Endogenous Metabolite
Activates G protein-coupled receptors (GPCRs), leading to intracellular signaling [1]

Incubation with Bax inhibiting peptide and deletion of the Bax gene partially protected retinal cells from atRAL toxicity in cultured neural retina. Necrosis was demonstrated not to be the main pathway in atRAL mediated cell death. Bcl-2-interacting mediator and Bcl-2 expression levels were not altered by atRAL in vitro. atRAL-induced oxidative stress results in DNA damage leading to the activation of Bax by phosphorylated p53 [1].

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Incubation of human ARPE-19 retinal pigment epithelial cells with all-trans-retinal at concentrations of 10, 20, and 30 µM for 16 hours caused a dose-dependent decrease in cell viability, as assessed by lactate dehydrogenase (LDH) assay and morphological disruption[1]
In ARPE-19 cells, all-trans-retinal (30 µM) induced a rapid increase in intracellular calcium concentration ([Ca²⁺]i) within 1 minute, which was sustained for at least 10 minutes[1]
Reactive oxygen species (ROS) generation was detected in ARPE-19 cells 10 minutes after incubation with 30 µM all-trans-retinal, reaching a plateau at 15 minutes[1]
Activation of the pro-apoptotic protein Bax was detected in ARPE-19 cells 10 minutes after incubation with 30 µM all-trans-retinal, reaching a plateau at 1 hour. This activation was attenuated by pretreatment with Bax inhibiting peptide (BIP)[1]
DNA damage, indicated by 8-hydroxy-2′-deoxyguanosine (8-OHdG) accumulation, was detected in the cytoplasm of ARPE-19 cells 30 minutes after incubation with 30 µM all-trans-retinal, with signal intensity increasing in a dose-dependent manner[1]
Phosphorylation of p53 at Ser46 was increased in both the cytoplasm and nucleus of ARPE-19 cells 30 minutes after incubation with 30 µM all-trans-retinal. This phosphorylated p53 co-localized with mitochondria[1]
A luciferase reporter assay showed that p53 gene expression levels in transfected ARPE-19 cells increased robustly until 10 minutes after incubation with 10 or 30 µM all-trans-retinal, plateauing around 30 minutes[1]
Immunoblot analysis showed increased phosphorylation levels of ERK1/2 and p38 in ARPE-19 cells treated with 30 µM all-trans-retinal[1]
The p53 inhibitor pifithrin-α decreased all-trans-retinal-induced Bax activation by more than 50% and increased cell viability in ARPE-19 cells in a dose-dependent manner[1]
The 661W photoreceptor cell line showed decreased viability to 51.1 ± 2.4% after 2 hours of incubation with all-trans-retinal, while ARPE-19 cell viability was 89.3 ± 2.9% after 4 hours of incubation with 30 µM all-trans-retinal[1]
The expression levels of Bcl-2-interacting mediator (Bim) and Bcl-2 did not change in ARPE-19 cells 60 minutes after all-trans-retinal treatment[1]
The necroptosis inhibitor Necrostatin-1 (Nec-1) did not show protective effects against all-trans-retinal-induced cell death in ARPE-19 cells or cultured mouse neural retinas, whereas BIP showed dose-dependent protection[1]

Other Bax-related cellular events were also evaluated by pharmacological and biochemical methods. Production of 8-OHdG, a DNA damage indicator, and the phosphorylation of p53 at Ser46 were detected prior to Bax activation in ARPE-19 cells incubated with atRAL. Light exposure to Abca4(-/-)Rdh8(-/-) mice also caused the above mentioned events in conditions of short term intense light exposure and regular room lighting conditions [1].

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In Abca4⁻/⁻ Rdh8⁻/⁻ double-knockout mice (a model of retinal degeneration with impaired all-trans-retinal clearance), exposure to intense light (10,000 lux for 30 minutes) caused a decrease in outer nuclear layer thickness observed by spectral-domain optical coherence tomography (SD-OCT) 1 hour post-exposure[1]
In light-exposed Abca4⁻/⁻ Rdh8⁻/⁻ mice, increased signals of 8-OHdG (DNA damage), phosphorylated p53 at Ser46, and activated Bax were observed in the outer nuclear layer and inner segments of the retina. These signals were not obvious in light-exposed wild-type mice or non-exposed Abca4⁻/⁻ Rdh8⁻/⁻ mice[1]
Similar molecular events (DNA damage, p53 phosphorylation, Bax activation) were observed in 6-month-old Abca4⁻/⁻ Rdh8⁻/⁻ mice kept under regular room lighting, though at milder levels compared to intense light exposure[1]

Lactate Dehydrogenase (LDH) Colorimetric Assay
LDH measurements were conducted using LDH-Cytotoxicity Colorimetric Assay Kit II (BioVision, Milpitas, CA) on cultured ARPE-19 cells and 661W cells in 96 well plates (104 cells/ well) following manufacturer’s instructions. Optical density values from the 96-well plate at 450 nm wavelength were measured with a microplate reader [1].

WST-1 assay [1]
WST-1 assay was conducted with Cell Proliferation Reagent WST-1 on ARPE-19 cells cultured in 96-well plates (1 × 104 cells/ well) following manufacturer’s instructions. Optical density values from the 96-well plate at 450 nm wavelength were measured with a microplate reader.

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In vitro detection of Bax activation[1]
ARPE-19 cells in 24 well cell plates (1 × 105 cells/well) were treated with atRAL at a final concentration of 10 – 30 μM in the cell culture medium for the indicated time with or without five mar Bax inhibiting peptide, BIP. Cells were washed in cold PBS and kept on ice after incubation. Cells were then fixed for 30 min with 4% paraformaldehyde prior to permeabilization with 0.5% Triton X at room temperature. Bax activation was detected via immunocytochemistry (ICC) with anti-Bax (6A7), a mouse monoclonal antibody at a dilution of 1:100. Cy-3 conjugated anti-mouse IgG antibody was the secondary antibody and was used at a dilution of 1:200. The signal intensity of activated Bax was monitored with an inverted fluorescence microscope.

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In vitro detection of phosphorylated p53 at Ser46[1]
ARPE-19 cells were cultured in 24 well plates (1 × 105 cells/well) and atRAL was added at a final concentration of 30 μM then incubated for 30 min. Phosphorylation of p53 at Ser46 was detected by ICC with anti-phosphorylated p53 (Ser46) specific antibody (rabbit polyclonal, at 1:50 dilution). Cy-3 conjugated anti-rabbit IgG antibody was used as the secondary antibody. Signal intensity measurements of phosphorylated p53 were also performed.

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In vitro detection of 8-OHdG[1]
ARPE-19 cells in 24 well plates (1 × 105 cells/well) were incubated with atRAL at the indicated concentration (10 – 30 μM) for 30 min. The marker of DNA injury by oxidative stress, 8-hydroxyhydroguanidine (8-OHdG) was monitored using ICC. Cells were incubated for 1 h with anti-8-OHdG antibody at a concentration of 10 μg/ml. Cy-3 conjugated anti-mouse IgG antibody was used as the secondary antibody.

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Mitochondria-selective Staining[1]
ARPE-19 cells in 96 well plates (1 × 104 cells/well) were incubated with Mitochondrion-Selective Probes, MitoTracker Orange CMTMRos at 100 nM for 30 min.

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Cell Viability Assay (LDH): ARPE-19 or 661W cells were seeded in 96-well plates at 1x10⁴ cells per well. After treatment with all-trans-retinal, cytotoxicity was measured using a colorimetric LDH assay kit according to the manufacturer's instructions. Optical density was measured at 450 nm with a microplate reader[1]
Cell Viability Assay (WST-1): ARPE-19 cells were seeded in 96-well plates at 1x10⁴ cells per well. After treatment, cell viability was measured using a WST-1 cell proliferation reagent according to the manufacturer's instructions. Optical density was measured at 450 nm[1]
Detection of Activated Bax by Immunocytochemistry (ICC): ARPE-19 cells in 24-well plates (1x10⁵ cells/well) were treated with all-trans-retinal (10-30 µM) for indicated times. Cells were fixed with 4% paraformaldehyde, permeabilized with 0.5% Triton X-100, and incubated with a mouse monoclonal anti-Bax (6A7) antibody (1:100 dilution). A Cy3-conjugated anti-mouse IgG antibody was used as the secondary. Signal intensity was monitored with a fluorescence microscope[1]
Detection of Phosphorylated p53 (Ser46) by ICC: ARPE-19 cells in 24-well plates were treated with 30 µM all-trans-retinal for 30 minutes. Cells were fixed and stained with a rabbit polyclonal anti-phosphorylated p53 (Ser46) antibody (1:50 dilution), followed by a Cy3-conjugated anti-rabbit IgG secondary antibody[1]
Detection of DNA Damage (8-OHdG) by ICC: ARPE-19 cells in 24-well plates were incubated with all-trans-retinal (10-30 µM) for 30 minutes. Cells were incubated with an anti-8-OHdG antibody (10 µg/mL) for 1 hour, followed by a Cy3-conjugated anti-mouse IgG secondary antibody[1]
Mitochondrial Staining: ARPE-19 cells in 96-well plates were incubated with 100 nM MitoTracker Orange CMTMRos for 30 minutes to stain mitochondria[1]
Luciferase Reporter Assay for p53 Expression: ARPE-19 cells in 96-well plates were transfected with a pF5A [CMV/p53-Nluc/Neo] vector using a lipid-based transfection reagent. After transfection, cells were treated with all-trans-retinal (10 or 30 µM) for various durations (5 to 60 minutes). Luciferase activity was measured using a commercial assay system, and luminescence was read with a microplate reader[1]
Immunoblotting: Cell extracts were prepared using ice-cold lysis buffer containing protease inhibitors. Proteins were separated by SDS-PAGE, transferred to a membrane, and probed with primary antibodies against Bim, Bcl-2, phospho-ERK1/2, and phospho-p38. Secondary antibodies conjugated with alkaline phosphatase were used for detection. β-actin or β-tubulin served as loading controls[1]

Animals[1]
Abca4 −/−Rdh8−/− mice and Bax−/− mice were generated and genotyped as previously described (Maeda et al., 2008). C57BL/6J mice were purchased from Jackson Laboratory (Bar Harbor Maine). All mice in this study were housed in the animal facility at the School of Medicine, Case Western Reserve University, where they were regularly maintained in a 12 h light (~10 lux) /12 h dark cycle environment. All animal procedures and experiments were approved by the Case Western Reserve University Animal Care Committees and conformed to both the recommendations of the American Veterinary Medical Association Panel on Euthanasia and the Association of Research for Vision and Ophthalmology.

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Induction of light damage[1]
Light damage was induced in mice by fluorescent light exposure at 10,000 lux (150 W spiral lamps, Commercial Electric) for 30 min in a white bucket as previously described (Maeda et al., 2012). The mice were dark-adapted overnight and pupils were dilated with 1% tropicamide eye solution in prior to light exposure.

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Measurement of spectral-domain optical coherence tomography (SD-OCT)[1]
SD-OCT was employed for in vivo retinal imaging of mice as previously described

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Mouse Model: Abca4⁻/⁻ Rdh8⁻/⁻ double-knockout mice and wild-type (C57BL/6J) mice were used[1]
Light Damage Induction: Mice were dark-adapted overnight, and pupils were dilated with 1% tropicamide. They were then exposed to fluorescent light at 10,000 lux for 30 minutes in a white bucket[1]
In Vivo Imaging: Retinal morphology was assessed in live mice using spectral-domain optical coherence tomography (SD-OCT)[1]
Tissue Collection and Analysis: After light exposure or at specified ages, mice were sacrificed. Retinas were harvested, embedded, and sectioned for immunohistochemical analysis[1]
Ex Vivo Cultured Neural Retina Assay: Neural retinas were dissected from 6-week-old mice. Eye cups were prepared, and the neural retina was carefully separated from the RPE/choroid using a piece of filter paper. Each retina on the paper was placed in a well of a 12-well plate with culture medium and incubated. Retinas were then treated with or without 30 µM all-trans-retinal for 6 hours. Retinal cell death was quantified using an LDH assay[1]

Metabolism / Metabolites
Retinaldehyde's known human metabolites include retinoic acid. Retinaldehyde is a known human metabolite of retinol.

[1]. All-trans-retinal induces Bax activation via DNA damage to mediate retinal cell apoptosis. Exp Eye Res. 2014 Jun;123:27-36.

All-trans-retinal is a type of retinal in which all four exocyclic double bonds are in the E-(trans) configuration. It functions as a gap-joint inhibitor of intercellular communication and is a metabolite in humans and mice. It is a type of retinal and also a form of vitamin A. Retinal is found in or produced by Escherichia coli (K12 strain, MG1655 strain). Retinal has also been reported in Corkia, humans, and other organisms with relevant data. Retinal is a diterpenoid compound derived from the carotenoid vitamin A, an active component of the visual cycle. It is the cofactor of rhodopsin (covalently linked to rod cell opsin in the form of 11-cis-retinal). When stimulated by visible light, rhodopsin converts the cis isomer of retinal to the trans isomer (11-trans-retinal). This conversion straightens the bent retinal molecule and causes a change in the shape of rhodopsin, thereby triggering the visual process. A series of energy-intensive enzymatic reactions convert 11-trans-retinal back to the cis isomer. See also: 13-cis-retinal (note moved to). All-trans-retinal is a major byproduct of the visual cycle, produced by photoisomerization of 11-cis-retinal [1]. Accumulation of all-trans-retinal due to impaired clearance (e.g. in Abca4⁻/⁻ Rdh8⁻/⁻ mice) is toxic and can lead to retinal degeneration [1]. The proposed toxicity mechanisms include: 1) activation of GPCRs, leading to increased intracellular calcium ion concentrations via the phospholipase C (PLC)/inositol 1,4,5-triphosphate (IP3) pathway. 2) Increased calcium ion concentrations activate NADPH oxidase, leading to excessive production of reactive oxygen species (ROS). 3) ROS cause DNA damage. 4) DNA damage leads to phosphorylation of p53 at the Ser46 site. 5) Phosphorylated p53 activates Bax, leading to mitochondrial-mediated apoptosis [1]
This study concludes that DNA damage and p53 phosphorylation activation of Bax are the main pathways of all-trans-retinal-mediated retinal cell apoptosis, a process confirmed in vitro and in vivo in mouse models of retinal degeneration [1]

C₂₀H₂₈O

284.44

284.214

C, 84.45; H, 9.92; O, 5.62

116-31-4

638015

Light yellow to yellow solid

0.9±0.1 g/cm3

421.4±14.0 °C at 760 mmHg

61-63°C

205.4±12.4 °C

0.0±1.0 mmHg at 25°C

1.541

Gut microbes

6.54

0

1

5

21

522

0

O=C([H])/C(/[H])=C(\C([H])([H])[H])/C(/[H])=C(\[H])/C(/[H])=C(\C([H])([H])[H])/C(/[H])=C(\[H])/C1=C(C([H])([H])[H])C([H])([H])C([H])([H])C([H])([H])C1(C([H])([H])[H])C([H])([H])[H]

NCYCYZXNIZJOKI-OVSJKPMPSA-N

InChI=1S/C20H28O/c1-16(8-6-9-17(2)13-15-21)11-12-19-18(3)10-7-14-20(19,4)5/h6,8-9,11-13,15H,7,10,14H2,1-5H3/b9-6+,12-11+,16-8+,17-13+

(2E,4E,6E,8E)-3,7-dimethyl-9-(2,6,6-trimethylcyclohex-1-en-1-yl)nona-2,4,6,8-tetraenal

Retinaldehyde; NSC-626581; Retinal; ARetinal; Vitamin A aldehyde; Retinaldehyde; NSC 122757; NSC626581; NSC122757; NSC-626581; NSC-122757; NSC 626581; all-trans-Retinal; retinene; axerophthal;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: (1). This product requires protection from light (avoid light exposure) during transportation and storage.  (2). Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture.  (3). This product is not stable in solution, please use freshly prepared working solution for optimal results.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~100 mg/mL (~351.57 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (7.31 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: 2.08 mg/mL (7.31 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.08 mg/mL (7.31 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)