Cytochrome P450

Both (R) and (S) 11-HETE induced cellular hypertrophic markers and cell surface area in RL-14 cells. Both enantiomers significantly upregulated CYP1B1, CYP1A1, CYP4F2, and CYP4A11 at the mRNA and protein levels, however, the effect of the S-enantiomer was more pronounced. Furthermore, 11(S)-HETE increased the mRNA and protein levels of CYP2J and CYP4F2, whereas 11(R)-HETE increased only CYP4F2. Only 11(S)-HETE significantly increased the catalytic activity of CYP1B1 in recombinant human CYP1B1, suggesting allosteric activation in an enantioselective manner.
Conclusion: Our study provides the first evidence that 11-HETE can induce cellular hypertrophy in RL-14 cells via the increase in CYP1B1 mRNA, protein, and activity levels. [1]

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Effect of (R) and (S) 11-HETE on cell viability [1]
MTT assay was used to assess the cytotoxicity of the 11-HETE concentrations used. RL-14 cells were treated with 2.5, 5, 10, and 20 μM of (R) and (S) 11-HETE for 24 h. All the concentrations tested did not significantly alter the cell viability (depicted by the viability above 90%) when compared to the control (data not shown). As a result, we used the 20 μM concentration in all the subsequent experiments.

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Effect of 11-HETE enantiomers on cellular hypertrophic markers in RL-14 cells [1]
To evaluate the potential effect of 11-HETE enantiomers in inducing cellular hypertrophy, RL-14 cells were treated with 20 μM of either 11(R)-HETE or 11(S)-HETE for 24 h. Thereafter, the expressions of the cardiac hypertrophic markers such as atrial natriuretic peptide (ANP), α-myosin heavy chain (α-MHC), β-MHC, skeletal α-actin (ACTA-1), and brain natriuretic peptide (BNP) were measured using RT-PCR. Figure 1 shows that 11(S)-HETE significantly increased the cardiac hypertrophic markers: ANP, β-MHC, and β/α-MHC by 231%, 499%, and 107%, respectively. 11(R)-HETE significantly increased the cardiac hypertrophic marker; β/α-MHC by 132%. Furthermore, ACTA-1 gene expression was increased by 46% in the 11(R)-HETE-treated group and was significantly increased by 282% in the 11(S)-HETE-treated group compared to the control (Figure 1). Both β-MHC and ACTA-1 gene expression were significantly increased in the 11(S)- compared to the 11(R) HETE-treated group.
It was established that the increase in the hypertrophic markers is associated with the increase in the cell surface area. As shown in Figure 2, our results showed that treating the RL-14 cells with 20 μM of either (R) or (S)- 11 HETE significantly increased the cell surface area by 29% and 34% compared to the control, respectively.

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Effect of 11-HETE enantiomers on CYP mRNA gene expression in RL-14 cells [1]
To examine the effect of 11-HETE enantiomers on CYP enzymes, RL-14 cells were treated with 20 μM (R) or (S) 11-HETE for 24 h. Thereafter, CYP1B1, CYP1A1, CYP4A11, CYP4F11, CYP4F2, CYP2J2, CYP2E1 and CYP2C8 mRNA were determined using RT-PCR. The CYP1B1, CYP1A1, CYP4A11, CYP4F11 and CYP4F2 mRNA were significantly increased in the cells treated with 11(R)-HETE by 116%, 112%, 70%, 238% and 167%, respectively, compared to the control group. Similarly, the 11(S)-HETE-treated group showed a significant increase in the gene expression of the same enzymes by 142%, 109%, 90%, 416% and 257% respectively, compared to the control (Figure 3). Albeit both (R) and (S) enantiomers have significantly increased the CYP2E1 mRNA gene expression by 146% and 163% respectively compared to the control group, only 11(S)-HETE increased the CYP2J2 mRNA gene expression by 47%.

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Effect of 11-HETE enantiomers on the protein level of CYP enzymes in RL-14 cells [1]
It was essential to assess the protein levels of the CYP enzymes of interest since the mRNA expression may not consistently align with the levels of these enzymes in terms of protein expression. The protein level of CYP1B1, CYP4F2, and CYP4A11 in the cells treated with 11(S-) HETE showed a significant increase by 186%, 153%, and 152%, respectively, compared to the control. While the 11(R)-HETE-treated cells did not affect the protein level to the same degree, it significantly increased the protein level of CYP1B1, CYP4F2, and CYP4A11 by 156%, 126%, and 141%, respectively, compared to the control (Figure 4).
Interestingly, the CYP2J protein level was significantly increased in the cells treated with 11(S-HETE) enantiomers by 135%, compared to the control group. Regarding CYP2C8, the increase in the protein level was not significant for both enantiomers (Figure 4). There was an increase in the gene expression of CYP4F11 for both enantiomers, however, CYP4F11 protein level was below the detection limit.

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Effect of 11-HETE enantiomers on recombinant human CYP1B1 enzyme activity [1]
The direct effect of R and S enantiomers of 11-HETE on rhCYP1B1 catalytic activity was assessed using rhCYP1B1-mediated EROD. The rate of resorufin formation (V) by rhCYP1B1 with various concentrations of 7-ER co-incubated with either S or R enantiomers of 11-HETE is shown in Figures 5A,C. 11(S)-HETE led to allosteric activation of CYP1B1 activity, causing a concentration-dependent increase in Vmax value, compared with control, by 1.03, 1.1, 1.5 and 1.4-fold for 0.5, 2.5, 10 and 40 nM of 11(S)-HETE, respectively (Table 2); whereas, 11(R)-HETE did not affect Vmax (Table 2). Km values of 7-ER hydrolysis of rhCYP1B1 did not change by either R or S enantiomers of 11-HETE; therefore, shared Km value was assumed in Michaelis-Menten model fitting, estimated to be 131.3 nM. The double reciprocal (Lineweaver-Burk) plots show intercepting lines for S and R enantiomers of 11-HETE, which in terms of allosteric interactions means changes in Vmax with no substantial effect on Km (Figures 5B,D).

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Effect of 11-HETE enantiomers on CYP1B1 activity in the human liver microsomes [1]
To further confirm the results obtained from rhCYP1B1, we have tested the possible effect of both 11-HETE enantiomers on the catalytic activity of CYP1B1 using EROD assay in the human liver microsomes. We used fixed concentrations of the substrate and varying concentrations of either 11(R) or 11(S) HETE (0, 10, 20, 40, and 100 nM). As shown in Figure 6, the results showed that incubation of human liver microsomes with increasing concentrations of 11(S)-HETE was associated with a concentration-dependent increase in the EROD formation rate when compared to the control group. 11(S)-HETE showed a stronger effect than 11(R)-HETE. A significant increase in the catalyzed EROD activity to 107%, 119%, 136%, and 183% was observed for the 10, 20, 40, and 100 nM of the 11(S)HETE compared to the control, respectively. Similarly, the concentrations of 40 and 100 nM 11(R)-HETE showed a significant increase to 87% and 145%, respectively (Figure 6).

Effect of 11-HETE enantiomers on human recombinant CYP1B1 enzymatic activities [1]
The rates of the O-dealkylation of 7-ethoxyresorufin catalyzed by recombinant human CYP1B1 were measured using the ethoxyresorufin-O-deethylase (EROD) assay. The measurements were conducted in the absence or presence of either (R) or (S) 11-HETE. The assay was done using a white 96-well microplate. Different concentrations of 7-ER (7-ethoxyresorufin; final concentration of 0, 5, 10, 20, 40, and 100 nM) were subjected to incubation with a reaction mixture containing 100 mM potassium phosphate (pH 7.4) buffer supplemented with 5 mM magnesium chloride hexahydrate and 1 pmol of human recombinant CYP1B1 enzyme. After that, various concentrations (final concentration of 0, 0.5, 2.5, 10, and 40 nM) of either (R) or (S) 11-HETE were added to the reaction mixture. Then 100 μL of this reaction mixture was added to each well of the 96-well plate followed by 100 μL of NADPH (2 mM) to start the reaction. The fluorescent reading of the plate related to the resorufin formation was measured by BioTek Synergy H1 Hybrid Reader every min for 30 min. The signal was recorded with 550/585 nm excitation/emission wavelengths, respectively. A resorufin standard curve was prepared and used to calculate the amount of the resorufin formed. The rate of resorufin formation was plotted versus the concentration of 7-ER for each concentration of 11-HETE.

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Effect of 11-HETE enantiomers on cytochrome P450 enzymatic activities in human liver microsomes [1]
Incubation with human liver microsomes was performed to test the effect of either (R) or (S) 11-HETE on modulating the enzymatic activities of the CYP1B1 enzyme. Human liver microsomes pooled from 25 different individuals (0.1 mg/mL) were incubated with different concentrations of (R) or (S) 11-HETE (0, 10, 20, 40, and 100 nM). 2 μM of EROD was used as the substrate in the reaction that also contains 100 mM potassium phosphate buffer (pH 7.4) supplemented with 5 mM magnesium chloride hexahydrate. 100 μL of the reaction mixture was added to the wells of 96-well polystyrene microplates. To start the reaction, 100 μL of 1 mM NADPH was added to the reaction mixture in each well. A BioTek Synergy H1 Hybrid Reader was used to measure the fluorescent signal generated due to the formation of resorufin every minute for 30 min under 37°C at 550/585 nm excitation/emission wavelengths, respectively.

Chemical treatments [1]
The cells in the control group were treated with the vehicle [serum-free DMEM/F-12 containing 0.5% dimethylsulfoxide (DMSO)]. The other groups were treated by adding 20 μM (R) or (S) 11-HETE to the serum free media (SFM) for 24 h. Both (R) and (S) 11-HETE were supplied as a stock solution in DMSO and were stored at −20°C until use. DMSO concentration didn’t exceed 0.5% in the treated groups during all the performed experiments.

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Measurement of cell viability [1]
Cell viability test was determined by using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay which measures the ability of the living cells to reduce the yellow tetrazolium salt to its water-insoluble purple formazan crystals. The optical density of the formazan crystals reflects the population of living cells. Cells were plated in a 48-well plate under 37°C temperature and 5% CO2 humidified condition until sufficient confluency was achieved. Then the cells were treated with 2.5, 5, 10, and 20 μM of either (R) or (S) 11-HETE for 24 h. The media containing the treatment was discarded and 100 μL of MTT reagent (1.2 mM) dissolved in SFM was added and incubated with the cells at 37°C. After incubation for 2 h, the medium was discarded and 200 μL of DMSO were added to solubilize the formed formazan crystals. Synergy™ H1 Hybrid Multi-Mode Reader was used to measure the color intensity at a wavelength of 570 nm.

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Measurement of cell surface area [1]
RL-14 cells were plated in a 6-well plate and treated with 20 μM of either (R) or (S) 11-HETE for 24 h. After that, cells were washed with 1x PBS (pH 7.4) 3 times and fixed with 4% paraformaldehyde for 15 min at 4°C. Then 10 μg/ml of wheat germ agglutinin, Alexa Fluor 488 conjugate was added, and the plates were incubated for 2 h in a dark place. The plates were washed again with 1x PBS (pH 7.4) 3 times each for 5 min using a shaker. Thereafter, the coverslips that have the stained cells were put on a glass slide with ProLong antifade reagent with DAPI. The slides were then imaged by an inverted microscope using the ×20 objective lens as described previously (Alammari et al., 2023) and the surface area was measured using Zeiss AxioVision Software. Sixty-five individual cells from each group were included in the analysis.

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RNA extraction and cDNA synthesis [1]
RNA extraction and cDNA synthesis were performed on the (R) and (S) 11-HETE-treated RL-14 cells according to the method described previously (Shoieb and El-Kadi, 2020). In brief, cells were plated in 12-well plates and treated with 20 μM (R) and (S) 11-HETE for 24 h. Thereafter, the total RNA was isolated with TRIzol reagent, and the concentration was determined by measuring the absorbance at 260 nm. The RNA purity was determined by measuring the 260/280 ratio (>1.8). The first strand of cDNA was performed according to the manufacturer’s instructions by mixing 1.25 µg of the total RNA isolated from each sample with high-capacity cDNA reverse transcription reagents (Applied Biosystems). Finally, the reaction mixture was inserted in a thermocycler and underwent the following conditions: 25°C for 10 min, 37°C for 120 min, 85°C for 5 min, and at last it was cooled to 4°C.

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Protein extraction from RL-14 cells and western blot analysis [1]
Protein extraction from the cells and Western blot analysis were performed as previously described by Shoieb and El-Kadi (2020). In brief, Rl-14 cells were grown in 6-well plates and incubated with 20 μM (R) or (S) 11-HETE for 24 h. Thereafter, the cell lysates were collected using 100 μL from the lysis buffer containing 50 mM HEPES, 1.5 mM magnesium chloride, 0.5 M sodium chloride, 10% (v/v) glycerol, 1 mM EDTA, 1% Triton X-100, and 5 μL/ml protease inhibitor cocktail. Subsequently, the Lowry assay was done to determine the concentration of the protein using bovine serum albumin as a reference standard (Lowry et al., 1951).
Western blot analysis was performed by separating 100 μg of the total cell lysate by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS–PAGE). The separated proteins were transferred to polyvinylidene difluoride membranes and were incubated with the specific primary antibody for the desired protein overnight at 4°C. The membranes were then incubated with secondary antibodies (anti-rabbit IgG HRP-linked secondary antibodies or anti-mouse IgG HRP-linked secondary antibodies) in a blocking solution for 45 min at room temperature.

[1]. 11-Hydroxyeicosatetraenoics induces cellular hypertrophy in an enantioselective manner. Front Pharmacol. 2024 Aug 12;15:1438567.

Background: R/S enantiomers of 11-hydroxyeicosatertraenoic acid (11-HETE) are formed from arachidonic acid by enzymatic and non-enzymatic pathways. 11-HETE is predominately formed by the cytochrome P450 1B1 (CYP1B1). The role of CYP1B1 in the development of cardiovascular diseases is well established.
Objectives: This study aimed to assess the cellular hypertrophic effect of 11-HETE enantiomers in human RL-14 cardiomyocyte cell line and to examine their association with CYP1B1 levels.
Methods: Human fetal ventricular cardiomyocyte, RL-14 cells, were treated with 20 µM (R) or (S) 11-HETE for 24 h. Thereafter, cellular hypertrophic markers and cell size were then determined using real-time polymerase chain reaction (RT-PCR) and phase-contrast imaging, respectively. The mRNA and protein levels of selected CYPs were determined using RT-PCR and Western blot, respectively. In addition, we examined the effect of (R) and (S) 11-HETE on CYP1B1 catalytic activity using human recombinant CYP1B1 and human liver microsomes. [1]

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To our knowledge this is the first study to investigate the cellular hypertrophic effect of 11-HETE enantiomers in RL-14 cells and the mechanisms involved. Both R and S enantiomers of the midchain 11-HETE could induce cellular hypertrophy in RL-14 cells. They significantly increased several hypertrophic markers as well as the protein level and the gene expression of various CYP enzymes. The S- enantiomer allosterically activates human recombinant CYP1B1 and both enantiomers significantly increased the EROD activity in human liver microsomes. The investigation of the role of high 11-HETE concentration in various cardiovascular diseases should be expanded, and strategies to inhibit its effect could be tested as potential therapeutic strategies. [1]

C20H32O3

320.47

320.235

C, 74.96; H, 10.07; O, 14.98

73804-65-6

Typically exists as solids at room temperature

CCCCCC=CC=CC(CC=CCC=CCCCC(=O)O)O

11-Hydroxy-5,8,12,14-eicosatetraenoic acid; 11-hydroxyicosa-5,8,12,14-tetraenoic acid; 73804-65-6; (?)11-HETE; 54886-50-9; ( inverted exclamation markA)11-HETE; 11(R)-hydroxy-5(Z),8(Z),12(E),14(Z)-eicosatetraenoic acid; SCHEMBL1479341; DTXSID40868247;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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