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]
Bongkrekic acid production depends on two distinct and sequential environmental conditions: those that support bacterial growth and proliferation, followed by those that favor Bongkrekic acid/BA production (Table 2). Bongkrekic acid is produced in warm environments (22–30 °C) with a neutral pH, the same conditions under which tempe is made. Production is also dependent on the presence of fatty acids, particularly those found in coconut and corn. Bacterial growth media containing oleic acid produced the highest concentrations of BA. When B. cocovenenans is cultured on coconut medium under ideal conditions, toxin production can reach 2–4 mg/g by the second day of culture. Lauric, myristic, and palmitic acids make up 71.5–74.5 % (by weight) of the fatty acids in coconut oil, and oleic acid can be found in varying concentrations in corn. Interestingly, R. oligosporum has a suppressing effect on BA production and can reduce BA concentration when allowed to form adequate numbers of fungal colonies.

Toxicokinetics [1]
There is scarce information on the toxicokinetics and lethal dose of Bongkrekic acid/BA in humans. One source suggests that 1–1.5 mg can be fatal in humans and another suggests an oral LD50 of 3.16 mg/kg. Studies on mice suggest an oral LD50 of 0.68–6.84 mg/kg and an intravenous LD50 of 1.41 mg/kg. Another study in rats showed that a 2 mg/100 g oral dose caused death within 2–5 h. In the same study, rats survived an initial 1 mg/100 g, but a repeat dose after 48 h caused death.
The absorption profile and volume of distribution for Bongkrekic acid/BA is unknown, although BA likely has a large volume of distribution because it is a highly unsaturated fat and is highly lipid soluble. We do not know how BA is metabolized. Early studies reported Flavotoxin A (a toxin also thought to be found in B. cocovenenans) and BA to be the same organic chemical compound according to nuclear magnetic resonance spectra, ultraviolet spectra, molar extinction coefficients, and mass spectra, but more recent studies theorize that flavotoxin A is possibly a metabolite of BA. The route of elimination of BA is unknown.

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Toxicity Summary
IDENTIFICATION AND USE: Bongkrekic acid (BA) is a white amorphous solid. BA is known to be produced by the bacterium Burkholderia gladioli pv. cocovenenans. It is used as a tool in biochemical research. HUMAN STUDIES: In a rural town in Mozambique, >230 persons became sick and 75 died of an illness linked to drinking pombe, a traditional alcoholic beverage. Toxic levels of BA were detected in the suspect pombe but not the control pombe. Burkholderia gladioli pathovar cocovenenans, the bacteria that produces BA, was detected in the flour used to make the pombe. BA is an inhibitor of adenine nucleotide translocase (ANT). Since inhibition of ANT is connected to the inhibition of cytochrome c release from mitochondria, which then results in the suppression of apoptosis, it has been used as a tool for the mechanistic investigation of apoptosis. BA has been implicated in outbreaks of food-borne illness involving coconut- and corn-based products in Indonesia and China. ANIMAL STUDIES: BA, a potent inhibitor of the mitochondrial ATP/ADP translocase, inhibits glucose-induced electrical activity in the pancreatic beta-cell through the stimulation of ATP-sensitive potassium channel (K-ATP-channel) activity.

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mouse LD50 intravenous 1410 ug/kg Tetrahedron., 26(5993), 1970
mouse LDLo oral 6840 ug/kg BEHAVIORAL: SOMNOLENCE (GENERAL DEPRESSED ACTIVITY); BEHAVIORAL: REGIDITY; LUNGS, THORAX, OR RESPIRATION: DYSPNEA Applied and Environmental Microbiology., 48(690), 1984 [PMID:6391376]

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Interactions
The in vitro effect of bongkrekic acid on stem bromelain, papain and ficin was studied. The hydrolysis of casein by these enzymes was inhibited by bongkrekic acid, but the inhibition was always incomplete even with a large excess of the effector. Using a fully activated specimen of stem bromelain, purified on an organomercurial agarose affinity column, the inhibition by bongkrekic acid was not stoichiometric. The SH group of cysteine remained intact after incubation with an excess of bongkrekic acid at 24 degrees C for 20 min. However, partial inhibition of stem bromelain by bongkrekic acid was reversed by incubation at 37 degrees C for 5 min with 5 mM cysteine or 2-mercaptoethanol. Ethylene glycol and glycerol had no such restorative effect. These results indicate that molecules of bongkrekic acid are non-covalently bound to a thiol protease, only partially and reversibly shielding its essential SH group. Murachi T et al; Toxicon 20 (6): 1011-7 (1982)

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Antidote and Emergency Treatment
/SRP:/ Immediate first aid: Ensure that adequate decontamination has been carried out. If patient is not breathing, start artificial respiration, preferably with a demand valve resuscitator, bag-valve-mask device, or pocket mask, as trained. Perform CPR if necessary. Immediately flush contaminated eyes with gently flowing water. Do not induce vomiting. If vomiting occurs, lean patient forward or place on left side (head-down position, if possible) to maintain an open airway and prevent aspiration. Keep patient quiet and maintain normal body temperature. Obtain medical attention. /Poisons A and B/

/SRP:/ Basic treatment: Establish a patent airway (oropharyngeal or nasopharyngeal airway, if needed). Suction if necessary. Watch for signs of respiratory insufficiency and assist ventilations if needed. Administer oxygen by nonrebreather mask at 10 to 15 L/min. Monitor for pulmonary edema and treat if necessary ... . Monitor for shock and treat if necessary ... . Anticipate seizures and treat if necessary ... . For eye contamination, flush eyes immediately with water. Irrigate each eye continuously with 0.9% saline (NS) during transport ... . Do not use emetics. For ingestion, rinse mouth and administer 5 mL/kg up to 200 mL of water for dilution if the patient can swallow, has a strong gag reflex, and does not drool ... . Cover skin burns with dry sterile dressings after decontamination ... . /Poisons A and B/

/SRP:/ Advanced treatment: Consider orotracheal or nasotracheal intubation for airway control in the patient who is unconscious, has severe pulmonary edema, or is in severe respiratory distress. Positive-pressure ventilation techniques with a bag valve mask device may be beneficial. Consider drug therapy for pulmonary edema ... . Consider administering a beta agonist such as albuterol for severe bronchospasm ... . Monitor cardiac rhythm and treat arrhythmias as necessary ... . Start IV administration of D5W TKO /SRP: "To keep open", minimal flow rate/. Use 0.9% saline (NS) or lactated Ringer's (LR) if signs of hypovolemia are present. For hypotension with signs of hypovolemia, administer fluid cautiously. Watch for signs of fluid overload ... . Treat seizures with diazepam (Valium) or lorazepam (Ativan) ... . Use proparacaine hydrochloride to assist eye irrigation ... . /Poisons A and B/

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Human Toxicity Excerpts /CASE REPORTS/ In January 2015, 75 people died and 177 were hospitalized in the Mozambique village of Chitima after attending a funeral. The deaths were linked to the consumption of a traditional African beverage called pombe. Samples of the suspect pombe were subjected to myriad analyses and compared to a control sample. Ultimately, non-targeted liquid chromatography-mass spectrometry screening revealed the presence of the potent toxin bongkrekic acid, and its structural isomer, isobongkrekic acid. Quantitative analysis found potentially fatal levels of these toxins in the suspect pombe samples. Bongkrekic acid is known to be produced by the bacterium Burkholderia gladioli pv. cocovenenans. This bacterium could not be isolated from the suspect pombe, but bacteria identified as B. gladioli were isolated from corn flour, a starting ingredient in the production of pombe, obtained from the brewer's home. When the bacteria were co-plated with the fungus Rhizopus oryzae, which was also isolated from the corn flour, synergistic production of bongkrekic acid was observed. The results suggest a mechanism for bongkrekic acid intoxication, a phenomenon previously thought to be restricted to specific regions of Indonesia and China. PMID:27823840

/CASE REPORTS/ BACKGROUND: On 9 January 2015, in a rural town in Mozambique, >230 persons became sick and 75 died of an illness linked to drinking pombe, a traditional alcoholic beverage. METHODS: An investigation was conducted to identify case patients and determine the cause of the outbreak. A case patient was defined as any resident of Chitima who developed any new or unexplained neurologic, gastrointestinal, or cardiovascular symptom from 9 January at 6:00 am through 12 January at 11:59 pm. We conducted medical record reviews, healthcare worker and community surveys, anthropologic and toxicologic investigations of local medicinal plants and commercial pesticides, and laboratory testing of the suspect and control pombe. RESULTS: We identified 234 case patients; 75 (32%) died and 159 recovered. Overall, 61% of case patients were female (n = 142), and ages ranged from 1 to 87 years (median, 30 years). Signs and symptoms included abdominal pain, diarrhea, vomiting, and generalized malaise. Death was preceded by psychomotor agitation and abnormal posturing. The median interval from pombe consumption to symptom onset was 16 hours. Toxic levels of bongkrekic acid (BA) were detected in the suspect pombe but not the control pombe. Burkholderia gladioli pathovar cocovenenans, the bacteria that produces BA, was detected in the flour used to make the pombe. CONCLUSIONS: We report for the first time an outbreak of a highly lethal illness linked to BA, a deadly food-borne toxin in Africa. Given that no previous outbreaks have been recognized outside Asia, our investigation suggests that BA might be an unrecognized cause of toxic outbreaks globally. PMID:29155976

/ALTERNATIVE and IN VITRO TESTS/ BACKGROUND/AIM: An in vitro cell model of long-term estrogen-deprived MCF-7 (LTED) cells has been utilized to analyze the re-growth mechanisms of breast cancers treated with blockers for estrogen receptor a (ERa) signaling. Bongkrekic acid (BKA) is a natural toxin isolated from coconut tempeh contaminated with the bacterium Burkholderia cocovenans. MATERIALS AND METHODS: LTED cells, MCF-7 cells and MDA-MB-231 cells were employed in the study. After treatment with BKA (chemically synthesized; purity: >98%), several biochemical analyses were carried out. RESULTS: LTED cells were categorized into an oxidative phenotype. When LTED cells were treated with BKA, lactate dehydrogenase A (LDH-A)/pyruvate dehydrogenase kinase 4 (PDK4) were down-regulated, thereby prompting the aggressive use of glucose via mitochondrial oxidative phosphorylation and induction of cell death responses. These effects of BKA were not observed in the other breast cancer cells analyzed. CONCLUSION: We suggest the potential of BKA as an experimental tool for the analysis of cancer biology in LTED cells. PMID:27798877

/ALTERNATIVE and IN VITRO TESTS/ Bongkrekic acid (BKA) is an inhibitor of adenine nucleotide translocase (ANT). Since inhibition of ANT is connected to the inhibition of cytochrome c release from mitochondria, which then results in the suppression of apoptosis, it has been used as a tool for the mechanistic investigation of apoptosis. BKA consists of a long carbon chain with two asymmetric centers, a nonconjugated olefin, two conjugated dienes, three methyl groups, a methoxyl group, and three carboxylic acids. This complicated chemical structure has caused difficulties in synthesis, supply, and biochemical mechanistic investigations. In this study, we designed and synthesized more simple tricarboxylic acids that were inspired by the molecular structure of BKA. Their cytotoxicity and apoptosis-preventing activity in HeLa cells and the effect on the mitochondrial inner membrane potential in HL-60 cells were then evaluated. All tested tricarboxylic acid derivatives including BKA showed little toxicity against HeLa cells. BKA and two of the synthesized derivatives significantly suppressed staurosporine (STS)-induced reductions in cell viability. Furthermore, STS-induced /mitochondrial potential/ collapse was significantly restored by pretreatment with BKA and a tricarboxylic acid derivative. Other derivatives, in which one of three carboxylic acids was esterified, exhibited potent toxicity, especially a derivative bearing a carbon chain of the same length as that of BKA. In conclusion, we have developed a new ... compound as an apoptosis inhibitor bearing three carboxylic acids connected with the proper length of a long carbon chain. PMID:22998163

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Non-Human Toxicity Excerpts
/ALTERNATIVE and IN VITRO TESTS/ Bongkrekic acid causes fatal food poisoning which is associated with hyperglycemia. Here we demonstrate that bongkrekic acid, a potent inhibitor of the mitochondrial ATP/ADP translocase, inhibits glucose-induced electrical activity in the pancreatic beta-cell through the stimulation of ATP-sensitive potassium channel (K-ATP-channel) activity. By comparison of its effects with those of oligomycin, we suggest that bongkrekic acid acts by the inhibition of glucose metabolism and may induce hyperglycemia by impairing beta-cell function. PMID:2037079

/ALTERNATIVE and IN VITRO TESTS/ The aim of this work was to characterize the effect of bongkrekic acid (BKA), atractyloside (ATR) and carboxyatractyloside (CAT) on single channel properties of chloride channels from mitochondria. Mitochondrial membranes isolated from a rat heart muscle were incorporated into a bilayer lipid membrane (BLM) and single chloride channel currents were measured in 250/50 mM KCl cis/trans solutions. BKA (1-100 uM), ATR and CAT (5-100 uM) inhibited the chloride channels in dose-dependent manner. The inhibitory effect of the BKA, ATR and CAT was pronounced from the trans side of a BLM and it increased with time and at negative voltages (trans-cis). These compounds did not influence the single channel amplitude, but decreased open dwell time of channels. The inhibitory effect of BKA, ATR and CAT on the mitochondrial chloride channel may help to explain some of their cellular and/or subcellular effects. PMID:17123460

/OTHER TOXICITY INFORMATION/ Two distinct conformations of the mitochondrial ADP/ATP carrier involved in the adenine nucleotide transport are called BA and CATR conformations, as they were distinguished by binding of specific inhibitors bongkrekic acid (BA) and carboxyatractyloside (CATR), respectively. To find out which amino acids are implicated in the transition between these two conformations, which occurs during transport, mutants of the Saccharomyces cerevisiae ADP/ATP carrier Anc2p responsible for resistance of yeast cells to BA were identified and characterized after in vivo chemical or UV mutagenesis. Only four different mutations could be identified in spite of a large number of mutants analyzed. They are located in the Anc2p transmembrane segments I (G30S), II (Y97C), III (L142S), and VI (G298S), and are independently enabling growth of cells in the presence of BA. The variant and wild-type Anc2p were produced practically to the same level in mitochondria, as evidenced by immunochemical analysis and by atractyloside binding experiments. ADP/ATP exchange mediated by Anc2p variants in isolated mitochondria was more efficient than that of the wild-type Anc2p in the presence of BA, confirming that BA resistance of the mutant cells was linked to the functional properties of the modified ADP/ATP carrier. These results suggest that resistance to BA is caused by alternate conformation of Anc2p due to appearance of Ser or Cys at specific positions. Different interactions of these residues with other amino acids and/or BA could prevent formation of stable inactive Anc2p BA complex. PMID:13678275

/OTHER TOXICITY INFORMATION/ The interactions between aflatoxin-producing fungi and bacteria have opened up a new avenue for identifying biological agents suitable for controlling aflatoxin contamination. In this study, we analyzed the interactions between A. flavus and the bacterium Burkholderia gladioli M3 that coexist in rice that is naturally contaminated with A. flavus. Our results showed that a cell-free culture filtrate (CCF) and the metabolite bongkrekic acid of the M3 strain potently suppressed the mycelial growth and spore production, and then affected the production of aflatoxin of A. flavus. Bongkrekic acid secreted by the M3 strain exhibited higher antifungal activity than did analogues. The CCF of the M3 strain and its metabolite bongkrekic acid can inhibit the growth of A. flavus, but the metabolites of A. flavus, aflatoxins, exerted no inhibitory effect on the growth of the M3 strain. Furthermore, we determined that the M3 cells could use the dead mycelia of A. flavus as energy sources for reproduction, while A. flavus could not grow in a solution containing dead M3 cells. In summary, these results indicated that B. gladioli has a competitive advantage in survival when it coexists with its fungal partner A. flavus. PMID:26058536

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Non-Human Toxicity Values
LD50 Mice iv 1.4 mg/kg
PMID:10435074
LD50 Mice oral 3.16 mg/kg /Purified Flavotoxin 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 that is docosa-2,4,8,10,14,18,20-heptaenedioic acid substituted at positions 2 ,5 and 17 by methyl groups, at positions 6 by a methoxy group and at position 20 by a carboxymethyl group. It is produced by the bacterium Burkholderia gladioli and implicated in outbreaks of food-borne illness involving coconut and corn-based products in Indonesia and China. It has a role as an apoptosis inhibitor, an EC 2.5.1.18 (glutathione transferase) inhibitor, a toxin, an ATP/ADP translocase inhibitor and a bacterial metabolite. It is a tricarboxylic acid, an ether and an olefinic compound. It is a conjugate acid of a bongkrekate(3-).
Bongkrekic acid has been reported in Burkholderia gladioli with data available.
An antibiotic produced by Pseudomonas cocovenenans. It is an inhibitor of MITOCHONDRIAL ADP, ATP TRANSLOCASES. Specifically, it blocks adenine nucleotide efflux from mitochondria by enhancing membrane binding.

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Mechanism of Action
Bongkrekic acid (BKA) is an inhibitor of adenine nucleotide translocase (ANT). Since inhibition of ANT is connected to the inhibition of cytochrome c release from mitochondria, which then results in the suppression of apoptosis, it has been used as a tool for the mechanistic investigation of apoptosis. BKA consists of a long carbon chain with two asymmetric centers, a nonconjugated olefin, two conjugated dienes, three methyl groups, a methoxyl group, and three carboxylic acids. This complicated chemical structure has caused difficulties in synthesis, supply, and biochemical mechanistic investigations.
Bongkrekic acid (BA) has a unique mechanism of toxicity among the mitochondrial toxins: it inhibits adenine nucleotide translocase (ANT) rather than the electron transport chain. Bongkrekic acid is produced by the bacterium Burkholderia gladioli pathovar cocovenenans (B. cocovenenans) which has been implicated in outbreaks of food-borne illness involving coconut- and corn-based products in Indonesia and China.

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Introduction: Bongkrekic acid (BA) has a unique mechanism of toxicity among the mitochondrial toxins: it inhibits adenine nucleotide translocase (ANT) rather than the electron transport chain. Bongkrekic acid is produced by the bacterium Burkholderia gladioli pathovar cocovenenans (B. cocovenenans) which has been implicated in outbreaks of food-borne illness involving coconut- and corn-based products in Indonesia and China. Our objective was to summarize what is known about the epidemiology, exposure sources, toxicokinetics, pathophysiology, clinical presentation, and diagnosis and treatment of human BA poisoning.
Methods: We searched MEDLINE (1946 to present), EMBASE (1947 to present), SCOPUS, The Indonesia Publication Index ( http://id.portalgaruda.org/ ), ToxNet, book chapters, Google searches, Pro-MED alerts, and references from previously published journal articles. We identified a total of 109 references which were reviewed. Of those, 29 (26 %) had relevant information and were included. Bongkrekic acid is a heat-stable, highly unsaturated tricarboxylic fatty acid with a molecular weight of 486 kDa. Outbreaks have been reported from Indonesia, China, and more recently in Mozambique. Very little is known about the toxicokinetics of BA. Bongkrekic acid produces its toxic effects by inhibiting mitochondrial (ANT). ANT can also alter cellular apoptosis. Signs and symptoms in humans are similar to the clinical findings from other mitochondrial poisons, but they vary in severity and time course. Management of patients is symptomatic and supportive.
Conclusions: Bongkrekic acid is a mitochondrial ANT toxin and is reported primarily in outbreaks of food-borne poisoning involving coconut and corn. It should be considered in outbreaks of food-borne illness when signs and symptoms manifest involving the liver, brain, and kidneys and when coconut- or corn-based foods are implicated. [1]

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The ADP/ATP carrier plays a key role in cell economy. Because of its unique properties, it has provided, in biochemical and genetic studies, advanced insights into the molecular basis of metabolite transport across biomembranes. A major progress in the knowledge of this carrier results from the recent determination at high resolution of the structure of the CATR-carrier complex. Solving the structure of the carrier in other conformational states would provide essential information to elucidate the molecular mechanism of adenine nucleotide exchange across the inner mitochondrial membrane and highlight the consequences of mutations involved in related genetic diseases.
In providing the cell with ATP generated by oxidative phosphorylation, the mitochondrial ADP/ATP carrier plays a central role in aerobic eukaryotic cells. Combining biochemical, genetic, and structural approaches contributes to understanding the molecular mechanism of this essential transport system, the dysfunction of which is implicated in neuromuscular diseases. [2]

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In the present study, we did not obtain direct evidence for the interaction between the Bongkrekic acid/BKA-induced down-regulation 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 modulator for the metabolic pathway in LTED cells (Figure 5B). BKA has potential as a therapeutic modality for the recurrence of breast cancers that have already been treated with blockers of 17β-estradiol/ERα signaling; however, further investigations are needed on the mechanism(s) responsible for BKA-mediated cell death in LTED cells.[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

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.)