Nivalenol
Nivalenol is a mycotoxin of the trichothecene group. In nature it is mainly found in fungi of the Fusarium species. The Fusarium species belongs to the most prevalent mycotoxin producing fungi in the temperate regions of the northern hemisphere, therefore making them a considerable risk for the food crop production industry.
The fungi are abundant in various agricultural products and their further processed products. "The Fusarium species invade and grow on crops, and may produce nivalenol under moist and cool conditions".
In pigs, the symptoms observed after nivalenol exposure are "feed refusal, vomiting, gastroenteric and dermal irritation or necrosis and immunological dysfunction", as well as haematotoxicity, resulting in a low leukocyte count.
History
In the period of 1946 to 1963, several cases of intoxication due to the ingestion of Fusarium infected grains were reported in Japan, Korea and India. There have been no reports of lethal cases and only mild symptoms like nausea, vomiting, diarrhea and abdominal pain. In these incidents F. graminaerum could be isolated, which hints at a nivalenol or deoxynivalenol contamination.In the same period two outbreaks involving over 100 cases were reported in India and China. These outbreaks were also non-lethal.
A well documented and acute outbreak in India in 1987 affected around 50,000 thousand people. Several Fusarium toxins under which nivalenol, deoxynivalenol and acetyldeoxynivalenol were found in rain-damaged wheat used for bread production. There were again no lethal cases and reported symptoms were abdominal pain, diarrhea, bloody stool and vomiting. These cases show that the main emerging danger of nivalenol comes from Fusarium infected cereals and is mainly via the route of digestion of uncontrolled wheat or other grains that are further processed or does enter the food chain via another route.
Weaponization and other instances of nivalenol poisoning
Nivalenol as well as deoxynivalenol and T-2 toxin have been used as biological warfare agents in Laos and Cambodia as well as in Afghanistan. The Soviet Union has been alleged to have provided the mycotoxins and to have used them themselves in Afghanistan. All three compounds could be identified in the vegetation at affected sites, whereas T-2 toxin could also be found in urine and blood samples of victims.The best documented use of trichothecenes in warfare is the yellow rain controversy, a number of attacks in Southeastern Asia, Laos and Afghanistan which used a "yellow rain" as described by witnesses. The toxins were delivered as a cloud of yellow dust or droplets. An article by L. R. Ember published in 1984 in Chemical Engineering News describes the use of trichothecene mycotoxins as biological weapons in Southeast Asia in a very detailed manner, covering reports of survivors, eyewitnesses, prisoners of war and Soviet informants along with information on the presence of Soviet technicians and laboratories. This led to the conclusion that these toxins have been used in Southeast Asia and Afghanistan. The Russian government however refuses to give a statement on these pieces of evidence. Furthermore, it has been shown that samples taken on the location of attacks contain these toxins, while sites that have not been attacked do not show any signs of toxins in them.
Even though it remains questionable if all witness reports are reliable sources of evidence, the symptoms recorded are typical for intoxication with trichothecenes.
There was a number of ways in which trichothecenes were weaponized, such as dispersion as aerosol, smoke, droplets or dust from aircraft, missiles, handheld devices or artillery.
Safety guidelines in the food industry
In 2000 a scientific opinion on nivalenol was issued by the Scientific Committee on Food. A temporary tolerable daily intake of 0–0.7 μg/kg bw per day was issued after evaluation of the general toxicity as well as the haematoxicity and the immunotoxicity. This t-TDI was reaffirmed by the SCF in 2002.In 2010, the Japanese Food Safety Commission issued a t-TDI of 0.4 μg/kg bw per day.
Between 2001 and 2011, the European Food Safety Authority collected data from 15774 nivalenol occurrences in 18 European countries to be assessed. This led to the establishment of a TDI of 1.2 μg/kg bw per day. Nivalenol was in this studies not found to be genotoxic, but well haematotoxic and immunotoxic.
Structure and reactivity
Nivalenol as part of the family of mycotoxins has the common structure which all members of this toxin family have. This includes the basic structure of a cyclohexene and a tetrahydropyran ring connected at C6 and C11. Additionally an ethyl-group connects the tetrahydropyran at C2 and C5 and a keto group is attached at the cyclohexene at C8. The epoxide group, responsible for the reactivity for most parts, is attached at C12 and C13 in the tetrahydropyran. Only the remaining groups at positions C3, C4, C7, C15 vary for the different mycotoxins. In case of nivalenol each of the four remaining groups is a substituted hydroxyl group which add up to the reactivity in presence of hydrophilic compounds or subgroups respectively thanks to their polar characteristics. In acidic medium the keto group is capable of reacting with a proton promoting polarity and reactivity as well. But altogether the epoxide group is crucial for the reactivity of the molecule.Available forms
Nivalenol, deoxynivalenol and T2-toxin are the three structural and similar synthesized mycotoxins naturally appearing in fungi.Synthesis
The synthesis of nivalenol is a 16 step process. It can differ in step 11 to step 14 depending on the order in which the reaction controlling trichodiene synthases TRI1, TRI13 and TRI7 are catalyzing. Farnesyl pyrophosphate is used as starting compound for the synthesis of nivalenol. Its cyclization reaction to trichodiene is catalyzed by terpene cyclase trichodiene synthase. This reaction is followed by several oxidation reactions catalyzed by cytochrome P450 monooxygenase. Thereby hydroxyl groups were substituted to the carbon atoms C2, C3 and C11 and one oxygen was added to C12 and C13 facilitating the formation of an epoxide group. This results in the intermediate isotrichotriol.In a further reaction trichotriol was gained through a shift of the C11 hydroxyl group of the isotrichotriol to the C9, similar the double bond shifted from C9=C10 to C10=C11. Trichotriol reacts in a non-enzymatic cyclization reaction to its isomere isotrichodermol. In the reaction the hydroxyl group on the C2 of the cyclopentane binds to the C11 of the cyclohexene forming a tetrahydropyran ring. The shifted OH-group at C9 is lost during the reaction. An acetyltransferase catalyzes the acetylation of the C3 OH-group of isotrichodermol forming isotrichdermin.
Isotrichodermin is converted to 15-decalonecitrin due to a substitution of one hydrogen by one hydroxyl at C15 which is then acetylated under help of TRI3. The same substitution and following acetylation reactions occur at C4 again under the control of TRI13 and TRI7. TRI1 in F. sporotrichiodies further catalyzes the addition of a fourth OH-group at C8 and a fifth OH-group at C7 at which then the hydrogen is eliminated and a keto group forms.
In a last step an esterase controlled by TRI8 catalyzes the deacetylation at C3, C4 and C15 resulting in the end product nivalenol. A partly alternative synthesis can occur when the catalysts TRI1 and TRI13, TRI7 are used in opposite order. Then the addition of the hydroxyl groups at C7 and C8 controlled by TRI1 are happening with calonectrin as reactant. In this reaction 7,8-dihydroxycalonectrin is formed. It further reacts spontaneously to 3,15-acetyl-deoxynivalenol via elimination of a hydrogen and formation of a keto-group at C8. The addition of a hydroxyl group at C4 controlled by TRI13 occurs and is acetylated under the help of TRI7. This yields 3,4,15-triacetylnivalenol from which it is than again the same synthesis as described above.
Mechanism of action
Nivalenol causes a change in a number of different biological pathways. The most well known and probably important, is the NF-κB pathway. NF-κB is a transcription factor that can be found in almost all human cells, and regulates the expression of its target genes by binding to specific motifs on the genomic DNA on regulatory elements. In vitro tests have shown, that nivalenol can change the expression of cytokines, which are important controller molecules of the immune system. Nivalenol induced the secretion of IL-8, a mediator of inflammation. When treated with an NF-κB inhibitor, IL-8 secretion was lowered. Another important factor influenced by nivalenol is MCP-1/CCL2, this cytokine plays a role in the mobility regulation of mononuclear leukocyte cells. Nivalenol causes CCL2 secretion to be lowered, and thus the mobility of monocytes to be reduced. This explains part of the immunosuppressive nature of nivalenol. Again, this effect is reduced by NF-κB inhibition which shows, that nivalenol and NF-κB interact to influence the cell.It was shown that while deoxynivalenol induces the secretion of chemokines, which are also immunorelevant messenger molecules, nivalenol does inhibit their secretion. Nivalenol also upregulates the expression of proinflammatory genes in macrophages, displaying a mixed effect on different cell types. It does so even at cytotoxic levels.
Another mechanism of cytotoxicity of nivalenol is the apoptotic cytotoxicity showing that nivalenol is more toxic than its often co-occurring mycotoxin partner deoxynivalenol, and does so by causing DNA damage and apoptosis. Nivalenol is also known to influence human leukocyte proliferation. It has been shown that nivalenol can change proliferation rates of human leukocytes in a dose dependent manner. Lower concentrations are known to enhance leukocyte proliferation, while higher concentrations decrease proliferation in a dose dependent manner.