Ochratoxin B   中文名称: 赭曲霉素

Microbial Metabolite; secondary metabolite from Aspergillus ochraceus

在NADPH存在下，大鼠肝微粒体部分由赭曲霉毒素B形成代谢产物。通过提取、薄层色谱、高压液相色谱和结晶从培养混合物中分离出来。根据质谱和核磁共振波谱，该结构被认为是4-羟基赭曲霉毒素B。确定了4-羟基赭霉毒素B形成的Km，赭曲霉毒素A的羟基化不会因赭曲霉毒素B.的存在而改变。大鼠腹腔注射赭曲霉毒素A.或B.或两者的混合物。在赭曲霉毒素B存在的情况下，尿液中排泄的三种代谢物赭曲霉毒素A、（4R）-4-羟基赭曲霉毒素A和赭曲霉毒素α的比例没有变化。当腹腔注射时，赭曲霉毒素β在24小时内排出[2]。

赭曲霉毒素B（OTB）是赭曲霉的次级代谢产物，是赭曲霉毒素a（OTA）的非氯化类似物，是啮齿动物中最强效的肾脏致癌物之一。尽管结构密切相关，但OTB被认为毒性要低得多。OTA代谢不良，消除缓慢，这可能在OTA毒性、致癌性和器官特异性中发挥重要作用。由于对OTB的生物转化和肾毒性知之甚少，本研究的目的是研究OTB在大鼠体内的生物转化，并表征OTB的肾毒性和细胞毒性。雄性F344大鼠接受单次剂量（10 mg/kg bw）或重复剂量（2 mg/kg bw，每周5天，持续2周）的OTB给药，并在最后一次给药后72小时实施安乐死。在用单次高剂量OTB治疗的动物的近端小管细胞中，观察到有丝分裂数略有增加，但在重复给药后，临床化学、肾功能和组织病理学方面没有明显的治疗相关变化。使用带荧光检测的HPLC和LC-MS/MS分析尿液和粪便中OTB和代谢物的排泄情况。除了少量4-羟基-OTB外，由肽键切割产生的赭曲霉毒素β是尿液中排泄的主要代谢物。总的来说，单次给药后72小时内，19%的给药剂量在尿液和粪便中以OTB和赭曲霉毒素β的形式回收。与OTA相比，单次和重复给药后，OTB没有明显的组织特异性保留。在LLC-PK1细胞中，一种保留了近端小管大部分特定特征的肾细胞培养系统，仅观察到OTA和OTB的细胞毒性程度存在微小差异。在低浓度（<25微M）下，OTA处理的毒性略高，而在高达100微M的浓度下，细胞存活率的降低相似。总之，这些数据表明，OTA和OTB在体外诱导细胞毒性的潜力相似，但在啮齿动物中诱导肾毒性的潜力存在很大差异。OTB比OTA代谢更广泛，消除速度更快。因此，OTB在肾脏中缺乏特异性保留以及毒代动力学的差异可能解释了OTB毒性较低的原因[1]

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在仅用赭曲霉毒素A治疗的大鼠中，食物摄入量减少了50%，近端小管出现了组织学上严重的病变、变性和坏死。当赭曲霉毒素A和B联合给药时，动物在临床上不受影响，组织学上仅对近端小管有轻微损伤。这些观察结果表明，赭曲霉毒素B显著降低了赭曲霉毒素A的毒性作用[2]。

HPLC/FLD和LC-MS/MS的样品制备[1]
组织匀浆是通过使用ultra-turrax在4倍体积的冰冷50 mM磷酸钠缓冲液（pH 6.5）中匀浆200-400 mg组织制备的。通过加入等体积的冰冷乙醇并在4°C下以14000 rpm离心30分钟来沉淀蛋白质。将血浆和尿液样本与1体积的冰冷酒精混合，并在4℃下以14000 rpm离心30分钟以沉淀蛋白质。所得上清液用H2O稀释或直接注入LC-MS/MS系统。从加标样品中回收率>90%。粪便样本被冻干，并用研钵和研杵均质化。为了定量粪便中排泄的赭曲霉毒素B（OTB）和OTbeta的量，按照之前对OTA的描述制备样品（Zepnik等人，2003）。简而言之，将等分试样（1 g）在10 ml H2O中超声处理30分钟。加入25%盐酸（100μl）后，用10 ml氯仿提取样品三次。将每个样品的氯仿相合并，混合并蒸发至干。残留物通过超声波重新溶解在水中，用于HPLC/FLD和LC-MS/MS分析。由于赭曲霉毒素B（OTB）的亲水性代谢产物，如谷胱甘肽和葡萄糖醛酸结合物，可能无法提取到氯仿相中，因此对冷冻干燥的粪便样本进行索氏提取。将0.5 g等分试样在50 ml甲醇中提取6小时。溶剂蒸发后，将提取物重新溶解在5 ml甲醇中，并将等分试样（10μl）注入LC-MS系统。

细胞培养[1]
LLC-PK1猪肾上皮细胞在37°C的标准条件下，在添加了10%胎牛血清（FCS）、2 mM谷氨酰胺、25 mM HEPES和2000 U/l青霉素和2 mg/l链霉素的Dulbecco改良Eagle培养基中，在加湿的5%CO2气氛中维持。OTA和赭曲霉毒素B（OTB）新鲜溶解在乙醇中，并在含有5%FCS的培养基中稀释。将细胞以1×104个细胞/孔的密度接种在96孔板中，在无赭曲霉毒素的培养基中生长24小时，随后与浓度在0至100μM之间的OTA或OTB一起孵育8、24和72小时。为了确定每孔的细胞数量，用PBS洗涤细胞，向每个孔中加入50μl 0.2%结晶紫的0.1M柠檬酸溶液，并用血细胞计数器计数染色的细胞核。根据制造商的说明，使用Cytotox 96非放射性细胞毒性试验测定乳酸脱氢酶（LDH）释放到培养基中的情况。基于MTT的体外毒理学检测试剂盒用于通过线粒体脱氢酶测量活细胞的活性。这种细胞毒性试验的一般原理是将四唑盐（MTT）转化为有色产物甲赞，可以通过完整的线粒体在570 nm下进行光度测量。每种试验都在至少两个独立的实验中进行，一式三份
将5×106个细胞与10μM OTA或赭曲霉毒素B（OTB）孵育24小时后，测定OTA和Ochratoxin B（OTB的细胞内水平。孵育后，用培养基洗涤细胞四次，胰蛋白酶化，以1000rpm离心4分钟，在PBS中反复洗涤，并在-20°C下储存过夜。将细胞裂解物重新悬浮在500μl H2O中，通过加入500μl乙醇沉淀蛋白质，并在4°C和12000 rpm下离心30分钟以清除样品。将所得上清液中的10μl注入LC-MS/MS系统。

Male F344 Fisher rats (8–9 weeks old, 150–170 g) were housed in metabolic cages with tap water and powdered diet ad libitum. Room temperature was maintained at 21 ± 2 °C with a relative humidity of 55 ± 10% and a day/night cycle of 12 h. Animals (n = 3/group) were treated with a single dose of Ochratoxin B (OTB) (10 mg/kg bw) in corn oil by oral gavage or repeatedly administered Ochratoxin B (OTB) (2 mg/kg bw) for 2 weeks (5 days per week). Control animals received an equal volume of the dosing vehicle.

Absorption, Distribution and Excretion
Methods for preparation of labelled ochratoxin A and B are described. The method for preparation of labelled ochratoxin B involves the synthesis of the azide of ochratoxin beta via the mixed anhydride and subsequent conjugation to labelled phenylalanine to yield (14)C-ochratoxin B. The labelled ochratoxins were injected into male Wistar rats and after different survival times they were sacrificed and subjected to whole body autoradiography. The distribution pattern of ochratoxin A in the rat did not differ from that earlier registered for mouse. The previously known, high susceptibility of rats (and not mice) to ochratoxin A-induced cancer could thus not be explained by an accumulation of the toxin in specific cells or organs. The distribution patterns of ochratoxin A and B were almost congruent--the only apparent difference being a much longer retention of the labelled ochratoxin A in the blood compared to ochratoxin B, which was much faster excreted. When analyzing tissue extracts for labelled metabolites only the extracts from the rats injected with ochratoxin B were found to contain easily detectable concentrations, while no metabolites of ochratoxin A were seen.
Mixture of ochratoxins A and B (0.38 and 0.13 mg/kg body wt) was fed to pigs daily for 8 days during early pregnancy. Ochratoxin B was poorly absorbed and preferentially hydrolyzed in intestinal tract.
... Since little is known regarding biotransformation ... of OTB, the aim of this study was to investigate biotransformation of OTB in rats... Male F344 rats were administered either a single dose of OTB (10 mg/kg bw) or repeated doses (2 mg/kg bw, 5 days/week for 2 weeks) and euthanized 72 hr after the last dosing. ... Excretion of OTB and metabolites in urine and feces was analyzed using both HPLC with fluorescence detection and LC-MS/MS. Ochratoxin beta, which results from cleavage of the peptide bond, was the major metabolite excreted in urine in addition to small amounts of 4-hydroxy-OTB. In total, 19% of the administered dose was recovered as OTB and ochratoxin beta in urine and feces within 72 hr after a single dose. In contrast to OTA, no tissue-specific retention of OTB was evident after single and repeated administration. ... OTB is more extensively metabolized and more rapidly eliminated than OTA. ...

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Metabolism / Metabolites
... Since little is known regarding biotransformation ... of OTB, the aim of this study was to investigate biotransformation of OTB in rats... Male F344 rats were administered either a single dose of OTB (10 mg/kg bw) or repeated doses (2 mg/kg bw, 5 days/week for 2 weeks) and euthanized 72 hr after the last dosing. ... Excretion of OTB and metabolites in urine and feces was analyzed using both HPLC with fluorescence detection and LC-MS/MS. Ochratoxin beta, which results from cleavage of the peptide bond, was the major metabolite excreted in urine in addition to small amounts of 4-hydroxy-OTB. In total, 19% of the administered dose was recovered as OTB and ochratoxin beta in urine and feces within 72 hr after a single dose. In contrast to OTA, no tissue-specific retention of OTB was evident after single and repeated administration. ... OTB is more extensively metabolized and more rapidly eliminated than OTA. ...
Ochratoxin B (0.13 mg/kg body wt) fed to pigs daily for 8 days was completely hydrolyzed to ochratoxin beta.
A metabolic product was formed from ochratoxin B by rat liver microsomal fractions in the presence of NADPH. It was isolated from the incubation mixture by extraction, thin-layer chromatography, high-pressure liquid chromatography, and crystallization. On the basis of mass and nuclear magnetic resonance spectroscopy, the structure is suggested to be 4-hydroxyochratoxin B. The Km for the formation of 4-hydroxyochratoxin B was determined, and the hydroxylation of ochratoxin A was not altered by the presence of ochratoxin B. Rats were given ochratoxin A or B, or a mixture of both intraperitoneally. The ratios of the three metabolites, ochratoxin A, (4R)-4-hydroxyochratoxin A, and ochratoxin alpha, excreted in the urine did not change in the presence of ochratoxin B. Ochratoxin B was metabolized to 4-hydroxyochratoxin B and ochratoxin beta, but in a different ratio than for the ochratoxin A metabolites. When given intraperitoneally, ochratoxin beta was excreted within 24 hr. In rats treated with ochratoxin A alone, the food intake was reduced by 50%, and histologically severe lesions, degeneration, and necrosis were observed in the proximal tubules. When ochratoxin A and B given in combination, the animals were clinically unaffected and histologically there was only slight damage of proximal tubules. These observations indicate that ochratoxin B considerably reduces the toxic effects of ochratoxin A.
The objectives of this study were to develop and evaluate procedures for the confirmation of ochratoxin A (OA), lactone opened OA (OP-OA), ochratoxin B (OB), hydroxy OA (OA-OH) and ochratoxin alpha (Oalpha) and metabolites formed in the rats from these toxins, and to demonstrate that many ochratoxin metabolites can be identified in the bile and urine of rats injected with the different ochratoxins. An esterification procedure in acidified methanol and a lactone hydrolysis procedure in strong base yielded two additional forms of most of the different ochratoxins. The esterification procedure provided a simple, fast and reliable method for the confirmation of the ochratoxins. A total of 20 different metabolites of OA, OP-OA, OB, OA-OH and Oalpha were detected in the urine and the bile of rats of which several were identified. Among these, OA and the recently discovered and toxic form of OA (OP-OA) were readily formed in vivo when either were injected. Procedures developed in this study can be used to confirm and isolate ochratoxins in biological samples and have shown that a new form of OA (OP-OA) along with many other metabolites are formed from OA and related ochratoxins in vivo.

Adverse Effects
Occupational hepatotoxin - Secondary hepatotoxins: the potential for toxic effect in the occupational setting is based on cases of poisoning by human ingestion or animal experimentation. Nephrotoxin - The chemical is potentially toxic to the kidneys in the occupational setting.

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chicken LD50 oral 54 mg/kg Applied Microbiology., 21(492), 1971 [PMID:4928604]

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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 the 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 /SRP: "To keep open", minimal flow rate/. Use 0.9% saline (NS) or lactated Ringer's 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 or lorazepam ... . Use proparacaine hydrochloride to assist eye irrigation ... . /Poisons A and B/

[1]. Biotransformation and nephrotoxicity of ochratoxin B in rats. Toxicol Appl Pharmacol. 2005 Aug 1;206(1):43-53.

[2]. Metabolism of ochratoxin B and its possible effects upon the metabolism and toxicity of ochratoxin A in rats. Appl Environ Microbiol. 1985 May;49(5):1108-12.

Crystals that exhibit blue fluorescence. (NTP, 1992)
Ochratoxin B is a phenylalanine derivative resulting from the formal condensation of the amino group of L-phenylalanine with the carboxy group of (3R)-8-hydroxy-3-methyl-1-oxo-3,4-dihydro-1H-2-benzopyran-7-carboxylic acid. Ochratoxin B differs from the more naturally abundant ochratoxin A in the absence of the dihydroisocoumarin chlorine atom. It has cytotoxic effects on kidney and liver cells in vitro but only minor effects in vivo, due to its rapid metabolism and excretion. It inhibits cell proliferation of human liver HepG2 cells at doses as low as 1 mug/ ml but lacks the genotoxic activity of ochratoxin A, even at higher concentrations. It has a role as an Aspergillus metabolite, a Penicillium metabolite, a mycotoxin and a calcium channel blocker. It is a phenylalanine derivative, a N-acyl-L-phenylalanine, a member of isochromanes and a monocarboxylic acid. It is a conjugate acid of an ochratoxin B(1-).
Ochratoxin B has been reported in Aspergillus ochraceus with data available.

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In summary, our data suggest that in rats, OTB is more extensively metabolized and more rapidly eliminated from plasma than OTA and does not accumulate in a tissue specific manner. Results from this study do not support the role of the chlorine atom as a structural requirement for ochratoxin-mediated toxicity and indicate that the lower toxicity of OTB may at least in part result from differences in toxicokinetics.[1]

C20H19NO6

369.37

369.121

C, 65.03; H, 5.19; N, 3.79; O, 25.99

4825-86-9

20966

White to off-white solid powder

1.4±0.1 g/cm3

632.4±55.0 °C at 760 mmHg

221ºC

336.2±31.5 °C

0.0±2.0 mmHg at 25°C

1.623

3.06

112.93

3

6

5

27

573

2

C[C@@H]1CC2=C(C(=C(C=C2)C(=O)N[C@@H](CC3=CC=CC=C3)C(=O)O)O)C(=O)O1

DAEYIVCTQUFNTM-ABAIWWIYSA-N

InChI=1S/C20H19NO6/c1-11-9-13-7-8-14(17(22)16(13)20(26)27-11)18(23)21-15(19(24)25)10-12-5-3-2-4-6-12/h2-8,11,15,22H,9-10H2,1H3,(H,21,23)(H,24,25)/t11-,15+/m1/s1

(2S)-2-[[(3R)-8-hydroxy-3-methyl-1-oxo-3,4-dihydroisochromene-7-carbonyl]amino]-3-phenylpropanoic acid

HSDB 3438; OCHRATOXIN B; 4825-86-9; CCRIS-5094; HSDB-3438; UNII-ECJ5WS94N2; ECJ5WS94N2; BRN 1300160; OCHRATOXIN B [MI]; CCRIS 5094; Ochratoxin B

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

注意： 本产品在运输和储存过程中需避光。

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

注意: 如下所列的是一些常用的体内动物实验溶解配方，主要用于溶解难溶或不溶于水的产品（水溶度<1 mg/mL）。 建议您先取少量样品进行尝试，如该配方可行，再根据实验需求增加样品量。

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注射用配方1: DMSO : Tween 80： Saline = 10 : 5 : 85 (如： 100 μL DMSO → 50 μL Tween 80 → 850 μL Saline)
*生理盐水/Saline的制备：将0.9g氯化钠/NaCl溶解在100 mL ddH ₂ O中，得到澄清溶液。
注射用配方 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL DMSO → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)
注射用配方 3: DMSO : Corn oil = 10 : 90 (如： 100 μL DMSO → 900 μL Corn oil)
示例: 以注射用配方 3 (DMSO : Corn oil = 10 : 90) 为例说明, 如果要配制 1 mL 2.5 mg/mL的工作液, 您可以取 100 μL 25 mg/mL 澄清的 DMSO 储备液，加到 900 μL Corn oil/玉米油中, 混合均匀。

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注射用配方 4: DMSO : 20% SBE-β-CD in Saline = 10 : 90 [如：100 μL DMSO → 900 μL (20% SBE-β-CD in Saline)]
*20% SBE-β-CD in Saline的制备（4°C，储存1周）：将2g SBE-β-CD (磺丁基-β-环糊精) 溶解于10mL生理盐水中，得到澄清溶液。
注射用配方 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (如： 500 μL 2-Hydroxypropyl-β-cyclodextrin (羟丙基环胡精)→ 500 μL Saline)
注射用配方 6: DMSO : PEG300 : Castor oil : Saline = 5 : 10 : 20 : 65 (如： 50 μL DMSO → 100 μL PEG300 → 200 μL Castor oil → 650 μL Saline)
注射用配方 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (如： 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
注射用配方 8: 溶解于Cremophor/Ethanol (50 : 50), 然后用生理盐水稀释。
注射用配方 9: EtOH : Corn oil = 10 : 90 (如： 100 μL EtOH → 900 μL Corn oil)
注射用配方 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL EtOH → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)

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口服配方 1: 悬浮于0.5% CMC Na (羧甲基纤维素钠)
口服配方 2: 悬浮于0.5% Carboxymethyl cellulose (羧甲基纤维素)
示例: 以口服配方 1 (悬浮于 0.5% CMC Na)为例说明, 如果要配制 100 mL 2.5 mg/mL 的工作液, 您可以先取0.5g CMC Na并将其溶解于100mL ddH2O中，得到0.5%CMC-Na澄清溶液；然后将250 mg待测化合物加到100 mL前述 0.5%CMC Na溶液中，得到悬浮液。

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口服配方 3: 溶解于 PEG400 (聚乙二醇400)
口服配方 4: 悬浮于0.2% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 5: 溶解于0.25% Tween 80 and 0.5% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 6: 做成粉末与食物混合

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注意: 以上为较为常见方法，仅供参考， InvivoChem并未独立验证这些配方的准确性。具体溶剂的选择首先应参照文献已报道溶解方法、配方或剂型，对于某些尚未有文献报道溶解方法的化合物，需通过前期实验来确定（建议先取少量样品进行尝试），包括产品的溶解情况、梯度设置、动物的耐受性等。

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
4、 如产品在配制过程中出现沉淀/析出，可通过加热(≤50℃)或超声的方式助溶;
5、为保证最佳实验结果，工作液请现配现用！
6、如不确定怎么将母液配置成体内动物实验的工作液，请查看说明书或联系我们;
7、 以上所有助溶剂都可在 Invivochem.cn网站购买。