Nivalenol (NIV) is a

mycotoxin A mycotoxin (from the Greek μύκης , "fungus" and τοξίνη , "toxin") is a toxic secondary metabolite produced by organisms of kingdom Fungi and is capable of causing disease and death in both humans and other animals. The term 'mycotoxin' ...

of the

trichothecene The trichothecenes are a large family of chemically related mycotoxins. They are produced by various species of ''Fusarium'', ''Myrothecium'', ''Trichoderma''/''Podostroma'', '' Trichothecium'', ''Cephalosporium'', '' Verticimonosporium'', and ''S ...

group. In nature it is mainly found in fungi of the ''

Fusarium ''Fusarium'' is a large genus of filamentous fungi, part of a group often referred to as hyphomycetes, widely distributed in soil and associated with plants. Most species are harmless saprobes, and are relatively abundant members of the soil mi ...

'' species. The ''

'' species belongs to the most prevalent

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 (cereal crops) and their further processed products (malt, beer and bread). "The ''

'' species invade and grow on crops, and may produce nivalenol under moist and cool conditions". The symptoms observed after being affected with nivalenol 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 1946-1963 several cases of intoxication due to the ingestion of ''

'' infected grains (Scrabby grain disease) were reported in Japan, Korea and India. There have been no reports of lethal cases and only mild symptoms like

nausea Nausea is a diffuse sensation of unease and discomfort, sometimes perceived as an urge to vomit. While not painful, it can be a debilitating symptom if prolonged and has been described as placing discomfort on the chest, abdomen, or back of the ...

, vomiting,

diarrhea Diarrhea, also spelled diarrhoea, is the condition of having at least three loose, liquid, or watery bowel movements each day. It often lasts for a few days and can result in dehydration due to fluid loss. Signs of dehydration often begin w ...

and

abdominal The abdomen (colloquially called the belly, tummy, midriff, tucky or stomach) is the part of the body between the thorax (chest) and pelvis, in humans and in other vertebrates. The abdomen is the front part of the abdominal segment of the torso ...

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 ''

'' toxins under which nivalenol (0.03–0.1 mg/kg in 2 of 24 samples), deoxynivalenol (0.34–8.4 mg/kg in 11 of 24 samples) and acetyldeoxynivalenol (0.6–2.4 mg/kg in 4 of 24 samples) were found in rain-damaged wheat used for bread production. There were again no lethal cases and reported symptoms were

pain,

, bloody stool and vomiting. These cases show that the main emerging danger of nivalenol comes from ''

'' infected cereals and is mainly via the route of digestion of uncontrolled wheat or other grains which is 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 T-2 Mycotoxin (pronounced as 'Tee-Two') is a trichothecene mycotoxin. It is a naturally occurring mold byproduct of ''Fusarium'' spp. fungus which is toxic to humans and animals. The clinical condition it causes is ''alimentary toxic aleukia'' ...

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

s and to have used them themselves in Afghanistan. All three compounds could be identified in the vegetation at affected sites, whereas

could also be found in urine and blood samples of victims. The best documented use of trichothecenes in warfare is the yellow rain controversy, this describes a number of attacks in Southeastern Asia as well as Laos and Afghanistan, which used a “

yellow rain Yellow rain was a 1981 political incident in which the United States Secretary of State Alexander Haig accused the Soviet Union of supplying T-2 mycotoxin to the Communist states in Vietnam, Laos and Cambodia for use in counterinsurgency warfare. ...

” as described by witnesses. The toxins were delivered as what has been described 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

s as biological weapons in Southeast Asia in a very detailed manner. In it reports of survivors and eyewitnesses as well as prisoners of war and soviet informants can be found together 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

s. There was a number of ways in which

s 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 (SCF). A temporary tolerable daily intake (t-TDI) 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 (FSCJ) issued a t-TDI of 0.4 μg/kg bw per day. Between 2001 and 2011 the European Food Safety Authority (EFSA) 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 An immunotoxin is an artificial protein consisting of a targeting portion linked to a toxin. When the protein binds to that cell, it is taken in through endocytosis, and the toxin kills the cell. They are used for the treatment of some kinds of c ...

Structure and reactivity

Nivalenol as part of the family of

s has the common structure which all members of this toxin family have. This includes the basic structure of a

cyclohexene Cyclohexene is a hydrocarbon with the formula C6H10. This cycloalkene is a colorless liquid with a sharp smell. It is an intermediate in various industrial processes. Cyclohexene is not very stable upon long term storage with exposure to light a ...

and a

tetrahydropyran Tetrahydropyran (THP) is the organic compound consisting of a saturated six-membered ring containing five carbon atoms and one oxygen atom. It is named by reference to pyran, which contains two double bonds, and may be produced from it by addi ...

ring connected at C6 and C11. Additionally an ethyl-group connects the

at C2 and C5 and a keto group is attached at the

at C8. The

epoxide In organic chemistry, an epoxide is a cyclic ether () with a three-atom ring. This ring approximates an equilateral triangle, which makes it strained, and hence highly reactive, more so than other ethers. They are produced on a large scale ...

group, responsible for the reactivity for most parts, is attached at C12 and C13 in the

. Only the remaining groups at positions C3, C4, C7, C15 vary for the different

s. 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

group is crucial for the reactivity of the molecule.

Available forms

Nivalenol, deoxynivalenol and T2-toxin are the three structural and similar synthesized

s naturally appearing in fungi (e.g. ''

'').

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 In chemistry, pyrophosphates are phosphorus oxyanions that contain two phosphorus atoms in a P–O–P linkage. A number of pyrophosphate salts exist, such as disodium pyrophosphate (Na2H2P2O7) and tetrasodium pyrophosphate (Na4P2O7), among othe ...

is used as starting compound for the synthesis of nivalenol. Its cyclization reaction to trichodiene is catalyzed by

terpene Terpenes () are a class of natural products consisting of compounds with the formula (C5H8)n for n > 1. Comprising more than 30,000 compounds, these unsaturated hydrocarbons are produced predominantly by plants, particularly conifers. Terpenes ...

cyclase trichodiene synthase (Tri5). This reaction is followed by several oxidation reactions catalyzed by

cytochrome P450 Cytochromes P450 (CYPs) are a superfamily of enzymes containing heme as a cofactor that functions as monooxygenases. In mammals, these proteins oxidize steroids, fatty acids, and xenobiotics, and are important for the clearance of various co ...

monooxygenase Monooxygenases are enzymes that incorporate one hydroxyl group (−OH) into substrates in many metabolic pathways. In this reaction, the two atoms of dioxygen are reduced to one hydroxyl group and one H2O molecule by the concomitant oxidation o ...

(encoded by TRI4). 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

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 Cyclopentane (also called C pentane) is a highly flammable alicyclic hydrocarbon with chemical formula C5H10 and CAS number 287-92-3, consisting of a ring of five carbon atoms each bonded with two hydrogen atoms above and below the plane. It occu ...

binds to the C11 of the

forming a

ring. The shifted OH-group at C9 is lost during the reaction. An acetyltransferase (encoded by TRI101) catalyzes the acetylation of the C3 OH-group of isotrichodermol forming isotrichdermin. Isotrichodermin is converted to 15-decalonecitrin due to a substitution (encoded by TRI11) 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 (3,4,15-triANIV) 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 Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) is a protein complex that controls transcription of DNA, cytokine production and cell survival. NF-κB is found in almost all animal cell types and is involved in cellular ...

pathway.

is a

transcription factor In molecular biology, a transcription factor (TF) (or sequence-specific DNA-binding factor) is a protein that controls the rate of transcription of genetic information from DNA to messenger RNA, by binding to a specific DNA sequence. The f ...

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

cytokine Cytokines are a broad and loose category of small proteins (~5–25 kDa) important in cell signaling. Cytokines are peptides and cannot cross the lipid bilayer of cells to enter the cytoplasm. Cytokines have been shown to be involved in autocrin ...

s, which are important controller molecules of the immune system. Nivalenol induced the secretion of IL-8, a mediator of

inflammation Inflammation (from la, inflammatio) is part of the complex biological response of body tissues to harmful stimuli, such as pathogens, damaged cells, or irritants, and is a protective response involving immune cells, blood vessels, and molec ...

. When treated with an

inhibitor, IL-8 secretion was lowered. Another important factor influenced by nivalenol is

MCP-1 ''For the ICAO airport code see Candle Lake Airpark, for the diradical compound see Dichlorocarbene.'' The chemokine (C-C motif) ligand 2 (CCL2) is also referred to as monocyte chemoattractant protein 1 (MCP1) and small inducible cytokine A2. CC ...

CCL2 ''For the ICAO airport code see Candle Lake Airpark, for the diradical compound see Dichlorocarbene.'' The chemokine (C-C motif) ligand 2 (CCL2) is also referred to as monocyte chemoattractant protein 1 (MCP1) and small inducible cytokine A2. C ...

, this cytokine plays a role in the mobility regulation of mononuclear leukocyte cells. Nivalenol causes

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

inhibition which shows, that nivalenol and

interact to influence the cell. It was shown that while deoxynivalenol induces the secretion of

chemokine Chemokines (), or chemotactic cytokines, are a family of small cytokines or signaling proteins secreted by cells that induce directional movement of leukocytes, as well as other cell types, including endothelial and epithelial cells. In additio ...

s, 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

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.

Metabolism

Nivalenol in mice is not only metabolized through the liver but also, for a lesser part through microbial detoxification in the intestines. Thereby especially the

group as most toxic part of the molecule is degraded. This happens by eliminating the oxygen of the

group resulting in a double carbon-carbon bond between C12 and C13. This double bond is nonpolar and very stable leading to a less reactive form of nivalenol called de-epoxynivalenol. The de-epoxinated nivalenol gained is therefore much less toxic, same as every de-epoxinated trichodiene, and can be segregated into the urine without having much toxic effects anymore (nearly non-toxic). In the urine of tested mice and pigs 80% of the de-epoxidated compound and only 7% of the actual nivalenol were found showing a high metabolising rate of the trichodienes. Thereby a low concentration of nitrogen in low proteins and urea were observed whereas the cholesterol concentration was observed to be higher than normal. This suggests that nivalenol is present and later degraded in the liver as the liver is responsible for the segregation of cholesterol into the bloodstream. The higher amount of cholesterol in the blood leads then to a higher amount of filtered cholesterol by the kidneys and eventually to an increased concentration in the urea. The lowered concentration of amides is assumed to be caused in the degradation process of the reactive

group. Therefore, the

s are often found to react with amides or amide groups by adding a hydroxyl group at a primary or secondary amine. As a consequence the epoxide group is degraded and less nitrogen is present for the synthesis of proteins or urea.

Adverse effects

Nivalenol did not yet find usage in medical treatments, and therefore it does not have known adverse effects besides the toxic effects described. It is however worth noting that it could be interesting for investigation due to its immunosuppressive effects.

Effects on animals and Efficacy

As nivalenol is a mycotoxic product of certain ''

'' species it is often found in infected wheat and grain. As unprocessed wheat and grain product are often used as feed for livestock animals these are at a higher risk of nivalenol intake. Toxicity studies in swine that received a dose of 0.05 mg nivalenol/kg body weight twice daily showed no lethal effects. Most nivalenol was secreted with the feces and did not reach the bloodstream despite the fact that there was still nivalenol upstage over the intestines after 16 hours of feeding. There were further no nivalenol metabolites found in feces or urine within the first three days. After a week of exposure to 2.5 or 5 mg nivalenol kg bw twice a day a microbiological adaptation was seen as nivalenol metabolites (de-epoxidated nivalenol) could be found in feces and urine. In rats and mice nivalenol showed to be toxic with adverse effects of

growth retardation Delayed milestone, also called developmental delays, is used to describe the condition where a child does not reach one of these stages at the expected age. However, in most cases, a wide variety of ages can be considered normal, and not a cause fo ...

and leukopenia already noticed at lowest doses of 0.7 mg/kg bw per day. Lethal doses were dependent on the route of administration/intake of nivalenol. As nivalenol is normally taken up with feed the LD₅₀ of oral administration which is 38.9 mg/kg bw per day in mice and 19.5 mg/kg bw per day in rats can be used as standard. The LD₅₀ of intravenous,

intraperitoneal The peritoneum is the serous membrane forming the lining of the abdominal cavity or coelom in amniotes and some invertebrates, such as annelids. It covers most of the intra-abdominal (or coelomic) organs, and is composed of a layer of mesothel ...

and subcutaneous (SC) is between 7 and 7.5 mg/kg bw per day.

Toxicity, indications and side effects

The toxicity of nivalenol in humans is for the most parts unknown yet, but it was investigated in mice, rats and hamster cells. Thereby the toxicity was divided in the following topics: acute/subacute, subchronic, chronic and carcinogenicity, genotoxicity, developmental toxicity studies and studies on reproduction, immunotoxicity/hematotoxycity and effects on nervous system.

Acute/subacute toxicity

The oral LD₅₀ of nivalenol was found to be 38.9 mg/kg bw in mice whereas the intraperitonal, subcutaneous and intravenous routes of exposure gave LD₅₀ values of 5–10 mg/kg bw. In mice already within 3 days the most deaths occurred after oral exposure through marked congestion and

haemorrhage Bleeding, hemorrhage, haemorrhage or blood loss, is blood escaping from the circulatory system from damaged blood vessels. Bleeding can occur internally, or externally either through a natural opening such as the mouth, nose, ear, urethra, v ...

in intestine, in acute toxicity also lymphoid organs are included. Nivalenol given over time periods of 24 days in lower doses (ca. 3,5 mg/kg bw) showed significant erythropenia and slight

leukopenia Leukopenia () is a decrease in the number of leukocytes (WBC). Found in the blood, they are the white blood cells, and are the body's primary defense against an infection. Thus the condition of leukopenia places individuals at increased risk of in ...

.{{Cite journal, date=2000, title=Opinion of the Scientific Committee on Food on Fusarium Toxins Part 41: Nivalenol, url=https://ec.europa.eu/food/sites/food/files/safety/docs/cs_contaminants_catalogue_out74_en.pdf, journal=Scientific Committee on Food, pages=2–6

Subchronic toxicity

The subchronic toxicity was tested by feeding mice with a daily dose of 0 to 3.5 mg nivalenol/ kg bw for 4 or 12 weeks. The observations after 4 weeks were reduced body weight and food consumption. The reduction in body weight can be explained by statistical decrease in organ weight in thymus, spleen and kidneys. Whereas the consumption time was less for female mice in comparison to male mice. After 12 weeks the toxin consumption resulted in reduction of relative organ weight in both males and females. Hereby only the liver was affected and no

histopathological Histopathology (compound of three Greek words: ''histos'' "tissue", πάθος ''pathos'' "suffering", and -λογία ''-logia'' "study of") refers to the microscopic examination of tissue in order to study the manifestations of disease. Spec ...

changes were observed.

Chronic toxicity and carcinogenicity

Female mice were fed with different doses of nivalenol (0, 0.7, 1.4 or 3.5 mg nivalenol /kg bw) for one or two years to investigate whether nivalenol is chronic toxic and/or carcinogenic. Also during this study a decrease in body weight and feed consumption was observed. The absolute weight of both liver and kidney was decreased through the two highest doses. The mice fed for one year with nivalenol (also with the lower doses) were affected with severe leukopenia whereas the mice fed for two years had no differences in count of white blood cells. Also "no histopathological changes including tumours were found in liver, thymus, spleen, kidneys, stomach, adrenal glands, pituitary glands, ovaries, bone marrow, lymph node, brain and small intestines with or without

Peyer's patch Peyer's patches (or aggregated lymphoid nodules) are organized lymphoid follicles, named after the 17th-century Swiss anatomist Johann Conrad Peyer. * Reprinted as: * Peyer referred to Peyer's patches as ''plexus'' or ''agmina glandularum'' (c ...

". The lowest doses (0.7 mg nivalenol /kg bw) inhibited the growth and caused leukopenia. "A no observable adverse effect level (

NOAEL The no-observed-adverse-effect level (NOAEL) denotes the level of exposure of an organism, found by experiment or observation, at which there is no biologically or statistically significant increase in the frequency or severity of any adverse effec ...

) could not be derived from these studies. IARC (1993) concluded that there is inadequate evidence of carcinogenicity of nivalenol in experimental animals. No human data were available. The overall conclusion was that the carcinogenicity was not classifiable (group 3)".

Genotoxicity

It was found that nivalenol effects the genes of Chinese hamster V79 (CHO) cells by slightly increased frequencies of chromosomal aberrations and sister chromatid exchange. The DNA was damaged in CHO cells as well as in mice. In mice (given 20 mg nivalenol /kg bw orally or 3.7 mg /kg bw ip) the DNA of kidney, bone marrow, stomach,

jejunum The jejunum is the second part of the small intestine in humans and most higher vertebrates, including mammals, reptiles, and birds. Its lining is specialised for the absorption by enterocytes of small nutrient molecules which have been previou ...

and colon was damaged. The DNA of the thymus and liver was not effected. In organs with DNA damage no

necrotic Necrosis () is a form of cell injury which results in the premature death of cells in living tissue by autolysis. Necrosis is caused by factors external to the cell or tissue, such as infection, or trauma which result in the unregulated dige ...

changes were found upon histopathological examination. It can be concluded that an adequate evaluation of the genotoxicity is not allowed based on the available data.

Developmental toxicity and studies on reproduction

For developmental and reproduction studies pregnant mice were injected with different amounts of purified nivalenol on days 7–15 of gestation and for one additional study with mouldy rice containing nivalenol. The studies showed that the toxin is embryotoxic in mice. No evidence of

teratogenicity Teratology is the study of abnormalities of physiological development in organisms during their life span. It is a sub-discipline in medical genetics which focuses on the classification of congenital abnormalities in dysmorphology. The related ...

was given. "The LOAEL in reproduction studies with nivalenol given by oral exposure was stated to be 1.4 mg/kg bw given in the feed throughout gestation and 5 mg/kg bw when given by gavage on days 7–15". Data from other species and on reproductive effects in adult males and females are not provided yet.

Immunotoxicity/haematotoxicity

Acute toxicity of nivalenol induces bone marrow toxicity and toxicity of lymphoid organs. Long-term exposure may result in erythropenia and/or

. In mice it was also observed that nivalenol increased the presence of serum IgA, "accompanied by immunopathological changes in kidneys analogous to human IgA-nephropathy". The blastogenesis in cultured human lymphocytes, proliferation of human male and female lymphocytes stimulated with phytoheamagglutin and

pokeweed ''Phytolacca americana'', also known as American pokeweed, pokeweed, poke sallet, dragonberries, and inkberry, is a poisonous, herbaceous perennial plant in the pokeweed family Phytolaccaceae. This pokeweed grows . It has simple leaves on green ...

and immunoglobulin production induced by pokeweed, are inhibited by nivalenol. The effects of nivalenol are in the same range as same doses of deoxynivalenol, whereas the

are 100 fold more toxic. An additive effect is gained by combination of nivalenol with

, 4,15-diacetoxyscirpenol or deoxynivalenol.

Effects on nervous system

About the nervous system no data has been provided yet.

References

External links

* http://ccinfoweb2.ccohs.ca/hsdb/records/3517.html * https://www.sigmaaldrich.com/catalog/product/sigma/n7769?lang=en®ion=NL * https://pubchem.ncbi.nlm.nih.gov/compound/430146#section=Top Fusarium Cyclohexenols Mycotoxins Epoxides Tetrahydropyrans Enones