mercury, pentachlorophenol, phenol, nicotine andparaquat, induce a rapid melanosome dispersion response, indi-cating that Xenopus melanophores may serve as a good toxicitysensor for screening toxicants. Environmental concentrations ofnitromusks, synthetic nitro-containing fragrances, are not

Table 1Summary of the toxicological studies performed in Xenopus tissues and organs.

Tissues/Organs Environmental factors Studies in Xenopus

Brain1,2,3,4,5

Aromatic amines; BTEX; nitromusks; pesticides; nanomaterials;methylmercury; alcohol; choline and nicotinic agonists; endosulfan;methylisothiazolinone; benomyl; DBP; CEP; triadimefon andtriadimenol; DHA; VPA; TNF-a; cis-bifenthrin and graphene oxide;thyroid hormone

(Davis et al., 1981); (Gao et al., 2016a); (Huyck et al., 2015); (Igarashiet al., 2015); (James et al., 2015); (Khakhalin et al., 2014); (Lee et al.,2010); (Li et al., 2020b); (Liao et al., 2020); (Preud’homme et al., 2015);(Shen et al., 2014); (Shi et al., 2014); (Spawn and Aizenman, 2012);(Thompson and Cline, 2016); (Titmus et al., 1999); (Yoon et al., 2003)

Embryo2,3,5,6

Benomyl; thyroid hormone; ethanol; polycyclic aromatic hydrocarbons;alcohol ethoxylate (AE) and alcohol ethoxy sulfate (AES); nitromusksatrazine; silver nanoparticles; methylmercury; rapamycin; cadmiumand zinc; regular cigarette butt (RCB), menthol (MCB) and electronic(ECB); Tributyltin (TBT) and triphenyltin (TPT); dibutyl phthalate andcitrate ester; quantum dots (QDs)

(Buryskova et al., 2006); (Cardellini and Ometto, 2001); (Chen et al.,2015); (Chou and Dietrich, 1999); (Colombo et al., 2017); (Galdieroet al., 2017); (Huyck et al., 2015); (Kim et al., 2020); (Moriyama et al.,2011); (Mouchet et al., 2015); (Parker and Rayburn, 2017); (Shi et al.,2014); (Shukrun et al., 2019); (Xu and Gye, 2018); (Yelin et al., 2007);(Yoon et al., 2003); (Yuan et al., 2011); (Zhu et al., 2018)

Eye6

Benomyl; BPA; thyroid hormone; ethanol; triphenyltin (TPT);butachlor; tire debris (TD); alcohol

(Baba et al., 2009); (Havis et al., 2006); (Li et al., 2016a); (Mantecca et al.,2007); (Peng et al., 2004); (Yelin et al., 2007); (Yoon et al., 2003); (Zhuet al., 2018)

Craniofacial3

Ethanol; thioridazine, ethanol and ICI 118,551; triadimefon; silvernanoparticles; Agþ; electronic cigarette (ECIG)

(Colombo et al., 2017); (Fainsod and Kot-Leibovich, 2018); (Fort et al.,2016); (Kennedy et al., 2017); (Li et al., 2017); (Li et al., 2020a);

Gonad7

BDE-47; atrazine; azocyclotin; boric acid; BPA; benzophenone-2; 17-ethinylestradiol (EE2); tebuconazole; triclosan

(Chen et al., 2015); (Fort et al., 2017); (Li et al., 2016b); (Haselman et al.,2016); (Hayes et al., 2010); (Pinet et al., 2019); (Poulsen et al., 2015);(Regnault et al., 2018); (Rimayi et al., 2018); (Sai et al., 2019); (Sai et al.,2020); (Tamschick et al., 2016)

Heart8,9

Chlorpyrifos; dichlorvos; diazinon; ketamine; dehydration; atrazine;thyroid hormone;

(Asouzu Johnson et al., 2019); (Guo et al., 2016); (Hawkins and Storey,2020); (Hwang et al., 2019); (Marshall et al., 2019); (Watson et al., 2014)

Intestine/gut5,6

Alcohol; thyroid hormone; aryl hydrocarbon receptor agonists; ethanol;TBBPA; nanoparticles; ZnO; polystyrene microspheres

(Bonfanti et al., 2015); (Bonfanti et al., 2020); (Colombo et al., 2017);(Hasebe et al., 2016); (Ikuzawa et al., 2006); (Ishizuya-Oka et al., 2001);(Ishizuya-Oka et al., 2014); (Hu et al., 2016); (Miller et al., 2013); (Taftet al., 2018); (Wang et al., 2017); (Yao et al., 2017); (Zhu et al., 2020);

Limb6

Dioxin; thyroid hormone; retinoic acid (Alsop et al., 2004); (Marsh-Armstrong et al., 2004); (Yokoyama et al.,2011)

Liver6

Triadimefon and triadimenol; azocyclotin; atrazine; benzophenone-2;benzo(a)pyrene and triclosan; paracetamol; tire debris organic extract(TDOE)

(Li et al., 2017); (Haselman et al., 2016); (Regnault et al., 2018); (Rimayiet al., 2018); (Saide et al., 2019); (Mantecca et al., 2007); (Zhang et al.,2020)

Skin4,6

Maltol; thyroid hormone; copper mercury; rapamycin; radiation;polystyrene nanoparticles

(Baba et al., 2009); (Carotenuto et al., 2016); (Dahora et al., 2020); (Iugaet al., 2009); (Moriyama et al., 2011); (Tussellino et al., 2015); (Yuanet al., 2011); (Zhang et al., 2014);

Spinal cord1,3,5,9

Muscarinic; thyroid hormone; aromatic amines; acridine; aniline;pyridine; quinoline; chlorpyrifos; dichlorvos; Methylisothiazolinone(MIT)

(Carotenuto et al., 2016); (Davis et al., 1981); (Marsh-Armstrong et al.,2004); (Munoz et al., 2015); (Porter and Li, 2018); (Schlosser et al.,2002); (Spawn and Aizenman, 2012); (Watson et al., 2014);

Tail3,4,9

BPA; thyroid hormone; hexabromocyclododecane (HBCD) and2,20 ,3,30 ,4,40 ,5,50 ,6-nona brominated diphenyl ether (BDE206);chlorpyrifos (CPF) and malathion (MTN); TrkA inhibitor; CuOnanoparticles; carbachol

(Bonfanti et al., 2004); (Das et al., 2002); (Helbing et al., 2003);(Iwamuro et al., 2006); (Iimura et al., 2020); (Li et al., 2020b); (Marsh-Armstrong et al., 2004); (Nations et al., 2015); (Porter and Li, 2018);(Schriks et al., 2006)

1, Behavior; 2, Mortality; 3, Malformation; 4, Metamorphosis; 5, Neurogenesis; 6, Embryogenesis; 7, Reproductive toxicity; 8, Heart dysfunction; 9, Regeneration.

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hazardous for early developing amphibians and fish (Chou andDietrich, 1999). However, the other study using an in vivo Xen-opus model has shown that maltol, a naturally occurring flavorenhancer and fragrance agent, induces pigment aggregation in bothdura and dermal melanophores, suggesting a putative toxicologicaleffect of maltol exposure (Dahora et al., 2020). Therefore, bothexposure time and concentration of chemicals and the sites of ac-tion are important parameters to be considered for comprehen-sively evaluating the toxic effects of environmental factors.

The different impact of traditional tobacco and electronic ciga-rette on environmental toxicity can be directly assessed withexposure butt leachates to tadpoles. Evaluation of the develop-mental toxicities of three different cigarette butt leachates, regular(RCB), menthol (MCB) and electronic cigarette butt (ECB), hasshown that RCB leachates comprise the most toxic compound,while MCB leachates have higher teratogenicity (Parker andRayburn, 2017). One study has reported that craniofacial forma-tion is adversely affected by aerosol mixtures produced by e-liquidaerosolization in e-cigarettes (ECIGs), which are often advertised asa safe alternative to traditional tobacco smoking (Kennedy et al.,2017). These results confirm that the Xenopus model can be use-ful in an integrated biological hazard assessment for preliminaryscreening.

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3.2. Pesticides

Nowadays, a variety type of pesticides, including herbicides,insecticides, fungicides, and rodenticides were proven efficient inpest control and crop productivity. However, it is known that due totheir toxicity, pesticides could have serious impacts on humanhealth as well as aquatic ecosystems (Christin et al., 2004). Thedevelopment of accurate and sensitive assessment of pesticidetoxicity is required to ensure public health. Several distinct chem-ical classes of pesticides have been primarily investigated in Xen-opus, such as chlorpyrifos (organophosphorus insecticide), atrazine(triazine herbicide), and triadimefon (conazole fungicide) (AsouzuJohnson et al., 2019; Bonfanti et al., 2004; Hayes et al., 2010;Rimayi et al., 2018; Sai et al., 2019; Watson et al., 2014; Zhang et al.,2020). However, theses pesticides exert differential toxic effects onembryogenesis, neurotransmitter release, and sexualdifferentiation.

Organophosphate pesticides, which act as a class of acetylcho-linesterase inhibitors, are used widely in agriculture to reduce in-sect populations. Chlorpyrifos (10 mM) and dichlorvos (0.1 mM),organophosphate insecticides, decrease heart rate, spine lengthand free-swimming activity of Xenopus (Bonfanti et al., 2004;Watson et al., 2014). Pyrethroid insecticide activates dopaminergic,noradrenergic, and serotonergic neurotransmitter systems, leading

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to interference in metamorphic development (Li et al., 2020). Theherbicide atrazine, one of the most commonly applied pesticidesworldwide, affects the growth of X. laevis and may cause repro-ductive toxicity through the disruption of tight junctions andmetabolism-related signaling pathways in germ cells and turnmalefrogs into female frogs, some of which are able to mate and produceviable eggs (Carr et al., 2003; Chen et al., 2015; Hayes et al., 2010;Sai et al., 2020). However, the other studies have reported contro-versial results that atrazine in 10 ppb causes no adverse effects onmetamorphosis but affects sex differentiation by inducing femini-zation using all-ZZ-male cohorts (Oka et al., 2008). The discrepancyof these results may result from different water quality, aquariaenvironment and exposure conditions. Moreover, the most severetoxic effects of benomyl fungicide were observed in the nerve tis-sues of the Xenopus embryo, inducing deficits in neuronal prolif-eration and migration (Yoon et al., 2003). And these detrimentaleffects on the central nervous system may eventually result indeficits in swimming activity and visually mediated avoidancebehaviors (Spawn and Aizenman, 2012; Zhang et al., 2020).Furthermore, pesticides may produce sex-linked differences intoxicokinetics between male and female frogs. For instance, tri-adimefon and triadimenol exposure induces more antioxidantenzyme activities in females than in males, which result in moredamaged liver histology and thyroid hormone levels in males thanin females (Zhang et al., 2020). These studies suggest that pesticidesmay have a direct causal relationship with amphibian sex ratio andpopulation decline worldwide (Mann et al., 2009). Nevertheless,the convenience of monitoring chemical toxicity by bath exposureto Xenopus is limited by the fact that environmental exposure tohuman is mainly through ingestion or inhalation. And the detri-mental effects of chemical toxicants on embryonic tissues or organsmay not apply to adult organisms, which should be cautiouslyrelating these findings to human health.

3.3. Plastics and nanoparticles

A growing number of studies have highlighted that contami-nation from diverse microplastics (<5 mm in size) negatively af-fects the health status of aquatic species in the seas and oceansworldwide. Xenopus has become one of the most studied andsuitable organisms out of 11 amphibian species for assessing theimpact of nanomaterials on ecotoxicity on freshwater ecosystemsdue to its extensive use in embryotoxic studies (do Amaral et al.,2019).

The Xenopus system is a valuable model to observe the distri-bution of nanoparticles (NPs) in live tissues/organs and assess thetoxic effects in vivo. Xenopus tadpoles take up microplastics anddebris through the digestive tract from the surrounding water (DeFelice et al., 2018; Hu et al., 2018; Mantecca et al., 2007), whichcould decrease the food taking by absorbing more polystyreneparticles (Hu et al., 2016). Engineered aluminum oxide nano-particles Al2O3 NPs are developmentally toxic and teratogenic toX. laevis and D. rerio, shown as decreased embryo length, inducedorgan malformations, and altered heart-and liver-specific genesexpression (Ismail et al., 2019). Quantum dots (QDs) and multi-walled carbon nanotubes (MWCNTs) have lower toxic effects onembryo survival and phenotypes, which were found in the gills,lung, and intestine (Galdiero et al., 2017; Zhao et al., 2020). Unlikethe experimental limitation of cell cultures and mammalianmodels, Xenopus embryos can evaluate NP biointeractions at theintestinal barrier to determine the safety of NPs (Bacchetta et al.,2014; Bonfanti et al., 2015, 2020; Colombo et al., 2017). Forexample, positively charged silver NPs induced severe effects onembryo development by disrupting embryo tissues of the intestine(Colombo et al., 2017). Therefore, the use of X. laevis as an in vivo

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toxicity model bridges the gap between in vitro culture assays androdent models, which allows for temporal control over the expo-sure to NPs at critical stages of embryogenesis, such as gastrulationand neurulation (Marin-Barba et al., 2018). Furthermore, Xenopusphenotypic scores with exposure to magnetite-cored NPs arehighly correlatedwith cell based in vitro assays as well as the test inmice, indicating that the Xenopus system provides a rapid andcheap identification of NP-induced toxicities (Webster et al., 2016).Nonetheless, the ability of tadpoles for taking up plastics and par-ticles is under the detection limit of the environmental particle size.The relevance between human risk assessment and detrimentaleffects on Xenopus tissues should be also carefully considered.

The toxic effects of NPs on TH-mediated amphibian meta-morphosis are dependent on the concentrations and exposuretime. Exposure to lower concentrations of CuO NPs stimulatesmetamorphosis and growth, whereas chronic exposure to 0.3 mg/LCuO elicits significant mortality and affects the rate of meta-morphosis (Nations et al., 2011, 2015). Environmentally-relevantlow concentrations (0.018 mg/L) of nanosilver particles (AgNPs)had developmental stage-specific endocrine disrupting effectsduring TH-dependent metamorphosis (Carew et al., 2015). Inparticular, microinjections of nanomaterials dispersed with Plur-onic F127 into one-to-two cell Xenopus embryos results in an activeexpulsion of nanomaterials due to the changes in membrane ac-tivity by Pluronic F127 (Holt et al., 2016). Thus, studies on X. laevisembryos allow us not only to better understand the toxicity,localization, and delivery efficacy of NPs but also to screen efficientreagents to react against the toxicity of NPs.

3.4. Endocrine-disrupting chemicals

Thyroid hormone regulates multiple aspects of neurogenesis,metamorphosis and metabolism in the early and later stages ofembryo development (Das et al., 2002; Havis et al., 2006; Marsh-Armstrong et al., 2004; Thompson and Cline, 2016). Abnormalthyrotropin-releasing hormone (TRH) secretion leads to thyroiddysfunction, which eventually leads to abnormal cell proliferationand differentiation, neuroendocrine disruption, and diseases(Wang et al., 2017). Xenopus has emerged as a powerful animalmodel for better understanding hormone balance and function dueto its high sensitivity to human reproductive hormones and thyroidhormone (TH)-dependent metamorphosis (Buchholz, 2017).

Endocrine-disrupting chemicals (EDCs) or endocrine disruptors(EDs) are exogenous chemicals or mixtures that attack the endo-crine system by interfering with endogenous hormones andconsequently cause adverse effects in reproductive developmentand sex differentiation. Some well-known EDCs, such as poly-chlorinated biphenyls (PCBs), atrazine, bisphenol A (BPA), anddibutyl phthalate (DBP), can cause feminizing effects on amphib-ians (Li et al., 2016). BPA, a major component of polycarbonateplastics, also induces various morphologic aberrations, includingscoliosis, eye dysplasia, otolith malformations and loss of pigmentsin the X. laevis tadpoles (Baba et al., 2009; Fini et al., 2009; Gibertet al., 2011; Tamschick et al., 2016). Flame retardant tetra-bromobisphenol A (TBBPA), a known TH signaling disruptor, showsvarious of neurotoxic, immunotoxic, reproductive toxic, and nu-clear receptor-mediated disruptive activities (Colnot et al., 2014;Schriks et al., 2006; Tamschick et al., 2016; Yu et al., 2019). FemaleX. tropicalis exposed to benzopyrene or triclosan EDCs at 50 ng/Ldisplayed a prediabetes state, leading to delayed metamorphosis,smaller individuals and reduced reproduction (Regnault et al.,2018).

Several studies have exerted their efforts to decipher the un-derlying mechanism for the signaling targets of EDCs. A relativelylow concentration of BPA (10�7 M) acts as an antagonist of T3

J. Gao and W. Shen Environmental Pollution 268 (2021) 115809

through suppression of TRa and TRb expression in Xenopus tailcultures (Iwamuro et al., 2006). Two EDC biocides, tributyltin (TBT)and triphenyltin (TPT), trigger severe malformations with defectiveeyes in X. tropicalis embryos by activation of the peroxisome pro-liferator activated receptor g (PPARg) (Yuan et al., 2011; Zhu et al.,2018). A recent study has shown that the environmental concen-tration of TBBPA affects intestinal development by altering cellproliferation and differentiation through Notch signaling (Zhuet al., 2020). Thus, Xenopus is considered an ideal model organ-ism for screening endocrine disruptors and understanding theintrinsic signaling pathways (Kloas et al., 1999).

3.5. Inhibitors and drugs

Xenopus has also been used to test the effects of chemicalcompounds or known reagents in treating tissues and organs,making Xenopus an excellent model to predict drug-inducedtoxicity (Maia et al., 2017). Rapamycin is commonly used as anantifungal agent, whereas it can cause developmental delay,pigmentation defects and gastrointestinal malformation in Xenopusembryogenesis (Moriyama et al., 2011). Valproic acid (VPA), ageneral histone deacetylase (HDAC) inhibitor, is one of the mostefficient antiepileptic drugs. However, it can induce neuro-developmental deficits and affect stem cell homeostasis (Gao et al.,2016b, 2020; Guo et al., 2015; James et al., 2015; Tao et al., 2015).Several other studies have tested the impacts of anti-toxic reagentson Xenopus embryos in addition to their protective effects. PXinduced deficits in the developing Xenopus tectum can be rescuedby GA, which is also beneficial to neural function and tadpolebehavior (Gao et al., 2016a; Liao et al., 2020). The effect of L-cysteinecan protect against the toxicity of acrylamide and furan, whereas itshows delayed embryo hatching within the first 24 h (Williamset al., 2014).

Currently, researchers tend to use Xenopus embryos for drugsafety assessment before performing expensive preclinical trials inmammals, such as antimalarial drug dihydroartemisinin (Longoet al., 2008; Richards and Cole, 2006; Wheeler and Brandli,2009). Decrease in glutathione levels by paracetamol, an antipy-retic and analgesic drug, has been used as an indicator ofparacetamol-induced liver injury, which helps predict the hep-atoxicity of the drug at early stages (Saide et al., 2019; Saide andWheeler, 2020). Celecoxib is a nonsteroidal anti-inflammatorydrug that is used to treat pain, fever, and inflammation by selec-tively inhibiting COX-2. However, celecoxib exposure inducesvarious malformations in Xenopus embryos as well as in humanumbilical vein endothelial cells, indicating its toxic and teratogeniceffects in developing vertebrates (Yoon et al., 2018). Ketamine, anoncompetitive inhibitor of NMDA receptors, is a commonly usedclinical anesthetic and sedative in humans. Studies have found thatketamine exposure has teratogenic effects on Xenopus embryoswith heart enlargement and ventricular shortening fraction (Guoet al., 2016). Waterborne antidepressants (amitriptyline, venlafax-ine, and sertraline) impact mRNA expression of genes related toheart, eye, brain, and bone development at environmentally rele-vant concentrations without changing Xenopus mortality(Sehonova et al., 2019). These studies provide an excellent oppor-tunity for in vivo assessing drug toxicity and screening potentialdrugs for the treatment of toxicant-induced diseases.

4. Disease modeling in Xenopus

Xenopus has served as a powerful tool for understanding humanphysiology in neurodevelopmental and biomedical research (Prattand Khakhalin, 2013; Tandon et al., 2017). Recent methodologicaladvances have allowed us to investigate genetic diseases in organ-

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specific contexts in Xenopus, leading to a further understanding ofthe mechanisms of human diseases (Hwang et al., 2019). In the pastfew years, CRISPR/Cas9 technology has been available for screeningcandidate disease genes and create patient-specific mutations forhuman disease, including congenital heart diseases (CHD), Holt-Oram syndrome, oculocutaneous albinism type 1A, and cataractin Xenopus (Aslan et al., 2017; Bhattacharya et al., 2015; Sakaneet al., 2018; Shi et al., 2019; Viet et al., 2020). Recently, next-generation sequencing technologies have allowed researchers toidentify putative disease-causing patient variants. Using Xenopus asa model system for studying human disorders, such asWolfeHirschhorn syndrome (WHS), ciliopathies and airway dis-eases, is beneficial in understanding the mechanism of develop-mental diseases (Lasser et al., 2019; Walentek and Quigley, 2017).

More evidence has shown that environmental factors havebecome a major cause for some human diseases (Table 2). Alcoholexposure during pregnancy results in developmental defects, calledFASDs (Fainsod and Kot-Leibovich, 2018). Tumor necrosis factor a(TNF-a), a pro-inflammatory cytokine, increases the susceptibilityto pentylenetetrazol-induced seizures (Lee et al., 2010). Whilechemicals alone are sufficient to induce some disease phenotypes,environmental factors in tandem with other underlying geneticmechanisms may lead to varying degrees of severity in these dis-orders. By adopting these toxicological and developmental ap-proaches to connect environmental factors with candidate genes,we may be better equipped to determine the roles of genes in pa-tient diseases. In the following section, we mainly discuss recentstudies on chemical-induced diseasemodeling in Xenopus, focusingon the understanding of disease mechanisms and drug screening(Table 2).

4.1. Fetal alcohol spectrum disorders (FASD)

Accumulating evidence has shown that alcohol is detrimental totissue and organ development. Prenatal alcohol exposure results inFASDs, which show a series of developmental abnormalities andbirth defects (Lichtig et al., 2020; Yelin et al., 2005, 2007). Ethanolexposure in Xenopus induces similar outcomes as in humans, suchas craniofacial malformations, growth retardation, and centralnervous system deficiencies (Dubey and Saint-Jeannet, 2017;Fainsod and Kot-Leibovich, 2018; Peng et al., 2005; Shukrun et al.,2019). Frog embryos, along with those of zebrafish, have beenused to demonstrate the molecular and biochemical understandingof the etiology and pathophysiology of FASDs (Fernandes et al.,2018).

Several studies have reported that some compounds couldrescue alcohol exposure-induced defects in the developing tad-poles. Catalase and peroxiredoxin 5 can protect Xenopus embryosagainst alcohol-induced ocular malformation (Peng et al., 2004).Ethanol-induced developmental malformations can be rescued byexposure to retinol/retinaldehyde (Yelin et al., 2005) or 5-methyltetrahydrofolate (5-MTHF), the most bioactive form of folicacid, which could be beneficial for treating FASDs (Shi et al., 2014).Further research showed that delayed growth and microencephalyinduced by ethanol could be reversed by vitamin C treatmentthrough NF-kB inactivation and ROS inhibition (Peng et al., 2005).These findings indicate that Xenopus offers many tools to elucidatethe etiology of FAS-associated anomalies, which can help to identifymolecular targets for therapeutic strategies.

4.2. Seizure/epilepsy

During brain development, interruption of the excitation-to-inhibition balance will induce neurological disorders, such as epi-lepsy and autism (Preud’homme et al., 2015; Wang et al., 2018).

Table 2Selected chemical-induced diseases studied in Xenopus.

Diseases Chemicals Observations Studies in Xenopus

ASD VPA; TNF-a Increases excitation and decreasesexcitability

(Fainsod and Kot-Leibovich, 2018); (Faulkner et al., 2015);(James et al., 2015); (Lee et al., 2010)

Craniofacialdisorder

Thioridazine; ethanol; BPA, Bisphenol AF(BPAF), DBP; cyclopamine; aerosol mixtures;triadimefon

Craniofacial malformations (Arancio et al., 2019); (Deniz et al., 2017); (Dubey and Saint-Jeannet, 2017); (Groppelli et al., 2005); (Pinet et al., 2019)

Epilepsy/seizure

PTZ, methylisothiazolinone (MIT); TNF-a; kainicacid; bicuculline; picrotoxin; 4-aminopyridine;pilocarpine

Increases seizure activity andsusceptibility

(Bell et al., 2011); (Hewapathirane et al., 2008); (James et al.,2015); (Lee et al., 2010); (Spawn and Aizenman, 2012)

FASD Alcohol Short stature, microcephally and facialdysmorphogenesis

(Kot-Leibovich and Fainsod, 2009); (Shi et al., 2014); (Shukrunet al., 2019); (Yelin et al., 2005); (Yelin et al., 2007)

Metabolicsyndrome

benzo(a)pyrene or/and triclosan; retinoic acid; Induces prediabetes state (Regnault et al., 2018); (Usal et al., 2019); (Zhang et al., 2020):(Zhu et al., 2017)

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Epilepsy is a common chronic central nervous system disease withclinical symptoms manifesting as repeated involuntary episodes.Seizures often cause aberrant and excessive neural activity, andmany antiepileptic drugs, such as phenobarbital, are intended topromote the release of GABA (g-aminobutyric acid) or enhanceGABA conduction (Bialer and White, 2010). Antiepileptic drugsprotect against seizures by regulating neuronal excitability throughNaþ and Ca2þ channels and GABAergic and glutamatergic neuro-transmission. The effects of various antiepileptic drugs wereexamined by using a Xenopus oocyte expression assay. Ethosux-imide selectively inhibits G protein-coupled inwardly rectifying Kþ

(GIRK) channels (Kobayashi et al., 2009). Carbamazepine (CBZ)produces a dose-dependent potentiation of the GABA current at aphysiological concentration in Xenopus oocytes expressing recom-binant GABAA receptors (Liu et al., 2006). Decanoic acid has beenfound to inhibit AMPAR-mediated excitatory neurotransmission,contributing to the anticonvulsant effect in Xenopus oocytes (Changet al., 2016). The oocyte screening system could facilitate researchon epilepsy mechanisms and therapeutic drug targets.

The members of the Hass lab have characterized an in vivoXenopus seizure model with bath application of several commonchemoconvulsants including pentylenetetrazol (PTZ), kainic acid,bicuculline, picrotoxin, 4-aminopyridine, or pilocarpine(Hewapathirane et al., 2008). PTZ-induced seizures in tadpoles aresimilar to the progressive nature of behavioral seizures and exhibitsimilar spike frequencies in zebrafish and rodent models, whichindicates that Xenopus is a powerful animal model for directlymonitoring seizure activity and studying the effects of seizures onneural function (James et al., 2015). VPA is a commonly prescribedantiepileptic drug with known teratogenic effects during earlydevelopment at the synaptic, cellular, circuit, and behavioral levels(Romoli et al., 2019). While tadpoles exposed to VPA have abnormalsensorimotor behaviors and increased seizure susceptibility (Jameset al., 2015), the delayed seizure onset times are due to the con-version of putrescine into GABA, which increase the frequency ofspontaneous GABAergic inhibitory postsynaptic currents. Onestudy showed that long-term developmental exposure to TNF-aresults in enhanced spontaneous excitatory synaptic transmission,increased AMPA/NMDA ratios, and decreased immature synapsesin tectal neurons, leading to increased incidences of epilepsy (Leeet al., 2010). The polyamines induced by a second PTZ exposurecan play a neuroprotective role in the developing brain (Bell et al.,2011). These findings will help researchers build a convenient andefficient Xenopus model for better understanding of the etiologyand pathology of seizure.

4.3. Autism spectrum disorder

Autism spectrum disorder (ASD) is a prevalent

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neurodevelopmental disorder that is characterized by deficits inverbal communication, impairments in social interests and ste-reotyped behaviors (Huguet et al., 2013). Environmental factorssuch as mercury may contribute to the process of ASD and theoccurrence of epilepsy (Bozzi et al., 2018), while prenatal exposureto VPA is associated with an increased risk of ASD (Wang et al.,2018). VPA-induced changes in synaptic function, circuit forma-tion and behavior outcomes are remarkably conserved betweenvertebrate species and Xenopus models (James et al., 2015; Roulletet al., 2013). Therefore, we may take advantages of Xenopus modelto expand the understanding of the basic cellular and circuit levelchanges induced by VPA in vivo, which is difficult to be performedin other model species.

Fragile X syndrome (FXS) represents a known monogenic genecause of ASD. Lines of evidence have shown that FMR1 is highlyconserved between fruit flies, fish, amphibians, and humans (Limet al., 2005). FMR1 mRNA and protein are developmentally upre-gulated throughout Xenopus embryonic development in the optictectum (Faulkner et al., 2015; Lim et al., 2005). Loss of fragile Xmental retardation protein (FMRP) leads to a decrease in neuronalproliferation and survival but an increase in neuronal differentia-tion and deficient dendritic arbor elaboration during Xenopus em-bryonic development (Faulkner et al., 2015). FMRP knockdownusing morpholino oligos in the optic tectum results in decreasedswimming behavior and increased local protein synthesis, sug-gesting that changes at the cellular level are sufficient to cause abehavioral phenotype by enhancing synaptic inhibition (Liu andCline, 2016; Truszkowski et al., 2016). These studies demonstratepromise in the use of Xenopus as an in vivo animal model to identifythe fundamental mechanism of FXS and ASD across animal species.

4.4. Cardiovascular disease

Several studies have found that environmentally relevant con-centrations of chemicals are correlated to the development andprogress of cardiovascular diseases. The severe contamination withhigher PM10, which mainly consists of tire debris, causes cardio-vascular diseases and respiratory diseases (Mantecca et al., 2007).Most chemical compounds exert their toxic effects on animalhearts, including atrazine (Asouzu Johnson et al., 2019), ketamine(Guo et al., 2016), anti-cancer drugs (Isidori et al., 2016) andorganophosphate pesticides (Watson et al., 2014). The otherchemicals cause detrimental impacts on both vasculature andhearts, such as celecoxib, leading to haemorrhage and oedemaduring Xenopus development (Yoon et al., 2018).

Congenital heart disease (CHD) is a common birth defect withhigh rates of morbidity and mortality. CHD modeling is the mostfrequently annotated human disease ontology (DO) term in thecardiovascular disease category in the Xenbase database (Fortriede

J. Gao and W. Shen Environmental Pollution 268 (2021) 115809

et al., 2020; Nenni et al., 2019). The functions of CHD-linked genes,such as galnt11, nek2, tbx20 and bcor, have been well studied inXenopus as an efficient human disease model (Duncan and Khokha,2016). Other cardiovascular disease-related conditions, includinghypertension, long QT syndrome and atrial fibrillation, have beenthoroughly investigated by expressing cardiac channel proteins inXenopus oocytes (Hoppler and Conlon, 2019; Steffensen et al.,2015). Hence, the fast and efficient Xenopus model system cancomplement the established mouse and zebrafish model, and thusto further understand the pathophysiology underlying CHD andscreen potential drugs.

5. Future perspectives

The Xenopus model organism, commonly used in risk assess-ment and life science research, is of great significance for exploringthe mechanisms of embryo development and revealing the toxic-ities of environmental factors and related diseases. This reviewsummarized the main findings in taking advantage of Xenopus as avertebrate model to explore embryo development, neuro-developmental toxicity, and human disease modeling. Studying theeffects of chemical compounds on Xenopus tissues/organs,including those on the heart, brain, spinal cord, kidneys, intestine,eyes, and so on, will allow us to unravel the mechanisms of diseaseetiology and pathogenesis. It is surely that the usage of Xenopusoocytes and embryos wouldmake complementary advantages withthe common mouse and zebrafish model to contribute the basicscience research and biomedical application. Next steps will gathermore potential association between chemical pollutants and dis-eases, which can be used to underpin research on the relatedmechanisms of exogenous factor-induced diseases. To furthercombine with the methodological advances of genome editing,proteomics, and sequencing, the Xenopus systemwill also facilitateresearch on the pharmacological screening of potential pharma-ceutical drugs to treat chemical pollutant-induced deficits anddiseases (Sater and Moody, 2017; Tandon et al., 2017). We also lookforwardmore in-depth studies that use Xenopus systems to developmore human disease models for unveiling the functionality andmechanistic basis of environmental factors related diseases(Vismara et al., 2001). Thus, the unique advantage of Xenopus sys-tem has rightfully earned a place in assessing chemical toxicity andhuman disease modeling.

Author contributions

All authors listed havemade a substantial, direct and intellectualcontribution to the work, and approved it for publication.

Declaration of competing interest

The authors declare that they have no known competingfinancial interests or personal relationships that could haveappeared to influence the work reported in this paper.

Acknowledgements

This work was supported by the National Nature SciencesFoundation of China (NSFC 31871041 to WS).

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