What Are Ochratoxins
Ochratoxin A (OTA) is a mycotoxin that has long been studied as a nephrotoxin, immunotoxin, teratogen and carcinogen. Molds associated with the production of OTA include Aspergillus ochraceus, Aspergillus niger and Aspergillus carbonarius, Penicillium verrucosum, and species of Penicillium, Petromyces, and Neopetromyces. Concerns regarding exposure to ochratoxin have primarily centered on exposure to food contaminated with OTA such as wine, beer, coffee, dried vine fruit, grape juices, pork, poultry, dairy, spices, and chocolate. However, ochratoxin has also been known to be inhaled from exposure in contaminated buildings. Toxicity from ochratoxin is considered serious enough that it is among approximately 20 mycotoxins monitored in food.
Ochratoxins are secondary metabolites of certain types of fungi. Ochratoxins occur in nature as Ochratoxin A (OTA), B (OTB), and C (OTC). Ochratoxin A is the most prevalent ochratoxin and the most toxic of the three, followed by OTB and OTC. Ochratoxin A (OTA), first discovered in 1965 in Aspergillus ochraceus has been subsequently reported in several Aspergillus species (such as carbonarius and niger), Penicillium species, and species of Petromyces and Neopetromyces. Ochratoxin A, B and C are all produced by Penicillium and Aspergillus species of mold. Two of the major producers are Aspergillus ochraceus and Penicillium verrucosum.
The chemical structure of OTA consists of weak organic acids with a dihydroisocumarin moiety joined by a peptide bond to 1-phenylalanine. The three ochratoxin forms, designated as A, B and C, have slight structural differences; however, ochratoxin A (OTA) is chlorinated and is the most toxic one. OTA is only slightly soluble in water, but it dissolves in ethanol, methanol and acetone. Ochratoxin B (OTB), a secondary metabolite of Aspergillus ochraceus, is the nonchlorinated analogue of the mycotoxin ochratoxin A (OTA). Despite the closely related structure, OTB is considered to be of much lower toxicity.
Ochratoxin A (OTA) is one of the most potent kidney carcinogens in rodents and has also caused kidney damage in humans. OTA is a proven carcinogen in animals and is classified as a class 2B, possible human carcinogen by the International Agency for Research on Cancer (IARC). The toxin has been classified as possibly carcinogenic to humans by the IARC due to an observed increase of the incidence of hepatocellular tumors and renal cell adenomas in rodents. OTA is poorly metabolized and slowly eliminated, and this may play an important role in OTA toxicity, carcinogenicity, and organ specificity. In contrast to OTA, in rats there was no tissue-specific retention of OTB evident after single and repeated administration.
Where Are Ochratoxins Found
Global exposure to ochratoxins is largely thought to be through food contamination, but exposure in contaminated buildings is also possible. Ochratoxin (OTA) is present in a large variety of foods because it is produced by several fungal strains of the Penicillium and Aspergillus species. Skin contact and inhalation are known to also cause toxicity. Inhalation is often from dust in commercial areas where grains are being stored and/or processed. Ochratoxin may also be found in water-damaged buildings. One clinical study identified OTA in 83% of over 100 individuals tested with chronic illness and a history of water-damaged building exposure.
Additionally, ochratoxin may be produced by Aspergillus growing in the respiratory or digestive tract. Many species of Aspergillus produce dry, hydrophobic spores that are easily inhaled. Due to their small size, about 70 % of spores of A. fumigatus are able to penetrate into the trachea and primary bronchi and close to 1 % into alveoli. When it becomes a human or animal pathogen the disease is called Aspergillosis. It is common in birds and is found in many animals as well as humans. Most people and animals with an intact immune system will be able to fend off Aspergillus. An x-ray of the chest will find a fungal mass (aspergilloma) if growing in the lungs. A sputm secretion can be tested for Aspergillus presence in the lungs or a nasopharyngeal swab can be taken to help ID a sinus or nasal colinization. If the person has an allergic response to Asperillus, skin, sputum or blood tests may reveal an allergic response. An organic acids test can be helpful to test for metabolites associated with Aspergillus and is used to help identify the fungus in the digestive tract. More recently, oxalic acid testing has been used to reveal Aspergillus since it is known to make oxalates which is thought to be made as part of the biofilm it creates to protect itself.
Exposure to ochratoxins is common through food contamination and the most common food to be contaminated is grains. Besides being found in grains, ochratoxins have been found in flour made from grains, coffee, beans, peas, and mixedanimal feeds (grains also), and are all too often present in wine, grape juice, and dried vine fruits.
It is most commonly found in wheat, corn and oats. Ochratoxin A is the most economically, and medically important form of ochratoxin; ochratoxins B and C are less toxic and less common. In addition to the most common foods that contain OTA, it also occurs in olives, beer, wine, coffee, cocoa products, raisins, figs, licorice, pulses, pumpkin seeds, and tea. It has been found on such food as dried and smoked fish, dried fruit and tree nuts. It has also been found on grapes and grape products such as wine, raisins, wine vinegars. The WHO in 2001 reported that the main sources are cereals, wine, grape juice, coffee and pork for human exposure at levels of 58%, 21%, 7%, 5% and 3% of ochratoxin intake respectively. Red wines typically contain higher ochratoxin levels than white wines. Although coffee bean roasting lowers ochratoxin levels by 80% - 96%, it was shown to be fairly stable in processing of wheat and barley via dry milling and heat processing. Wet milling of corn resulted in reductions of corn grits and germ by 49% and 96 % respectively. Penicillium verrucosum is the leading cause of ochratoxin contamination of cereal grains in temperate climates. Grapes, raisins, and even wines may become contaminated with ochratoxins produced by Aspergillus carbonarius, the principal causal agent of grape black mold. A number of Aspergillus spp. may cause ochratoxin contamination in green and processed coffee, including A. ochraceus, A. carbonarius, and A. niger. Tree nuts and figs may be infested with A. ochraceus and A. melleus, the leading producers of ochratoxins in these commodities.
Another mycotoxin called Citrinin is produced by the genera Penicillium, Aspergillus and Monascus, and contaminates the same staple foodstuffs as OTA. Citrinin and Ochratoxin are thought to act synergistically together.
Zearalenone also occurs on the same grains as ochratoxin and is additionally thought to have a synergistic activity with OTA.
Details On Growth
OTA-producing Aspergillus species, A. carbonarius and A. niger (which produces OTA more rarely), grow well at high temperatures and produce dark colored hyphae and spores, making these species resistant to UV light. Therefore, Aspergillus carbonarius is commonly found in grapes and similar types of fruits that mature in sunlight and at high temperatures.
Aspergillus ochraceus grows better in oilseeds (peanuts and soybeans), while Penicillium verrucosum has been shown to grow better in wheat and corn.
Although Aspergillus is found more commonly in conditions of high humidity and temperature, Penicillium species are a source of ochratoxin in cool temperate regions.
Dairy, Meat And Eggs May Contain Ochratoxins
Ochratoxins may be transferred through milk, blood, and meat, so you will find it in dairy and meat products from animals that have consumed contaminated grains. Ochratoxins are even found in human milk. Ochratoxin levels were significantly higher in the milk of habitual consumers of bread, bakery products and cured pork meat. It has also been detected in chicken eggs. The Balkan Endemic Nephropathy that took place in the 1950s was thought to be caused by consumption of pork. The pigs had consumed feed contaminated with ochratoxin, it accumulated in the animals, and some of those individuals who ate the pork ended up with kidney damage.
In the European Union, the overall contribution of products of animal origin to human exposure has been found on average to not be more than 3 % of the total ochratoxin A burden (Miraglia and Brera, 2001).
Skin Contact And Inhalation Exposure
Skin contact and inhalation are known to also cause toxicity, and I think there are more people exposed to ochratoxin from water-damaged buildings than is currently realized by practitioners. Testing has found the presence of ochratoxin A from samples of air filters, refrigerator filters, dust from air vents, in wallpaper, and a towel and sandals from water-damaged buildings. As already mentioned, one clinical study identified OTA in 83% of over 100 individuals tested with chronic illness who had a history of water-damaged building exposure. The authors of one study concluded that airborne dust can be a significant source of OTA and that peak exposures and absorptions from this route can be considerable in some environments, especially given the efficiency with which OTA is absorbed through the lung. Inhalation can also be from dust in commercial areas where grains are being stored and/or processed. Researchers found people working in environments where coffee, cocoa beans and spices are processed are at risk since these are foods that tend to be more likely to have ochratoxins on them. The other working environment that would lead to exposure would be those environments where farmers, or processors are working with grains, or in enclosed animal shelters where grain is often fed, such as has been found in cow sheds and in poultry houses.
Preventing Ochratoxin Exposure
The Role Of The Farmer
OTA is not visible to the eye, so it can be consumed by unaware humans. The farmer is key in preventing Aspergillus ochraceus and Penicillium verrucosum as well as other mold (the molds that make OTA) growth during farming, as well as by following good harvesting methods. Farming practices such as supporting the soils healthy microbial/fungal/parasitic biome, rotating crops, and supplying nutrients to the soil, will all go a long way to supporting a healthy plant, that will do better in droughts, and not attract insects (insects damage plants allowing for fungal entry into plant). This helps to decrease fungal growth and mycotoxin production. Keeping the plants healthy and happy will decrease plant stress, and decrease the plants susceptibility to all disease, including fungal disease.
High humidity and warm temperatures and low altitudes have been shown to support the production of OTA by Aspergillus spp. in coffee while growing. However, it is not the growing stage, but the harvesting, drying and storage stage of coffee that usually sees growth of Aspergillus and OTA. In a study of OTA in coffee it was found that frequent mist, infrequent turning of the coffee bean while drying and a thick layer of drying beans are issues that support OTA. Moisture from the floor and ceiling as well as bird access was a recipe for Aspergillus growth. Harvesting beans from off the ground increases the likelihood of OTA. Heaping the beans in piles to wait until there is room to dry them also is a bad practice. In one case a storage room had hot, humid air being injected into the room from another room with a coffee dryer and the stored beans were getting hot and moistened, leading to Aspergillus growth.
Luckily, in regards to coffee beans, studies have shown that coffee roasting can remove a very significant percentage of OTA. Depending on the roasting process, 65 to 100% reduction of OTA can be achieved. Not enough data is available on the effects of roasting, grinding and beverage preparation and stability in relation to OTA. Additionally modified mycotoxins can be formed during processing but their toxicity remains unclear. It appears that they are less cytotoxic than OTA.
OTA is very stable during feed-processing, hence it cannot be eliminated from the feed industry, and remains intact and biologically active in the finished feed.
How Processing And Storage Affects Ochratoxin In Food
Ochratoxin A is partially destroyed by baking. Baking and roasting have been reported to reduce the toxin by only 20%. Treatment of grain by scouring while cleaning the grain for milling has resulted in as much as 50% reduction of ochratoxin A in wheat flour. Milling has little effect on OTA. Unfortunately ochratoxin is found in foods that go through high heat processing. Therefore, it is found in cereal products, roasted coffee and beer. Although it can be partially destroyed, it is simply not destroyed well by heat.
It seems one of the key points for growth of ochratoxins in grain is at the point where grain is being dried and stored. If grain is sufficiently dry with moisture content below 14.5% it is considered safe to prevent OTA production (Magan and Aldred, 2007). The fungus responsible for the production of ochratoxin can invade starchy cereal grains such as corn and wheat with a moisture content starting around 15.5-16%.
Regulation Of Ochratoxin
There are numerous countries that limit the amount of ochratoxin A allowed in cereals (grains) and cereal products. The limits range from 3 micrograms/kilogram to 50 micrograms/kilogram. At the time of this writing I saw suggested limits on the FDA website but not any regulated limits.
The European Commission has established a list for the maximum tolerable limits of OTA in food commodities, such as grains (5 µg/kg ), grain products (3 µg/kg ), wine and dried fruits (10 µg/kg ) and foodstuff for baby and children below three years of age (0.5 µg/kg).
The average concentration of OTA is reported to range from 0.1 to 100 ng/g. OTA concentration in black pepper, cayenne pepper, caraway, cardamom, coriander, chili powder, turmeric, and dried red pepper ranges from 1 to 100 ng/g.The levels of OTA in animal products are the levels of OTA range from 0.1 to 1 ng/g. I was reading through a list of European Union alerts regarding ochratoxin and saw that some pumpkin seeds from China were listed as having 20,000 ng/g and licorice root from Turkey had 434 ng/g.
For Ochratoxin A:
- The U.S. FDA has no regulations around Ochratoxins.
- The European Union allows 2-10 micrograms/kilogram of Ochratoxin A in food.
- The foods more commonly assumed may have these mycotoxins and therefore more likely to be tested are grains, dried vine fruits, wine, grapes, coffee, cocoa and cheese.
- The countries that currently have legal limits on ochratoxin are the European Union, Bosnia, China, Egypt, Kenya, Herzegovina, India, Nigeria, and Russia.
Levels In Recent Harvests
According to the recent Biomin World Mycotoxin Survey Report, OTA contamination in Europe in the year 2018, was 40% in the finished (complete) feeds and 12% in cereals, with the average of positive samples containing 4 μg/kg in finished feeds and 19 μg/kg in cereals, respectively.
Health Effects of Ochratoxins
The tolerable dosage in humans was estimated to be 0.2 to 4.2 ng/kg (0.0002 to 0.0042 μg/kg) body weight based upon NTP carcinogenicity study in rats. Following intravenous administration, OTA is eliminated with a half-life from body in vervet monkeys in 19-21 days (Stander et al, 2001).
The oral LD50 of OTA ranges from 3 to 20 mg/kg in different animals.
Ochratoxin is known to be acutely toxic to the kidneys and the liver.
There is one case on record of an acute kidney failure due to inhalation of OTA over an 8 hour period. The Aspergillus was growing on wheat in a granary building that had been closed up for a few months. The woman effected had worked 8 hours in this granary prior to succumbing to the mycotoxin. She and her husband who also worked in the granary for 8 hours both had respiratory distress, but she, unlike her husband, developed kidney failure due to tubulonecrosis from OTA exposure.
The International Agency For Research On Cancer has listed Ochratoxin as a Group 2B (possible human carcinogen). It is also thought to cause Balkan Endemic Nephropathy.
Balkan endemic nephropathy (BEN) is a chronic disease associated with ochratoxin exposure. It is a chronic interstitial nephropathy that initially shows changes in kidney epithelial cells without any noticable anatomical changes to the eye. After chronic exposure, kidneys have interstitial fibrosis and they are smaller. Towards the end of this process the impairment of kidney function leads to polyuria (pee a lot) accompanied with red tongue, thirst, and a bitter taste. Neither edema (water retention) nor hypertension that is noticed with other kidney diseases is observed. Other symptoms such as headaches, lower back pain, weakness, and anemia (iron deficiency) have been noted.
Ochratoxin, as with many mycotoxins is fat soluble and has been shown to accumulate in tissues of animals, especially pigs.
Ochratoxin has a similar molecular structure as the essential amino acid phenylalanine. It has been shown to interfere with phenylalanine hydroxylase activity in the liver and kidney, which inhibits normal creation of proteins and inhibits creation of RNA/DNA.
The primary long term damage is chronic nephropathy (kidney damage). The lesions include tubularatrophy, interstitial fibrosis and, at later stages, hyalinized glomeruli. It has produced nephrotoxic effects in all species of monogastric animals studied so far, even at the lowest level tested (200 μg/kg feed in rats and pigs).
Several studies in animals have confirmed a causal connection between OTA exposure and cancers of the urinary tract, liver, and mammary glands. A correlation of consumption of foods known to contain OTA and the incidence of testicular cancer in 20 countries has suggested the possibility of OTA being related to an increased incidence of testicular cancer. There is also a correlation of pork and coffee intake with testicular carcinoma. In addition, animals exposed to OTA contain OTA in the testes and OTA causes adducts in testicular DNA.
There are reports of Ochratoxin being toxic to the kidney, Liver, nervous system, immune system, the DNA, to embryos and causing birth defects in humans and animals both.
Organ Damage Due To Ochratoxin A
OTA is a nephrotoxic, genotoxic, immunotoxic, and neurotoxic mycotoxin which is a known carcinogen in animals and a class 2B, possible human carcinogen. There are both species and sex differences related to the toxicities. Associations have been found with human kidney disease including Balkan endemic nephropathy and focal segmental glomerulosclerosis.
- Kidney toxicity
- Liver toxicity
- Nervous system toxicity including blood-brain barrier damage and CNS effects
- Testicular toxicity
The fact that OTA is such a potent mycotoxin relies on its ability to disturb cellular physiology in multiple ways, particularly by inhibiting the phenylalanyl-tRNA-synthetase. (Baudrimontetal., 1997; Creppyet al., 1983a)
OTA acts as a nephrotoxin and an urothelial carcinogen as a result of both the oxidative stress and direct genotoxic mechanisms. Strikingly, chronic exposure to low OTA doses could be even more damaging than acute exposure to a high dose.
Where Ochratoxin Is Found In The Body
The amount of absorbed OTA has been shown to be species‐dependent, and, although the results present a high level of variability, humans have shown the highest level of OTA absorption (62% to 100%) (Versantvoort, Oomen, Van De Kamp, Rompelberg, & Sips, 2005). Other species had a lower absorption levels, such as pigs (66%), rabbits (56%), and chickens (40%) (Galtier, Alvinerie, & Charpenteau, 1981).
Tissue distribution after exposure of animals to OTA has consistently revealed that the greatest concentration is in the kidneys followed by either liver or muscle and then fat. It has also been found in the skin, gastric mucosa, the heart muscles bone marrow, as well as the adrenal medulla and cortex in animals.
In humans, OTA has been detected in blood, urine, sweat and breast milk as well as renal cell carcinomas, breast cancer, astrocytoma, inflamed bladder tissue and transitional cell carcinoma of the bladder, and a skin biopsy sample. In one family case study where there the family was exposed to a water-damaged building, OTA was even found in the placenta and umbilical cord. Studies with radiolabeled OTA in mice showed OTA to cross the placenta, preferentially at specific times during gestation. Ochratoxin has been found to be embryotoxic in rats and mice. In one human study OTA concentration in fetal serum was reported to be twice that of the mothers indicating an active transfer of OTA across the placenta. Ochratoxin poisoning is thought to be the cause of a chronic kidney disease in humans known as Balkan endemic nephropathy. Recent studies have provided a link between ochratoxin exposure and human testicular cancer in Europe.
in 50 lactating mothers and their infants in Egypt, the presence of OTA was associated with significantly higher levels of urine microglobulin and microalbuminuria in the infants, which is consistent with early renal injury. Moreover, the level of OTA in the infants sera correlated with the degree of microalbuminuria.
More Likely To Affect Monogastric Animals
Ochratoxicosis is more likely to affect monogastric animals than ruminant animals. Pigs are thought to be the most sensitive and like humans they are monogastric animals. Kidney damage is seen in pigs eating 1 mg OTA/kg feed but not on diets containing 0.2 mg OTA/kg feed for two years. Some degenerative changes were seen in the kidneys on a diet of OTA at 0.8 mg/kg for six months. There were changes in the immune system in pigs eating 2.5 mg/kg in another study and on 25 g·kg-1 there was decreased feed efficiency, decreased daily gain of weight and final body weight.
OTA Effect On Digestive Tract
Studies have shown gut microbiota are able to elminate mycotoxins from the host naturally if the host has a healthy gut microbiota. Unfortunately, mycotoxins have also demonstrated an ability to modulate the gut microbiota too and usually it is not beneficial to the host and eventually leaves the host without the protective microbiota to remove mycotoxins. This means in those individuals exposed for a longer period of time, there is more liklihood of loss of a healthy gut microbiota and more chance the mycotoxins will end up getting into the blood stream from the gut. The fact that animal studies show ochratoxin can damage the intestinal barrier means the lack of protective bacteria and the leaky gut barrier will ensure that both the mycotoxins and other antigens will cross the intestinal wall and end up inside the body.
OTA was studied in pig intestinal epithelial cells to get an idea of how it might affect the intestinal barrier. OTA induced intestinal barrier dysfunction indicated by the reduction in transepithelial electrical resistance and elevation in paracellular permeability to 4 kDa dextran. The barrier dysfunction was accompanied with tight junction disruption including a down-regulation in ZO-1 expression and redistribution of Occludin and ZO-1. Moreover, OTA exposure increased reactive oxygen species generation, elevated the intracellular calcium level and activated myosin light chain kinase. Simultaneously, n-acetyl cysteine, a reactive oxygen species (ROS) scavenger, blocked OTA-induced ROS generation, intracellular calcium level elevation, barrier dysfunction and tight junction disruption, suggesting that OTA-induced ROS generation may act as a trigger.
OTA Effect On Immune System
Ochratoxin has been shown to be immunospuressive in many studies. It can increase risk for a vareity of infectious diseases. Studies on animals show immunosuppressant activity including reduction of the size of the thymus, spleen and lymph nodes, as well as a decrease in antibody response, and changes in number as well as function of immune cells and changes in cytokine production. In broiler chicks even low levels of ochratoxin caused immune suppression. In the broiler chickes there was a decrease in size of thymus, spleen and bursa of Fabricius, as well as lowered white blood cells. In a dose dependent manner there was a decrease in lymphocytes and heterophils with an increase in monocytes. There was also a decrease is IgY and IgA serum concentrations. Severe lymphocytopenia as well as decreased antibody response have been obeserved in poulty exposed to OTA. Chicks have shown decreased number of macrophages and decreased phagocytic activity.
In pigs immunosuppression and delayed response to immunization against Salmonella Cholera-suis was seen. In mice there has been a decrease in phagocytosis of E. coli by neutrophils and monocytes.
Rats have shown increase of NF-KappaB expression, and in mice high expression of heat shock protein 72. Rodents have also shown increase TNF-alpha release, increase in IL-1 synthesis, IL-6 and IL-8 release and increased cyclooxygenase-2https://www.researchgate.net/figure/Continued_tbl2_273511420
Mechanism of Injury
- Oxidative stress
- Bio-energetic compromise
- Mitochondrial impairment
- Inhibition of protein synthesis
- DNA single strand breaks
- Formation of OTA-DNA adducts
OTA has a similar structure to the amino acid phenylalanine, therefore it may impair protein synthesis. The phenylalanine moiety of OTA has been discussed as the responsible substructure for competitive inhibition of enzymes needed for protein biosynthesis.
The generation of reactive oxygen species (ROS) has furthermore been considered responsible for OTA-induced toxicity and carcinogenicity. Several studies imply that the mode of action of ochratoxin is the formation of covalent DNA adducts and the increase of reactive oxygen species, which would explain the genotoxic and mutagenic activity of OTA. Oxidative metabolism of OTA resulting in OTA metabolites such as reactive quinone structures has also been observed (Dai et al. 2002; Calcutt et al. 2001; Gillman et al. 1999). Chemical reaction products of these compounds such as glutathione conjugates have been detected in cell culture and in rodents.
in rat liver and kidney after feeding the rats with OTA. The results suggest that OTA undergoes metabolism to generate electrophiles that can react with glutathione in rat liver and kidney.
OTA affects the expression of several genes related to cell damage, apoptosis (cell death), cellular stress and antioxidant defence systems. The mechanism of OTA toxicity includes the formation of oxygen free radicals and consequently peroxidation of polyunsaturated fatty acids. The oxidation theory is further supported by the fact that low molecular weight antioxidants, such as vitamin E or vitamin C, decrease the formation of lipid peroxides and the corresponding toxic effects of ochratoxin A.
Many Nrf2-regulated enzymes are involved in the antioxidant defence and expression of most of them is inhibited by OTA treatment in the kidney of chickens.
In the blood, OTA binds to albumin and the bound fraction constitutes a mobile reserve of OTA . The relative contribution of each excretory route is influenced by the degree of serum macromolecular binding, as well as differences in the enterohepatic recirculation of OTA. Elimination of OTA in urine and feces is felt to be relatively slow, and has been shown to vary by species and gender, as well as specific genotype that may affect the biotransformation of OTA.
OTA causes decreased 5-oxoproline. Decreased 5-oxoproline is seen with Mild glutathione synthetase deficiency. Moderate deficiency can cause hemolytic anemia, elevated acidity – metabolic acidosis. Severe deficiency can cause neurological symptoms such as seizures, decreased physical reactions, movements and speech, intellectual disability and loss of coordination. Some people with severe deficiency experience recurrent bacterial infections.
Interest in the mechanism of action of mycotoxins and especially OTA has increased with the availability of a Clinical Laboratory Improvement Amendments (CLIA) regulation-compliant registered laboratory test, which has identified OTA in the urine of humans with chronic illness. One of the clinical studies identified OTA in 83% of over 100 individuals tested with chronic illness and a history of water-damaged building exposure.
Factors Involved In The Mechanisms Of Injury
- Individual genetic differences affect the biotransformation and relative toxicity of OTA, with enzymatic hydrolysis and cytochrome P450 induction felt to play a role in toxicity.
- DNA adducts also occur in animals exposed to OTA in all available studies.
- Oxidative stress is an important component of OTA toxicity.
- In rodents that mTOR/AKT pathways are significantly deregulated after exposure to OTA, possibly contributing to carcinogenicity in kidney cells.
- Limited information is available on the metabolic disposition of OTA in humans, although it has been suggested that it has a long serum half-life due to strong binding to human serum macromolecules.
- Intestinal microflora also appear to contribute significantly to the metabolism of OTA via hydrolyzation to the less toxic ochratoxin alpha in rats
- After initial exposure from any source, the urinary and fecal excretory routes of OTA are both important with the relative contribution of each dependent upon factors such as route of administration and dose
- Glutathione conjugation and glucuronidation as well as n-acetyl cysteine may be involved in removal, decreasing amount available to cause injury.
- In the blood, OTA binds to albumin and the bound fraction constitutes a mobile reserve of OTA
- Levels of dopamine and its metabolites have been shown to decrease in a dose dependent manner in the striatum of the brain. This might induce Parkinsons Disease or induce an earlier onset when mixed with the normal age-dependent decline. OTA has been shown in mice to kill dopaminergic neurons. It may also act through it's ability to inhibit protein synthesis by competition with phenylalanine and phenylalanine hydroxylase activity leading to impaired synthesis of DOPA, dopamine and catecholamines or enzymes involved in the metabolism of DNA. Parkinson's patients were tested for ochratoxin A and found to have much more ochratoxin than people without Parkinsons. Patients with Parkinsons should be checked for ochratoxin A.
Oxidative stress may be a major mechanism for the toxicity of Ochratoxin A (OTA). Oxidative DNA damage has been seen in vitro with OTA and in the kidney and liver of rats. OTA was shown to increase the formation of oxidative products of lipids, with increased production of malondialdehyde, increased reactive oxygen species levels, as well as 8-oxoguanine formation and deplted cellular glutathione levels. A study showed OTA could be decreased by maintaining glutathione production through pretreating the in vitro cell with the glutathione limiting amino acid, N-acetyl cysteine, which decreased reactive oxygen species and 8-oxoguanine formation. These are in vitro studies but they lead us to believe glutathione may be protective in ochratoxin oxidative damage. It is thought that both DNA adduct-mediated damage by ochratoxin as well as general oxidatative stress are causes of OTA induced carcinogenicity. Aflatoxin appears to have similar mechanisms where-by it induces cancer.
OTA lowers the level of phosphoenolpyruvate carboxykinase, a key enzyme in gluconeogenesis. The toxin also enhances lipid peroxidation both in vivo and in vitro, which is probably responsible for its adverse affects on mitochondrial function. OTA also forms DNA adducts in the kidney, liver and spleen that results in single-strand breaks (OTA, 2002).
How The Body Transforms And Removes The Mycotoxin Ochratoxin
Ochratoxin A is partially absorbed from the gastrointestinal tract in monogastrics mammels. Consequently ochratoxin A has been found in edible tissues and products of monogastric animals, particularly pork products in Europe (Krogh et al., 1974). In ruminants, ochratoxin A is mainly metabolised by the rumen microbiota to ochratoxin α before absorption. This major metabolite appears less toxic than ochratoxin A (Creppy et al., 1983). Ochratoxin A is excreted in the urine and feces. How much OTA is excreted by these different routes in various species is influenced by the extent of enterohepatic recirculation of ochratoxinA and its binding to serum macromolecules. Elimination of OTA in urine and feces is felt to be relatively slow and has been shown to vary by species and sex, as well as specific genotype that may affect the biotransformation of OTA.
Ochratoxin A is only slightly soluble in water and is absorbed in the upper sections of the gastrointestinal tract in a passive manner in the non-ionized form, and is subjected to secretion and reabsorption via enterohepatic recycling (Leeson et al., 1995). Absorption is faster where pH is low. In mammals, ochratoxin A is absorbed primarily in the stomach and proximal jejunum, although absorption through the lungs into the systemic circulation has also been documented. Urine is the main excretion route for both OTA and OTα, which is a metabolic degradation product of OTA. The amount excreted in the urine is thought to be dependent of the free OTA concerntration. Since OTA binds to serum albumin, many other things that bind to albumin could change how much free OTA is available to be excreted. Anything affecting serum albumin would also affect the amount of OTA free to be excreted after ingestion. This may be why some studies find the ingested amount of OTA is not related to the amount that is found in the urine. The efficient binding of OTA to the serum albumin decreases its elimination by limiting its transfer from the blood stream to the liver and kidney cells. It serves as a mobile reserve of mycotoxin that can be available for release to the tissues for a long time. Men tend to have 27% more positive urine samples than women when groups of people are studied. Women tend to have high serum albumin levels, although they decrease and come closer to men's levels at age 60.
Wide species differences in the serum half life of OTA have been reported. After oral administration in the monkey (Macaca mulata), 510 hr (Hagelberg et al., 1989), in the pig, 72-120 hours (Galtieret al., 1981; Mortensen et al., 1983a), in the pre-ruminant calf 77 hours (Sreemannarayana et al., 1988), in rats 55-120 hours (Galtier et al., 1979; Ballinger et al., 1986; Hagelberg et al., 1989), in mice 24-39 hours (Fukui et al., 1981), in quail 6.7 hours (Hagelberg et al., 1989) and in chickens 4.1 hour (Galtier et al., 1981).
The disappearance rate of OTA from blood was slower than from kidney, liver and other tissues in the pig (Hult et al., 1979).
Little data is available on the metabolic disposition of OTA in humans. Some researchers believe OTA in humans may have a long serum half life, based on the strong binding of OTA to human serum macromolecules.
Ochratoxin is also excreted in human milk. Yes, this means moms exposed to OTA will expose their breast fed infants. The amount in milk is less than that found in blood though. Sudies report as much as ten times less.
Ruminant animals are not as sensitive to ochratoxin A as are non-ruminants. In fact only 2-6.5% of the toxin appears to be absorbed into the animals system. The highest measured amount has been 10%. This protection appears to be due to rumen microflora (good gut bug) and some studies show largely due to protozoan content of their rumen. It also appears that the protozoans will decrease in number if the ruminant is fed grains. When I read the research on protozoans assisting in ochratoxin removal from the ruminants, I immediately started wondering about the role of blastocystis which is now being called a commensual. Is it possible that people who are eating ochratoxin contaminated food on a regular basis, (too many of these folks) and have blastocystis in their intestine might be getting assistance from the blastocystis in removing the ochratoxin?
For those of you who want to see the science behind this, here it is: In ruminant animals such as cows, OTA goes through hydrolysis to a non-toxic ochratoxin alpha that takes place in their digestive system in the presence of the ruminant protozoa. This appears to remove the negative effects of the toxin. In one study aflatoxin A and DON were not degraded by rumen microorganisms, but ochratoxin A was cleaved mostly into ochratoxin alpha and phenylalanine. (Ochratoxin alpha has been shown to be non harmful to pig kidneys.)
The next thing that was identified was that feeding concentrates (largely grains) to ruminants could decrease this protective feature and increase the amount of absorbed mycotoxin. Feeding sheep grains along with their hay decreased the ability to degrade the ochratoxin A by 20%. There are two possible things that might be happening here. One is that the grains may have a lot of ochratoxin on them or the other is that the grains may make the rumen less hospitable for the protozoans. (When I use to have organic dairy goats, all of us raising goats organically knew that grains were not healthy to feed the animal and we kept them to a minimum. It is kind of like feeding candy to kids.) So, in comes another study which found feeding sheep grains decreased the protozoan in the rumen and caused an increase in OTA that was not degraded and ended up being absorbed by the sheep where it was detected in their serum.
Excretion is both biliary into the feces as well as through the kidney in the urine. This is not well studied but has been found to change with species. It appears with the current data that much more is excreted via the urine.
Removal By Conjugation
There is known formation of OTA-glutathione conjugates, as well as the occurrence of N-acetyl-l-cysteine (NAC) conjugates as the most probable renal excretion products. Besides glutathione conjugates, other phase 2 metabolites of OTA such as glucuronides have only been detected by indirect methods, so glucuronidation may be partly reasonable for biotransformation of OTA. Their occurrence in human urine has been assumed based on an increasing OTA concentration in urine after enzymatic incubation with β-glucuronidase/arylsulfatase (Muñoz et al. 2017; Duarte et al. 2011). The formation of three OTA-glucuronides after incubation of OTA with rat microsomes has also been proposed based on mass spectrometric data.
Testing For Ochratoxin Exposure
Ochratoxin can be measured in blood serum, in urine and in human kidneys (post-mortem of course). OTA in blood serum is a useful biomarker of OTA exposure due to its high-affinity binding to serum albumin, or to other small proteins, which should result in higher serum OTA levels and long persistence of OTA in blood serum. Indeed, the OTA blood levels give a better idea of exposure over longer periods of time. Urine biomarkers are more relevant for day to day exposure. OTA derivatives have also been measured in the blood and urine.
In one study, The correlation between the blood plasma ochratoxin A levels and ochratoxin A consumption was not significant (95% confidence limit). However, a significant correlation was found between ochratoxin A consumption and the urine ochratoxin A concentration expressed as the total amount excreted.
Testing for ochratoxin in the urine can be obtained at these labs.
Urine testing should be 24 hour testing as the amount of toxin can change through-out the day.
PKU - Ochratoxin A Inhibits Phenylalanyl-tRNA Synthetase
The variation of the gene frequency for phenylketonuria (PKU) between races and geographical areas suggests that some regional environmental conditions may confer a selective advantage to the heterozygous state, as is the case with sickle cell anemia and glucose-6-phosphate dehydrogenase deficiency in regions with endemic malaria. Researchers have proposed that the heterozygote advantage in PKU consists in protection from the fungal toxin ochratoxin A, which is produced by some Aspergillus molds that cause food to rot.
Ochratoxin A competitively inhibits the coupling of phenylalanine to its cognate tRNA by the corresponding aminoacyl transferase and thereby disrupts protein synthesis. It is more toxic to fetuses than to adults. This appears to be due to the fetus having low levels of the enzymes that inactivate xenobiotics and toxins such as ochratoxin. Mothers who are heterozygous for PKU will have a somewhat higher level of phenylalanine, which will be shared with the fetus via the placenta. This will counter the inhibition of tRNA aminoacylation in the fetus and thereby afford it some measure of protection.
PKU is quite high in Ireland. This country has had repetative episodes of severe famine. Starving people will tend to eat rotten food rather than discard it. In Irish women, lower rates of abortion were found in those who were heterozygous for PKU.
Deotoxification of Ochratoxins With Biotransformation And Binders
Biotransformation of Ochratoxins
Glucurondiation - At the cellular level, endogenous glucuronic acid can be conjugated (attached) to the ochratoxin (OTA) phenolic group, or carboxylic group under the catalytic reaction of uridine-diphosphate glucuronosyltransferase (UGT). OTA-glucuronide conjugates have been detected in liver (8%–17%) and intestinal tissue (6%). The presence of glucuronide conjugates were also reported in the bile of pigs that were fed OTA contaminated feed. However, in all these reports, OTA-glucuronides were determined indirectly by using β-glucuronidase hydrolysis. No direct or definite evidence for the formation of OTA-glucuronides as well as complete chemical configurations are available.
The degradation of OTA to OTα (7-carboxy-5-chloro-8-hydroxy-3,4-dihydro-3-R-methylisocoumarin) is the most important mechanism of OTA biodegradation. Degradation products OTα and L-β-phenylalanine are formed by the hydrolysis of the amide bond via hydrolytic enzymes, such as carboxypeptidase A. OTα is considered much less toxic than OTA. One researcher reports it is 500 times less toxic. Some strains of bacteria are known to degrade OTA into OTα. Here are a few of them that have been studied.
Bacteria that change OTA to OTα
- Cupriavidus basilensis
- Brevibacterium spp. strains (B. casei DSM 20657, DSM 9657, DSM 20658, RM101; B. linens DSM 20425; B. iodinum DSM20626; B. epidermidis DSM 20660) The strain, B. casei RM101, could completely cleave OTA even at 40 μg/mL, which is 1,000 times greater than the OTA concentration commonly found in foodstuffs
- Aspergillus niger GX312
- A. japonicus AX35
- A. carbonarius SA332 (a weak OTA producer)
- Bacillus subtilis CW 14 could degrade 97.6% of OTA (6 μg/mL) within 24 h, but no degradation product was detected.Furthermore, 66.6 and 87.9% of OTA (6 μg/mL) was adsorbed by viable and dead B. subtilis CW 14 cells, respectively
Personally, the idea of giving someone A. niger or other fungi that I perceive as being problematic bothers me. I feel safer with Brevibacterium spp.
Our Normal Gut Flora: Research has shown that all 29 strains of Lactobacillus and Lactococcus bacteria reduce OTA content in growth media. The greatest toxin elimination ability has been found for the enteric bacteria Lactobacillus rhamnosus and L. acidophilus as well as for the plant-associated bacteria, such as L. plantarum, L. brevis, and L. sanfranciscensi. This means that any food that is fermented such as home made sour-dough bread or sour vegetables or fruit will likely be less toxic even if ochratoxin may be on the food material. It is thought that these bacteria may also be beneficial to use as a probiotic to remove ochratoxin in the gut from food or during enterohepatic circulation of ochratoxin with the bile. It appears that besides the biotransformation of the mycotoxin there is also a binding affinity of the bacteria cell walls to the ochratoxin. We don't know enough about the binding ability or the biotransformative ability in the bodies of humans or animals at this time. Fermented foods can be really useful to people with CIRS or "mold" issues, but for those that have histamine issues, they need to be aware that some bacteria will increase histamine levels in the gut while others will lower it. A product called Probiota Histaminx was made to specifically lower histamine in the gut and is useful to these people.
Enzymes are being studied, both natural and synthetic in their ability to biotransform and biodetoxify OTA. Bovine pancreatic carboxypetidase A has been used to cleave OTA to OTα (Pitout, 1969). A crude enzyme isolated from A. niger called ANcex was able to degrade 99.8% of OTA (1 μg/mL) to OTα after 25 h incubation at pH 7.5 and 37°C. Abrunhosa et al. (2006) Commercially purified enzymes have not worked as well. Purified protease A and Pancreatin degraded 87.3 and 43.4% of OTA after 25 h incubation at pH 7.5 and 37°C, respectively.
Binders To Remove Ochratoxins
As is found with some mycotoxins, many binders work in a petri dish, but inside an animal or human the results are not so positive. Bentonite and charcoal do not look like they have been very helpful in animal studies so far.
Cholestyramine does have research in rats that shows it reduced plasma levels of the toxin and prevented ochratoxin-induced kidney toxicity. A different rat study showed that OTA exposed rats that were fed a diet with cholestyramine experienced decreased OTA concentration in plasma as well as decreased excretion of OTA and its metabolites (ochratoxin alpha and hydroxylated ochratoxin A) in bile and urine. This was associated with an increased excretion of OTA in feces which was felt to reduce the potential nephrotoxicity of OTA. It seems to currently be the best binder of choice to reduce enterohepatic recirculation of OTA. The cholestyramine will reduce levels that are filtered through the kidneys and shifting excretion to the stool where it is presumably bound to cholestyramine resin. Cholestyramine is not absorbed systemically allowing it to be safe even for those with advanced kidney disease. Many patients tolerate the pure resin better than the commercial prescription prepared with sugar, artificial colors, and additives. All binders can slow down the digestive tract, so make sure the digestion is supported as needed. If the person starts to get constipated or gets reflux, immediately support them or they will need to stop the chlolestyramine. All binders can do this.
Using Bacteria or their cell walls: The most important influence factor of OTA adsorption capacity of microorganisms is cell wall components. However, there are controversies among different scholars on specific cell wall components, such as glucogalactans and β-glucans (Ben Taheur et al., 2017), mannoproteins (Caridi et al., 2012), β-glucans and mannans (Pereyra et al., 2015).
Saccharomyces cerevisiae Syrena LOCK 0201 and S. cerevisiae Malaga LOCK 0173 removed 85.1 and 82.8% of OTA (1 μg/mL) in white grape juice as well as 65.2 and 10.7% of OTA (1 μg/mL) in blackcurrant juice after 10 days incubation, respectively. Saccharmocyes boulardii (closely related to cerevisiae) has shown to be helpful in ochratoxicosis in chickens.
The OTA binding ability of S. cerevisiae RC008 and RC009 was significantly stronger in simulated mammalian gastrointestinal conditions than that in YPD broth (pH 7), while the OTA binding ability of S. cerevisiae RC012 and RC016 was not significantly different between simulated mammalian gastrointestinal conditions and YPD broth (pH 7). This appears to show that the normal gastrointestinal conditions would enhance adsorption ability of S. cerevisiae to OTA. Both viable and dead S. cervisiae Lalvin rone 2056 has been shown to be able to remove OTA. Bejaoui et al. (2004) demonstrated that viable and dead S. cerevisiae LALVIN Rhône 2056 was able to remove 17 and 75% of OTA (2 μg/mL) within 2 h in liquid yeast peptone glucose (YPG) medium, respectively.
A study on two commercial yeast cell walls examined the different content of polysaccharides and β-glucans/mannans. The results showed the OTA adsorption ability was not significantly influenced by different percentages of polysaccharides or β-glucans/mannans (Pereyra et al., 2015).
Dead Lactobacillus plantarum LOCK 0862, L. brevis LOCK 0845, and L. sanfranciscensis LOCK 0866 could reduce 46.29–59.82% of OTA (1 μg/mL) within 30 min in PBS buffer, but the alive L. plantarum LOCK 0862, L. brevis LOCK 0845, and L. sanfranciscensis LOCK 0866 needed 24 h to remove 14.80–26.42% of OTA (1 μg/mL) in PBS buffer. Lactobacillius plantarum is found in sauerkraut.
I would point out that this binding is often partially reversible and is not studied much.
Souring Grain & Other Foods - Some bacteria are being studied for use in food and feed industries. I laughed as I read about the various bacterias being studied as additives into grain flours as it sounds like they are recreating the method that many of us already use for our grains and other foods when we use the souring/fermentation process. After spending a lot of money on research they may simply prove the old methods of souring grains are a necessity for good health. Their approach is a little different. Rather than allowing the grains to naturally sour, they add dried bacteria to the dry grain and feed it to animals to allow it to take affect in their intestinal tract. Companies are trying to come up with strains of bacteria they can sell as an additive to human food or animal feed. They are also using some different methods with yeast to remove OTA from liquids such as grape juice and wine. Some of the bacteria and yeast they use have come from sourdough, fermented flour, sausage, and wine. Others have come from pig feces, and soil. They are also isolating enzymes made by these yeast and bacteria to reproduce and sell.
Protein - Due to the high affinity of ochratoxin A for albumin, some studies have increased dietary protein to decrease the damaging effects of ochratoxin and found in broilers the damage decreased at levels of 22-26% protein in the diet.
Sweating - Ochratoxin A has been shown to be found in the sweat in one study, so supporting sweating through saunas or other means should increase excretion through sweat of OTA. Sweating is a common method used for removal of all mycotoxins.
Antioxidants, have been shown to decrease OTA toxicity in several species of animals. Melatonin exhibits a preventive effect against OTA-induced oxidative stress, and structural damage in the kidney through its role in the scavenging of free radicals and/or the prevention of lipid peroxidation. Alpha-tocopherol (vitamin E) in the diet decreased by 58% the total DNA adduct provoked in kidney by a single administration of OTA in mouse and rat kidney. Both Artichoke extract and sesame seeds given to laying hens in their diet showed protection agains decreased egg production and toxic effect on various ogans due to ochratoxin.
Vitamin C has been shown to be beneficial in laying hens exposed to ochratoxin.
Vitamin E, but not vitamin C was found to be beneficial in male leghorn chickens.
Vitamin A, C and E as pretreatment before OTA administration in rats show significant decreased number of DNA adducts formed in the kidney by 70% for vitamin A, 90% for Vitamin C and 80% for Vitamin E.
Selenium has shown promise in protection from toxicity by OTA to pig kidney epithelial cells. In vivo research is necessary. Additionally, selenium was shown to block the increases of DNMT1, DNMT3a and HDAC1 mRNA and protein expression, reversed the decreases of glutathione peroxidase 1 (GPx1) mRNA and protein expression, and promoted the increases of SOCS3 mRNA and protein expression induced by OTA.
Lycopene in a rat study proteted rats from OTA toxcity. Male Sprague-Dawley rats (<200 g, n=6) were treated with OTA (0.5 mg/kg/day) and/or lycopene (5 mg/kg/day) by gavage for 14 days. Lycopene supplementation with OTA increased GPx1 activity and GSH levels, and decreased apoptotic cell death in both cortex and medulla vs. control. Lycopene is found in red, pink and orange colored foods, including tomatoes, watermelon, pink grapefruit, apsaragus, red cabbage, guava, papapya, red bell pepper, persimmon, and mango.
Green Tea - Camellia sinensis catechins, epigallocatechin gallate (EGCG) and epicatechin gallate (ECG) are antioxidants, and have been shown in vitro to be protective against OCTA. This means they would be helpful in the digestive tract, but to know if they would be helpful systemically, we need invivo testing. The likelihood is yes as they have been shown to be useful as an antioxidant to protect animals and people from other mycotoxins.
The cyanidin 3-O-β-D-glucoside (C3G), an anthocyanin found in beans, fruits, vegetables and red wine, might counteract damage induced by chronic exposure to OTA in rats. The rats had significant reduction in damage from OTA in this study. This study confirmed that the effects of OTA are mediated by oxidative stress and demonstrated that C3G efficiently counteracted the deleterious effects of OTA because of its antioxidant effect.
Licorice - Glycyrrhiza glabra has strong antioxidant activity and has shown a protective effect from OTA induced kidney toxicity. A study with rats given OTA showed damage such as testicular degeneration, seminiferous tubule atrophy, dissociation of germinative epithelium, vasodilatation with vascular thrombosis, perivascular immune cell infiltration, hypertrophied leydic cells, giant cell formation, and negative tubular differentiation index (TDI) . A Licorice extract showed a protective effect from these deleterious changes in the rats exposed to OTA for 28 days. Licorice alleviated most of the biochemical abnormalities associated with the exposure. The same study also looked at Melatonin. (see below)
Melatonin demonstrated a protective effect in rats exposed to OTA for 28 days and alleviated most of the biochemical abnormalities associated with the exposure. In this study Melatonin exerted a protective effect from testicular degeneration, seminiferous tubule atrophy, dissociation of germinative epithelium, vasodilatation with vascular thrombosis, perivascular immune cell infiltration, hypertrophied leydic cells, giant cell formation, and negative tubular differentiation index (TDI) that was induced in the rats given only OTA without melatonin or Licorice as a protective feature .
Green Coffee Powder was shown to be protective of albino wistar rats. Rats of Group 1 designated Vehicle Control (only water), Group2 (10 mg/kg Ochratoxin A); Group 3 designated Low dose (2000 mg/kg Coffee+10 mg/kg Ochratoxin A); Group 4 designated High dose (4000 mg/kg Coffee+10 mg/kg Ochratoxin A); Group 5 designated Coffee Control (1000 mg/kg Coffee) and orally administered with the above test materials repeatedly every day for 28 days. Treatment of Ochratoxin A alone (group 2 rats) significantly increased malondialdehyde content, catalase, and glutathione reductase activities with a decrease in the activity of superoxide dismutase enzyme and reduced glutathione level in brain, kidney and liver. Whereas, low dose coffee (group 3) and high dose coffee (group 4) rats showed dose-dependent increase in antioxidant and less histopathological alterations. Concomitant treatment of Yemeni green coffee powder and Ochratoxin A brought dose-dependent protective effects against oxidative stress which was induced using Ochratoxin A in rats and after death examining liver, brain, and kidney tissues of female rats.
Catalase and Super Oxide Dismutase (SOD) demonstrated protective effect in rats exposed to OTA. Catalase and SOD (20 mg/kg body weight each) were given to rats by subcutaneous injection, every 48 h, 1 h before gavage by OTA (289 micrograms/kg b.w. every 48 h), for 3 weeks. SOD and catalase prevented most of the nephrotoxic effects induced by ochratoxin A, observed as increased enzymuria, proteinuria, creatinemia and increased urinary excretion of OTA.
N-acetyl Cysteine has been shown to combine directly with OTA or metabolites and is removed in the urine.
Resveratrol is used by grapes to control Aspergillus carbonarius growth and ochratoxin A biosynthesis, and has also been used in research to attenuate damage from OTA.
Herbal Research Shown To Be Beneficial
Three herbs, Silybum marianim - Milk thistle, Withania somnifera - Ashwaghanda, and Centella asiatica - Gotu kola were all shown to protect young chicks from ochratoxin toxicty by protecting them from the immunosuppressive effect. TheMilk thistle and Ashwagandha had a hepatoprotective effect and the Milk thistle had a kidney protective effect.
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