- The primary target of 3-Nitropropanoic acid (3-NPA) is mitochondrial respiratory Complex II (succinate dehydrogenase). It acts as a suicide inhibitor, forming a covalent adduct with a catalytic arginine residue in the active site of Complex II after oxidation by the enzyme [1]

- Incubation of 3-NPA with mitochondrial extracts leads to inhibition of Complex II activity. The inhibition mechanism involves oxidation of 3-NPA by Complex II, followed by covalent binding to a catalytic arginine residue in the enzyme’s active site, which irreversibly inactivates Complex II [1]
- 3-NPA exhibits potent antimycobacterial activity against mycobacterial strains (specific strains not specified in the abstract); it inhibits the growth of mycobacteria in in vitro culture systems [2]
- Treatment of cells with 3-NPA induces autophagy. This autophagy induction is mediated by the formation of mitochondrial permeability transition pores (mPTP) rather than activation of the mitochondrial fission pathway; 3-NPA does not alter the expression or localization of mitochondrial fission-related proteins [3]
- Exposure of goose granulosa cells to 3-NPA induces oxidative stress, as evidenced by increased reactive oxygen species (ROS) production. It also triggers apoptosis of granulosa cells, characterized by changes in apoptotic marker expression and nuclear fragmentation [4]
- In synaptosomal preparations from an animal model of Huntington's disease, 3-NPA induces oxidative stress, including increased lipid peroxidation and reduced antioxidant enzyme activity; this oxidative stress is attenuated by melatonin treatment [6]

- Administration of 3-NPA in animal models (specific animal species not specified in the abstract) induces two distinct modes of cell death: acute excitotoxic necrosis (occurring within hours of administration) and delayed apoptosis (developing over days after administration). The acute necrosis is associated with excitatory amino acid release, while delayed apoptosis involves mitochondrial dysfunction [5]
- In an animal model of Huntington's disease, systemic administration of 3-NPA leads to oxidative stress in brain synaptosomes, manifested by elevated lipid peroxidation and decreased activities of superoxide dismutase (SOD) and glutathione peroxidase (GPx). Co-administration of melatonin reverses these 3-NPA-induced oxidative stress markers [6]
- Intraperitoneal injection of 3-NPA in mice evokes seizures. The seizure latency, duration, and severity are dose-dependent (specific doses not specified in the abstract); 3-NPA-induced seizures are associated with mitochondrial dysfunction in the brain [7]

- Prepare mitochondrial fractions from tissues or cells. Incubate the mitochondrial fractions with different concentrations of 3-NPA (concentrations not specified in the abstract) for a specific period at 37°C. Measure Complex II activity by monitoring the reduction of 2,6-dichlorophenolindophenol (DCPIP) in the presence of succinate (the substrate of Complex II). Compare the activity of Complex II in 3-NPA-treated groups with that in the control group to calculate the inhibition rate. Additionally, perform mass spectrometry or immunoblotting to detect the formation of covalent adducts between 3-NPA and the arginine residue in Complex II [1]

- Autophagy Detection Assay: Culture mammalian cells (specific cell line not specified in the abstract) in appropriate medium. Treat the cells with 3-NPA (concentration not specified in the abstract) for 24–48 hours. Detect autophagy by immunofluorescence staining of LC3 (an autophagy marker) to observe the formation of LC3-positive puncta. Measure the expression levels of autophagy-related proteins (e.g., LC3-I/LC3-II, Beclin-1) using western blotting. To exclude the involvement of mitochondrial fission, assess the expression and mitochondrial localization of fission-related proteins (e.g., Drp1) via western blotting and immunofluorescence [3]
- Goose Granulosa Cell Oxidative Stress and Apoptosis Assay: Isolate goose granulosa cells from ovarian follicles and culture them in serum-containing medium. Treat the cells with 3-NPA (concentration not specified in the abstract) for different time points (not specified in the abstract). Measure ROS production using a fluorescent probe (e.g., DCFH-DA) and detect lipid peroxidation by measuring malondialdehyde (MDA) levels. Assess apoptosis via flow cytometry (using Annexin V-FITC/PI staining) and western blotting (detection of cleaved caspase-3 and Bax/Bcl-2 ratio) [4]

- Cell Death Induction Protocol: Administer 3-NPA to animals (specific species not specified in the abstract) via intraperitoneal injection (frequency and total duration not specified in the abstract). The dose of 3-NPA is adjusted to induce acute necrosis (high dose, single administration) or delayed apoptosis (low dose, repeated administration). At different time points after administration, sacrifice the animals, collect brain tissues (or other target tissues), and perform histological staining (e.g., hematoxylin and eosin staining) to observe tissue damage, as well as TUNEL staining to detect apoptotic cells [5]
- Huntington's Disease Model Protocol: Use rodents (specific species not specified in the abstract) as the Huntington's disease model. Administer 3-NPA via intraperitoneal injection at a fixed dose (not specified in the abstract) once daily for 7–14 days. For the intervention group, co-administer melatonin with 3-NPA at the same time. After the treatment period, sacrifice the animals, isolate brain synaptosomes, and measure oxidative stress markers (lipid peroxidation, SOD, GPx activity) [6]
- Mouse Seizure Induction Protocol: Use adult mice (specific strain not specified in the abstract). Administer 3-NPA via intraperitoneal injection at doses ranging from low to high (specific doses not specified in the abstract). Place the mice in an observation chamber immediately after injection, and record seizure latency (time from injection to first seizure), seizure duration, and seizure severity (using a standard seizure scoring system). Sacrifice the mice after the observation period (24 hours) and collect brain tissues for mitochondrial function analysis [7]

Toxicity Summary
3-Nitropropionic acid is a suicide inhibitor of succinate dehydrogenase, an enzyme essential for the tricarboxylic acid cycle (TCA cycle) and the electron transport chain activity of mitochondrial respiratory chain complex II. It forms a covalent adduct with the side chain of Arg297, inactivating succinate dehydrogenase. This affects neurons, leading to NMDA receptor activation, excessive calcium influx, and reactive oxygen species formation, ultimately resulting in neuronal cell death. (A2967, A2968) Interactions Systemic administration of 3-nitropropionic acid (3-NPA, a fungal toxin) can cause brain damage accompanied by disruption of the blood-brain barrier (BBB). Since endothelial cells are a crucial component of the blood-brain barrier and a primary target of systemic poisoning, this study used imaging techniques to detect changes in intracellular calcium ion concentration ([Ca2+]i) to investigate the effects of 3-nitrophenylalanine (3-NPA) on primary cultured rat brain endothelial cells (rBECs). The results showed that medium to high concentrations of 3-NPA induced rBEC damage, manifested as the accumulation of [Ca2+]i; however, combined treatment with 17β-estradiol or tamoxifen alleviated this damage, suggesting that estrogen may play a protective role against cerebrovascular injury through estrogen receptors. This study also investigated the role of mitochondrial dysfunction in the development of epilepsy in mice. The mitochondrial complex III inhibitor 3-nitropropionic acid is known to induce seizures; in this study, its use at subthreshold doses enhanced both current- and 4-aminopyridine-induced seizures. Conversely, 3-nitropropionic acid has no effect on seizures induced by γ-aminobutyric acid (GABA) receptor antagonists (such as bispuline, pentylenetetrazol, and picric acid), glycine antagonists (such as strychnine), cholinergic drugs (such as pilocarpine), and kynurenine aminotransferase inhibitors (such as aminooxyacetic acid). It is speculated that mitochondrial metabolic disorders make the central nervous system more susceptible to factors that induce seizures through direct depolarization. Currently, there is no effective treatment for Huntington's disease (HD), a progressive and fatal neurodegenerative disease characterized by motor and cognitive decline. The close association between Huntington's disease and mitochondrial energy metabolism disorders is well-established. Tauroursodeoxycholic acid (TUDCA), a naturally occurring bile acid, can stabilize mitochondrial membranes, inhibit mitochondrial permeability transitions, reduce free radical generation, and block apoptosis pathways. This study found that tauroursodeoxycholic acid (TAA) significantly reduced 3-nitropropionic acid (3-NPA)-mediated striatal neuronal cell death in cell culture. Furthermore, rats treated with TAA showed an 80% reduction in 3-nitropropionic acid-induced apoptosis and lesion volume. More importantly, rats treated with TAA in combination with 3-nitropropionic acid showed no significant difference in sensory/motor and cognitive task performance compared to the control group, and this effect lasted for at least 6 months. Bile acids have historically been used to treat certain liver diseases. However, this is the first time that bile acids have been demonstrated to be transported to the brain and exert neuroprotective effects, thus potentially offering therapeutic benefits for certain neurodegenerative diseases.

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Non-human toxicity values
Rat intraperitoneal injection LD50: 67 mg/kg
Mouse intravenous injection LD50: 50 mg/kg

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- 3-NPA can induce acute excitatory toxic necrosis and delayed apoptosis in animal tissues (mainly brain tissue), leading to tissue damage and dysfunction[5]
-In goose granulocytes, 3-NPA can cause oxidative stress (increased ROS and MDA) and apoptosis, leading to decreased granulocyte viability and functional impairment[4]
-In mice, 3-NPA can induce dose-dependent seizures, which are associated with brain mitochondrial dysfunction[7]

[1]. 3-nitropropionic acid is a suicide inhibitor of mitochondrial respiration that, upon oxidation by complex II, forms a covalent adduct with a catalytic base arginine in the active site of the enzyme. J Biol Chem. 2006 Mar 3;281(9):5965-72.

[2]. 3-Nitropropionic acid (3-NPA), a potent antimycobacterial agent from endophytic fungi: is 3-NPA in some plants produced by endophytes? J Nat Prod. 2005 Jul;68(7):1103-5.

[3]. 3-Nitropropionic acid induces autophagy by forming mitochondrial permeability transition pores rather than activating the mitochondrial fission pathway. Br J Pharmacol. 2013 Jan;168(1):63-75.

[4]. Effect of 3-nitropropionic acid inducing oxidative stress and apoptosis of granulosa cells in geese. Biosci Rep. 2018 Sep 12;38(5):BSR20180274.

[5]. Pang Z, Geddes JW. Mechanisms of cell death induced by the mitochondrial toxin 3-nitropropionic acid: acute excitotoxic necrosis and delayed apoptosis. J Neurosci. 1997 May 1;17(9):3064-73.

[6]. Protective effect of melatonin on 3-nitropropionic acid-induced oxidative stress in synaptosomes in an animal model of Huntington's disease. J Pineal Res. 2004 Nov;37(4):252-6.

[7]. A. Mitochondrial toxin 3-nitropropionic acid evokes seizures in mice. Eur J Pharmacol. 1998 Oct 16;359(1):55-8.

3-Nitropropionic acid is a golden-yellow crystal (soluble in chloroform). (NTP, 1992)
3-Nitropropionic acid is a C-nitro compound formed by replacing a methyl hydrogen atom in the propionic acid molecule with a nitro group. It functions as a neurotoxin, an EC 1.3.5.1 [succinate dehydrogenase (quinone)] inhibitor, an antimycobacterial drug, and a fungicide. Functionally, it is related to propionic acid. It is the conjugate acid of 3-nitropropionic acid ester. It is a tautomer of 3-acid nitropropionic acid.
3-Nitropropionic acid has been reported to exist in Phomopsis velata, Penicillium atrovenetum, and several other organisms with relevant data.
Bovine toxin was isolated from Aspergillus sp. and food contamination molds.
Bovine toxin belongs to the β-amino acid and its derivatives family. These amino acids have a (-NH2) group attached to the β carbon atom.

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Mechanism of Action
3-Nitropropionic acid (3-NPA) is a naturally occurring mycotoxin and an irreversible inhibitor of succinate dehydrogenase. It leads to the depletion of adenosine triphosphate (ATP) in cortical explants and is associated with motor disorders in livestock and humans consuming contaminated food.
Previous studies have shown that the neurotoxicity of 3-nitropropionic acid (3-NPA) involves excitotoxic activation of N-methyl-D-aspartate (NMDA) receptors. Therefore, this study investigated the effects of oxyphenadrine on cultured rat cerebellar granule cells (CGCs) and N-methyl-D-aspartate (NMDA) neurotoxicity in rats. The results showed that oxyphenadrine could protect cerebellar granule cells from NMDA-induced cell death, a conclusion confirmed by neutrophil viability assays and laser scanning cytology (using propidium iodide staining). In rats, this study employed two indirect markers of neuronal injury: the binding of ((3)H)-PK 11195 to the peripheral benzodiazepine receptor (PBR, a microglial marker) and the expression of 27 kDa heat shock protein (HSP27, an activated astrocyte marker). Systemic administration of N-methyl-D-aspartate (30 mg/kg/day for 3 consecutive days) increased ((3)H)-PK 11195 binding by 170% and upregulated the expression of 27 kDa heat shock protein. Pre-administration of phenadrine (30 mg/kg/day for 3 consecutive days) inhibited the increase in ((3)H)-PK 11195 and HSP27 expression. Lower doses (10 and 20 mg/kg) had no protective effect. Phenadrine also reduced N-methyl-D-aspartate-induced mortality in a dose-dependent manner. ...Olfenadrine or similar drugs can be used to treat neurodegenerative diseases mediated by overactivation of N-methyl-D-aspartate receptors. This study investigated the mechanism of striatal cell degeneration following administration of the mitochondrial toxin 3-nitropropionic acid (3-NP). Typical apoptosis-associated internucleosome breaks were observed in the DNA of rat striatal cells treated with 3-nitropropionic acid (3-NP). TUNEL assays mediated by terminal deoxynucleotidyl transferase (dUTP-biotin nick end labeling) also confirmed these DNA breaks. The data indicate that striatal cells die via apoptosis after 3-NP administration. 3-nitropropionic acid blocks energy metabolism before causing neurotoxic damage, and the degree of energy depletion determines the detrimental effects of 3-nitropropionic acid. In this study, we also demonstrated that glutamate and glutamine levels, as well as astrocyte function, may play a crucial role in 3-nitropropionic acid-induced striatal damage. For more complete data on the mechanisms of action of 3-nitropropionic acids (15 in total), please visit the HSDB record page. 3-NPA is a mitochondrial toxin that exerts its biological effects primarily by irreversibly inhibiting mitochondrial complex II, leading to mitochondrial dysfunction and subsequent cell damage [1,5]. 3-NPA is a potent antimycobacterial drug isolated from endophytic fungi. This study hypothesizes that 3-NPA present in certain plants may be produced by endophytic fungi colonizing the plants [2]. 3-NPA is widely used in animal models of Huntington's disease to induce mitochondrial dysfunction and oxidative stress, thereby mimicking the pathological features of the disease [6].

C3H5NO4

119.0761

119.021

504-88-1

1678

White to yellow solid powder

1.4±0.1 g/cm3

303.0±25.0 °C at 760 mmHg

68-70ºC(lit.)

148.4±11.6 °C

0.0±1.4 mmHg at 25°C

1.463

-0.08

1

4

2

8

104

0

WBLZUCOIBUDNBV-UHFFFAOYSA-N

InChI=1S/C3H5NO4/c5-3(6)1-2-4(7)8/h1-2H2,(H,5,6)

3-nitropropanoic acid

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 (e.g. under nitrogen), avoid exposure to moisture and light.

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

DMSO : ~125 mg/mL (~1049.71 mM)
H2O : ~100 mg/mL (~839.77 mM)

Solubility in Formulation 1: ≥ 2.17 mg/mL (18.22 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 21.7 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 (17.47 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in 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 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 (17.47 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.)