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
BETA-AMINOPROPIONITRILE (BAPN) WAS FOUND IN URINE WITHIN 1 HR OF ORAL ADMIN. ORAL 250 MG BAPN AT 6 HR INTERVALS EACH DAY FOR 21 DAYS RESULTED IN URINARY BAPN RECOVERIES APPROXIMATING 16% OF TOTAL DOSE. BAPN WAS NOT DETECTED IN SPECIMENS COLLECTED LATER THAN 7 HR AFTER CESSATION OF BAPN DOSAGE. URINARY CYANOACETIC ACID APPEARED MORE SLOWLY THAN BAPN & INCR GRADUALLY TO APPROX 3 TIMES THAT OF URINARY BAPN. AFTER BAPN WAS DISCONTINUED, THERE WAS PROLONGED URINARY EXCRETION OF BAPN-DERIVED CYANOACETIC ACID.
AFTER APPLICATION TO THE SKIN OF RATS, (14)C-BAPN FREE BASE WAS ABSORBED MORE RAPIDLY AND TO A GREATER EXTENT THAN THE FUMARATE SALT. SIX HOURS AFTER TOPICAL ADMINISTRATION OF THE FREE BASE ONLY TRACES OF (14)C WERE FOUND ON THE SKIN AND LESS THAN 1% OF THE DOSE WITHIN THE SKIN SECTION SUGGESTING RAPID DRUG ABSORPTION.

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Metabolism / Metabolites
...BETA-AMINOPROPIONITRILE /IS METABOLIZED/ INTO CYANOACETIC ACID...
BETA-AMINOPROPIONITRILE (BAPN) WAS FOUND IN URINE WITHIN 1 HR OF ORAL ADMIN. ORAL 250 MG BAPN AT 6 HR INTERVALS EACH DAY FOR 21 DAYS RESULTED IN URINARY BAPN RECOVERIES APPROXIMATING 16% OF TOTAL DOSE. BAPN WAS NOT DETECTED IN SPECIMENS COLLECTED LATER THAN 7 HR AFTER CESSATION OF BAPN DOSAGE. URINARY CYANOACETIC ACID APPEARED MORE SLOWLY THAN BAPN & INCR GRADUALLY TO APPROX 3 TIMES THAT OF URINARY BAPN. AFTER BAPN WAS DISCONTINUED, THERE WAS PROLONGED URINARY EXCRETION OF BAPN-DERIVED CYANOACETIC ACID.
Organic nitriles are converted into cyanide ions through the action of cytochrome P450 enzymes in the liver. Cyanide is rapidly absorbed and distributed throughout the body. Cyanide is mainly metabolized into thiocyanate by either rhodanese or 3-mercaptopyruvate sulfur transferase. Cyanide metabolites are excreted in the urine. (L96)

Toxicity Summary
Organic nitriles decompose into cyanide ions both in vivo and in vitro. Consequently the primary mechanism of toxicity for organic nitriles is their production of toxic cyanide ions or hydrogen cyanide. Cyanide is an inhibitor of cytochrome c oxidase in the fourth complex of the electron transport chain (found in the membrane of the mitochondria of eukaryotic cells). It complexes with the ferric iron atom in this enzyme. The binding of cyanide to this cytochrome prevents transport of electrons from cytochrome c oxidase to oxygen. As a result, the electron transport chain is disrupted and the cell can no longer aerobically produce ATP for energy. Tissues that mainly depend on aerobic respiration, such as the central nervous system and the heart, are particularly affected. Cyanide is also known produce some of its toxic effects by binding to catalase, glutathione peroxidase, methemoglobin, hydroxocobalamin, phosphatase, tyrosinase, ascorbic acid oxidase, xanthine oxidase, succinic dehydrogenase, and Cu/Zn superoxide dismutase. Cyanide binds to the ferric ion of methemoglobin to form inactive cyanmethemoglobin. (L97)

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Interactions
USE OF BAPN (1 G/KG/DAY) FOR PERIOD OF 8 WK CAUSED SIMULTANEOUS CHANGES IN SKIN & AORTIC CONNECTIVE TISSUES OF RATS. IN SKIN, COLLAGEN TISSUE WAS DISLOCATED & BROKEN INTO FRAGMENTS, ELASTIC TISSUE DISAPPEARED & FIBROBLASTS WERE VACUOLIZED. ADDITION OF PYRIDINOL CARBAMATE (PDC) TO BAPN PREVENTS FORMATION OF LESIONS OF ELASTIC TISSUE & OF FIBROBLASTS. WHEN GIVEN AFTER CESSATION OF LATHYROGEN TREATMENT, PDC ARRESTED FORMATION OF LESIONS & ACCELERATED THEIR REGRESSION.
A DOSE OF 2,500 MG/KG BAPN GIVEN BY GAVAGE ON DAY 11 TO PREGNANT HAMSTERS PRODUCED 69.5% SKELETAL ANOMALIES IN THE OFFSPRING. ADMINISTRATION OF BETA-HYDROXYETHYLRUTOSIDES (WHICH PROTECT AGAINST COLLAGEN DAMAGE FROM LATHYROGENS) IMMEDIATELY AFTER BAPN TO THE PREGNANT ANIMALS RESULTED IN SIGNIFICANTLY DECREASED TERATOGENIC RESPONSE. THIS SUPPORTS THE VIEW THAT THE MECHANISM FOR BAPN-INDUCED SKELETAL DYSMORPHOGENESIS IS THE INHIBITION OF CROSS-LINKING DURING THE MATURATION OF COLLAGEN FIBERS.

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Non-Human Toxicity Values
LD50 Mouse ip 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.

Beta-aminopropionitrile is an aminopropionitrile carrying an amino group at the beta-position. It has a role as a plant metabolite, an antineoplastic agent, an antirheumatic drug and a collagen cross-linking inhibitor. It is a conjugate base of a beta-ammoniopropionitrile.
beta-Aminopropionitrile is a metabolite found in or produced by Escherichia coli (strain K12, MG1655).
3-Aminopropanenitrile has been reported in Euglena gracilis with data available.
beta-Aminopropionitrile is a toxic amino-acid derivative. On an unusual case of the Cantrell-sequence in a premature infant with associated dysmelia, aplasia of the right kidney, cerebellar hypoplasia and circumscribed aplasia of the cutis, maternal history suggested an occupational exposure to aminopropionitriles prior to pregnancy. The characteristic features of the Cantrell-sequence--anterior thoraco-abdominal wall defect with ectopia cordis and diaphragm, sternum, pericardium, and heart defects--have been observed in animals following maternal administration of beta-aminopropionitrile. Some species of lathyrus (chickling pea, Lathyrus sativus- related), notably Lathyrus odoratus, are unable to induce human lathyrism but contain beta-aminopropionitrile, that induces pathological changes in bone (osteolathyrism) and blood vessels (angiolathyrism) of experimental animals without damaging the nervous system. The administration of beta-aminopropionitrile has been proposed for pharmacological control of unwanted scar tissue in human beings. beta-Aminopropionitrile is a reagent used as an intermediate in the manufacture of beta-alanine and pantothenic acid. (A11439, A11440, A11441)
Reagent used as an intermediate in the manufacture of beta-alanine and pantothenic acid.
See also: ... View More ...

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Mechanism of Action
The mechanism of the effect is unknown, but it is thought to be by some action on growth of certain mesodermal tissues. It is not due to one of its major metabolites, cyanoacetic acid, and both the free amino group and the cyano group seem essential for activity. It is not produced if the amino group is in the alpha position, or if in the gamma position in butyronitrile.
IT HAS BEEN SUGGESTED THAT LATHYROGENIC AGENTS ACT BY BLOCKING CERTAIN CARBONYL GROUPS NORMALLY PRESENT IN COLLAGEN, & THUS INTERFERING WITH FORMATION OF CROSS LINKAGES. THEIR ACTION MAY BE RETARDED BY RESERPINE OR BY CALCIUM SALTS. /LATHYROGENIC AGENTS/

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Therapeutic Uses
EXPTL USE: ADMIN OF LYSYL OXIDASE INHIBITOR, BAPN, PREVENTED DEVELOPMENT OF HYPERTENSION & DECR AMT OF VASCULAR COLLAGEN IN RATS IN WHICH HYPERTENSION HAD BEEN INDUCED. HISTOLOGICAL EXAM REVEALED THAT ARTERIOSCLEROTIC CHANGES WERE PREVENTED BY BAPN.
EXPTL USE: IN YOUNG HYPERTENSIVE RATS, BAPN (20 MG, IP DAILY, FOR 2 WK) PREVENTED DEVELOPMENT OF HYPERTENSION. IN ADULT SPONTANEOUS HYPERTENSIVE RATS (50 MG, IP, DAILY FOR 2 WK) DECR BLOOD PRESSURE.
EXPTL USE: RATS WITH SC IMPLANTED POLYVINYL ALCOHOL SPONGES AND WITH INFLICTED SKIN INCISION WOUNDS RECEIVED A SINGLE INJECTION OF β-Aminopropionitrile (BAPN) AT 4 DOSAGES RANGING FROM 1-40 MG/100 G. EVEN THE LOWEST DOSE OF BAPN INHIBITED LYSYL OXIDASE ACTIVITY FOR 6 HOURS; WITH LARGER DOSAGES THE INHIBITION LASTED LONGER, AT 40 MG BAPN, AT LEAST 48 HOURS. THE MAGNITUDE AND DURATION OF INHIBITION WERE REFLECTED IN THE EXTRACTABILITY OF COLLAGEN AND BURSTING STRENGTH OF THE WOUND. THE DATA SUGGEST THAT A MINIMAL DOSE OF BAPN WOULD BE CLINICALLY EFFECTIVE IF EITHER THE METABOLISM OF THE DRUG WERE REDUCED (BY MONOAMINE OXIDASE INHIBITORS) OR A SUSTAINED-RELEASE PREPARATION OF BAPN WERE USED.
EXPTL USE: BETA-AMINOPROPIONITRILE (BAPN) WAS TESTED FOR ABILITY TO PREVENT EXCESS COLLAGEN FORMATION IN BLEOMYCIN-INDUCED PULMONARY FIBROSIS IN THE HAMSTER. TWO GROUPS RECEIVED 1 ENDOTRACHEAL DOSE OF BLEOMYCIN; ONE OF THESE WAS INJECTED WITH BAPN TWICE DAILY FOR 30 DAYS. A 3RD GROUP RECEIVED SALINE AND BAPN. THE BLEOMYCIN INCREASED COLLAGEN CONTENT, DECREASED LUNG VOLUME, AND PRODUCED FIBROSIS AND A MORTALITY RATE OF 51%. ADMINISTRATION OF BAPN TO BLEOMYCIN-TREATED ANIMALS PREVENTED EXCESS COLLAGEN ACCUMULATION, PRODUCED LESS FIBROSIS, AND LESSENED MORTALITY RATE TO 24%; BAPN ALONE HAD NO EFFECT ON LUNG MECHANICS OR COLLAGEN CONTENT.

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Extracellular matrix (ECM) remodelling of the adipose tissue plays a pivotal role in the pathophysiology of obesity. The lysyl oxidase (LOX) family of amine oxidases, including LOX and LOX-like (LOXL) isoenzymes, controls ECM maturation, and upregulation of LOX activity is essential in fibrosis; however, its involvement in adipose tissue dysfunction in obesity is unclear. In this study, we observed that LOX is the main isoenzyme expressed in human adipose tissue and that its expression is strongly upregulated in samples from obese individuals that had been referred to bariatric surgery. LOX expression was also induced in the adipose tissue from male Wistar rats fed a high-fat diet (HFD). Interestingly, treatment with β-Aminopropionitrile (BAPN), a specific and irreversible inhibitor of LOX activity, attenuated the increase in body weight and fat mass that was observed in obese animals and shifted adipocyte size toward smaller adipocytes. BAPN also ameliorated the increase in collagen content that was observed in adipose tissue from obese animals and improved several metabolic parameters - it ameliorated glucose and insulin levels, decreased homeostasis model assessment (HOMA) index and reduced plasma triglyceride levels. Furthermore, in white adipose tissue from obese animals, BAPN prevented the downregulation of adiponectin and glucose transporter 4 (GLUT4), as well as the increase in suppressor of cytokine signaling 3 (SOCS3) and dipeptidyl peptidase 4 (DPP4) levels, triggered by the HFD. Likewise, in the TNFα-induced insulin-resistant 3T3-L1 adipocyte model, BAPN prevented the downregulation of adiponectin and GLUT4 and the increase in SOCS3 levels, and consequently normalised insulin-stimulated glucose uptake. Therefore, our data provide evidence that LOX plays a pathologically relevant role in the metabolic dysfunction induced by obesity and emphasise the interest of novel pharmacological interventions that target adipose tissue fibrosis and LOX activity for the clinical management of this disease. [1]

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Tumor invasion and migration are major causes of mortality in patients with cervical carcinoma. Tumors under hypoxic conditions are more invasive and have a higher metastasic activity. Lysyl oxidase (LOX) is a hypoxia-responsive gene. LOX has been shown to be essential for hypoxia-induced metastasis in breast cancer. However, the direct impact of LOX on cervical cancer cell motility remains poorly understood. Our study revealed that LOX expression at protein and catalytic levels is upregulated in cervical cancer cells upon exposure to hypoxia. Hypoxia induced mesenchymal-like morphological changes in HeLa and SiHa cells which were accompanied by upregulation of α-SMA and vimentin, two mesenchymal markers, and downregulation of E-cadherin, an epithelial marker, indicating the epithelial-mesenchymal transition (EMT) of cervical cancer cells occurred under hypoxic conditions. Treatment of tumor cells with β-Aminopropionitrile (BAPN), an active site inhibitor of LOX, blocked the hypoxia-induced EMT morphological and marker protein changes, and inhibited invasion and migration capacities of cervical carcinoma cells in vitro. Collectively, these findings suggest LOX enhances hypoxia-induced invasion and migration in cervical cancer cells mediated by the EMT which can be inhibited by BAPN. [2]

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Thoracic aortic dissection (TAD) is a catastrophic disease with high mortality and morbidity, characterized by fragmentation of elastin and loss of smooth muscle cells. However, the underlying pathological mechanisms of this disease remain elusive because there are no appropriate animal models, limiting discovery of effective therapeutic strategies. We treated mice on C57BL/6 and FVB genetic backgrounds with β-Aminopropionitrile monofumarate (BAPN), an irreversible inhibitor of lysyl oxidase, for 4 wk, followed by angiotensin II (Ang II) infusion for 24 h. We found that the BAPN plus Ang II treatment induced formation of aortic dissections in 100% of mice on both genetic backgrounds. BAPN without Ang II caused dissections in few FVB mice, but caused 87% of C57BL/6 mice to develop TAD, with 37% dying from rupture of the aortic dissection. Moreover, a lower dose of BAPN induced TAD formation and rupture earlier with fewer effects on body weight. Therefore, we have generated a reliable and convenient TAD model in C57BL/6 mice for studying the pathological process and exploring therapeutic targets of TAD.[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.)