Ochratoxin

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What Are Ochratoxins

Ochratoxin A structure

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Ochratoxin Ochratoxins occur in nature as Ochratoxin A (OTA), B (OTB), and C (OTC). Ochratoxin A is the most prevalent toxin and the most toxic followed by OTB and OTC. Ochratoxin A, B and C are produced by Penicillium and Aspergillus species.

Ochratoxins are described as consisting of a para-chlorophenolic moiety containing a dihydroiso-coumarin group that is amide-linked to L–phenylalanine.

Ochratoxin B (OTB), a secondary metabolite of Aspergillus ochraceus, is the nonchlorinated analogue of the mycotoxin ochratoxin A (OTA), which is one of the most potent kidney carcinogens in rodents. Despite the closely related structure, OTB is considered to be of much lower toxicity. OTA is poorly metabolized and slowly eliminated, and this may play an important role in OTA toxicity, carcinogenicity, and organ specificity.

OTA and OTB have a similar potential to induce cytotoxicity in vitro, but large differences in their potential to induce nephrotoxicity in rodents. OTB is more extensively metabolized and more rapidly eliminated than OTA. The lack of specific retention of OTB in the kidneys and the differences in toxicokinetics may therefore provide an explanation for the lower toxicity of OTB. Biotransformation and nephrotoxicity of ochratoxin B in rats. PMID: 15963343 [PubMed - indexed for MEDLINE]

Ochratoxin A is produced by Aspergillus ochraceus,  research shows oxidative stress may be a major mechanism for the toxicity of OTA - Ochratoxin A (OTA) is a mycotoxin produced by species of Aspergillus and Penicillium 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 [1]. Toxicity from ochratoxin is considered serious enough that it is among approximately 20 mycotoxins monitored in food.

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 sxs such as seizures, decreased physical reactions, movements and speech, intellectual disability and loss of coordination. Some people with sevre deficiency experience recurrent bacterial infections.

https://www.ncbi.nlm.nih.gov/medgen/97988

Ochratoxin A (OTA), first discovered in 1965 in Aspergillus ochraceus has been subsequently reported in several Aspergillus and Penicillium species.

Aspergillus niger, Aspergillus, and species of Penicillium, Petromyces, and Neopetromyces. OTA is a nephrotoxic, genotoxic, immunotoxic, and  neurotoxic mycotoxin which is a known carcinogen in animals and a class 2B, possible human carcinogen. Associations have been found with human kidney disease including Balkan endemic nephropathy and focal segmental glomerulosclerosis.

Ochratoxin A is preferentially distributed in liver, kidney, muscle and fat. Pharmacokinetics of ochratoxin A in animals. PMID: 1820333 [PubMed - indexed for MEDLINE]

 

 

Where Are Ochratoxins Found

Exposure to ochratoxins is largely through food contamination and the most common food to be contaminated is grains. Skin contact and inhalation are known to also cause toxicity. Inhalation is usually 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 found in environments where farmers or processers are working with grains. There are also ochratoxins that have been associated with water-damaged buildings.

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

Although Aspergillus is found more commonly in conditions of high humidity and temperature, Penicillium species are a source of ochratoxin in cool temperate regions.

Ochratoxin (OTA) is an other mycotoxin produced by Aspergillus. It is also produced by Penicillium. It is present in a large variety of foods because it is produced by several fungal strains of the Penicillium and Aspergillus species. It is most commonly found in wheat, corn and oats. Ochratoxin A is the most economically important form of ochratoxin; ochratoxins B and C are less toxic and less common. 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.  OTA occurs in olives, beans, 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, raisens, 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.

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 produciton (Magan and Aldred, 2007).

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, curcuma, 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.

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.

Health Effects of Ochratoxins

Kidney toxicity

Liver toxicity

Immunosuppressive

Carcinogen

Teratogen

The fact that OTA is such a potent mycotoxin relies on its ability to disturb cellular physiology in multiple ways, particularly byinhibiting 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

OTA is nephrotoxic, hepatotoxic, embryotoxic, teratogenic, neurotoxic, immunotoxic, genotoxic, and carcinogenic in many species with species and sex-related differences Ochratoxin A (OTA) is a known nephrotoxic, immunotoxic, and carcinogenic mycotoxin in animals [1, 2] that has long been studied for its role in animal and human disease. Molds associated with the production of OTA include Aspergillus ochraceus, Aspergillus niger and Aspergillus carbonarius, Penicillium verrucosum, and species of Penicillium, Petromyces, and Neopetromyces [3]. 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 [1]. Toxicity from ochratoxin is considered serious enough that it is among approximately 20 mycotoxins monitored in food.

In the blood, OTA binds to albumin and the bound fraction constitutes a mobile reserve of OTA [6]. 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 [7]. 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.

 

Ochratoxin has been found in liver, muscle, fat, skin, gastric mucosa, the heart muscles bone marrow, as well as the adrenal medulla and cortex in animals. Ochratoxin is considered toxic to the kidneys, immune system, damages the growing fetus and is a possible carcinogen. 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.

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.

Oxidative stress may be a possible key facctor in the toxicity and genotoxicity of OTA. Oxidative DNA damage has been seen in vitro with OTA adn in the kidney and liver of rats. OTA was shown to increase the formation of oxidative products of lipds, with increased production of malondialdehyde, in creased reactive oxygen species levels as well as 8-oxoguanine formation and deplted cellular glutathione levels. A study showed OTA could be decreased by maitining glutathione production through pretreating the in vitro cell with the glutathione rater 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 glutahione may be protective in ochratoxin damage. It is thought that both DNA adduct-mediated damage by ochratoxin as well as 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).

OTA has been detected in human blood and milk.

Acute Toxicity

The estimated tolerable dosage in humans was estimated at 0.2 to 4.2 ng/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).

Chronic Toxicity

Balkan endemic nephropathy (BEN) is a chronic disease associated with ochratoxin exposure. It is a chronic interstitial nephropathy that initially has 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 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.

Excretion Route

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 availble 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. men tend to have 27% more positive urine samples than women when groups of people are studied.

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 has been 10%. This protection appears to be largely due to protozoan content of their rumen. It also appears that the protozoans will decrease in number if the ruminant is fed grains. My brain is wired oddly, so when I read the research on protozoans assisting in ochratoxin removal from the ruminants, I immediately started wondering about blastocystis which is now being called a commensual. Is it possible that people who are eating ochratoxin conatminated 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 dand phenylalanine. (Ochratoxin alpha has been shown to be non harmful to pig kidneys) more than 90% of ZEA was degrade to zearalenol and about twice as much alpha zea (has 3-4 times more estrogenic activity as does the parent OTA compound although it may be easier to remove) and beta zea was formed. DAS was quickly and completely deacetylated to monoacetoxyscirpenol and T-2 toxin was deacetylated to HT-2 toxin.

Now 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.

 

Preventing Ochratoxins Exposure

The USA does not seem to have limits on ochratoxin expsoure although it does require all corn being exported to be tested for alfatoxin and lists the same amount of allowable ochratoxin A in foods being tested as it does aflatoxin: 5-100 ppb. DON is .5-5 ppm, fumonisin .5-5 ppm and zearalenone 100-1000 ppb.

I found a USA mycotoxin regulation form that lists aflatoxin maximum level at 20 ppb in all foods but milk. However, in other foods there were higher levels allowed, for instance some of the food given to animals was allowed to have as much as 300 ppb in the food. For patulin the maximum level in any food was listed as 50 ppb. Deoxynivalenol was suggested to have below 1000 ppb but it is not regulated and fumonisin was suggested at 2000, 3000 and 4000 ppb depending on the type of food and again was not regulated. Ochratoxin had no mention on the reuglatory sheet. However, if you check the regulations in the European Union,

 

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.

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. the correlation between the 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

Binders and Bile to Remove Ochratoxins

 Antioxidants

Interaction of 2′R-ochratoxin A with Serum Albumins: Binding Site, Effects of Site Markers, Thermodynamics, Species Differences of Albumin-binding, and Influence of Albumin on Its Toxicity in MDCK Cells

Received: 18 July 2018 / Revised: 17 August 2018 / Accepted: 27 August 2018 / Published: 1 September 2018

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Abstract

Ochratoxin A (OTA) is a nephrotoxic mycotoxin. Roasting of OTA-contaminated coffee results in the formation of 2′R-ochratoxin A (2′R-OTA), which appears in the blood of coffee drinkers. Human serum albumin (HSA) binds 2′R-OTA (and OTA) with high affinity; therefore, albumin may influence the tissue uptake and elimination of ochratoxins. We aimed to investigate the binding site of 2′R-OTA (verses OTA) in HSA and the displacing effects of site markers to explore which molecules can interfere with its albumin-binding. Affinity of 2′R-OTA toward albumins from various species (human, bovine, porcine and rat) was tested to evaluate the interspecies differences regarding 2′R-OTA-albumin interaction. Thermodynamic studies were performed to give a deeper insight into the molecular background of the complex formation. Besides fluorescence spectroscopic and modeling studies, effects of HSA, and fetal bovine serum on the cytotoxicity of 2′R-OTA and OTA were tested in MDCK kidney cell line in order to demonstrate the influence of albumin-binding on the cellular uptake of ochratoxins. Site markers displaced more effectively 2′R-OTA than OTA from HSA. Fluorescence and binding constants of 2′R-OTA-albumin and OTA-albumin complexes showed different tendencies. Albumin significantly decreased the cytotoxicity of ochratoxins. 2′R-OTA, even at sub-toxic concentrations, increased the toxic action of OTA. Full article

Low Doses of Ochratoxin-A Decrease IgY and IgA Production in Broiler Chicks

Received: 4 July 2018 / Revised: 31 July 2018 / Accepted: 1 August 2018 / Published: 6 August 2018

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Abstract

The mycotoxin, ochratoxin-A (OTA), produced by some fungi, and is a natural contaminant of many foods and animal feeds worldwide. Due to its toxic effects, the recommended maximum daily intake of OTA for poultry feeds is 0.1 mg OTA/kg (ECR2006/575/EC); this dose does not induce changes in hepatic/renal parameters, but decreases thymus size and serum globulin concentrations. Accordingly, in this study, we assessed quantitatively the total circulating IgY and IgA serum levels, in chicks consuming a 0.1 mg OTA/kg diet (limit) and higher doses (0.3–1.1 mg OTA/kg diet) for 14 or 21 days. We also evaluated other immunological parameters (thymus, bursa of Fabricius, and spleen weights and leukocyte profiles) at day 21. Decreased IgY serum levels were observed in all OTA-treated groups (p < 0.05). In the low-dose group, IgA levels were decreased on day 21, but not on day 14. The size of the thymus and the bursa of Fabricius was decreased in all OTA-treated groups (p < 0.05), whereas reduced spleen size and altered leukocyte profiles were detected only in the high-dose group (p < 0.05). We concluded that chronic exposure to OTA, even at the recommended highest dose, affected IgY and IgA production in chicks. Full article

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