Цитринин: Токсичность, происхождение и изучение микотоксина.

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.

Citrinin was one of the many mycotoxins discovered by H. Raistrick and A. C. Hetherington in the 1930s. 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. As a result, the interest in citrinin decreased, but there still was a lot of research. 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. 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. 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. 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.

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

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. 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.

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. Intravenous administration required the lowest dose for lethality. The LD50 is 19 mg/kg b. w. in rabbits. Intraperitoneal the LD50 is 50 mg/kg b. w. for rabbit.

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.

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.

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.

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.

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. 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.

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

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.

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.