ROS; NOS

An ROS blocker reverses ANE-induced ROS generation [1]
To confirm the results obtained above, we explored if inhibition of oxidative stress with the antioxidant aminoguanidine hemisulfate (AGH) would affect the role of the ANE on inducing ROS/RNS and superoxide. As shown in Fig. 2C, we found that the ROS level was similar to the vehicle-treated HepJ5 and Mahlavu cells. ROS production dramatically increased in ANE-treated HepJ5 and Mahlavu cells. However, pretreatment with AGH with subsequent ANE exposure induced fewer ROS compared with ANE-treated only cells (Fig. 2C). These results indicated that ROS induction by ANE treatment could be reversed by antioxidant compounds.

NO Content in the Larvae on Exposure to L-arginine and aminoguanidine hemisulfate/AGH [2]
To verify the function of NOS in M. coruscus, pediveliger larvae were treated with L-arginine and aminoguanidine hemisulfate/AGH. In comparison to the control (AFSW), NO levels showed a significant increase after L-arginine exposure for 48 h, whereas the inhibition of NOS activity by AGH led to a decrease in NO levels irrespective of subsequent L-arginine treatment (Figure 1). It is notable that NOS activity inhibition by AGH followed by L-arginine exposure did not increase NO levels (Figure 1). These results suggested that NOS can synthesize NO using the exogenous substrate L-arginine.

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Behavior of the Larvae on the Exposure to L-arginine and aminoguanidine hemisulfate/AGH [2]
To determine the effects of NO and NOS on larval behavior and metamorphosis, the larvae were stimulated with EPI (positive control), L-arginine, and aminoguanidine hemisulfate/AGH. Neither of them had a significant effect on the survival rate of the larvae (Figure 3A). With an increase in exposure time, as compared to the control (AFSW), larval metamorphosis showed an increase; the least increase was observed on L-arginine exposure. The proportion of the larvae in metamorphosis induced by EPI was reduced in the presence of L-arginine. When the larvae were exposed to L-arginine alone, more larvae remained in the crawling stage, representing the beginning of metamorphosis preparation. Meanwhile, the proportion of metamorphotic larvae showed an increase from 48 to 72 h after AGH exposure, while L-arginine reduced larval metamorphosis in the presence of AGH (Figure 3B–D).

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Metamorphosis of Pediveliger Larvae on Exposure to L-arginine and aminoguanidine hemisulfate/AGH [2]
The larval metamorphosis rate showed a significant increase after 48 h of EPI (up to 18.63%) and aminoguanidine hemisulfate/AGH (up to 3.82%) exposure compared to the control (AFSW). The addition of L-arginine after the induction with EPI significantly inhibited the larval metamorphosis rate, with the metamorphosis rate decreasing to 10.17% (Figure 4A). After 72 h, the larval metamorphosis rate reached 32.78% under EPI and 14.85% under AGH induction. However, the induction rate of EPI and AGH was only 19.81% and 7.65%, respectively, in the presence of L-arginine (Figure 4B). After 96 h of induction, the larval metamorphosis rate reached 36.52% under EPI and 18.73% under AGH induction. In the presence of L-arginine, the induction rate of EPI was still only 18.73%, but that of AGH increased to 13.95% (Figure 4C).

Total reactive oxygen species (ROS)/superoxide detection [1]
ROS were measured using a total ROS/Superoxide Detection Kit according to the manufacturer's instructions. Cells were stained with the two-color ROS Detection Kit and analyzed using a NucleoCounter® NC-3000TM system. In brief, cells (2.4×105) were seeded in six-well plates overnight and then exposed to the ANE or the vehicle for 24 hours. Cells were harvested, and ROS and oxidative stress were detected by staining with the two fluorescent dyes from the ROS-ID® Total ROS/Superoxide detection kit. In addition, harvested cells were stained with Hoechst-33342, which was used to detect the total cell population.

Metamorphosis Assays of Pharmacological Treatments [2]
Larvae were treated with epinephrine (EPI), which can induce larval metamorphosis; L-arginine, the substrate for NO synthesis; and aminoguanidine hemisulfate (AGH), which can inhibit NOS activity. Stock solutions of all chemicals were prepared as explained in the relevant manuals (EPI, 10−3 M; L-arginine, 10−3 M; and AGH, 10−3 M) using distilled water or 1 M HCl and diluted to desired final concentrations with autoclaved FSW (pH 7.8–8.2). All stock solutions and test chemical compounds were used immediately after preparation. The larval metamorphosis experiment was performed in a sterile glass petri dish (64.0 mm (Φ) × 19.0 mm) containing 20 pediveliger larvae (335.75 ± 16.5 μm (n = 100)) and 20 mL autoclaved filtered seawater (AFSW) mixed with chemicals. Larvae were treated with 10−4 M EPI, 10−4 M L-arginine, and 10−3 M AGH for 96 h. Larval behavior, the metamorphosis rate, and the survival rate were detected 24, 48, 72, and 96 h after chemical exposure. In case of mixed exposure experiments, 10−3 M aminoguanidine hemisulfate/AGH was added to the dish after treating the larvae with 10−4 mol/L L-arginine for 15 min. All bioassays were performed at 17 °C ± 1 °C in the dark with six replicates (n = 120). EPI was used as a positive control, and AFSW served as a negative control. The larvae were not fed any food during the experiments.

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Determination of NO Levels in the Larvae [2]
Each treatment group (n = 300 pediveliger larvae) was exposed to EPI, L-arginine, and aminoguanidine hemisulfate/AGH at the same concentrations as those stated in Section 2.2; the larvae were collected 72 h after chemical treatments. Four assessments were independently performed for each treatment group (n = 1200). The total protein was extracted from the larvae using a kit, and the protein concentration was determined with the modified BCA Protein Assay Kit. The NO levels in the larvae were measured using a kit. All procedures were performed as recommended by the manufacturer.

2146 rat LD50 subcutaneous 1258 mg/kg Journal of Pharmacology and Experimental Therapeutics., 119(444), 1957 [PMID:13417100]
2146 mouse LD50 subcutaneous 963 mg/kg Journal of Pharmacology and Experimental Therapeutics., 119(444), 1957 [PMID:13417100]

[1]. Areca nut extract (ANE) inhibits the progression of hepatocellular carcinoma cells via activation of ROS production and activation of autophagy. Int J Med Sci. 2021 Aug 9;18(15):3452-3462.

[2]. Effects of L-arginine on Nitric Oxide Synthesis and Larval Metamorphosis of Mytilus coruscus. Genes (Basel). 2023 Feb 9;14(2):450.

Hepatocellular carcinoma (HCC) is a worldwide health problem. Currently, there is no effective therapeutic strategy for HCC patients. Chewing areca nut is closely associated with oral cancer and liver cirrhosis. The therapeutic effect of areca nut extract (ANE) on HCC is unknown. Our results revealed that ANE treatment caused a reduction in cell viability and an increase in cell apoptosis and suppressed tumor progression in xenograft models. ANE-treated didn't induce liver tumor in nude mice. For mechanism dissection, ANE treatment caused ROS-mediated autophagy and lysosome formation. Pretreatment with an ROS inhibitor, aminoguanidine hemisulfate (AGH), abolished ANE-induced ROS production. ANE treated cells caused an increase in light chain 3 (LC3)-I to -II conversion, anti-thymocyte globulin 5+12 (ATG5+12), and beclin levels, and apoptosis related-protein changes (an increases in BAX, cleaved poly(ADP-ribose) polymerase (c-PARP), and a decrease in the Bcl-2 level). In conclusion, our study demonstrated that the ANE may be a new potential compound for HCC therapy. [1]

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To investigate the regulatory functions of L-arginine and nitric oxide (NO) on Mytilus coruscus metamorphosis, M. coruscus larvae were exposed to an inhibitor of nitric oxide synthase (NOS), aminoguanidine hemisulfate (AGH), and a substrate for NO synthesis, L-arginine. We observed that NO levels showed a significant increase, and this trend continued with L-arginine treatment. When NOS activity was inhibited, the larvae could not synthesize NO, and metamorphosis was not inhibited even in the presence of L-arginine. On transfecting pediveliger larvae with NOS siRNA followed by L-arginine exposure, we found that the larvae did not produce NO and that the larval metamorphosis rate was significantly increased, suggesting that L-arginine regulates M. coruscus larval metamorphosis by promoting NO synthesis. Our findings improve our understanding of the effects of marine environmental factors on larval metamorphosis of mollusks.[2]

CH6N4.1/2H2O4

123.13

246.085

996-19-0

Aminoguanidine hydrochloride;1937-19-5;Aminoguanidine sulfate-13C,15N2;Aminoguanidine sulfate-13C,15N4

2734952

White to pink solid powder

261.4ºC at 760mmHg

200 °C

111.9ºC

0.896

8

8

0

15

123

0

S(=O)(=O)(O[H])O[H].N([H])([H])/C(=N/N([H])[H])/N([H])[H].N([H])([H])/C(=N/N([H])[H])/N([H])[H]

VKGQPUZNCZPZKI-UHFFFAOYSA-N

InChI=1S/2CH6N4.H2O4S/c2*2-1(3)5-4;1-5(2,3)4/h2*4H2,(H4,2,3,5);(H2,1,2,3,4)

2-aminoguanidine;sulfuric acid

Aminoguanidine hemisulfate; 996-19-0; Hydrazinecarboximidamide, sulfate (2:1); pimagedine hemisulfate; R13YB310MU; DTXSID0046016; Di(carbazamidine) sulphate; Bis(Aminoguanidinium) Sulfate;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

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

H2O: 100 mg/mL (812.15 mM)

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.)