Mitochondrial toxin; mitochondrial adenine nucleotide translocase (ANT)

With EC50s of 34.14 μM, >50 μM, and 2.58 μM, respectively, Bongkrekic Acid (0-50 μM; 48 hours) induces formazan production in MDA-MB-231, MCF-7, and LTED cells. In LTED cells and parental MCF-7 cells, bongkrekic acid (0.1–25 μM; 48 hours) decreases viable cell numbers in a dose-dependent manner [1].

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Effects of Bongkrekic acid/BKA on formazan formation in ERα-positive (MCF-7 and LTED cells) and ERα-negative MDA-MB-231 cells. [3]
We first performed an MTS assay using a reagent of MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethophenyl)-2-(4-sulfophenyl)-2H-tetrazolium; Owen's reagent] to analyze whether Bongkrekic acid/BKA (Figure 1) and FAs affect the formation of formazan in breast cancer cells. The MTS assay utilizes reducing equivalents, such as the co-enzyme nicotinamide adenine dinucleotide phosphate reduced form (NADH) to convert MTS into a colored formazan product; MTS may be selectively cleaved by mitochondrial succinate dehydrogenase, which is a component of complex II (Figure 2). The MTS assay is generally used as an alternative method for [3H]thymidine incorporation. As shown in Figure 3, we investigated the effects of BKA on the formation of formazan in three breast cancer cell lines: MDA-MB-231, MCF-7 and LTED. Although BKA positively enhanced the formation of formazan in MCF-7 cells (EC50=34.14 μM), the molecule weakly stimulated its formation in MDA-MB-231 cells with an EC50 value of >50 μM (Figure 3A). When MCF-7 cells were compared with LTED cells, the basal formation of formazan was approximately 1.5-fold higher in LTED cells than in parent MCF-7 cells (Figure 3B, left panel), even though these cells were seeded at the same cell number (5×103 cells/well). In the trial with LTED cells, BKA effectively stimulated formazan formation with an EC50 value of 2.58 μM (Figure 3B, right panel). Thus, BKA appears to preferentially accelerate the formation of formazan in breast cancer cells, particularly ERα-positive LTED cells.

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BKA/Bongkrekic acid-mediated decreases in living cells: a comparison of LTED cells and parent MCF-7 cells. [3]
The enhanced formation of formazan is generally considered to reflect the number of living cells (i.e., cell viability). If this is the case in LTED cells treated with BKA, results using other approaches to examine living cells may be the same or similar to those obtained by the MTS assay. We utilized a calcein (calcein AM) probe and trypan blue dye to detect live cells. This calcein AM probe readily passes through the membranes of “viable cells”. After being transported into live cells, calcein is hydrolyzed by cytosolic esterases into a green-fluorescent calcein. Since dead cells lack esterases, live cells are selectively marked with the probe. Although BKA had no observable effects on the viability of MDA-MB-231 cells, even at 25 μM (data not shown), it slightly reduced the number of live MCF-7 cells at concentrations up to 25 μM (Figure 4A) and significantly reduced the number of live LTED cells in a concentration-dependent manner (Figure 4B). The BKA-mediated suppression of cell growth was detected with the trypan blue exclusion test (data not shown). In support, the morphology of LTED cells was affected (implicating cell death responses) more by BKA than vehicle-treated control cells (Figure 4C). Furthermore, an analysis of the cell death type using an EthD-III probe indicated that BKA-mediated death was not dependent on necroptotic cell death (Figure 4D).

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Expression status of LDH-A in breast cancer cells and effects of Bongkrekic acid/BKA on the expression of LDH-A. [3]
LDH-A is one of the major isoforms of LDH expressed in breast tissues that regulates the conversion of pyruvate to lactate in the cellular glycolytic process. MDA-MB-231 and MCF-7 breast cancer cell lines have been used to study the Warburg effect; highly aggressive MDA-MB-231 cells are known to obtain ATP by the glycolytic pathway, even in the presence of O2 (i.e., the “glycolytic” phenotype), which converts glucose to lactate and reduces OXPHOS activity (Figure 5A). In contrast to MDA-MB-231 cells, non-invasive/less tumorigenic MCF-7 cells utilized the OXPHOS pathway to produce ATP with lower glycolytic activity (i.e., the “oxidative” phenotype) (Figure 5B). In support of the above-described phenomena, decreases in LDH-A levels correlated with increases in cytosolic NADH/NAD+, which is consistent with a higher mitochondrial respiration rate (Figures 5A and 4B). Thus, we focused on the expression status of LDH-A in the three breast cancer cells. Semiquantitative and real-time RT-PCR analyses indicated that LDH-A expression was detected in the following order: MDA-MB-231 cells (5.79-, 2.3-fold) >> MCF-7 cells (1.0-fold)=LTED cells (0.76-, 1.3-fold) (Figure 6A). Therefore, LTED cells were categorized into the “oxidative phenotype”, similar to the parent MCF-7 cells. We then investigated the effects of BKA and FAs (PA and AA) on the expression of LDH-A in breast cancer cells; BKA and FA concentrations were fixed at 25 μM based on the results shown in Figure 6. AA did not exert any modulating activity on any of the breast cancer cells examined. In spite of the cell type, PA up-regulated the expression of LDH-A, whereas BKA significantly down-regulated its expression in LTED cells (0.66-fold) compared to control (1.0-fold) (Figures 6B–D).

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Expression status of PDK4 in breast cancer cells and effects of Bongkrekic acid/BKA on the expression of PDK4. [3]
Previous studies reported that cellular glucose oxidation is decreased by FAs due to the induction of PDK4, a key enzyme for the regulation of glucose (carbohydrate) oxidation via the inhibition of the pyruvate dehydrogenase complex (PDH) in mitochondria (see Figure 5A). Taken together with the results described in Figure 6, BKA appears to modulate the expression status of “key enzymes” responsible for the metabolic fate of glucose. The results of the real-time RT-PCR analysis demonstrated that the basal expression of PDK4 varied markedly among the three breast cancer cells tested; MDA-MB-231 cells (2.18-fold) expressed the highest levels of PDK4, followed by MCF-7 cells (1.0-fold) and LTED cells (0.08-fold) (Figure 7A). Thus, LTED cell growth appears to be highly dependent on glucose (i.e., glucose oxidation) as an energy source. We then investigated the effects of BKA and two FAs (PA and AA) on the three breast cancer cells. As expected, the expression of PDK4 was significantly stimulated by the two FAs in MDA-MB-231 cells. However, AA up-regulated the expression of PDK4, whereas PA down-regulated it in MCF-7/LTED cells (Figures 7B-D). It is important to note that, in contrast to the effects of FAs, BKA “selectively” down-regulated the expression of PDK4 in LTED cells (Figure 7D) and, as shown in Figure 6D, LDH-A in LTED cells was identified as a target of BKA. Thus, BKA-mediated decreases in PDK4/LDH-A levels may relieve the inhibition of PDH, thereby prompting the aggressive use of glucose.

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Expression status of Topo IIα in breast cancer cells and effects of Bongkrekic acid/BKA on the expression of Topo IIα. [3]
We measured the expression pattern of the proliferation marker Topo IIα in the three breast cancer cells. Real-time RT-PCR analyses revealed that the strongest expression of Topo IIα was detected in MDA-MB-231 cells (1.3-fold), followed by MCF-7 cells (1.0-fold) > LTED cells (0.54-fold). Although the reason why Topo IIα was more weakly expressed in LTED cells than in parent MCF-7 cells remains currently unclear, it is speculated that highly aggressive MDA-MB-231 breast cancer cells express the highest levels of Topo IIα (Figure 8A). This expression order of Topo IIα was similar to that of PDK4 (Figure 7A). Consistent with the results shown in Figure 8B, PA at 25 μM decreased the viability of MDA-MB-231 cells. When the effects of BKA were investigated in more detail, we found that this molecule down-regulated the expression of Topo IIα in LTED cells only; this modulative effect by BKA was only observed for the expression of PDK4 in LTED cells (Figures 8B-D; see also Figures 6 and 7). In order to further support the modulation of Topo IIα by BKA in LTED cells, we focused on another proliferation marker of cancer cells, Ki-67. Real-time RT-PCR results for the expression of Ki-67 were similar to those of Topo IIα (0.41±0.014 vs. Ctl.=1.0, p<0.05), indicating BKA-selective inhibitory effects on the proliferation of LTED cells. Inversely related to the formation of formazan by BKA (Figure 2B), proliferation markers' (Topo IIα and Ki-67) levels and living cell numbers were reduced by BKA in LTED cells (Figures 3B and 8D).

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Effects of simplified Bongkrekic acid/BKA analogs on formazan formation and PPARγ-mediated transcriptional activity. [3]
As we reported previously, BKA has been established as a selective activator for PPARγ, coupled with the up-regulation of its regulated gene, adiponectin (ADIPOQ), but not with fatty acid 2-hydroxylase (FA2H), a PPARα-regulated gene. After switching parent MCF-7 cells into LTED cells cultured under reduced E2 conditions, growth signaling mediated by IGF-1 (i.e., mitogenic and anti-apoptotic effects) was observed and, in some settings, the activation of PPARγ by rosiglitazone suppressed the IGF regulatory system in vitro and in vivo. Furthermore, a strong positive association has been reported between IGF-1 levels and breast cancer. Based on these findings, we hypothesized that the selective anti-proliferative effects of BKA on LTED cells are based on its association with PPARγ. As shown in Figure 9A, we originally synthesized two simplified BKA analogs, BKA-1’ and BKA-4, and applied them in addition to BKA, BKA-2 and BKA-3 to the formazan formation assay at 10 and 50 μM. Of these, only BKA-3 exhibited significant, but weaker stimulating activity at 50 μM than that of parent BKA (Figures 3B and 9B). Transcriptional experiments on PPARγ revealed that BKA-3 (4.86-fold) and BKA-4 (4.18-fold) in addition to BKA (2.07-fold) activated PPARγ (Figure 9C). Although the PPARγ activation potentials of BKA-3/BKA-4 were approximately 2-fold stronger than that of BKA, they exhibited very weak or negative effects on formazan formation. Thus, the stimulatory effects of BKA on PPARγ are not assertively involved in LTED-directed cell death.

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BKA/Bongkrekic acid is insensitive to Cu2+-mediated oxidation. [3]
Fatty acids are oxidized by 15-LOX and/or Cu2+-mediated reaction in the body; for example, LA (C18:2) may be metabolized into (±)13-HpODE (Figure 10A), a positive stimulator for breast cancer cell growth. When the structure of BKA was examined, it was found to have a dienylmethylene (-CH=CH-CH=CH-CH2-), a possible oxidizable moiety (Figure 1). Thus, we speculated that BKA may be oxidized by Cu2+; however, the results shown in Figure 10B revealed that BKA is insensitive to Cu2+, whereas LA was oxidized. The resistance of BKA to 15-LOX was also demonstrated. Furthermore, we did not detect any absorption peaks at wavelengths ranging between 200 nm and 800 nm (data not shown). Collectively, these results suggest that BKA is more stable than LA against Cu2+-mediated oxidation.

The ADP/ATP carrier can also be very efficiently and specifically inhibited by Bongkrekic acid (BA), a natural poison secreted by the bacteria Pseudomonas cocovenenans. BA is a polyunsaturated long-chain fatty acid derivative (FIGURE 2B) that interacts with high affinity (Kd in the nanomolar range) with carrier sites accessible from the matrix compartment. Therefore, in contrast to ATRs, BA has to cross the mitochondrial inner membrane to produce its inhibitory effect on ADP/ATP transport.

It was shown that binding of ATRs and Bongkrekic acid/BA to the ADP/ATP carrier are mutually exclusive. This behavior demonstrated the existence of two conformational states of the carrier, referred to as the CATR and BA conformations since they are able to bind CATR and BA, respectively. These two conformations exist in equilibrium in the mitochondrial membrane, but in the presence of CATR or BA this equilibrium is shifted and results in the formation of very stable distinct CATR- or BA-carrier complexes that can be differentiated on the basis of their chemical and immunochemical reactivity and on their sensitivity to proteases. In the absence of inhibitors, only ADP and ATP are able to trigger the rapid interconversion between the CATR and the BA conformations, suggesting that this transition is involved in the transport process. This peculiar feature of the carrier has been especially advantageous for the study of the transport mechanism at a molecular level. [2]

Tempe bongkrek is a locally produced, inexpensive protein source in Java, Indonesia. It is made by pressing the coconut meat by-product from coconut milk or oil production into a cake that is then inoculated with R. oligosporum mold for fermentation. The final product is sliced or cubed for frying or cooking in soup. If fermentation is incomplete, B. cocovenenans and Bongkrekic acid/BA can proliferate. Deaths from BA poisoning related to tempe bongkrek consumption were first reported in 1895. Since 1975, consumption of contaminated tempe bongkrek has resulted in almost 3000 cases of BA toxicity, including at least 150 deaths. In Indonesia, the reported mortality rate averages 60 % among those affected by BA toxicity. After an outbreak in 1988, production of tempe bongkrek was banned, but production and occasional outbreaks continue to occur.

In northeastern China, fermented corn products used to make breads, noodles, and dumplings appear to be the primary source of Bongkrekic acid/BA poisoning. In southern China, diaojiangba (hanging syrup cake) has been linked to BA poisoning events. In addition, half of the Tremella fuciformis mushrooms consumed in China and other Asian countries might be contaminated with B. cocovenenans possibly from the soil. Outbreaks due to BA usually occur during warm summer months in both Indonesia and China.

In 2015, the first outbreak of Bongkrekic acid/BA toxicity outside of Asia was reported. An outbreak in 2015 in northwestern Mozambique killed 75 people and sickened many who drank pombe, a homemade, fermented corn flour-based beverage (Table 1).

In the earliest studies investigating the cellular pathophysiology of Bongkrekic acid/BA, Welling et al. showed dose-dependent decreases in glucose content and cellular oxygen uptake in sheep heart tissue, along with lactate accumulation and acidosis. These findings led them to hypothesize that BA inhibits mitochondrial enzymes. Later research demonstrated that BA is a specific ligand for ANT, and inhibits the translocase by freezing ANT in its “m” (matrix-oriented) conformation. Just 1 μmol of BA per 1 mg of mitochondrial protein is sufficient to block phosphorylation of ADP completely. About 10 μmol of BA per 1 mg of mitochondrial protein at 6 mmol ATP is required to block hydrolysis of ATP completely. Other natural toxins that also inhibit ANT include atractyloside, apoatractyloside, apocarboxyatractyloside, epiatractyloside, carboxyatractyloside, aryl azido atractyloside, n-ethyl maleimide, agaric acid, and isobongkrekic acid.

Diagnostic Testing: Detecting B. cocovenenans and Bongkrekic acid/BA can be difficult and unreliable. B. cocovenenans has been isolated from contaminated food and vomit. It can be identified using commercial test kits such as the Biologic GN2 System. The most commonly used method for B. cocovenenans identification is 16S rDNA sequencing, but it can sometimes falsely identify other Burkholderia pathovars for B. cocovenenans. B. cocovenenans can be identified using capillary electrophoresis-single strand conformation polymorphisms (CE-SSCP), microarray analysis, or probe-based cell fishing. The most reliable method might be the multiplex PCR protocol. B. cocovenenans was isolated from lymphoadenoid and lung tissue from a man in Thailand and identified by 16s rDNA sequencing. We found no other reports of B. cocovenenans isolation and detection from biological media.

We could not locate any published reports of testing biological media for Bongkrekic acid/BA, but the presence and quantification of BA in environmental samples can be tested using liquid thin layer chromatography, chromatography-mass spectroscopy, and high-pressure liquid chromatography [1].

Formazan formation analysis (MTS assay). [3]
In the MTS assay, cells were seeded on 96-well plates at a density of 5×103 cells/well and FAs and Bongkrekic acid/BKA (individual concentrations were indicated in the Figures) were introduced 4 h after plating. After a 48-h incubation, cell viability was analyzed using the CellTiter 96® Aqueous One Solution Cell Proliferation Assay, according to the manufacturer's instructions. Test chemicals were prepared in appropriate organic solvents, including dimethyl sulfoxide (DMSO) or ethanol. Control incubations contained equivalent additions of solvents with no measurable influence of vehicle on the formation of formazan at the final concentrations used.

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Live/dead cell analysis. [3]
MCF-7 and LTED cells were seeded on 96-well plates at densities of 5×103 cells in 200 μl cell culture medium and Bongkrekic acid/BKA (0.01, 1 and 25 μM) was introduced 4 h after seeding. After a 48-h incubation, live and necrotic cells were analyzed using Live/Dead Cell Staining Kit II, according to the manufacturer's instructions. The fluorescence of calcein-AM and ethidium homodimer III (EthD-III) was measured using the GloMax-Multi Detection System. In the morphological examination of LTED cells, images were obtained using a Leica DMIL inverted microscope and captured with a Pixera® Penguin 600CL Cooled CCD digital camera. Data were processed using Pixera Viewfinder 3.0 software. Breast cancer cells were plated on 6-well plates. Three areas with approximately equal cell densities were identified in each well and images of each of these areas were captured.

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Transfection and luciferase reporter assay (dual-luciferase assay). [3]
The day before transfection, MCF-7 cells were seeded (5×104 cells/well) on 24-well plates containing MEMα medium. The transfection of each expression plasmid was performed using Lipofectamine® LTX with PLUS™ reagent according to the manufacturer's instructions. The maximal transcriptional efficiencies of the human PPARγ expression plasmid in combination with the human retinoid X receptor α (RXRα) plasmid were 100 ng and 100 ng, respectively, in the transfections. DNA mixtures of 300 ng of the PPRE-Luc plasmid containing the rat acyl-CoA oxidase PPRE were co-transfected with 20 ng of the Renilla luciferase reporter plasmid (pRL-CMV) in 24-well plates. All plasmid concentrations were equalized with the pcDNA3.1 vector. The expression plasmids of human PPARγ, RXRα and the PPRE reporter construct were gifts from Dr. Curtis J. Omiecinski. At 24 h post-transfection, cells were washed with phosphate-buffered saline and changed to MEMα without phenol red supplemented with 5% serum, followed by a treatment with Bongkrekic acid/BKA and its derivatives (BKA-1’, BKA-2, BKA-3 and BKA-4) for 24 h. After being treated with the compounds, cell extracts were prepared with 100 μl of passive lysis buffer and 20 μl of the extracts was used for the firefly luciferase and Renilla luciferase assays by the GloMax-Multi Detection System. The ratio of firefly luciferase activity (expressed from reporter plasmids) to Renilla luciferase activity (expressed from pRL-CMV) in each sample served as a measure of normalized luciferase activity.

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Assay for the formation of conjugated dienes. [3]
Experiments were performed based on previously described procedures. Bongkrekic acid/BKA or LA in the presence of copper (Cu2+, CuSO4) was incubated for various times in spectrophotometric cuvettes (1.0-cm light path) in 100 mM borate buffer at pH 9.0 at room temperature. Absorbance at 234 nm was periodically recorded after the addition of copper. Results were expressed as an increase in absorbance from the reaction mixture at time zero.

Exposure[1]
The production of Bunkleic acid depends on two distinct and sequential environmental conditions: first, an environment that supports bacterial growth and proliferation, and second, an environment conducive to the production of Bunkleic acid/BA (Table 2). Bunkleic acid is produced in warm (22–30 °C) and neutral pH environments, which are the same as those used in tempeh production. Its production also depends on the presence of fatty acids, particularly those found in coconut and corn. Bacterial culture media containing oleic acid produce the highest concentrations of BA. When Colocasia esculenta is cultured on coconut medium under ideal conditions, the toxin yield reaches 2–4 mg/g on the second day of culture. Lauric acid, myristic acid, and palmitic acid account for 71.5–74.5% (by weight) of the fatty acids in coconut oil, while corn also contains varying concentrations of oleic acid. Interestingly, R. oligosporum has an inhibitory effect on BA production, reducing BA concentrations when a sufficient number of fungal colonies are allowed to form.

Toxicokinetics [1] Limited information is available regarding the toxicokinetics and lethal dose of Bongkrekic acid (BA) in humans. Some studies indicate that doses of 1–1.5 mg can be fatal, while others show an oral LD50 of 3.16 mg/kg. Mouse studies have shown oral LD50 of 0.68–6.84 mg/kg and intravenous LD50 of 1.41 mg/kg. Another rat study showed that an oral dose of 2 mg/100 g could lead to death within 2–5 hours. In the same study, rats survived an initial dose of 1 mg/100 g, but died after a second dose 48 hours later. Although BA may have a large volume of distribution due to its high unsaturated fat content and high lipid solubility, the absorption curve and volume of distribution of Bongkrekic acid (BA) remain unclear. The metabolic pathway of Bongkrekic acid (BA) is also unknown. Early studies reported that, based on NMR spectroscopy, UV spectroscopy, molar extinction coefficient, and mass spectrometry analysis, flavotoxin A (a toxin also believed to be found in Burkholderia cocovenenans) and BA are the same organic compound. However, recent studies speculate that flavotoxin A may be a metabolite of BA. The elimination pathway of BA is unclear.

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Toxicity Overview
Identification and Uses: Bonklein acid (BA) is a white amorphous solid. BA is known to be produced by Burkholderia cocovenenans. It is used as a tool in biochemical research. Human Studies: In a rural town in Mozambique, more than 230 people fell ill, and 75 died from illnesses related to drinking pombe (a traditional alcoholic beverage). Toxic levels of BA were detected in suspected contaminated cracked fruit, but not in control cracked fruit. The bacterium that produces BA, Burkholderia gladioli pathovar cocovenenans, was detected in the flour used to make cracked fruit. BA is an inhibitor of adenine nucleotide translocase (ANT). Because the inhibition of ANT is associated with the inhibition of mitochondrial release of cytochrome c, which in turn leads to the inhibition of apoptosis, it has been used as a tool to study the mechanisms of apoptosis. BA has been associated with foodborne illness outbreaks involving coconut and corn products in Indonesia and China. Animal studies: BA is a potent mitochondrial ATP/ADP translocase inhibitor that inhibits glucose-induced electrical activity in pancreatic β-cells by stimulating the activity of ATP-sensitive potassium channels (K-ATP channels).

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Intravenous LD50 in mice is 1410 ug/kg, Tetrahedron, 26(5993), 1970.
Intravenous LD50 in mice is 6840 ug/kg. Behavior: lethargy (overall activity inhibition); Behavior: rigidity; Lung, pleural, or respiratory: dyspnea, Applied and Environmental Microbiology, 48(690), 1984 [PMID:6391376]

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Interactions
The in vitro effects of bromelain, papain, and figase on bromelain stem enzymes were investigated. Bunklelain inhibited the hydrolysis of casein by these enzymes, but the inhibition was incomplete even with large excesses of the effector. The inhibitory effect of bromelain was not stoichiometric using fully activated bromelain stem enzyme samples purified by organomercury agarose affinity column. The sulfhydryl (-SH) group of cysteine remained intact after 20 min of incubation with excess bromelain at 24°C. However, incubation with 5 mM cysteine or 2-mercaptoethanol at 37°C for 5 min reversed the partial inhibition of bromelain stem enzymes by bromelain. Ethylene glycol and glycerol did not have this reversing effect. These results indicate that the bolklein molecule binds non-covalently to thiol proteases, partially and reversibly shielding them only from their essential sulfhydryl groups (-SH). Murachi T et al.; Toxicon 20 (6): 1011-7 (1982)

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Antidotes and First Aid Treatment
/SRP:/ Immediate First Aid Measures: Ensure adequate decontamination has been performed. If the patient stops breathing, begin artificial respiration immediately, preferably using a ventilator on demand, bag-valve-mask, or simple breathing mask, and follow the training instructions. Perform cardiopulmonary resuscitation if necessary. Immediately flush contaminated eyes with running water. Do not induce vomiting. If vomiting occurs, tilt the patient forward or place them in the left lateral decubitus position (head down if possible) to maintain an open airway and prevent aspiration. Keep the patient calm and maintain normal body temperature. Seek immediate medical attention. /Class A and Class B Poisoning/
/SRP:/ Basic Treatment: Establish an open airway (using an oropharyngeal or nasopharyngeal airway if necessary). Suctioning may be necessary. Observe for signs of respiratory failure and provide assisted ventilation if necessary. Administer oxygen via a non-invasive ventilation mask at a flow rate of 10 to 15 liters per minute. Monitor for pulmonary edema and treat as necessary… Monitor for shock and treat as necessary… Anticipate seizures and treat as necessary… If eyes are contaminated, flush with water immediately. During transport, continuously flush each eye with 0.9% saline… Do not use emetics. In case of ingestion, rinse mouth and dilute with 5 mL/kg to 200 mL of water, provided the patient is able to swallow, has a strong gag reflex, and does not drool… After decontamination, cover skin burns with a dry, sterile dressing… /Class A and Class B Poisons/
/SRP:/ Advanced Treatment: For patients with impaired consciousness, severe pulmonary edema, or severe respiratory distress, consider oropharyngeal or nasopharyngeal endotracheal intubation to control the airway. Positive pressure ventilation via bag-valve-mask may be effective. Consider medical treatment for pulmonary edema… Consider using a beta-agonist (such as salbutamol) to treat severe bronchospasm… Monitor heart rhythm and treat arrhythmias if necessary… Start intravenous infusion of 5% glucose solution (D5W TKO) /SRP: “Keep it patent”, minimum flow rate/. If signs of hypovolemia appear, use 0.9% normal saline (NS) or lactated Ringer's solution (LR). Use caution with fluid administration for hypotension accompanied by signs of hypovolemia. Watch for signs of fluid overdose… Treat seizures with diazepam (Valium) or lorazepam (Atifan)… Use promecaine hydrochloride to assist eye irrigation… /Toxins A and B/

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Human Toxicity Excerpt/Case Report/ In January 2015, 75 people died and 177 were hospitalized in the village of Chitima, Mozambique, after attending a funeral. The deaths were linked to the consumption of a traditional African beverage called pombe. Multiple analyses were performed on suspected pathogenic pombe samples and compared with control samples. Ultimately, non-targeted liquid chromatography-mass spectrometry (LC-MS) screened and detected the presence of the potent toxin bunkelic acid and its structural isomer, isobkelic acid. Quantitative analysis revealed that the levels of these toxins in the suspected fissile yeast sample could reach lethal levels. Bunkelic acid is known to be produced by Burkholderia gladioli pv. cocovenenans. Although this bacterium could not be isolated from the suspected fissile yeast, bacteria identified as Burkholderia gladioli were isolated from corn flour (the starting material for fissile yeast) obtained from a brewer's home. Co-production of bunkelic acid was observed when these bacteria were co-cultured with Rhizopus oryzae, also isolated from corn flour. These findings suggest a mechanism of bunkelic acid poisoning, a phenomenon previously thought to be limited to specific regions of Indonesia and China. PMID: 27823840
/Case Report/ Background: On January 9, 2015, in a rural town in Mozambique, more than 230 people fell ill after consuming a traditional alcoholic beverage called “pombe,” with 75 deaths. Methods: We conducted an investigation to identify cases and determine the cause of the outbreak. Cases were defined as residents of Chitima who developed any new or unexplained neurological, gastrointestinal, or cardiovascular symptoms between 6:00 a.m. on January 9 and 11:59 p.m. on January 12. We conducted medical record reviews, healthcare worker and community surveys, anthropological and toxicological investigations of local medicinal plants and commercial pesticides, and laboratory testing of “pombe” for suspected cases and controls. Results: We identified a total of 234 cases; of these, 75 (32%) died and 159 recovered. Overall, 61% of the cases were female (n = 142), with an age range of 1 to 87 years (median age 30 years). Signs and symptoms included abdominal pain, diarrhea, vomiting, and malaise. Psychomotor agitation and abnormal postures were observed prior to death. The median time interval from pombe consumption to symptom onset was 16 hours. Toxic levels of basculic acid (BA) were detected in the pombe from suspected cases but not in control pombe. The BA-producing bacterium Burkholderia gladioli pathovar cocovenenans was detected in the flour used to make the pombe. Conclusion: We report for the first time a highly lethal disease outbreak in Africa associated with the deadly foodborne toxin BA. Given that no such outbreaks have been previously reported outside of Asia, our investigation suggests that BA may be a contributing factor to a previously under-recognized toxicity outbreak globally. PMID:29155976
/Alternatives and In Vitro Assays/ Background/Objective: This study used a long-term estrogen-deprived MCF-7 (LTED) cell model in vitro to analyze the regeneration mechanism of breast cancer treated with estrogen receptor α (ERα) signaling blockers. Bunkelkaic acid (BKA) is a natural toxin isolated from coconut fermented soybeans contaminated with Burkholderia cocovenenans. Materials and Methods: LTED cells, MCF-7 cells, and MDA-MB-231 cells were used in this study. Multiple biochemical analyses were performed after treatment with BKA (chemically synthesized; purity: >98%). Results: LTED cells were classified as oxidative phenotype. When LTED cells were treated with BKA, lactate dehydrogenase A (LDH-A)/pyruvate dehydrogenase kinase 4 (PDK4) expression was downregulated, thereby promoting the overuse of glucose by mitochondrial oxidative phosphorylation and inducing cell death. These effects of BKA were not observed in other breast cancer cells analyzed. Conclusion: We believe that BKA can serve as an experimental tool for cancer biological analysis in LTED cells. PMID: 27798877
/Alternatives and In Vitro Assays/ Bunkelkaic acid (BKA) is an inhibitor of adenine nucleotide translocase (ANT). Because ANT inhibitors are associated with the inhibition of mitochondrial release of cytochrome c, leading to inhibition of apoptosis, they are used as a tool to study the mechanisms of apoptosis. BKA consists of a long carbon chain containing two asymmetric centers, a non-conjugated olefin, two conjugated dienes, three methyl groups, one methoxy group, and three carboxylic acid groups. This complex chemical structure presents challenges for its synthesis, supply, and biochemical mechanism studies. In this study, inspired by the molecular structure of BKA, we designed and synthesized a series of simpler tricarboxylic acid derivatives. Subsequently, we evaluated the cytotoxicity and anti-apoptotic activity of these derivatives in HeLa cells, as well as their effects on the mitochondrial inner membrane potential of HL-60 cells. All tested tricarboxylic acid derivatives (including BKA) showed low toxicity to HeLa cells. BKA and two of its synthetic derivatives significantly inhibited astrosporin (STS)-induced cell viability decline. Furthermore, pretreatment with BKA and its tricarboxylic acid derivatives significantly restored STS-induced mitochondrial membrane potential collapse. Other derivatives (in which one of the three carboxylic acids was esterified) exhibited potent toxicity, particularly one derivative with the same carbon chain length as BKA. In summary, we have developed a novel compound as an apoptosis inhibitor containing three carboxylic acids linked by appropriately long carbon chains. PMID:22998163

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Non-human toxicity excerpt
/Alternatives and in vitro studies/ Bunkelliic acid can cause fatal food poisoning accompanied by hyperglycemia. This paper demonstrates that bunkelliic acid, a potent inhibitor of mitochondrial ATP/ADP translocases, inhibits glucose-induced electrical activity in pancreatic β-cells by stimulating the activity of ATP-sensitive potassium channels (K-ATP channels). By comparing its effects with those of oligomycins, we suggest that the mechanism of action of bunkelliic acid is through inhibition of glucose metabolism and, possibly, through impaired β-cell function, the induction of hyperglycemia. PMID:2037079
/Alternatives and In Vitro Assays/ This study aimed to characterize the effects of bunkelevic acid (BKA), atrazine (ATR), and carboxy-atrazine (CAT) on the single-channel properties of mitochondrial chloride channels. Mitochondrial membranes isolated from rat myocardium were integrated into a lipid bilayer (BLM), and single chloride channel currents were measured in 250/50 mM KCl cis-trans solutions. BKA (1–100 μM), ATR, and CAT (5–100 μM) inhibited chloride channels in a dose-dependent manner. The inhibitory effects of BKA, ATR, and CAT were most pronounced on the trans side of the BLM and increased with increasing time and negative voltage (trans-cis). These compounds did not affect the current amplitude of individual channels but shortened the channel opening time. The inhibitory effects of BKA, ATR, and CAT on mitochondrial chloride channels may help explain some of their cellular and/or subcellular effects. PMID:17123460
/Other Toxicity Information/ The mitochondrial ADP/ATP carrier involved in adenine nucleotide transport exists in two distinct conformations, termed the BA conformation and the CATR conformation, distinguished by their binding to the specific inhibitors basalicylic acid (BA) and carboxyatraside (CATR), respectively. To determine which amino acids are involved in the conversion between these two conformations during transport, we identified and characterized mutants of the Saccharomyces cerevisiae ADP/ATP carrier Anc2p, responsible for yeast cell resistance to BA, through in vivo chemical or UV mutagenesis. Despite analyzing a large number of mutants, only four distinct mutations were identified. These are located in transmembrane segments I (G30S), II (Y97C), III (L142S), and VI (G298S) of Anc2p and independently enabled cell growth in the presence of BA. Immunochemical analysis and atrazine binding assays showed that the expression levels of mutant and wild-type Anc2p in mitochondria were nearly identical. In the presence of BA, the efficiency of ADP/ATP exchange mediated by the Anc2p mutant in isolated mitochondria was higher than that of wild-type Anc2p, confirming that BA resistance in mutant cells is related to the functional properties of the modified ADP/ATP carrier. These results indicate that BA resistance is caused by conformational changes in Anc2p due to the presence of serine or cysteine residues at specific positions. Different interactions of these residues with other amino acids and/or BA may prevent the formation of a stable, inactive Anc2p-BA complex. PMID:13678275
/Other Toxicity Information/ Interactions between aflatoxin-producing fungi and bacteria open new avenues for identifying biopharmaceuticals suitable for controlling aflatoxin contamination. This study analyzed the interaction between Aspergillus flavus and Burkholderia M3, which coexists with naturally occurring Aspergillus flavus-contaminated rice. The results showed that cell-free culture filtrate (CCF) of strain M3 and its metabolite ponkelic acid effectively inhibited mycelial growth and spore production, thereby affecting aflatoxin production. Ponkelic acid secreted by strain M3 exhibited higher antifungal activity than other analogues. CCF and its metabolite bonclamide of strain M3 inhibited the growth of Aspergillus flavus, but aflatoxin, a metabolite of Aspergillus flavus, did not inhibit the growth of strain M3. Furthermore, we found that M3 cells could utilize dead Aspergillus flavus mycelium as an energy source for reproduction, while Aspergillus flavus could not grow in solutions containing dead M3 cells. In conclusion, these results indicate that Gladiolus has a competitive advantage when coexisting with its fungal partner, Aspergillus flavus. PMID:26058536

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Non-human toxicity values
Mice intravenous LD50: 1.4 mg/kg
PMID:10435074
Mice oral LD50: 3.16 mg/kg /Purified flavin toxin A/

[1]. Bongkrekic Acid-a Review of a Lesser-Known Mitochondrial Toxin. J Med Toxicol. 2017 Jun;13(2):173-179.

[2]. Molecular, functional, and pathological aspects of the mitochondrial ADP/ATP carrier. Physiology (Bethesda). 2006 Aug:21:242-9.

[3]. Bongkrekic Acid as a Warburg Effect Modulator in Long-term Estradiol-deprived MCF-7 Breast Cancer Cells. Anticancer Res. 2016 Oct;36(10):5171-5182.

Bongkrekic acid is a tricarboxylic acid with the structure 2,4,8,10,14,18,20-hepteneic acid, with methyl groups at positions 2, 5, and 17, a methoxy group at position 6, and a carboxymethyl group at position 20. It is produced by Burkholderia gladioli and has been linked to foodborne illness outbreaks involving coconut and corn products in Indonesia and China. Bongkrekic acid has multiple functions, including inhibiting apoptosis, inhibiting EC 2.5.1.18 (glutathione transferase), acting as a toxin, inhibiting ATP/ADP translocases, and serving as a bacterial metabolite. It is a tricarboxylic acid, an ether, and an olefin. It is the conjugate acid of (3-)Bongkrekic acid. Bongkrekic acid has been reported in Burkholderia gladioli, and data exist on its presence. It is also an antibiotic produced by Pseudomonas cocovenenans. It is an inhibitor of mitochondrial ADP-ATP translocases. Specifically, it blocks the outflow of adenine nucleotides from mitochondria by enhancing membrane binding. Mechanism of Action Bunkellyl acid (BKA) is an inhibitor of adenine nucleotide translocase (ANT). Because the inhibition of ANT is associated with the inhibition of mitochondrial cytochrome c release, leading to the inhibition of apoptosis, it has been used as a tool to study the mechanisms of apoptosis. BKA consists of a long carbon chain with two asymmetric centers, a non-conjugated olefin, two conjugated dienes, three methyl groups, one methoxy group, and three carboxylic acids. This complex chemical structure presents challenges to the study of its synthesis, supply, and biochemical mechanisms. Among numerous mitochondrial toxins, bunkellyl acid (BA) has a unique toxic mechanism: it inhibits adenine nucleotide translocase (ANT), rather than the electron transport chain. Bunkelleic acid is produced by Burkholderia gladioli pathovar cocovenenans (B. cocovenenans), a bacterium associated with foodborne illness outbreaks involving coconut and corn products in Indonesia and China. Introduction: Bunkelleic acid (BA) is a unique mitochondrial toxin with a mechanism of toxicity distinct from other mitochondrial toxins: it inhibits adenine nucleotide translocase (ANT) rather than the electron transport chain. Our aim is to summarize current information regarding the epidemiology, sources of exposure, toxicokinetics, pathophysiology, clinical manifestations, diagnosis, and treatment of BA poisoning in humans. Methods: We searched MEDLINE (1946–present), EMBASE (1947–present), SCOPUS, the Indonesian Publications Index (http://id.portalgaruda.org/), ToxNet, book chapters, Google searches, Pro-MED alerts, and references from previously published journal articles. A total of 109 references were retrieved and reviewed. Of these, 29 (26%) contained relevant information and were therefore included in the study. Bunkelleic acid is a heat-stable, highly unsaturated tricarboxylic acid fatty acid with a molecular weight of 486 kDa. Outbreaks of bunkelleic acid poisoning have been reported in Indonesia, China, and most recently, Mozambique. The toxicokinetics of bunkelleic acid are poorly understood. Bunkelleic acid exerts its toxic effects by inhibiting mitochondria (ANT). ANT can also alter apoptosis. Human signs and symptoms are similar to those of other mitochondrial toxins, but the severity and course of the disease differ. Treatment is primarily symptomatic and supportive. Conclusion: Bonklein is a mitochondrial ANT toxin that is primarily found in foodborne poisoning outbreaks involving coconut and corn. The possibility of Bonklein should be considered when signs and symptoms involving the liver, brain, and kidneys are present and involve coconut or corn products. [1] ADP/ATP carriers play a key role in cellular metabolism. Due to their unique properties, they provide in-depth insights into the molecular basis of metabolite transport across biological membranes in biochemical and genetic studies. Recent high-resolution resolution of the CATR-carrier complex has greatly enhanced our understanding of this carrier. Resolution of the structure of this carrier in other conformational states will provide important information for elucidating the molecular mechanisms of adenine nucleotide exchange across the inner mitochondrial membrane and will help reveal the consequences of mutations in related genetic diseases. Mitochondrial ADP/ATP carriers play a central role in providing cells with ATP produced by oxidative phosphorylation in aerobic eukaryotic cells. Combining biochemical, genetic, and structural biological approaches can help understand the molecular mechanisms of this important transport system, whose dysfunction is closely related to neuromuscular diseases. [2]

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In this study, we did not obtain direct evidence of an interaction between Bongkrekic acid/BKA-induced downregulation of LDH-A/PDK4 (Topo IIα/Ki-67) and LTED cell death; however, this is the first study to identify BKA as a highly selective regulator of LTED cell metabolic pathways (Figure 5B). BKA holds promise as an effective treatment for recurrent breast cancer that has been treated with 17β-estradiol/ERα signaling pathway blockers; however, further research is needed on the mechanisms by which BKA mediates LTED cell death. [3]

C28H38O7

486.59712

486.262

11076-19-0

6433556

Colorless to light yellow liquid

1.114g/cm3

715.1ºC at 760mmHg

50-60°

231ºC

1.545

5.885

3

7

17

35

898

2

OC(C/C(/C=C/[C@H](C/C=C/CC/C=C/C=C/C[C@H](/C(=C/C=C(/C(=O)O)\C)/C)OC)C)=C\C(=O)O)=O

SHCXABJSXUACKU-WUTQZGRKSA-N

InChI=1S/C28H38O7/c1-21(15-18-24(19-26(29)30)20-27(31)32)13-11-9-7-5-6-8-10-12-14-25(35-4)22(2)16-17-23(3)28(33)34/h6,8-12,15-19,21,25H,5,7,13-14,20H2,1-4H3,(H,29,30)(H,31,32)(H,33,34)/b8-6+,11-9+,12-10-,18-15+,22-16-,23-17+,24-19+/t21-,25+/m0/s1

(2E,4Z,6R,8Z,10E,14E,17S,18E,20Z)-20-(carboxymethyl)-6-methoxy-2,5,17-trimethyldocosa-2,4,8,10,14,18,20-heptaenedioic acid

BONGKREKIC ACID; 11076-19-0; Flavotoxin A; Bongkrek acid; L7V4I673D2; (2E,4Z,6R,8Z,10E,14E,17S,18E,20Z)-20-(carboxymethyl)-6-methoxy-2,5,17-trimethyldocosa-2,4,8,10,14,18,20-heptaenedioic acid; (-)-BONGKREKIC ACID; BONGKREKIC ACID [MI];

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: 1. Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture and light; 2. This product requires protection from light (avoid light exposure) during transportation and storage.

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

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

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

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

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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

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

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

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