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Table 1 Examples of the impacts of retinoic acid signalling effects on morphology, phenotype, and/or development. Explanations and abbreviations are at the end of this table

From: Highlighting the gaps in hazard and risk assessment of unregulated Endocrine Active Substances in surface waters: retinoids as a European case study

Apical Effect/Key mechanism/Endpoint

Morphological alteration level

Developmental stage

Test system (species)

Main relevant and related pathways, genes, enzymes

Endpoint/hallmark affected cross-reactions

Substances associated with key mechanism/adverse outcome

Associated human pathology

Reference(s)

Axial development, Anterior-posterior patterning, osteogenesis

Tissue

Embryo, larvae

Branchiostoma floridae (amphioxus), zebrafish (stocksteif mutant), chicken, mouse

ADH/ RALDH, Cyp26b1, Cyp26a1, RARα, RARβ, RARγ, Wnt, FGF, Hox (esp. 1&3), BMP

RA acts as an early developmental morphogen along the anterior-posterior axis; it coordinates the position of endoderm-derived organs along the anterior-posterior axis. Cyp26/ RA concentration drives the osteogenesis in the vertebral column (in osteoblasts); posteriorization of gills and mouth in invertebrate chordates. Hyperactive RAR induces higher expression levels of RA-metabolizing Cyp26a1 and acts in a paracrine way

RA, retinol

 

[76, 148, 313,314,315,316,317,318,319,320,321,322]

     

Symmetric somite development is mediated by RA

  

[323,324,325]

   

Human, zebrafish, rat, mouse

Dhrs3, Cyp26(a1)

Axial skeletal and craniofacial defects upon exposure

Triazole fungicides (flusilazole, triadimefon)

 

[165, 326]

Neural tube formation

Organ/organism

Embryo

Quail

FGF and Wnt gradients/signaling, CYP26A1, RALDH activity

Mesodermal segmentation, somite formation, and neurogenesis in caudal neural tube (future spinal cord) are RA dependent

RA

 

[73, 315, 327,328,329]

   

Human, zebrafish, rat

Dhrs3, Cyp26(a1)

Neural tube (and axial) defects; proposed Adverse Outcome Pathway: “for neural tube and axial defects mediated by modulation of retinoic acid homeostasis”

Triazole fungicides (flusilazole)

 

[165]

Neural differentiation and spinal cord formation

Tissue/organ

Embryo, (adult)

Mouse, zebrafish, Xenopus

FGF and Wnt, CYP26A1, RALDH, RARβ, RARα, Hox

The nervous system develops sequentially along this axis, starting anteriorly (CNS/brain), continuing via hindbrain to spinal cord. Determination of cell fate and differentiation of ventral neuronal cell types in developing spinal cord. Neurite outgrowth in embryos and adults is dependent on RARβ expression; RARα knockdown abolishes atRA-mediated dendritic growth

RA (endogenous)

 

[76, 196, 314, 327, 330,331,332,333,334,335,336]

Early neural differentiation

Tissue/organ system

Embryo

Mouse

RAR, TR, MCT8

Organization of the (central) nervous system; RA signalling precedes TRα expression/TR signaling; RA induces MCT8 expression in the developing brain allowing TH transport

RA, TH

 

[135, 136]

   

Zebrafish

 

RA co-administration (1 nM) prevents adverse effects (behavioural and histological) of ethanol (150 mM) exposure during gastrulation

RA, ethanol

 

[337]

Hindbrain segmentation, Ear development and hearing recovery

Tissue/organ

Embryo, adult

Mouse, rat, zebrafish, VAD quail model, chicken, Xenopus

RARα, RARβ, (CRABP), SHH, Wnt, FGF, Hox; CYP26A1, CYP26C1, RALDH activity

RA guides the formation of 8 segments (rhombomeres) that give rise to e.g. otic vesicle, sensory tract. RA determines the forebrain-hindbrain and hindbrain-spinal cord boundary (excess leads to posteriorization); midbrain-hindbrain boundary is unaffected (in mouse and Xenopus). RA directly influences the differentiation of branchiomotor neurons (zebrafish)

RA

Congenital hearing loss [338, 339]

[91, 124, 148, 321, 331, 340,341,342,343,344,345,346,347,348,349,350,351,352,353,354,355,356,357,358,359]

   

Mouse, zebrafish

 

Development of the olfactory region requires RA. RA stimulates regeneration of auditory hair cells

  

[360,361,362,363]

       

Fetal alcohol syndrome; cerebellar maldevelopment

[364, 365]

Head and forebrain development; Eye development

Tissue/organ

Embryo

Mouse, VAD quail model, pig, rabbit, cattle, sheep, rat, zebrafish

 

Formation of optic vesicle (retina precursor; invagination of neuroepithelium); micro-/anophthalmia in absence of RA. However: head development in general requires absence of RA [76]

RA

 

[90, 125, 127, 129, 163, 363, 366, 367]

   

Rat, human

RALDH, CYP26, AhR

Craniofacial malformations are linked to RA deregulation

atRA, 13cRA

Cleft palate and lip

[368,369,370,371,372]

Telencephalon differentiation

Tissue

Embryo

Mouse

 

Changed population of ganglia; altered precursor population; RA stimulates production of dopaminergic neurons

RA

 

[373,374,375,376,377]

Cerebral cortex

Tissue

Embryo, postnatal

Mouse

RAR

Influence on neurogenesis/migration/ differentiation in other brain regions/ at other developmental stages. Sensitivity to RA is retained in the mature cortex

RA (endogenous), 13cRA

Affective liability and behavioural disinhibition upon 13cRA treatment, depression [177]

[73, 374, 378,379,380]

Hippocampus, neuronal plasticity

Cell/ tissue

Adult

Mouse, rat, zebra finch

RAR/RXR, esp. RARβ, RXRγ

Defects in spatial learning and memory, and recognition working memory (RXRγ) upon vitamin A deprivation; restoration of cognitive impairment with vitamin A supply; cognitive impairment also in excess RA scenario (13cRA). Decreased ability to learn mating song in zebra finches

RA, vitamin A

Learning and memory impairment, depression

[150,151,152,153, 177, 192, 381,382,383]

Hippocampus

Tissue/organ

Adult

Rat, human (Alzheimer’s disease patients), mouse

Calbindin D28K, neurogranin, somatostatin, CHAT; RARα, RARβ, RXRγ, amyloid pathway, RBP4, CYP26, RALDH2 (in adjacent meninges)

Regulation of memory and spatial learning; RA acts as a proamnesic molecule

Deprivation leads to amyloid-β accumulation, RARα downregulation, CHAT expression loss in forebrain cortical neurons

RA regulates proteins linked to protection from Alzheimer’s disease

Vitamin A

Alzheimer’s disease, ageing

[82, 151, 178, 192, 205, 211, 384,385,386,387,388,389,390,391,392]

Hippocampus

Organ

Postnatal

Mouse, rat

RARs (esp. RARα), RXRs, GR, somatostatin, RALDH2 (in adjacent meninges)

RARβ-/- and RXRγ-/-: deficiency in spatial learning and memory, like VAD rats (rescue by RA treatment). Degradation of hippocampal function in aging mice via proliferation/differentiation of hippocampal stem cells

VAD increases GR binding capacity and modulates the somatostatinergic and acetylcholinergic hippocampal system

Vitamin A, atRA, 13cRA

Memory/learning impairment, dementia, Alzheimer’s disease, depression

[73, 151,152,153, 173, 188, 192, 193, 203, 374, 381, 391, 393,394,395,396,397,398]

   

Rat

 

Hippocampal volume is reduced after 3 weeks of 13cRA treatment

  

[190]

Learning

Organism

Embryo

Rat

RALDH, PC2

Exposure during gestation (day 11–13 in rat) impaired amphetamine-stimulated activity and avoidance learning, but not performance in complex spatial maze or auditory startle response in offspring. A signalling decline is observed in aging and associated with cognitive impairment, decreased acquisition of new memories; reversible by RA administration

atRA, 13cRA, 9cRA

Possibly: affective disorders, neurodegenerative disorders, schizophrenia, autism

[172, 186, 188, 203, 220, 395, 399,400,401]

  

Postnatal

Rat, Mouse

RARβ, RXRγ

Vitamin A deprivation and RARβ-/- mutants show spatial learning and memory impairment

  

[151,152,153]

Behavioural changes

Organism

Adult

Mouse, rat

 

Extended low-dose exposure in mice induced depression-like behaviour. This was partially confirmed in 91 days-old rats, but not in older rats.

13cRA

Depression

[177, 187, 232, 234]

   

Human

D2, (D1), Ser1A

13cRA use/treatment is associated with depression and suicidal behaviour with longer onset (~4–8 weeks; long-term effect).

 

Case studies are reviewed in the reference

[177]

Striatum

Tissue/organ

Adult

Mouse, rat, human

D2, RAR(α)β/RXRβγ; Nurr1/RXR, RALDH1, RALDH3, neurogranin, GAP43

The striatum shows the highest endogenous RA concentrations in the adult brain. Dopaminergic neurons; autocrine action on neurotransmission, paracrine action on striatal cells; locomotor impairment in RAR/RXR and Nurr1/RXR mutant mice. RXRγ-/- mutants: increased despair behaviour, anhedonia (reversible by re-expression of RXRγ). Induction of Parkinsonism and catatonia by lesions in basal ganglia

RA (endogenous), disulphirama

Depression, (potentially: Parkinson’s and Huntington’s disease), mood disorders

[177, 226,227,228,229,230,231, 374, 395, 402,403,404]

     

Extended 13cRA dose (in rat) increases dopamine and serotonin metabolites

  

[187]

GABAergic (inter-)neurons

Cell/ tissue

Embryo/foetus

Mouse

Raldh3

Enhanced differentiation; migration to olfactory bulb and cortex. Raldh3 activity is required for efficient differentiation of GABAergic interneurons, while Raldh2 is not

RA (endogenous)

 

[163, 380]

  

Adult

  

GABAergic interneurons of the olfactory bulb are RA sensitive

  

[405]

Branchial arches

Tissue/organ

Embryo

Mouse

RAR, Hoxa1, Hoxb1, Pax1/9

3rd–6th arch are RA responsive, give rise to: endodermal pouches, thymus, parathyroid glands, aorta and associated large blood vessels, nerves etc. Linked to rhombomeric (hindbrain) origin of mesenchyme/neural crest cells

RA

DiGeorge syndrome, CATCH22 syndrome

[343, 358, 406,407,408,409,410]

Heart development

Organ

Embryo

Mouse, chicken, zebrafish

RARs, RXRs (esp. RXRα), Hoxb1, Hoxb5b, Tbx1, RALDH2, STRA6, CYP26A1, FGF8, NR2F5

Congenital heart disease, incl. conotruncal and aortic arch artery malformations (patterning defects); defects in RA synthesis can be, in some cases, partly rescued by maternal RA levels/RA supplementation. STRA6 mutations (vitamin A transport/ cellular uptake) may result in developmental defects in atrial and venous vessels. Later in development, RA is cardiotoxic (in zebrafish)

RA

Rarely observed/undocumented, maybe due to embryonic death. Matthew-Wood syndrome (STRA6 mutation) [411, 412], DiGeorge syndrome [413]

[89, 138, 140, 164, 196, 369, 414,415,416,417,418,419,420,421,422,423,424,425]

   

Zebrafish, rat

RARα, SHH, AhR

 

Flame retardants (miTP, TPP, PBDE), TCDD

 

[169, 170, 370, 426]

Lung development and regeneration

Tissue/organ

Embryo, adult

Mouse; embryonic explants, rat

RALDH2, Wnt, TGF-β, FGF10, BMP, SHH

Lack of RA/RAR activity prevents induction and growth of primary lung buds; RA induces regeneration of alveoli in rat and rescues lung functionality in experimental hypoplasia. RA is not required for endodermal lung cell fate, but essential for primordial lung bud formation

RA

 

[141,142,143, 427,428,429,430,431,432,433]

    

VEGF, FGF18

RA regulates angiogenesis and elastin production in the maturing lung

  

[434]

Pancreas formation

Tissue/organ

Embryo

Zebrafish, Xenopus, mouse

Cyp26a1, Cdx4, RALDH

Formation of dorsal pancreatic bud (pancreatic and hepatic endoderm); specification of pancreatic endocrine cell lineages. RA is required for ventral pancreas patterning. In mouse and human pancreas, β-cell differentiation may be influenced by RA

RA

 

[148, 334, 435,436,437,438,439,440,441,442]

Kidney formation

Organ

Embryo

Xenopus, zebrafish, mouse

RARα, RARβ, RALDH2, Notch signalling, mecom

Inactivation of RARα and RARβ results in renal malformation (mouse); ureteric bud cell signalling depends mainly on RALDH2-generated atRA. Specification of renal progenitor cells depends on RA signaling (Xenopus, zebrafish)

RA

 

[149, 443,444,445,446,447,448]

     

Competition for RALDH2

Ethanol

 

[449, 450]

Limb (and tail) development and regeneration

Organ

Embryo, postnatal

Amphibians, chicken, mouse, zebrafish

FGF8, SHH, FGF4, RALDH2, Cyp26(b1), Hoxb8

RA can induce development (embryonic) or regeneration (postnatal) of supernumerary limbs or digits, when locally applied to the limb bud. In mice, RA exposure on gestational day 12 (33–41 somite pairs) affected rather forelimbs, on gestational day 13 (40–51 somite pairs) rather hindlimbs. Also, tail and tail vertebrae development is impaired by RA exposure

RA, 9cRA

 

[130, 134, 363, 451,452,453,454,455,456,457,458,459]

Gene expression repression by unliganded receptors (RARs)

Cellular

Embryo

Xenopus, mouse

RAR, NCoR-1/2, SMRT, CYP26. (further: Fgf, Wnt, Hox genes)

Head development in Xenopus and skeletal development in mice requires gene repression by unliganded RAR (otherwise: malformation of the head, anterior/posterior shift)

RA

 

[460,461,462,463]

       

Acute promyelocytic leukemia

[462]

Invertebrate development

Organ/organism

 

Chordata, Arthropoda, Mollusca, Porifera

RAR, RXR, CYP26, RALDH

Conservation of the retinoid signalling pathway and involvement in invertebrate development; incl.: all body, digestive glands, gonads, limb buds, regeneration of body parts

RA, retinol

 

Reviewed in [322]

   

Crustacea, Drosophila

EcR, USP

Ecdysone is the driver of invertebrate molting. EcR dimerizes with USP (RXR-homolog), which increases dimer stability and affinity towards target DNA sequences

Ecdysone, tributyltin

 

[464,465,466,467]

 

Organ

 

Mollusca, Gastropoda

RXR

Imposex (gastropods), shell thickening, reproduction perturbation (Crassostrea gigas); RAR/RXR heterodimers have a repressive function (instead of activating)

Tributyltin, HX630 (only linked to imposex in gastropods), 9cRA

 

[111, 468,469,470]

Reproductive tract development

Organ

 

Molluscs

RXR; potential crosstalk with PPARγ

Induction of outgrowth of male reproductive structures (T. clavigera), regulation of male/female seasonality (reproductive tract recrudescence; Ilyanassa obsolete)

9cRA

 

[111, 144, 322, 469, 471,472,473]

Reproductive organ development, testes; male fertility

Organ/organism

Embryo, adult

Mouse

RARα, RARγ, RXRβ, RALDH2, Cyp26b1, SHH, BMP4, STRA8

Degeneration of testes after knockout of all RARα isoforms; sterility of male mice after knockout of RARγ; male sterility in RXRβ knockout mice. RA is required for spermatogenesis

RA

Sterility in adult men in absence of RA [124, 160]

[13, 76, 145,146,147, 474,475,476,477,478]

   

Mouse, in vitro (P19 and C3H10T1/2), human

RALDH, Cyp26 Hoxa1, HDAC-I, AR (via SHH)

 

Phthalate esters (esp. containing aryl and cyclohexane groups), valproic acid

 

[168, 479,480,481,482]

Peripheral nervous system, regeneration

Organ system

Adult

Human

RARα, RARβ, RBP

Regeneration of spinal cord motor neurons depends on RA-induced RARβ(2) expression; RA peaks 4-7 days after encountering the injury

RA, 9cRA

 

[82, 154,155,156, 158, 483]

Nervous system

Organ system

Adult

Human

RARα, RALDH2, RBP(1)

Motor neurons: neurofilament accumulation, astrocytosis, decrease in neuron number, elimination of RARα and reduction of RALDH2 expression, reduction in retinol binding protein levels in spinal cord

Vitamin A, RA

Amyotrophic lateral sclerosis (ALS)

[82, 484,485,486]

  

Embryo

Rat

 

Gestational exposure (day 11–13) led to difficulty to swallow milk (motor control); delayed righting reflex at 35 days. Decreased locomotor activity, motor coordination, and learning (90 days)

 

Depression, suicidal behaviour [223]

[177, 186, 395, 487,488,489,490]

Vision

 

Pregnancy

Human (pregnant women)

  

Vitamin A

Night blindness; associated with miscarriage

[131]

  

Pre-school age children, adult

Human

 

Retinal is required in the retina

Vitamin A, RA

Blindness; 50% fatal in children within 1 year if untreated; impaired vision

[131, 133, 491]

Keratinization of epithelia

Tissue

Adult, embryo

Human, rat

 

Mucous epithelia (as with the trachea/respiratory/gastrointestinal tract) become keratinized in absence of RA; RA is required for continuous renewal of skin epithelia.

Treatment of cystic and nodular acne with “Accutane” (13cRA)

RA, 13cRA

 

[124, 139, 160, 221, 492]

   

Human (in vitro)

AhR, RA-signaling

TCDD alters matrix protein (esp. collagen) deposition; atRA shows an additive effect. The increased protein deposition is due to promoter activation and increased mRNA stability

TCDD, atRA

 

[29]

Immune function

Organ system

Adult

Human, mouse

RARα

Immune function severely compromised in absence of RA. RA is required for (CD4+) T cells in the thymus

RA

 

[124, 126, 157,158,159,160, 493,494,495,496]

  1. Bold indicates high level of confidence. Gene and protein nomenclature has been adapted to human, though homologous genes/proteins in other species may have been assessed in the original studies
  2. ADH: alcohol dehydrogenase, AR: androgen receptor, BMP: borne morphogenetic protein, 9cRA: 9-cis retinoic acid, 13cRA: 13-cis retinoic acid, Cdx4: Homeobox protein transcription factor, CHAT: choline acetyl transferase, CRABP: cellular retinoic acid-binding protein, Dhrs3: short-chain dehydrogenase/reductase 3, EcR: ecdysone receptor (homologous to vertebrate farnesoid X receptor, FXR, though endogenous ligand is the ecdysone steroid), FGF: fibroblast growth factor, GR: glucocorticoid receptor, HDAC-I: class I histone deacetylase, Hox: Homeobox gene family, HC630: selective RXR agonist, MCT8: monocarboxylate transporter 8, mecom: mds1/evi1 complex transcription factor, miTP: monosubstituted isopropylated triaryl phosphate, NCoR-1/2: nuclear receptor corepressor 1/2, NR2F5: nuclear receptor 2F5 (COUP-transcription factor family), PBDE: polybrominated diphenyl ether, RA: retinoic acid, PPARγ: peroxisome proliferator activator receptor γ, RALDH: retinal dehydrogenase, RAR: retinoic acid receptor, RBP: retinol-binding protein, RXR: retinoid X receptor, Ser1A: serotonin receptor 1A, SHH: sonic hedgehog gene family, SMRT: silencing mediator of RAR and thyroid hormone receptor, TCDD: 2,3,7,8-tetrachlorodibenzo-p-dioxin, TH: thyroid hormone, TPP: triphenylphosphate, TR: thyroid hormone receptor, USP: ultraspiracle (nuclear receptor in Drosophila, homologous to vertebrate RXR), VAD: vitamin A deficiency
  3. aDisulfiram acts a selective antagonist to RALDH1, hence prevents endogenous RA synthesis. Therefore, effects are often reported as RA-dependent rather than disulfiram-sensitive