Lysyl oxidase (LOX)

β-Aminopropionitrile (BAPN) enhances glucose absorption in an in vitro model of insulin resistance and normalizes the expression of GLUT4 and adiponectin[1]. In vitro cervical cancer cell invasion and migration are inhibited by β-Aminopropionitrile (500 μM; 72 h), which also suppresses the hypoxia-induced EMT morphological and marker protein changes[2].

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BAPN/β-Aminopropionitrile inhibition of hypoxia-induced invasion of cervical cancer cells [2]
The first step of the cancer metastatic process is cell invasion. To study the effect of LOX inhibition with cervical carcinoma cell behavior, we examined the invasion of HeLa and SiHa cells in vitro. Both cell lines were incubated on Matrigel-coated filters of transwells for 48 h under normoxia or hypoxia in the presence or absence of 500 μM BAPN, an inhibitor of LOX. Following incubation, trans-filter (invasion) cells were stained (Fig. 2A and B) and numbers counted (Fig. 2C and D). As shown, two cell lines showed strong invasion phenomenon under hypoxia in comparison to normoxia. Notably, BAPN that inactivates LOX activity significantly reduced hypoxia-elicited cell invasion in both cell models (Fig. 2A and B). Counting of invasive cell number further illustrated morphological conclusion. As shown (Fig. 2C and D), hypoxia enhanced cancer cell invasion to 220 and 250% of the control, which were suppressed to 50 and 60% of the control in HeLa and SiHa cells, respectively, in the presence of 500 μM BAPN. Thus, LOX plays a key role in the development of invasion of cervical carcinoma cells.

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BAPN/β-Aminopropionitrile abolishment of the EMT morphological alterations in HeLa and SiHa cells [2]
To answer whether BAPN inhibits hypoxia-induced EMT in cancer cell models, we examined BAPN effects on morphological changes in cervical carcinoma cells under hypoxia with phase contrast microscopy. HeLa and SiHa cells exposed to hypoxia for 48 h displayed morphological changes towards a mesenchymal-like appearance (Fig. 5). Anoxic HeLa and SiHa cells were no longer able to form ‘cobblestone’ clusters typical for epithelial cells, but acquired a more elongated, spindle-like morphology, a critical marker of EMT. These morphological changes were involved in cervical carcinoma cell invasion and migration under hypoxia (33). This is based on findings that BAPN inhibited tumor cell invasion and migration (Figs. 2 and 3) antagonized morphological changes in cancer cells under hypoxia, and reversed cell phenotypes similar to those under normoxia. Thus, BAPN prevented HeLa and SiHa cells from hypoxia-induced morphological changes toward the EMT.

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BAPN/β-Aminopropionitrile antagonism of hypoxia-induced changes of EMT marker proteins in HeLa and SiHa cells [2]
Finally, we detected the effects of BAPN on the expressions of E-cadherin, α-SMA, vimentin, MMP-2 and MMP-9 proteins, the EMT markers, in HeLa and SiHa cells exposed to hypoxia for 48 h. As shown, BAPN effectively prevented hypoxia-induced downregulation of E-cadherin (Fig. 6A and B) and strongly inhibited hypoxia-induced upregulation of α-SMA and vimentin (Fig. 6C and D). Although MMP-2 in HeLa and SiHa cells was not changed significantly under hypoxia or hypoxia plus BAPN, MMP-9 in both cell lines was markedly changed in response to experimental conditions (hypoxia and BAPN treatment). MMP-9 expression was increased by hypoxia in SiHa cells up to 1.65-fold of the control (47.5 density units) and turned to 0.88 of the control (47.5 density units) in the presence of 500 μM BAPN. Furthermore, BAPN also reduced MMP-9 expression to 0.41 of the control (128 density units) in HeLa cells in response to hypoxia. Densitometry data of BAPN effects on EMT marker proteins are presented in Table I. These results are consistent with BAPN effects on tumor cell invasion and migration, strongly supporting the conclusion that LOX is an essential factor for modulation of hypoxia-induced EMT, invasion and migration of cervical cancer cells.

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BAPN/β-Aminopropionitrile normalises the expression of GLUT4 and adiponectin, and improves glucose uptake in an in vitro model of insulin resistance [1]
To determine whether BAPN directly affects adipocyte function, we performed in vitro experiments in the TNFα-induced insulin resistance model in differentiated 3T3-L1 adipocytes. As shown in Fig. 5 and as previously described (Stephens et al., 1997; Sethi and Hotamisligil, 1999), TNFα reduced expression of GLUT4 and adiponectin and increased SOCS3 protein levels in these cells. Interestingly, these effects were prevented by BAPN (Fig. 5A-C). Accordingly, BAPN normalised the TNFα-induced decrease in insulin-stimulated glucose uptake observed in differentiated 3T3-L1 adipocytes (Fig. 5D).

In rats with diet-induced obesity, β-Aminopropionitrile (BAPN) (100 mg/kg/day; po; 6 weeks) improves the metabolic profile and decreases body weight gain[1]. C57BL/6 mice are given β-Aminopropionitrile monofumarate (1 g/kg/day; po; 4 weeks) to induce thoracic aortic dissection[3].

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BAPN/β-Aminopropionitrile ameliorates the increase in body weight, adiposity and adipose tissue fibrosis in HFD-fed rats [1]
To investigate whether LOX contributes to the adipose tissue dysfunction associated with obesity, rats subjected to a HFD were treated with BAPN, an irreversible and specific inhibitor of LOX activity. As shown in Fig. 3A, the HFD induced a significant increase in body weight that reached a significant difference from the third week onwards. After three weeks of treatment, BAPN significantly prevented the rise in body weight in HFD rats, but not in animals that were fed a standard diet (Fig. 3A). These differences were sustained until the end of the study (Table 1, Fig. 3A). Similarly, BAPN reduced the increase in the weight of white adipose tissue (both epididymal and lumbar) in obese animals (Table 1) and attenuated their enhanced adiposity (Table 1). It should be noted that the changes in body weight triggered by BAPN in HFD-fed rats were not related to differences in food intake (Table 1).

The changes in adipose tissue mass elicited by β-Aminopropionitrile/BAPN in HFD-fed rats prompted us to determine whether LOX inhibition could modulate the adipocyte area. Histological analysis of epididymal adipose tissue revealed an increased adipocyte area in the HFD group. A trend towards an attenuation of this parameter was observed in obese animals that had been treated with BAPN (Fig. 3B,C). Furthermore, a shift toward smaller adipocytes was detected in BAPN-treated obese animals compared with HFD-fed rats (Fig. 3D). Interestingly, LOX inhibition prevented the increase in pericellular collagen content observed in obese rats, as analysed by using Picrosirius red staining (Fig. 3E,F).

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BAPN/β-Aminopropionitrile improves the metabolic alterations observed in obese rats [1]
Next, we examined whether the inhibition of LOX activity could modify metabolic parameters in obese animals. Treatment with BAPN improved fasted glucose and insulin levels and consequently reduced HOMA index in the HFD group (Table 1). LOX inhibition also reduced plasma triglycerides in obese animals, but no significant differences were observed in the total cholesterol levels (Table 1).

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BAPN/β-Aminopropionitrile improves insulin signalling in adipose tissue from obese animals [1]
In order to understand how BAPN improves insulin sensitivity in obese animals, we analysed the levels of proteins involved in the control of insulin sensitivity in epididymal adipose tissue. The reduction in both glucose transporter 4 (GLUT4) and adiponectin expression observed in the HFD group was normalised through the inhibition of LOX activity (Fig. 4A,B). Furthermore, the increase in the protein levels of DPP4 and suppressor of cytokine signaling 3 (SOCS3) triggered by the HFD was completely prevented by BAPN (Fig. 4C,D).

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Basal characterization of mice with β-Aminopropionitrile/BAPN treatment, with or without Ang II [3]
It was reported that treatment with the LOX inhibitor BAPN plus Ang II induced TAD in FVB mice8. We recently found that, on a C57BL/6 background, the same dose of BAPN caused sudden death in approximately 56% of mice, prior to Ang II administration, and that this was caused by thoracic aortic ruptures4. We thus compared the effects of BAPN in mice with the two genetic backgrounds. Male mice (3 wk old) on FVB or C57BL/6 backgrounds were administered BAPN in drinking water at a dose of 1 g per kg body weight for 4 wk. BAPN treatment reduced diastolic (Fig. 1a) with no effect on systolic (Fig. 1b) blood pressure, indicating increased aortic stiffness. BAPN treatment also attenuated body weight gains (Fig. 1c,d) and significantly decreased plasma triglyceride and cholesterol levels (Fig. 1e,f) in both FVB and C57BL/6 mice.

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Induction of TAD with BAPN/β-Aminopropionitrile treatment [3]
We next examined the effects of BAPN treatment on the incidence of TAD. To determine whether Ang II was also required for TAD development, the mice were sacrificed for autopsy after 4 wk of BAPN treatment, with or without 24 h of Ang II infusion. Consistent with the previous report8, administration of BAPN plus Ang II induced TAD in all mice, while approximately 75% of FVB mice treated with BAPN alone did not develop TAD (Fig. 2a and Table 1). However, the incidence of TAD in C57BL/6 mice treated with only BAPN reached 87% (Fig. 2a and Table 1) and 45% of mice in this group died of aortic rupture. TAD was also observed in all C57BL/6 mice treated with BAPN plus Ang II, with 50% of these having aortic rupture within 24 h of Ang II infusion. Aortas of C57BL/6 mice given BAPN, with or without Ang II infusion, were enlarged from the root to the thoracic segment and, in some cases, the abdominal segment was also involved. Hematomas were observed in the lesions, indicating thrombosis (Fig. 2a).

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BAPN/β-Aminopropionitrile dose optimization for TAD induction [3]
To further investigate the causal effects of medial degeneration on TAD formation, we applied different doses of BAPN by feeding 3-wk-old C57BL/6 male mice with diets containing 0, 0.4, 1.0 or 1.5 g BAPN per 100 g mouse chow for 4 wk. Body weights were lower with increased BAPN doses (Fig. 3a). All six mice fed with the 0.4 g BAPN per 100 g diet developed TAD and five died of dissection ruptures by 2 to 4 wk after BAPN administration. Of six mice fed with the 1.0 g BAPN diet, two had TAD at the end of the treatment, but no ruptures occurred. Most surprisingly, no TAD formation was observed in mice fed with the 1.5 g BAPN diet (Fig. 3b).

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Molecular phenotypic features of β-Aminopropionitrile/BAPN-induced TAD [3]
Because BAPN-induced TAD exhibited typical histological features of the human disease, we next examined whether expression of TAD-related genes were also changed in the media of aortas. A panel of genes known to be dysregulated during medial degradation in TAD formation were selected for analysis. These were matrix metalloproteinases (MMPs, MMP2/3/9)5,8,11,12 and cathepsins (cathepsin S/K/L)13 (that degrade extracellular matrix), collagen I α1 (COL1α1) and connective tissue growth factor (CTGF) (target genes indicating activation of the TGF-β signalling pathway in LDS)14, α-smooth muscle actin (α-SMA) and β-myosin heavy chain (β-MHC) (associated with familial thoracic aortic aneurysm and dissection syndrome)15. Expression of these genes was compared in control and BAPN-treated C57BL/6 mice. In the BAPN-treated group, compared with the control, MMP2 was significantly upregulated (Fig. 4a), while MMP3 and MMP9 were downregulated (Fig. 4b,c). Cathepsin S and cathepsin K levels were no different in the two groups (Fig. 4d,e), while cathepsin L was significantly decreased in the BAPN group (Fig. 4f). Both COL1α1 and α-SMA expression were dramatically decreased with BAPN treatment (Fig. 4g,h), while CTGF and β-MHC levels were not changed (Fig. 4i,j). These results revealed that BAPN-induced TAD was associated with typical ECM degradation, possibly via MMP2, and loss of SMC leading to decreased α-SMA, effects consistent with previous observations in humans and mouse models.

Glucose uptake measurements [1]
Fully differentiated 3T3-L1 adipocytes were insulin-deprived and pre-treated with β-Aminopropionitrile/BAPN in the presence or absence of TNFα for 24 h. Adipocytes were serum-deprived for 3 h in DMEM supplemented with 2% of fatty-acid-free bovine serum albumin (BSA) with or without TNFα and BAPN. Serum-free medium was then removed and the cells were washed with 1 ml of Krebs-Ringer-HEPES (KRH) buffer pH 7.4 plus 0.2% BSA. Glucose uptake was initiated with 0.9 ml of KRH buffer containing 100 mM insulin for 30 min followed by the addition of 100 µM 2-deoxy-d-glucose and 1 µCi/ml of [3H]-2-deoxy-d-glucose/ml. After 10 min, cells were washed with an ice-cold solution of 50 mM d-glucose in PBS three times. Cells were lysed with a buffer containing 0.5 N NaOH and 0.1% sodium dodecyl sulfate (SDS), and the radioactivity retained by the cell lysate was measured using a liquid scintillation counter. Measurements were made in triplicate and corrected for nonspecific diffusion. Counts of [3H]-2-deoxyglucose were normalised to protein levels.

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LOX activity assay [2]
Cervical carcinoma cells in phenol red-free DMEM were incubated in normoxia or hypoxia. The conditioned medium was collected and LOX activity was assayed using diaminopentane as a substrate in the Amplex Red fluorescence assay. The reaction mixture consisted of 1.2 mol/l urea, 0.05 mol/l sodium borate (pH 8.2), 10 mmol/l diaminopentane, 10 μmol/l Amplex red and 1 U/ml horseradish peroxidase in a final volume of 1 ml. Conditioned medium (500 μl) was added to the reaction mixture in the presence or absence of 0.5 mmol/l β-Aminopropionitrile (BAPN), an active site inhibitor of LOX. Samples were incubated at 37°C for 30 min, placed on ice, and then recorded at an excitation wavelength of 563 nm and emission wavelength of 587 nm (31). All enzyme activities were calculated as the increase of fluorescent units above background levels of BAPN controls and normalized to total cell protein.

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In vitro invasion and migration assay [2]
Cells were serum-deprived for 24 h, seeded at a density of 50,000 cells/well on the top of Matrigel-coated filters, moved to chambers containing 600 μl of 10% FBS as a chemo-attractant and incubated under normoxia or oxygen-deprived conditions for 48 h. β-Aminopropionitrile/BAPN (500 μM) was added to the culture 24 h before oxygen deprivation and continued throughout the experiment. At the same time, equal cells were plated into 96-well plates for cell number assay (MTT). The cells (treated and untreated) were incubated at 37°C under normoxia or oxygen-deprived conditions for 48 h and then the Matrigel was removed with a cotton bud. The invaded cells were fixed, stained with hematoxylin and counted. The invasiveness of cervical carcinoma cells was determined by the percentage-of-invasion score (invaded cell number/total cell number 100%). Experiments were repeated three times. The in vitro cellular migration assay was based on the described membrane invasion culture system, but differed in the use of filters not Matrigel-coated.

Western Blot Analysis[1]
Cell Types: 3T3-L1 adipocytes
Tested Concentrations: 200 μM with 1.15 nM and 2.87 nM TNFα
Incubation Duration: 72 h
Experimental Results: TNFα decreased expression of GLUT4 and adiponectin, and increased SOCS3 protein levels in these cells. And these effects were prevented. Cell Invasion Assay[2]
Cell Types: HeLa and SiHa cells
Tested Concentrations: 500 μM
Incubation Duration: 72 h
Experimental Results: Dramatically decreased hypoxia- elicited cell invasion in both cell models.

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Cell Migration Assay [2]
Cell Types: HeLa and SiHa cells
Tested Concentrations: 500 μM
Incubation Duration: 72 h
Experimental Results: diminished hypoxia-induced migration from 180 and 240% to 60 and 70% in HeLa and SiHa cells, respectively.

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Western Blot Analysis[2]
Cell Types: HeLa and SiHa cells
Tested Concentrations: 500 μM
Incubation Duration: 72 h
Experimental Results: Effectively prevented hypoxia-induced downregulation of E-cadherin and strongly inhibited hypoxia-induced upregulation of α-SMA and vimentin.

Animal/Disease Models: Male Wistar rats of 150 g, high-fat diet (HFD) model[1]
Doses: 100 mg/kg/day
Route of Administration: In the drinking water, 6 weeks
Experimental Results: Dramatically prevented the rise in body weight in HFD rats, but not in animals that were fed a standard diet. decreased the increase in the weight of white adipose tissue (both epididymal and lumbar) in obese animals and attenuated their enhanced adiposity. Improved fasted glucose and insulin levels and consequently decreased HOMA index in the HFD group. Improved insulin signaling in adipose tissue from obese animals.

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Animal/Disease Models: C57BL/6 mice[3]
Doses: 1 g/kg/day
Route of Administration: In the drinking water, 4 weeks
Experimental Results: Induce thoracic aortic dissection (TAD) in all mice with 24 h of Ang II infusion. Caused 87% of C57BL/6 mice to develop TAD without Ang II.

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Male Wistar rats of 150 g were fed either a HFD (33.5% fat) or a standard diet (3.5% fat) for 6 weeks. Half of the animals of each group received the irreversible inhibitor of LOX activity β-Aminopropionitrile/BAPN (100 mg/kg/day) in the drinking water for the same period, as previously described (Brasselet et al., 2005). The amount of β-Aminopropionitrile/BAPN effectively taken daily per animal was calculated from the amount of water consumed on a daily basis. Animal weight was periodically controlled to adjust the target dose of BAPN. Food and water intake were determined throughout the experimental period. Animals were fasted the day before euthanasia by anaesthesia with a cocktail of ketamine (70 mg/kg; intraperitoneal) and xilacine (Rompun 2%, 6 mg/kg). Serum and plasma were collected and abdominal adipose tissue was dissected for further analysis. Adiposity index was calculated as: sum of fat pads/[(body weight-fat pad weight)×100]. [1]

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Animal model and ethics statement [3]
Three-week-old male mice were fed a normal diet and administered freshly prepared β-Aminopropionitrile/BAPN solution dissolved in the drinking water (1 g/kg/d) for 4 wk, as described previously8. Blood pressure was measured before and after BAPN administration for 4 wk, using the tail-cuff method. Interventions lasted 4 wk and body weights were measured weekly. As previously reported, at 7 wk old, osmotic mini pumps administering 1 μg/kg per min Ang II were implanted subcutaneously and mice were euthanized 24 h after implantation17. All mice died before expected end time of the experiment were autopsied immediately, and Blood clots were found in the thoracic cavities of these mice. Mice surviving at the end of the experiment were sacrificed by an overdose of sodium pentobarbital and their blood and tissue samples were collected for further analyses. Histopathological analysis [3]
Complete gross and histopathological evaluations were performed with samples from control and β-Aminopropionitrile/BAPN-treated mice. After euthanasia, normal and dissected aortas were harvested from the ascending aorta to the iliac artery and were fixed in 10% buffered formalin, as were human tissues. Fixed, paraffin-embedded tissues were cut at 5 μm thickness, stained with haematoxylin and eosin following standard procedures and examined under light microscopy, as previously described4.

Absorption, Distribution and Excretion
β-aminopropionitrile (BAPN) was detectable in urine within one hour of oral administration. After oral administration of 250 mg BAPN every 6 hours for 21 days, approximately 16% of the total dose was recovered in urine. BAPN was not detected in urine samples collected 7 hours after BAPN discontinuation. The appearance of cyanoacetic acid in urine was slower than that of BAPN, gradually increasing to approximately three times the level of BAPN in urine. After BAPN discontinuation, BAPN-derived cyanoacetic acid was continuously excreted in urine. When the (14)C-BAPN free base was applied to the skin of rats, its absorption rate and extent were higher than that of fumarate. Six hours after topical application of the free base, only trace amounts of (14)C were detected on the skin, and the dose in the skin was less than 1% of the total dose, indicating rapid drug absorption.

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Metabolism/Metabolites
……β-Aminopropionitrile/Metabolism/Cyanoacetic Acid…
β-Aminopropionitrile (BAPN) is detectable in urine within 1 hour after oral administration. After oral administration of 250 mg BAPN every 6 hours for 21 days, the recovery rate of BAPN in urine was approximately 16% of the total dose. BAPN was undetectable in samples collected 7 hours after discontinuation. The appearance of urinary cyanoacetic acid was slower than that of BAPN, gradually increasing to approximately 3 times the level of BAPN in urine. After discontinuation of BAPN, BAPN-derived cyanoacetic acid was continuously excreted in urine.
Organic nitriles are converted to cyanide ions in the liver by cytochrome P450 enzymes. Cyanide is rapidly absorbed and distributed throughout the body. Cyanide is primarily metabolized to thiocyanate by thiocyanate esterase or 3-mercaptopyruvate thiotransferase. Cyanide metabolites are excreted in urine. (L96)

Toxicity Summary
Organic nitriles can decompose into cyanide ions both in vivo and in vitro. Therefore, the main toxic mechanism of organic nitriles is the production of toxic cyanide ions, or hydrogen cyanide. Cyanide ions are inhibitors of cytochrome c oxidase in the fourth electron transport chain complex (located on the mitochondrial membrane of eukaryotic cells). It forms a complex with the ferric atom in this enzyme. The binding of cyanide ions to this cytochrome prevents electrons from being transferred from cytochrome c oxidase to oxygen. As a result, the electron transport chain is disrupted, and the cell can no longer perform aerobic respiration to produce ATP for energy. Tissues that rely primarily on aerobic respiration, such as the central nervous system and the heart, are particularly susceptible to this. Cyanide can also produce some toxic effects by binding to catalase, glutathione peroxidase, methemoglobin, hydrocobalamin, phosphatase, tyrosinase, ascorbic acid oxidase, xanthine oxidase, succinate dehydrogenase, and copper/zinc superoxide dismutase. Cyanide binds to the iron ions in methemoglobin to form inactive methemoglobin cyanide. (L97)

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Interactions
Continuous administration of BAPN (1 g/kg/day) for 8 weeks resulted in simultaneous changes in the skin and aortic connective tissue of rats. In the skin, collagen tissue shifted and fragmented, elastic tissue disappeared, and fibroblasts vacuolated. The addition of pyridinium carbamate (PDC) to BAPN prevented the formation of elastic tissue and fibroblast damage. Administration of PDC after discontinuation of sensitizer treatment prevented the formation of damage and accelerated its regression.
Administering BAPN at a dose of 2500 mg/kg via gavage to pregnant hamsters on day 11 of gestation resulted in skeletal malformations in 69.5% of their offspring. Immediate administration of β-hydroxyethylrutin (which protects collagen from teratogenic damage) after BAPN injection in pregnant animals significantly reduced the teratogenic response. This supports the view that the mechanism by which BAPN induces skeletal dysplasia is the inhibition of cross-linking during collagen fiber maturation.

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Non-human Toxicity Values
Intraperitoneal LD50 in mice: 1152 mg/kg

[1]. The lysyl oxidase inhibitor β-aminopropionitrile reduces body weight gain and improves the metabolic profile in diet-induced obesity in rats. Dis Model Mech. 2015 Jun;8(6):543-51.
[2]. Inactivation of lysyl oxidase by β-aminopropionitrile inhibits hypoxia-induced invasion and migration of cervical cancer cells. Oncol Rep. 2013 Feb;29(2):541-8.
[3]. β-Aminopropionitrile monofumarate induces thoracic aortic dissection in C57BL/6 mice. Sci Rep. 2016 Jun 22;6:28149.

β-Aminopropionitrile is an aminopropionitrile with an amino group at the β-position. It is a plant metabolite and also an antitumor drug, antirheumatic drug, and collagen cross-linking inhibitor. It is the conjugate base of β-aminopropionitrile. β-Aminopropionitrile is a metabolite found or produced in Escherichia coli (K12 strain, MG1655 strain). 3-Aminopropionitrile has been reported in Euglena gracilis, and relevant data are available. β-Aminopropionitrile is a toxic amino acid derivative. A rare case of preterm infant Cantrell syndrome was reported, with limb dysplasia, right renal dysplasia, cerebellar dysplasia, and localized cutaneous dysplasia. The mother's medical history suggested occupational exposure to aminopropionitrile before pregnancy. The characteristic manifestations of Cantrell syndrome—anterior thoracoabdominal wall defects with ectopic heart, diaphragm, sternum, pericardium, and cardiac defects—were also observed in animals after maternal administration of β-aminopropionitrile. Some Vitex species (such as Vitex negundo, and related to cultivated Vitex negundo), especially Vitex amaranth, while not inducing Vitex negundo poisoning in humans, contain β-aminopropionitrile, a substance that can induce pathological changes in the bones (bone Vitex negundo poisoning) and blood vessels (vascular Vitex negundo poisoning) of experimental animals without damaging the nervous system. β-Aminopropionitrile has been proposed for use in the pharmacological control of unwanted scar tissue in humans. β-Aminopropionitrile is a reagent used as an intermediate in the production of β-alanine and pantothenic acid. (A11439, A11440, A11441)
Used as an intermediate in the production of β-alanine and pantothenic acid.
See also: …See more…

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Mechanism of Action
Its mechanism of action is not yet clear, but it is presumed to be achieved through some effect on the growth of certain mesodermal tissues. Its action is not caused by its main metabolite, cyanoacetic acid; both the free amino and cyano groups appear to be essential for its activity. If the amino group is located at the α-position, or at the γ-position of the butyronitrile, the substance will not be produced. Studies have shown that the mechanism of action of dizziness inducers is to interfere with cross-linking by blocking certain carbonyl groups normally present in collagen. Toxapeptide or calcium salts can delay its effect. /Dizziness Inducers/

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Therapeutic Uses
Experimental Applications: Administration of the lysyl oxidase inhibitor BAPN can prevent the occurrence of hypertension and reduce the collagen content in the blood vessels of rats with induced hypertension. Histological examination showed that BAPN can prevent atherosclerotic changes.
Experimental Applications: In young hypertensive rats, BAPN (20 mg, intraperitoneal injection daily for 2 weeks) can prevent the occurrence of hypertension. In adult spontaneously hypertensive rats (50 mg, intraperitoneal injection once daily for 2 weeks), blood pressure decreased.
Experimental Applications: Rats with subcutaneous polyvinyl alcohol sponge implants and skin incisions received a single injection of β-aminopropionitrile (BAPN) at doses ranging from 1-40 mg/100 g, for a total of 4 doses. Even the lowest dose of BAPN inhibited lysyl oxidase activity for 6 hours; the higher the dose, the longer the duration of inhibition, with 40 mg BAPN maintaining inhibition for at least 48 hours. The degree and duration of inhibition were reflected in collagen extraction and wound rupture strength. Data indicate that the minimum dose of BAPN can achieve clinical efficacy if drug metabolism is reduced (through monoamine oxidase inhibitors) or a sustained-release formulation is used. Experimental Application: In a hamster bleomycin-induced pulmonary fibrosis model, the ability of β-aminopropionitrile (BAPN) to prevent excessive collagen production was tested. Two groups of animals received a single intratracheal injection of bleomycin; one group received BAPN twice daily for 30 days. A third group received saline and BAPN. Bleomycin increased collagen content, reduced lung volume, and led to fibrosis with a mortality rate of 51%. Administration of BAPN to animals treated with bleomycin prevented excessive collagen accumulation, reduced fibrosis, and lowered mortality to 24%. BAPN alone had no effect on lung mechanics or collagen content.

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Extracellular matrix (ECM) remodeling in adipose tissue plays a crucial role in the pathophysiology of obesity. Amine oxidases of the lysyl oxidase (LOX) family, including LOX and LOX-like (LOXL) isoenzymes, control ECM maturation, and upregulation of LOX activity is essential in fibrosis; however, its role in adipose tissue dysfunction in obesity remains unclear. In this study, we observed that LOX is the major isoenzyme expressed in human adipose tissue, and its expression was significantly upregulated in samples from obese individuals who underwent bariatric surgery. LOX expression was also induced in the adipose tissue of male Wistar rats fed a high-fat diet (HFD). Interestingly, treatment with β-aminopropionitrile (BAPN)—a specific and irreversible inhibitor of LOX activity—reduced the increase in body weight and fat mass in obese animals and induced a reduction in adipocyte volume. BAPN also improved the increase in collagen content in adipose tissue of obese animals and improved a variety of metabolic indicators, including reducing glucose and insulin levels, reducing homeostasis model assessment (HOMA) index and reducing plasma triglyceride levels. In addition, in white adipose tissue of obese animals, BAPN also prevented the downregulation of adiponectin and glucose transporter 4 (GLUT4) induced by high-fat diet (HFD), as well as the increase in the levels of cytokine signaling inhibitor 3 (SOCS3) and dipeptidyl peptidase 4 (DPP4). Similarly, in the TNFα-induced insulin-resistant 3T3-L1 adipocyte model, BAPN prevented the downregulation of adiponectin and GLUT4 and the increase in SOCS3 levels, thereby restoring insulin-stimulated glucose uptake to normal. Therefore, our data suggest that LOX plays a pathological role in obesity-induced metabolic dysfunction and highlight the importance of novel pharmacological interventions targeting adipose tissue fibrosis and LOX activity in the clinical treatment of obesity. [1]

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Tumor invasion and metastasis are the main causes of death in patients with cervical cancer. Tumors under hypoxic conditions are more aggressive and have higher metastatic activity. Lysyl oxidase (LOX) is a hypoxia-responsive gene. Studies have shown that LOX is crucial for hypoxia-induced metastasis in breast cancer. However, the direct effects of LOX on cervical cancer cell motility remain poorly understood. Our study shows that LOX protein and catalytic activity levels are upregulated in cervical cancer cells under hypoxic conditions. Hypoxia induces mesenchymal-like morphological changes in HeLa and SiHa cells, accompanied by upregulation of mesenchymal markers α-SMA and vimentin, and downregulation of the epithelial marker E-cadherin, indicating that cervical cancer cells undergo epithelial-mesenchymal transition (EMT) under hypoxic conditions. Treatment of tumor cells with the LOX active site inhibitor β-aminopropionitrile (BAPN) blocks hypoxia-induced EMT morphological and marker protein changes and inhibits the in vitro invasion and migration of cervical cancer cells. In summary, these results indicate that LOX enhances hypoxia-induced cervical cancer cell invasion and migration through EMT, while BAPN can inhibit this process. [2] Thoracic aortic dissection (TAD) is a catastrophic disease with high mortality and disability rates, characterized by elastin rupture and smooth muscle cell loss. However, the underlying pathological mechanisms of the disease remain unclear due to the lack of suitable animal models, which limits the discovery of effective treatment strategies. We treated mice with C57BL/6 and FVB genetic backgrounds for 4 weeks with β-aminopropionitrile monofumarate (BAPN), an irreversible inhibitor of lysyl oxidase, followed by an infusion of angiotensin II (Ang II) for 24 hours. We found that BAPN combined with Ang II treatment induced aortic dissection in 100% of mice in both genetic backgrounds. BAPN without angiotensin II caused aortic dissection in only a minority of FVB mice, but caused TAD in 87% of C57BL/6 mice, of whom 37% died from aortic dissection rupture. In addition, lower doses of BAPN induced TAD formation and rupture earlier and had less impact on body weight. Therefore, we have established a reliable and convenient TAD model in C57BL/6 mice, which can be used to study the pathological process of TAD and explore its therapeutic targets. [3]

C₃H₆N₂

70.09

70.053

151-18-8

1647

Colorless to light yellow Liquid

0.9±0.1 g/cm3

186.3±13.0 °C at 760 mmHg

< 25 °C

66.5±19.8 °C

0.7±0.4 mmHg at 25°C

1.430

-1.02

1

2

1

5

49.2

0

C(CN)C#N

AGSPXMVUFBBBMO-UHFFFAOYSA-N

InChI=1S/C3H6N2/c4-2-1-3-5/h1-2,4H2

3-aminopropanenitrile

β-Aminopropionitrile; 3-aminopropionitrile; 3-Aminopropanenitrile; 151-18-8; 2-Cyanoethylamine; Aminopropionitrile; BETA-AMINOPROPIONITRILE; Propanenitrile, 3-amino-; beta-Cyanoethylamine; β Aminopropionitrile

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.

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 (~1426.74 mM)
H2O : ~50 mg/mL (~713.37 mM)

Solubility in Formulation 1: ≥ 3.25 mg/mL (46.37 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 32.5 mg/mL clear DMSO stock solution to 400 μL of PEG300 and mix evenly; then add 50 μL of Tween-80 to the above solution and mix evenly; then add 450 μL of 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: ≥ 3.25 mg/mL (46.37 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 32.5 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: ≥ 3.25 mg/mL (46.37 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 32.5 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 100 mg/mL (1426.74 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.

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