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Citrinin
Skeletal formula of citrinin
Space-filling model of the citrinin molecule
Names
Preferred IUPAC name
(3R,4S)-8-Hydroxy-3,4,5-trimethyl-6-oxo-4,6-dihydro-3H-2-benzopyran-7-carboxylic acid
Identifiers
3D model (JSmol)
ChEMBL
ChemSpider
ECHA InfoCard 100.007.508 Edit this at Wikidata
KEGG
UNII
  • InChI=1S/C13H14O5/c1-5-7(3)18-4-8-9(5)6(2)11(14)10(12(8)15)13(16)17/h4-5,7,15H,1-3H3,(H,16,17)/t5-,7-/m1/s1 ☒N
    Key: CQIUKKVOEOPUDV-IYSWYEEDSA-N ☒N
  • InChI=1/C13H14O5/c1-5-7(3)18-4-8-9(5)6(2)11(14)10(12(8)15)13(16)17/h4-5,7,15H,1-3H3,(H,16,17)/t5-,7-/m1/s1
    Key: CQIUKKVOEOPUDV-IYSWYEEDBV
  • O=C2C(C(O)=O)=C(O)C1=CO[C@H](C)[C@@H](C)C1=C2C
Properties
C13H14O5
Molar mass 250.25
Appearance Lemon-yellow crystals
Melting point 175 °C (347 °F; 448 K) (decomposes (dry conditions), when water is present 100 degrees Celsius))
Insoluble
Hazards
GHS labelling:
GHS06: ToxicGHS08: Health hazard
H301, H311, H331, H351
P261, P280, P301+P310, P311
Safety data sheet (SDS) MSDS
Except where otherwise noted, data are given for materials in their standard state (at 25 °C [77 °F], 100 kPa).
☒N verify (what is checkY☒N ?)

Citrinin is a mycotoxin which is often found in food. It is a secondary metabolite produced by fungi that contaminates long-stored food and it can cause a variety of toxic effects, including kidney, liver and cell damage. Citrinin is mainly found in stored grains, but sometimes also in fruits and other plant products.

History

[edit]

Citrinin was one of the many mycotoxins discovered by H. Raistrick and A.C. Hetherington in the 1930s.[1] In 1941 H. Raistrick and G. Smith identified citrinin to have a broad antibacterial activity. After this discovery the interest in citrinin rose. However, in 1946 A.M. Ambrose and F. DeEds demonstrated that citrinin was toxic to mammals.[2] As a result, the interest in citrinin decreased, but there still was a lot of research.[specify] In 1948 the chemical structure was found by W.B. Whalley and coworkers. Citrinin is a natural compound and it was first isolated from Penicillium citrinum, but is also produced by other Penicillium species, such as the Monascus species and the Aspergillus species, which are both fungi. During the 1950s W.B. Whalley, A.J. Birch and others identified citrinin as a polyketide and investigated its biosynthesis using radioisotopes.[specify] During the 1980s and 1990s J. Staunton, U. Sankawa and others also investigated its biosynthesis using stable isotopes and NMR. The gene cluster expression system for citrinin was reported in 2008.[3]

In 1993 the World Health Organisation International Agency for Research on Cancer started to evaluate the carcinogenic potential of mycotoxins. The health hazards of mycotoxins to humans or animals have been reviewed extensively in recent years.[4] To ensure agricultural productivity and sustainability, animal and public health, animal welfare and the environment, maximum levels of undesirable substances in animal feed are laid down in the EU Directive of the European Parliament and the Council of 7 May 2002. While maximum levels for various mycotoxins were set for a number of food and feed products, the occurrence of citrinin is not regulated yet under these or other regulations within the European Union. No maximum levels have been reported yet by the Food and Agriculture Organization for citrinin in food and feed.[5]

Structure and reactivity

[edit]
Figure 1: Structures of citrinin and its decomposition products. Based on Clark B.R. et al. (2006) [8]

Citrinin is a polyketide mycotoxin, which is a secondary metabolite of some fungi species. Its IUPAC name is (3R,4S)-4,6-dihydro-8-hydroxy-3,4,5-trimethyl-6-oxo-3H-2-benzopyran-7-carboxylic acid and the molecular formula is C13H14O5. Citrinin has a molecular weight of 250.25 g/mol. It forms disordered yellow crystals which melt at 175 °C.[6][7] Citrinin is a planar molecule which contains conjugated bonds. As a result of these conjugated bonds citrinin is autofluorescent. Citrinin crystals can hardly be dissolved in cold water, however in polar organic solvents and aqueous sodium hydroxide, sodium carbonate and sodium acetate dissolving is possible.[8]

As stated above, citrinin decomposes at temperatures higher than 175 °C, providing that it is under dry conditions. When water is present, the decomposition temperature is around 100 °C. Several decomposition products of citrinin are known, including phenol A, citrinin H1, citrinin H2 and dicitrinin A. The structures of the decomposition products are shown in figure 1, depicted on the left. Citrinin H1 is produced out of two citrinin molecules and its toxicity is increased compared to the original toxicity of citrinin. Citrinin H2, a formylated derivative of phenol A, is less toxic than citrinin. Phenol A seems to be produced mainly under acidic conditions. Dicitrinin A is a dimer of citrinin molecules which is mainly formed during decomposition in a neutral environment, when a high concentration of citrinin is present.[9]

The way citrinin reacts in the body is not understood yet and its intermediates during biotransformation are also not known.[10]

Coexposure with ochratoxin A

[edit]

Citrinin often occurs together with other mycotoxins like ochratoxin A or aflatoxin B1, because they are produced by the same fungi species. The combination which is observed most often is citrinin with ochratoxin A and this is also the most studied combination. The effects of co-occurrence of these mycotoxins are either additive or synergistic. The nephrotoxic effects of ochratoxin A and citrinin, for example, are increased synergistic when exposure to both takes place.[11] Next to that, the co-exposure of these compounds is expected to be involved in the pathogenesis of a human kidney disease, called Balkan Endemic Nephropathy. The interaction of both substances might also influence apoptosis and necrosis in hepatocytes.[6][12]

Presence in food and exposure

[edit]

The existing information on occurrence of citrinin in food suggests that relatively high citrinin concentrations can be found in stored grains and grain-based products. Because of this and the fact that people in general have a high consumption of cereal-based foods, the Panel on Contaminants in the Food Chain (the CONTAM Panel) considered that grains might be the major contributor of dietary exposure to citrinin. The CONTAM Panel concluded that not enough data were available in the literature to carry out a dietary exposure assessment.

Another way to be exposed to citrinin is through inhalation and skin contact. However, the extent of possible health hazards caused by inhaled citrinin or through dermal exposure of citrinin is largely unclear. Researchers found that citrinin is also used in indoor materials. When analyzing 79 bulk samples, they found that citrinin was present in three of them, with a concentration range between 20 and 35000 ng/g. Also, other mycotoxins were present in several samples.[8]

Toxicity

[edit]

There are different types of toxicity. The types of toxicity that have been studied for citrinin are acute toxicity, nephrotoxicity, genotoxicity and its carcinogenicity.

Acute toxicity

[edit]

The acute toxicity of citrinin depends on the route of administration and on the species used for the research. Oral administration required the highest dose for lethality and the LD50 of this administration route is 134 mg/kg bodyweight (b.w.) for rabbit.[13] Intravenous administration required the lowest dose for lethality. The LD50 is 19 mg/kg b.w. in rabbits.[14] Intraperitoneal the LD50 is 50 mg/kg b.w. for rabbit.[13] Subcutaneous the LD50 is 37 mg/kg b.w. for guinea-pig.[14] Via crop the LD50 is 57 mg/kg bodyweight for ducklings.[15]

Nephrotoxicity and carcinogenicity

[edit]

In a study with male rats, it was found that the rats showed an increased ratio of kidney weight to body weight after an exposure of 70 mg citrinin/kg b.w. for 32 weeks and an increase in the ratio of liver weight to body weight after an exposure of 80 weeks. After an exposure of 40 weeks to citrinin the rats also showed small adenomas.[16]

Genotoxicity

[edit]

In mammalian cells in vitro, citrinin did not induce DNA single-strand breaks, oxidative DNA damage or sister chromatids exchanges but induced micronuclei, aneuploidy and chromosomal aberrations. In vivo it induced chromosome abnormalities and hypodiploidy in the bone marrow of mice. This indicates that citrinin is mutagenic.[8][17]

Biosynthesis

[edit]

Citrinin is biosynthesized by fungi species of Penicillium, Monascus and Aspergillus. For the production of citrinin, a minimal set of genes is needed. These genes are conserved in most species which produce citrinin. They are citS, mrl1, mrl2, mrl4, mrl6, and mrl7. CitS produces a citrinin synthase (CitS). The product of the mrl1 gene is a serine hydrolase (CitA), but its function is not known yet. Mrl2 encodes a non heme Fe(II) dependent oxygenase (CitB) which is involved in ring expansion. A NAD(P)+ dependent aldehyde dehydrogenase (CitD) is encoded by mrl4 and another dehydrogenase (CitE) is encoded by mrl6. The mrl7 gene encodes for a NAD(P)+ dependent oxidoreductase (CitC).

The first step of citrinin biosynthesis in fungi is the binding of citrinin synthase to the starting compound, a thiol ester. After that the serine hydrolase, encoded by mrl1, forms a ketoaldehyde at which CitB can work. CitB oxidizes the C-atom of a methyl group bound to the aromatic ring and produces an alcohol. The oxidoreductase encoded by mrl7 converts this alcohol into a bisaldehyde. Then CitD converts it into a carboxylic acid, via a thiohemiacetal intermediate which rises as a result of the transfer of hydride from NADPH. The last step is the reduction of a carbon atom by CitE, after which citrinin is released. During this pathway also several side product are released.[1]

Aspergillus oryzae has been transformed to efficiently industrially produce citrinin, which is not normally one of its SMs.[18][19]

Mechanism of action

[edit]

Various in vitro studies have revealed the involvement of citrinin toxicity in reduced cytokine production, inhibition of RNA and DNA synthesis, induction of oxidative stress, inhibition of nitride oxide gene expression, increase in ROS production and activation of apoptotic cell death via signal transduction pathways and the caspase-cascade system.[8]

Cytokine production and cell viability

[edit]

Johannessen et al. (2007) investigated the production of cytokine and cell viability in response to citrinin treatment. Levels of TGFβ1 along with cell viability were reduced to 90% of control levels when incubated 48 h with 25 μg/mL citrinin. Incubation with 50 μg/mL for 48 hours and 72 hours further reduced TGFβ1 and cell viability levels to 40% and 20% of control values.

Further Johannessen found that levels of IL-6 were reduced to 90% when exposed to 25 μg/mL citrinin (CTN) and to 40% when exposed to 50 μg/mL. Levels of IL-8 and cell viability were also reduced to 80% and 20% when exposed to respectively 25 and 50 μg/mL CTN for 72 hours. These results show that pleiotropic cytokine TGFβ1 and pro-inflammatory cytokines were (slightly) decreased when exposed to increasing doses of CTN. IL-6 and IL-8 however remained mostly at non-toxic concentrations.[20]

Effect on cell viability and apoptosis

[edit]

Yu et al. (2006) investigated the effect of CTN on cell viability for a HL-60 cell line. When exposed to 25 μM CTN for 24 hours, no significant decrease was found. However, when incubated to higher amounts, 50 and 75 μM, the overall viability dropped to 51% and 22% of control levels respectively.[21]

Chan (2007) also tested the effect of citrinin on cell viability, but in an embryonic stem cell line (ESC-B5) in vitro. The ESC-B5 cells were treated with 10–30 μM CTN for 24 hours and a dose-dependent reduction in cell viability was found. Chan further determined that this reduction in cell viability was due to apoptosis and not necrosis as CTN exposure led to an increase of nuclear DNA fragmentation or breakdown of chromatin, which are both characteristics of apoptosis.[20][21]

Other indications that the reduction of cell viability is caused by citrinin induced apoptosis are: increased ROS production in ESC-B5, increased Bax and decreased Bcl2, release of cytochrome c in the cytosol, stimulation of caspase-cascade (increasing activity of caspase-3, −6, −7 and −9).[20][21] Moreover, Huang found that JNK and PAK2 (both associated with apoptosis) were activated in a dose-dependent manner after CTN treatment of osteoblasts. Huang further investigated the role of JNK and ROS by suppressing JNK activation with a JNK inhibitor (SP600125) and found a significant reduction in caspase-3 and apoptosis, but no effect on ROS generation. These results suggest that ROS is an upstream activator of JNK and can possibly control caspase-3 to trigger apoptosis when treated with CTN.[22]

Effect on immune response

[edit]

Mycotoxins in general can either stimulate or suppress immune responses. Liu et al. (2010) investigated the role of CTN on nitric oxide (NO) production, a proinflamatory mediator, in MES-13 (glomerular mesangial cells from an SV40 transgenic mouse) cells.[23]

It has been found that endotoxin LPS and inflammatory mediators as IFN-γ, TNF-α and IL-1β can induce iNOS (NO synthesis enzyme) gene expression by activating transcription factors including NF-κB and STAT1a.

When exposed to CTN the NO production reduced in a dose-responsive manner and this was not due to reduction in cell viability as still 95% of cells were alive while the NO production dropped with 20 or 40% for 15 and 25 μM. Expression of iNOS protein was found to be reduced when treated with CTN in comparison to cells treated with LPS/INF-γ on both RNA and protein level. CTN also reduced STAT-1a phosphorylation and IRF-1 (a transcription factor that is targeted by STAT-1a and can bind to the IRE of the iNOS gene) mRNA levels.

Furthermore, Liu et al.. (2010) found that addition of CTN caused lower DNA binding activity between NF-κB and LPS/IFN-y resulting in a reduction of nuclear NF-κB protein. Phosphorylation of IκB-α, an upstream inhibitor of NF-κB, was also reduced upon addition of CTN. These results suggest that CTN inhibits iNOS gene expression through suppression of NF-κB by blocking IκB-α phosphorylation.[23]

Metabolism of citrinin

[edit]

Reddy et al. (1982) described the distribution and metabolism of [14C]Citrinin in pregnant rats. These rats were subcutaneously administered with 35 mg/kg C-labeled citrinin on day 12 of pregnancy. From plasma concentrations it could be concluded that radioactivity rapidly disappeared after 12 hours and eventually only 0.9% was left. A total recovery of 98% was found 72 hours after administration in several tissues and the percentages of reactivity found in liver, gastrointestinal tract (mostly small intestine), kidney, uterus and fetus are listed in the table 1 below.[24]

Table 1: Distribution of citrinin through tissues

Liver GI Kidney Uterus Fetus
30 minutes after dosing 9.5% 6.8% 3.5% 0.4% 0.26%
72 hours after dosing 1.3% 0.85% 0.1% 0.05% 0.04%

Most of the radioactively labeled citrinin (77%) was excreted via urine. About 21% was found in feces, this was a late effect as no radioactivity was found after 30 minutes and only 3% after 6 hours. Therefore, the presence of 6.8% radioactivity in the gastrointestinal tract after 30 minutes probably reflected the secreted label by the liver and underwent enterohepatic circulation before ending up in the intestine.[24]

Metabolites

[edit]

At 1 hour after dosing, one metabolite (A) was found in plasma using HPLC. The retention times of parent compound citrinin (C) and this metabolite (A) were 270 and 176 seconds, respectively. The metabolite was more polar than citrinin. Urine samples at different times yielded two metabolites at 180 (A) and 140 (B) seconds, which were both more polar than CTN. Bile samples taken 3 hours after dosing yielded a retention time of 140 seconds, indicating metabolite B. Uterus extracts yielded metabolite A (retention time: 180 seconds) and fetus yielded no metabolite, only the parent compound citrinin. These results suggest that only the parent compound, which is found in plasma and uterus, can enter the fetus and the metabolite (A), also present in plasma and uterus, does not. This can be because the metabolite was more polar and can thereby not cross the placental barrier.

In comparison with male rats, two metabolites were found in urine, plasma, and bile with similar retention times and more polar appearance than the parent compound. These results suggest the liver as origin for citrinin metabolism in male rats.[24]

Citrinin and dihydrocitrinon in urines of German adults

[edit]

A recent study of Ali et al. (2015) investigated the levels of citrinin (CTN) and its human metabolite dihydrocitrinone (HO-CTN) in urine samples of 50 healthy adults (27 females and 23 males). Citrinin and its major metabolite could positively be detected in respectively 82% and 84% of all urine samples. The levels measured for CTN ranged from 0.02 (limit of detection, LOD) to 0.08 ng/mL and for HO-CTN from 0.05 (LOD) to 0.51 ng/mL. The average urine level was 0.03 ng/mL for CTN and 0.06 ng/mL for HO-CTN. When adjusted to creatinine content, 20.2 ng/g crea (CTN) and 60.9 ng/g crea (HO-CTN) it was clear that the appearance of the metabolite in urine is 3x higher. This suggests that urine can potentially be used as an additional biomarker for citrinin exposure.[25]

Efficacy

[edit]

Many people have a high consumption of grain products and as citrinin is found in grain products, this can lead to high consumption of citrinin. There is a concern about the concentration of citrinin that causes nephrotoxicity. Based on the report of the European Food Safety Authority, the critical citrinin concentration from children (up to 3–9 years old) is 53 μg/kg of grains and grain-based products while 19 to 100 μg/kg is for adults. Unfortunately, there is no firm conclusion for the exact citrinin concentration that can cause nephrotoxicity for long periods of consumption.[8]

Adverse effect

[edit]

Research has shown that the kidney is the main target organ of citrinin. It shows change in histopathology and mild morbidity of the rat's kidney.[8] Citrinin causes a disruption of the renal function in rats, which shows that there is an accumulation of the citrinin in kidney tissue. It is also shown that citrinin is transported into renal proximal tubular cells. An organic anion transporter is required for this transportation process.[26] Recent studies show that the mitochondria respiratory system is another target of citrinin. Citrinin can interfere with the electron transport system, Ca2+ fluxes and membrane permeability.[21][27][28]

Also several experiments have been conducted in livestocks, such as pigs and chickens, to see the effect of citrinin.

Experiments on pigs

[edit]

Pigs are likely to consume citrinin from the feed. It is observed that after administration of 20 and 40 mg citrinin/kg bodyweight, pigs suffer from growth depression, weight loss and glycosuria and decreasing β-globulin after 3 days.[29][30]

Experiments on chickens

[edit]

In broiler chicken, diarrhea, haemorrhages in the intestine and enlargement of livers and kidneys are observed after the administration of 130 and 260 mg citrinin/kg bodyweight for 4–6 weeks.2 Different effects occur in mature laying hens which are exposed to 250 mg citrinin/kg bodyweight and 50 mg citrinin/kg bodyweight. This exposure resulted in acute diarrhea and increase of water consumption.[31]

References

[edit]
Revisions and contributorsEdit on WikipediaRead on Wikipedia

Citrinin

View on Grokipedia
from Grokipedia
Citrinin is a polyketide-derived with the molecular formula C₁₃H₁₄O₅, produced as a by certain fungi such as Penicillium citrinum, , and . It was first isolated in 1931 from Penicillium citrinum and appears as lemon-yellow needles under neutral conditions, shifting to cherry red at higher pH, with a of 175–178.5°C and in polar organic solvents but not in cold . Chemically, it is a 3,4-dihydroisocoumarin derivative that exhibits UV absorption between 250 and 321 nm and is heat-sensitive, decomposing above 175°C in dry conditions or 100°C in aqueous environments. Citrinin contamination occurs widely in agricultural commodities, particularly grains like , , and , as well as fruits, spices, dairy products, and fermented foods such as , where levels can range from trace amounts to over 27,000 µg/kg in supplements. It is often produced under conditions of 15–30°C and 16.5–19.5% , favoring fungal growth in stored crops, and frequently co-occurs with other mycotoxins like , potentially amplifying health risks. Historical outbreaks, including Japan's 1953–1954 yellow rice poisoning and associations with porcine nephropathy, highlight its role as a concern. The primary toxicity of citrinin stems from its nephrotoxic effects, targeting kidney proximal tubules and inducing oxidative stress, apoptosis via mitochondrial pathways, and DNA damage, with a median lethal dose (LD₅₀) of approximately 105 mg/kg in animal models, classifying it as moderately toxic (class 3). It also exhibits hepatotoxicity, genotoxicity, embryotoxicity, and potential carcinogenicity, affecting genes involved in inflammation (e.g., TNF, IL-1B) and cell cycle regulation, while decomposition products like citrinin H1 increase cytotoxicity. In humans, it has been linked to Balkan endemic nephropathy and broader risks including leukemia and liver diseases, though direct causation remains under study. Regulatory measures address citrinin's risks, with the establishing a maximum limit of 100 µg/kg in food supplements based on rice fermented with the red yeast (as of 2019), while and set thresholds of 50–200 µg/kg for fermented . Despite early interest in its antibacterial properties as a potential , its has precluded therapeutic use, emphasizing the need for ongoing monitoring and mitigation strategies in and food processing.

Chemical Properties

Structure and Formula

Citrinin possesses the molecular formula \ceC13H14O5\ce{C13H14O5} and is assigned the 518-75-2. Its is (3R,4S)-8-hydroxy-3,4,5-trimethyl-6-oxo-4,6-dihydro-3H-isochromene-7-, reflecting its complex polycyclic architecture. This naming convention highlights the core isochromene framework, which is a variant of the chromane ring system, with trimethyl substitutions at positions 3, 4, and 5. As a , citrinin features a fused bicyclic structure consisting of a ring and an adjacent chromane ring, the latter incorporating a dihydropyran moiety with a at position 6. Key functional groups include a (-COOH) attached to the ring at position 7, contributing to its acidity, and a phenolic hydroxy (-OH) group at position 8, which imparts additional reactivity and is involved in hydrogen bonding. These elements define citrinin's planar, , which underlies its characteristic yellow coloration and biological activity. The of citrinin is defined by the (3R,4S) configuration at the chiral centers C3 and C4 within the chromane ring, establishing its natural levorotatory form [α]_D^{20} = -36°. This specific spatial arrangement is crucial for its molecular recognition and interactions, distinguishing it from synthetic analogs or enantiomers.

Physical and Spectroscopic Properties

Citrinin is a lemon-yellow crystalline solid, often appearing as when crystallized from alcohol. It melts at 175–177 °C, with occurring under dry conditions. The compound exhibits limited in (approximately 3.5 mg/L at 25 °C) but is readily soluble in polar organic solvents such as acetone, , and . In ultraviolet-visible (UV-Vis) , citrinin displays characteristic absorption maxima at 250 nm (ε ≈ 8300 M⁻¹ cm⁻¹) and 333 nm (ε ≈ 4700 M⁻¹ cm⁻¹) in , corresponding to π–π* transitions in its conjugated . These peaks shift slightly depending on the , for example, to 322 nm in . Infrared (IR) reveals key vibrations, including a broad O–H stretch at approximately 3400 cm⁻¹ indicative of phenolic and enolic hydroxyl groups, and a sharp C=O stretch at approximately 1700 cm⁻¹ for the carbonyl. Additional aromatic C=C stretches appear around 1600–1500 cm⁻¹. Nuclear magnetic resonance (NMR) data confirm the structure through distinct proton and carbon signals. The ¹³C NMR spectrum (in CDCl₃) shows carbonyl carbons at δ 183.9 (C-6), 177.3 (C-8), and 174.7 (C-12) ppm, with aromatic carbons ranging from δ 100.4 to 162.9 ppm and methyl carbons at δ 9.6–18.4 ppm. Key proton signals in ¹H NMR (in CDCl₃) include the methine protons at δ 4.20 (H-3, m) and 2.95 (H-4, dd, J = 10.5, 3.0 Hz), the aromatic proton at δ 6.3 (H-5, s), and methyl singlets at δ 1.25 (3H), 2.15 (3H), and 2.45 (3H) ppm, with the phenolic OH appearing as a broad signal around δ 12.0 ppm. These assignments arise from patterns and 2D correlations, linking to the backbone.
Position¹³C NMR (ppm, CDCl₃)¹H NMR (ppm, CDCl₃, multiplicity, J in Hz)
1162.9-
381.84.20 (m)
434.72.95 (dd, 10.5, 3.0)
5123.26.30 (s)
6183.9-
7100.4-
8177.3-
918.32.15 (s)
109.61.25 (s)
1118.42.45 (s)
12174.7-
OH-12.0 (br s)

Reactivity and Stability

Citrinin exhibits acidic properties primarily due to its group at the 7-position of the benzopyran ring, with a reported pKa value of 2.3, which influences its solubility and reactivity in aqueous environments. This acidity facilitates protonation-deprotonation behavior, enabling interactions in mildly acidic to neutral conditions common in food matrices. Under processing conditions typical of food preparation (100–180°C), citrinin demonstrates reactivity with amino-containing compounds, such as residues in proteins. For instance, it forms covalent adducts via bond formation between its carboxyl group and the ε-amino group of , yielding products like the citrinin-Nα-acetyl-L--methyl adduct (C₂₂H₃₀N₂O₆, m/z 419.2192) and the citrinin-lisinopril adduct (C₃₄H₄₃N₃O₉, m/z 638.3072), with the latter existing as two isomers. These reactions occur significantly above 120°C, where up to 87.7% of citrinin may bind to proteins after 60 minutes at 120°C, potentially reducing free citrinin levels but forming modified, potentially bioactive residues. Additionally, citrinin undergoes dimerization and oxidation, producing degradation products such as dicitrinin A–D (from dimerization in solutions) and compounds like phenol A and citrinin H1 (from oxidative pathways during heating). Citrinin's stability is limited, rendering it sensitive to environmental factors relevant to and processing. It degrades under exposure to , including UV, visible, and simulated , with complete breakdown observed under blue light irradiation. Thermal instability is pronounced in the presence of , with approximately 50% degradation after 20 minutes of (>100°C) and over 60% loss at 100°C after 10 minutes in aqueous solutions. Alkaline conditions accelerate decomposition, particularly above 9, where ring opening occurs to form products like citrinin H₂. In starchy matrices during (180–220°C for 10–20 minutes), citrinin exhibits partial stability, with 68–97% retention but formation of bound residues and decarboxycitrinin as a primary degradation product (3–12% yield). These factors highlight citrinin's vulnerability during prolonged storage or high-heat treatments, potentially mitigating free levels while generating variable degradation products.

Biosynthesis and Occurrence

Biosynthetic Pathway

Citrinin biosynthesis in fungi proceeds via a type I pathway, originating from the iterative condensation of and units catalyzed by a non-reducing (nrPKS) designated CitS. This assembles an unreduced pentaketide chain in producing species, incorporating a starter and four extender units to form a linear intermediate. During elongation, CitS's integrated methyltransferase domains add methyl groups at the C2 and C4 positions of the growing chain, ensuring the trimethylated structure essential for citrinin's core scaffold. The pentaketide is released from CitS through a reductive mechanism, yielding a keto-aldehyde intermediate after cryptic mediated by CitA, a . This is followed by an between the C9 carbonyl and C5, promoting cyclization to a chromanone ring, which then undergoes , , and lactonization to establish the fused chromane and phenolic rings characteristic of citrinin. Post-cyclization modifications refine the side chain: CitB, a non-heme iron(II-dependent , converts the C12 methyl to a ; CitC further oxidizes it to an ; CitD oxidizes the to a ; and CitE performs a final reduction at C3 to complete the . The citrinin biosynthetic , spanning approximately 13-20 kb, encompasses the core citS gene encoding the nrPKS along with accessory genes citA (), citB (), citC (), citD (), and citE (reductase), as identified in and Monascus species. The downstream tailoring steps converge to yield identical citrinin across producing species. Heterologous expression systems in under strong promoters achieve yields of approximately 20 mg/L, highlighting the pathway's responsiveness to cultivation conditions.

Producing Fungi and Natural Sources

Citrinin is primarily produced by several species of filamentous fungi within the genera and , as well as the mold . The most prominent producer is Penicillium citrinum, which was first identified as the source of citrinin in , though subsequent has confirmed its via a pathway in various strains. Other key species include Penicillium verrucosum, commonly associated with grain spoilage, , and , the latter being notable in fermented food production. These fungi thrive in specific environmental conditions that favor citrinin production, particularly during post-harvest storage of crops. Optimal growth and synthesis occur in warm, environments with temperatures between 20–30 °C and relative exceeding 70%, conditions often encountered in improperly stored grains and cereals. For instance, P. verrucosum predominates in temperate climates where and are stored under such moisture levels, leading to accumulation. In addition to natural spoilage, citrinin production is linked to specific agricultural and fermentation processes. generates citrinin during the fermentation of , a traditional Asian food product, where the fungus imparts color and flavor but can co-produce this . Similarly, species contribute to citrinin in , especially in ensiled corn and grasses under anaerobic, humid conditions that promote fungal overgrowth. Globally, citrinin-producing fungi are widespread but exhibit regional variations in prevalence. They are most common in temperate regions of Europe and , where P. verrucosum dominates in storage, but incidence is notably higher in due to the extensive use of in Monascus-based fermentations. These distributions reflect climatic factors and agricultural practices that support fungal proliferation. Non-fungal sources of citrinin are negligible in natural settings.

Exposure and Detection

Presence in Food and Feed

Citrinin contamination is prevalent in various food and feed commodities, particularly those of plant origin. In cereals such as wheat and barley, levels can reach up to 1–3 mg/kg in moldy samples, though mean concentrations in surveyed grains are typically below 10 μg/kg. Fruits like apples and grapes, as well as dairy products including cheese, have also been reported as matrices for citrinin presence, often at trace levels up to several hundred μg/kg. Red yeast rice supplements exhibit notably higher contamination, with levels up to 10 mg/kg or more in some products. Co-occurrence of citrinin with other mycotoxins, such as in grains and in fruits, is common, observed in 20–50% of contaminated samples depending on the commodity and region. This frequent association arises from shared fungal producers like species during storage. Global surveys, including (EFSA) data from 2012 onward, indicate mean citrinin levels below 10 μg/kg in grains intended for human consumption, with higher concentrations up to 998 μg/kg reported in stored . A 2025 study analyzing 70 food samples, including spices, , dried fruits, vegetables, and nuts, detected citrinin in 71% of samples. These findings highlight grains and grain-based products as primary sources, though occurrence data remains limited for comprehensive exposure modeling. Key factors influencing citrinin contamination include harvest exceeding 14%, which promotes fungal growth, and improper storage conditions such as elevated and . Seasonal variations contribute, with higher detections often linked to autumn harvests in temperate regions due to increased post-harvest exposure. As of 2025, is exacerbating contamination, including citrinin, in grains through altered precipitation and patterns that favor fungal proliferation during cultivation and storage.

Human Exposure Assessment

The primary route of human exposure to citrinin is dietary, accounting for the vast majority of intake through contaminated grains, cereals, fruits, and dietary supplements such as products, while inhalation and dermal exposures are considered negligible in most scenarios. This dietary pathway predominates due to citrinin's production by fungi in stored agricultural commodities, leading to widespread in the food supply chain. Mean dietary intake estimates for in range from 0.6 to 16.5 ng/kg body weight per day across various population groups, based on occurrence data and consumption patterns assessed by the (EFSA) and subsequent studies. In , exposure levels are generally comparable but can reach up to 5 ng/kg body weight per day from consumption in certain regions, with worst-case scenarios for children exceeding 187 ng/kg body weight per day due to higher in staple foods. These estimates highlight regional variations driven by dietary habits and agricultural practices. High-risk groups for citrinin exposure include infants, who may ingest higher relative amounts through contaminated and cereal-based foods; vegetarians and vegans relying heavily on grains and plant-based products; and consumers of supplements, where citrinin contamination can exceed regulatory limits, leading to elevated intake. efforts using urinary biomarkers, such as citrinin itself and its dihydrocitrinone (DH-CIT), reveal detectable levels in the general population, with median urinary citrinin concentrations around 0.08–0.15 μg/L and DH-CIT up to 1.13 μg/L in European cohorts, indicating low but ubiquitous exposure.

Analytical Methods

Sample preparation for citrinin analysis typically involves extraction from food matrices using a mixture of and water, often in a ratio of 84:16 (v/v), followed by to separate the supernatant. This solvent combination effectively disrupts matrix interferences and solubilizes the polar citrinin molecule. Cleanup is commonly achieved through (SPE) columns, such as immunoaffinity or molecularly imprinted polymer-based cartridges, which selectively retain citrinin while removing co-extractants like pigments and , improving overall method . High-performance liquid chromatography (HPLC) coupled with (UV) or (FLD) detection serves as a standard confirmatory method for citrinin quantification, leveraging its strong at 220–340 nm and native at excitation/emission wavelengths of 331/501 nm. These techniques achieve limits of detection (LOD) around 0.1 μg/kg in various food samples, with linearity up to several hundred μg/kg and recoveries typically exceeding 90%. For enhanced specificity and multi-mycotoxin analysis, liquid chromatography-tandem (LC-MS/MS) is preferred, offering LODs as low as 0.05 μg/kg through multiple reaction monitoring of precursor ions at m/z 251 and product ions at m/z 205 and 233. This method provides superior selectivity in complex matrices, with trueness values between 85% and 110% across fortified levels from 0.5 to 200 μg/kg. Immunoassays, particularly enzyme-linked immunosorbent assays () kits, enable rapid screening of citrinin in field or high-throughput settings, with sensitivities reaching approximately 1 μg/kg and quantification limits of 5–6 μg/kg in grains and feeds. These antibody-based kits exhibit cross-reactivity primarily with citrinin (100%) and minimal interference from related mycotoxins like (<5%), allowing semi-quantitative results within 45–60 minutes without extensive sample cleanup. While suitable for initial triage, ELISA results often require chromatographic confirmation due to potential matrix effects. Recent advances in citrinin detection include nontarget screening approaches using high-resolution mass spectrometry (HRMS) integrated with machine learning algorithms for multi-mycotoxin profiling in foods, as demonstrated in 2024–2025 studies on cereals and nuts. These methods employ unsupervised clustering and supervised models, such as random forests, to predict retention times and identify unknown citrinin congeners or degradation products from HRMS data, achieving detection rates >95% for low-level contaminants (0.1–10 μg/kg) without prior standards. Such innovations facilitate comprehensive risk assessment in diverse matrices like fruits and supplements. Validation of these analytical methods adheres to reference protocols, such as CEN standard EN 17203:2021 for LC-MS/MS determination of citrinin in cereals and , ensuring accuracy >95% through inter-laboratory proficiency tests with relative standard deviations <15%. These standards mandate ruggedness testing for stability and recovery efficiency, confirming method reliability for at maximum limits of 100 μg/kg in supplements.

Metabolism

Metabolic Pathways in Mammals

Citrinin is rapidly absorbed from the in mammals, with oral ranging from 37% to 44% in pigs and complete absorption (113–131%) in chickens, though intervention trial indicate partial absorption with a gastrointestinal absorption fraction of 0.25 (90% CI: 0.09–0.65). In rats, leads to quick distribution to plasma, liver, , and other tissues, indicating efficient uptake even if oral routes show species variation. Phase I metabolism of citrinin primarily occurs in the liver and kidney via enzymes, involving and reduction. CYP3A4 catalyzes to form metabolites such as 3-hydroxy-citrinin, while other isoforms like , , and produce additional hydroxylated and dehydrogenated forms, including 5-hydroxymethyl-citrinin and 7-hydroxy-citrinin. Reduction to dihydrocitrinone represents a major pathway, observed in both pigs and humans, where it constitutes up to 73% of the area under the curve for citrinin exposure in some species. The proceeds as follows: citrinin undergoes enzymatic reduction to dihydrocitrinone, followed by further CYP450-mediated to more polar forms that facilitate . These phase I reactions enhance for subsequent elimination, primarily through renal routes. Phase II conjugation, including and sulfation, occurs to a limited extent in mammals, with recent human data showing approximately 6% of the dose as conjugates; polar metabolites in and are primarily phase I products. Overall kinetics reveal a biphasic elimination profile, with an initial of approximately 2 hours in rats and around 9 hours in humans, dominated by renal clearance where 74% of the dose is excreted in within 24 hours in rats.

Key Metabolites and Excretion

Citrinin undergoes metabolic transformation primarily to dihydrocitrinone (DHC), its major metabolite, which results from the reduction of the C4-C5 in the pyran ring. This reduction represents a key step, as DHC exhibits significantly lower compared to the parent compound, with values of 200–320 μM versus 62–70 μM in renal cell lines. Minor metabolites include 3-hydroxy-citrinin, 5-hydroxymethyl-citrinin, and 4,5-ene-citrinin, formed via cytochrome P450-mediated and dehydrogenation in liver microsomes. In mammals, excretion is primarily renal; in rats, approximately 80% of an administered dose of citrinin is excreted in within 24 hours (primarily as phase I metabolites such as DHC and unchanged citrinin, with minor conjugates), while fecal elimination accounts for approximately 20%. In humans, urinary predominates, with cumulative excretion of citrinin plus DHC reaching 33–71% within 24 hours. In human studies from the , DHC has been detected in adult samples from German cohorts at concentrations ranging from 0.04 to 7.44 ng/mL (0.04–7.44 μg/L), indicating widespread low-level exposure and efficient .

Toxicity

Acute Toxicity Profiles

Citrinin exhibits moderate acute toxicity in rodents, with oral LD50 values ranging from 50 mg/kg body weight in rats to 112 mg/kg in mice. Subcutaneous and intraperitoneal LD50 values are generally lower, falling between 35 and 89 mg/kg across rats, mice, guinea pigs, and rabbits. Intravenous administration results in even lower lethality thresholds, with reported LD50 values of 19 mg/kg in rabbits, highlighting the compound's rapid systemic effects via direct bloodstream exposure. In single-dose studies, acute exposure to citrinin in manifests as gastrointestinal distress, including and , accompanied by and progressive weakness within hours of administration. Higher doses lead to rapid onset of renal dysfunction, culminating in , , and death primarily from acute renal failure, typically within 48-72 hours. These effects underscore citrinin's primary targeting of the kidneys even in short-term exposures. No-observed-adverse-effect levels (NOAELs) for single oral doses in models range from 1 to 5 mg/kg body weight, based on the absence of clinical signs or histopathological changes at these thresholds in assays. Doses exceeding this range elicit dose-dependent increases in mortality and renal biomarkers, establishing a steep dose-response curve for citrinin's nephrotoxic potential. Human cases of are rare and typically result from accidental ingestion of heavily contaminated foodstuffs, with reported symptoms including severe , , and at estimated doses above 1 mg/kg body weight. Such incidents have been linked to mycotoxin-contaminated grains or fermented products, though definitive attribution to citrinin alone is challenging due to frequent with other mycotoxins. As of 2025, no new reports of acute incidents involving citrinin have emerged, but experimental models continue to confirm its short-term toxic profile, with ongoing studies from 2024-2025 reinforcing the LD50 and symptomatic data from earlier .

Nephrotoxicity and Other Organ Effects

Citrinin exerts pronounced effects, primarily targeting the proximal tubules of the , where it induces degeneration and of the tubular epithelium. In rats, histopathological examinations reveal swelling, , and eventual in the proximal convoluted tubules following oral or parenteral administration, with effects observable within days of exposure. serves as a key of this renal damage, with studies in rats showing a significant increase in urinary protein levels (from <2+ to 3+) within 48 hours of citrinin dosing, reflecting impaired tubular reabsorption and barrier function. The derived a chronic (NOAEL) of 20 µg/kg body weight per day from a 90-day oral study in rats, with no renal histopathological changes at this dose. Based on this NOAEL and an uncertainty factor of 100 to account for inter- and intraspecies differences, EFSA established a level of no concern for of 0.2 µg/kg body weight per day. Hepatotoxicity from citrinin exposure is less severe than but involves disruptions to liver function and structure. In mice, doses exceeding 10 mg/kg body weight lead to elevated serum levels of (ALT) and aspartate aminotransferase (AST), indicative of hepatocellular injury and . These enzyme elevations correlate with histopathological findings of swelling and inflammatory infiltration. Links to fatty liver have been suggested in broader toxicological profiles, where citrinin exposure contributes to accumulation and metabolic disturbances in hepatic tissue. Effects on other organs include and . In animal models, citrinin at doses of 5–20 mg/kg body weight has been associated with cardiac arrhythmias and structural changes, such as pericardial observed in embryos at equivalent concentrations of 50 µM. manifests as reduced , with male mice exposed to 10 mg/kg body weight showing decreased sperm count, impaired , and lower rates in mating trials. Human correlations to citrinin exposure include historical associations with , a chronic tubulointerstitial prevalent in rural areas of the ; studies detected co-occurrence of citrinin and in cereals from high-risk Bulgarian villages, supporting a potential etiological role in this nephropathy.

and Carcinogenicity

Citrinin exhibits controversial genotoxic potential, with results varying across assays. In bacterial mutagenicity tests, such as the using typhimurium strains, citrinin is generally negative both with and without metabolic activation (S9 mix), though one study reported positive results specifically when using rat hepatocytes as the activation system. In mammalian cells, citrinin induces chromosomal aberrations, including in Chinese hamster ovary (CHO) cells and V79 cells following metabolic activation by rat or human liver microsomes, as well as sister chromatid exchanges in Hep3B cells at picomolar concentrations. These effects occur indirectly, primarily through the generation of (ROS), which aligns with observations in the broader mechanisms of cellular . , the no-observed-effect level (NOEL) for genotoxicity is below 30 μM, as micronuclei formation was induced at concentrations of 30 μM or higher in V79 cells. A 2024 review by the Committee on Toxicity confirms these mixed genotoxicity findings with limited new evidence altering the profile. In vivo genotoxicity studies show mixed outcomes. Oral administration of citrinin to mice at doses of 5–20 mg/kg body weight per day for 8 weeks induced chromosomal abnormalities and hypodiploidy in cells. Conversely, in rats dosed at 20–40 mg/kg body weight for 2–28 days, no genotoxic effects were observed in assays for mutations, DNA strand breaks (), or micronuclei. Mechanisms underlying these genotoxic effects involve minimal direct formation by citrinin alone, with oxidative damage to DNA being the predominant pathway, as evidenced by increased and in exposed cells. Regarding carcinogenicity, citrinin administered at 0.1% in the diet (approximately 70 mg/kg body weight per day) to male F344 s for 80 weeks resulted in renal adenomas in 73% of surviving animals after 40 weeks, with progressive histopathological changes suggesting potential for malignant progression. The International Agency for Research on Cancer (IARC) classifies citrinin as Group 3, not classifiable as to its carcinogenicity to humans, due to limited evidence in experimental animals and inadequate data in humans. A 2024 review by the on Toxicity highlights limited overall evidence for carcinogenicity, noting proliferative effects in rat kidneys at 20–40 mg/kg body weight for 28 days, including increased PCNA-positive cells and upregulation of genes. Synergistic effects with (OTA) exacerbate risks, as combined exposure (0.75 mg/kg OTA + 15 mg/kg citrinin in feed for 60 days) in rabbits induced enhanced renal and beyond individual effects. The (EFSA) concludes that concerns for and carcinogenicity cannot be excluded at exposures below the nephrotoxicity NOAEL of 20 μg/kg body weight per day.

Mechanisms of Action

Cellular and Oxidative Stress Effects

Citrinin induces in cellular systems primarily through the generation of (ROS), which disrupts normal cellular function, particularly in renal cells. Studies have shown that exposure to citrinin leads to a rapid increase in ROS levels, including (H₂O₂) and anions, in renal epithelial cells such as HK-2 human cells. This ROS accumulation occurs within hours of exposure and is linked to interference with the mitochondrial respiratory chain, where citrinin promotes electron leakage and production. Endoplasmic reticulum (ER) stress represents an upstream mechanism contributing to citrinin's oxidative effects, as it promotes ROS generation, mitochondrial dysfunction, and in renal cells. As of 2024, ER stress activation by citrinin has been linked to renal tubule damage and dysfunction in cellular models. Mitochondrial dysfunction is a key mechanism underlying citrinin's oxidative effects, as the inhibits the activity of respiratory chain complexes I and III, leading to uncoupled respiration and reduced ATP production. In rat renal cortical mitochondria, citrinin decreases the transmembrane potential (ΔΨm) and phosphorylation efficiency at concentrations of 10–50 μM, contributing to further ROS generation via disruption. These changes result in a dose-dependent drop in ΔΨm, exacerbating oxidative damage in affected cells. As a cellular response to citrinin-induced , upregulation of defense pathways occurs, including activation of the and increased expression of genes such as (mitochondrial ) and GRE2 (a homolog). In models, which mirror mammalian stress responses, citrinin rapidly upregulates these promoters in a dose-dependent manner at 50–400 ppm, enhancing enzymatic activities to counteract ROS. Similar Nrf2-mediated responses have been observed in mammalian renal cells, helping to mitigate initial oxidative insults but often overwhelmed at higher doses. The cytotoxicity of citrinin exhibits dose-dependency, with an IC50 of approximately 70 μM in MDCK (Madin-Darby canine kidney) cells after 24 hours exposure, reflecting impaired cell viability due to oxidative stress.

Apoptosis and Cell Viability Impacts

Citrinin triggers apoptosis primarily through the intrinsic mitochondrial pathway, involving upregulation of the pro-apoptotic protein Bax and downregulation of the anti-apoptotic protein Bcl-2, which elevates the Bax/Bcl-2 ratio and promotes cytochrome c release from mitochondria to the cytosol. This mechanism is evident in embryonic stem cells exposed to citrinin concentrations exceeding 25 μM, such as 30 μM for 24 hours, leading to subsequent activation of downstream caspases. In addition to apoptotic signaling, citrinin induces arrest at the /M phase in fibroblasts, driven by activation that halts progression to prevent propagation of damaged cells. This arrest is observed following topical exposure to citrinin in mouse models, where expression is significantly elevated alongside accumulation of cells in the /M compartment. Citrinin markedly reduces cell viability, as demonstrated by MTT assays in human hepatocytes ( cells), where exposure to 30 μM for 24 hours results in approximately 50% inhibition of metabolic activity. The execution of apoptosis involves time-dependent activation of caspase-3, which peaks at 24 hours post-exposure in affected cells. Upstream (ROS) generation contributes to these apoptotic and viability impacts. Recent studies in yeast models, such as fission yeast (), confirm that citrinin-induced ROS-dependent mechanisms underlie G2/M cell cycle arrest and subsequent processes.

Immune Response Modulation

Citrinin exhibits immunotoxic effects by inhibiting the production of key cytokines in T-cells, particularly those associated with Th1 responses. In human peripheral blood mononuclear cells stimulated with , citrinin at a concentration of 8.3 μg/mL (approximately 33 μM) reduced interferon-γ (IFN-γ) secretion by 50%, with less pronounced effects on interleukin-4 (IL-4), suggesting a preferential suppression of Th1-mediated immunity. This selective inhibition contributes to a Th1/Th2 imbalance, potentially shifting immune responses toward Th2 dominance and impairing cell-mediated defenses. In splenocytes, citrinin modulates proliferation and cell populations in a dose-dependent manner. in mice led to decreased numbers of macrophages (F4/80+) and B cells (+) in the , alongside increased concanavalin A-induced proliferation of splenocytes, indicating complex immunoregulatory activity rather than uniform suppression. Although some studies report enhanced proliferative responses, higher exposures may indirectly limit overall splenocyte function through induction via altered Bax/ ratios. Citrinin impairs function, notably by reducing and (NO) production. In murine s exposed to citrinin prior to infection with , infectivity increased to 77.5% compared to 59% in controls, implying diminished phagocytic clearance or intracellular killing. Similarly, in RAW 264.7 cells, citrinin suppresses lipopolysaccharide-induced NO and inducible (iNOS) expression, critical for activity, at concentrations of 3–25 μM without affecting cell viability. This inhibition likely involves suppression of signaling, as observed in related where citrinin attenuates IκB-α phosphorylation and nuclear translocation of , thereby downregulating pro-inflammatory pathways. Recent reviews highlight citrinin's role in elevating susceptibility through immune modulation, as mycotoxin-induced suppression of signaling and function compromises host defenses against pathogens. may contribute briefly to these effects by amplifying inflammatory dysregulation, though primary mechanisms remain - and NF-κB-centric. As of August 2025, citrinin has been shown to induce ER stress-mediated immune dysfunction in the and , further impairing lymphoid organ function and contributing to overall .

Animal Studies and Interactions

Effects in Animal Models

In rodent models, citrinin administration has demonstrated pronounced nephrotoxic effects, particularly in rats. In long-term studies, male F344 rats fed a diet containing 0.1% citrinin (approximately 70 mg/kg body weight per day initially) for up to 80 weeks developed renal adenomas, with tumors observed in 72.9% of survivors beyond 40 weeks and the first tumor appearing at week 52; no such tumors occurred in controls. In mice, manifests as reduced sperm motility and count following exposure to doses of 5-20 mg/kg body weight, alongside histopathological damage to testicular tissues and decreased serum testosterone levels. exhibit sensitivity to citrinin's nephrotoxic and growth-impairing effects, with renal lesions including tubular degeneration and observed at dietary levels as low as 1 mg/kg feed, accompanied by reduced body weight gain and feed efficiency in subchronic feeding trials. In , dietary citrinin at 100 mg/kg feed induces lesions such as cortical and tubular in chickens. Citrinin also causes acute in dogs and rabbits at doses of 20-50 mg/kg body weight, leading to renal swelling and .

Coexposure with Other Mycotoxins

Citrinin (CIT) frequently co-occurs with (OTA) in contaminated grains, leading to synergistic in animal models. In male Dark rats fed diets containing 26 µg/kg OTA and 100 µg/kg CIT for three weeks, the combination resulted in a 10-fold increase in OTA-DNA adducts in tissue compared to OTA alone, indicating enhanced genotoxic potential. This interaction is attributed to CIT's inhibition of OTA , prolonging its renal accumulation and amplifying DNA damage. The mechanisms underlying CIT-OTA synergy involve shared renal uptake via organic anion transporters and amplified (ROS) production. In human HK-2 cells exposed to nanomolar concentrations (10 nM OTA + 1 nM CIT), the combination induced a 1459% increase in ERK1/2 , promoting and epithelial-to-mesenchymal transition far beyond additive effects. In models, combined oral administration of OTA (0.125-0.250 mg/kg body weight for 21 days) and CIT (20 mg/kg for 2 days) depleted glutathione levels and elevated , confirming ROS-mediated oxidative damage in kidneys and liver. Coexposure with (PAT) exhibits additive effects on in neuronal cell lines. In undifferentiated cells, a 60:1 CIT:PAT mixture (e.g., 60 µM CIT + 1 µM PAT) increased intracellular ROS by up to 132% after 120 minutes, comparable to the sum of individual exposures at 24 hours, while shifting to antagonistic at 48 hours. This suggests shared pathways of mitochondrial dysfunction and contributing to without pronounced synergy. In , low-dose combined exposure worsens renal . Subacute dosing via stomach tube (0.02 mg/kg OTA + 0.01 mg/kg CIT daily for 57 days) in pigs induced clinical mycotoxicosis, with histopathological findings of severe tubular degeneration, , and interstitial in kidneys, more pronounced than individual toxins at equivalent doses. These changes occurred at levels approximating regulatory limits (e.g., 0.05 mg/kg feed equivalent), highlighting amplified renal vulnerability. Recent multi-mycotoxin models underscore 2- to 5-fold potency increases with CIT involvement. In 2024 analyses of co-contaminated feeds, CIT-OTA mixtures in cell-based assays (e.g., MDCK renal cells) demonstrated synergistic , reducing the effective dose for 50% by 2-5 times compared to OTA alone, driven by enhanced ROS and transporter inhibition. Such interactions emphasize the need for assessing combined exposures in risk evaluations.

Regulation and Management

Regulatory Standards

In the , the maximum level for citrinin is established at 100 μg/kg in food supplements based on rice fermented with the fungus , as specified in Commission Regulation (EU) 2023/915, which replaced earlier regulations to address contamination risks in these products. No general maximum levels have been set for citrinin in other food categories, reflecting limited occurrence data and low overall dietary exposure estimates that do not warrant broader restrictions. The (EFSA) has derived a tolerable daily intake (TDI) of 0.2 μg/kg body weight for citrinin, based on a of 20 μg/kg body weight per day from a 90-day study on , applying an uncertainty factor of 100 to account for inter- and intraspecies differences. In , the maximum limit for citrinin in products is 50 μg/kg. In the United States, the (FDA) has not promulgated specific regulatory limits or action levels for citrinin in or . Instead, citrinin is addressed under broader FDA guidance for poisonous or deleterious substances, where contaminants are evaluated on a case-by-case basis during and compliance activities, with enforcement discretion applied based on health risks and exposure contexts. Japan has set a maximum permitted level of 200 μg/kg for citrinin in red fermented rice products, aimed at controlling contamination from Monascus fermentation processes commonly used in traditional foods. This standard aligns with efforts to mitigate nephrotoxic risks in staple and supplement items. The (WHO), in alignment with EFSA assessments, references a provisional TDI of 0.2 μg/kg body weight for citrinin to guide global risk management, emphasizing protection against renal effects while noting that dietary exposures in most populations remain below this threshold. The Joint FAO/WHO Expert Committee on Food Additives (JECFA) has not established a specific provisional maximum tolerable daily intake (PMTDI) for citrinin, deferring to regional authorities for regulatory implementation.

Control and Mitigation Approaches

Control and mitigation of citrinin contamination focus on preventive measures during cultivation and storage, post-harvest techniques, genetic improvements in crops and production strains, and integrated monitoring systems to minimize exposure risks in and feed chains. These strategies aim to interrupt fungal growth, degrade or the , and predict contamination hotspots, drawing from established agricultural and biotechnological practices. Prevention strategies emphasize environmental controls to inhibit citrinin-producing fungi such as and species. Maintaining grain moisture content below 14% during storage is critical, as higher levels exceeding 16% promote fungal proliferation and citrinin production in cereals like and . Chemical fungicides, such as applied to damp grains, suppress mold growth and reduce citrinin levels by altering and inhibiting toxin biosynthesis, particularly effective at concentrations of 0.5-1% in corn storage. Biological controls using , including Lactobacillus plantarum, offer a natural alternative by producing metabolites that inhibit citrinin-producing molds. Post-harvest processing methods target citrinin removal or inactivation, though their efficacy varies with food matrix and conditions. Thermal treatments alone, such as heating above 175°C in dry conditions or 100°C with , lead to partial but often result in bound residues that retain and evade detection, rendering them insufficient without complementary approaches. Adsorption using clays like is more reliable; organophilic variants bind citrinin with efficiencies approaching 99% in aqueous solutions, while standard reduces levels by approximately 50% in grain feeds by sequestering the in the of animals. Breeding programs develop crop varieties resilient to environmental stresses that exacerbate fungal infections. Drought-tolerant wheat cultivars, such as those engineered for low-water regimes, indirectly lower citrinin contamination by maintaining plant vigor under arid conditions, reducing fungal entry points and toxin production by up to 30-50% compared to susceptible lines in field trials. For fermented products like , genetic engineering of Monascus strains minimizes citrinin output. techniques targeting the ctnA or ctnE loci in the citrinin biosynthetic pathway yield strains with 78-96% reduced production, enabling safer and monacolin yields without compromising efficiency. Integration of Hazard Analysis and Critical Control Points (HACCP) ensures proactive monitoring, with critical controls at where visual fungal assessment and rapid testing identify contamination risks early. Recent 2025 advancements incorporate AI-driven predictive modeling, using algorithms on climatic and agronomic data to forecast citrinin hotspots in cereals with over 85% accuracy, allowing targeted interventions like adjusted timing.

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