Penicilloneines A and B, Quinolone-Citrinin Hybrids from a Starfish-Derived Penicillium sp.

Penicilloneines A (1) and B (2) are the first reported quinolone-citrinin hybrids. They were isolated from the starfish-derived fungus Penicillium sp. GGF16-1-2, and their structures were elucidated using spectroscopic, chemical, computational, and single-crystal X-ray diffraction methods. Penicilloneines A (1) and B (2) share a common 4-hydroxy-1-methyl-2(1H)-quinolone unit; however, they differ in terms of citrinin moieties, and these two units are linked via a methylene bridge. Penicilloneines A (1) and B (2) exhibited antifungal activities against Colletotrichum gloeosporioides, with lethal concentration 50 values of 0.02 and 1.51 µg/mL, respectively. A mechanistic study revealed that 1 could inhibit cell growth and promote cell vacuolization and consequent disruption of the fungal cell walls via upregulating nutrient-related hydrolase genes, including putative hydrolase, acetylcholinesterase, glycosyl hydrolase, leucine aminopeptidase, lipase, and beta-galactosidase, and downregulating their synthase genes 3-carboxymuconate cyclase, pyruvate decarboxylase, phosphoketolase, and oxalate decarboxylase.

Nucleic acid demethylase MpAlkB1 regulates the growth, development, and secondary metabolite biosynthesis in Monascus purpureus.

Nucleic acid demethylases of α-ketoglutarate-dependent dioxygenase (AlkB) family can reversibly erase methyl adducts from nucleobases, thus dynamically regulating the methylation status of DNA/RNA and playing critical roles in multiple cellular processes. But little is known about AlkB demethylases in filamentous fungi so far. The present study reports that Monascus purpureus genomes contain a total of five MpAlkB genes. The MpAlkB1 gene was disrupted and complemented through homologous recombination strategy to analyze its biological functions in M. purpureus. MpAlkB1 knockout significantly accelerated the growth of strain, increased biomass, promoted sporulation and cleistothecia development, reduced the content of Monascus pigments (Mps), and strongly inhibited citrinin biosynthesis. The downregulated expression of the global regulator gene LaeA, and genes of Mps biosynthesis gene cluster (BGC) or citrinin BGC in MpAlkB1 disruption strain supported the pleiotropic trait changes caused by MpAlkB1 deletion. These results indicate that MpAlkB1-mediated demethylation of nucleic acid plays important roles in regulating the growth and development, and secondary metabolism in Monascus spp.

Citrisorbicillinol, an undescribed hybrid sorbicillinoid with osteogenic activity from Penicillium citrinum ZY-2.

Citrisorbicillinol (1), along with six other known compounds (2-7), was isolated from an endphyte Penicillium citrinum ZY-2 of Plantago asiatica L. Citrisorbicillinol (1) was characterized as a skeletally unprecedented hybrid sorbicillinoid, and its unique framework is likely formed by intermolecular [4 + 2] cycloaddition between intermediates derived from citrinin and sorbicillinoid biosynthetic gene clusters. Compounds 1 and 2 demonstrated to promote osteoblastic differentiation in MC3T3-E1 cells, and to be osteogenic in the prednisolone induced osteoporotic zebrafish. Compounds 3-7 exhibited moderate cytotoxicity against four human cancer cell lines.

Five previously undescribed citrinin derivatives from the endophytic fungus Penicillium citrinum GZWMJZ-836.

Penicillium citrinum GZWMJZ-836 is an endophytic fungus from Drynaria roosii Nakaike. Five previously undescribed citrinin derivatives (1-5) and six intermediates related to their biosynthesis (6-11) were obtained from the extract of this strain's solid fermentation using multiple column chromatography separations, including high-performance liquid chromatography. The structures of these compounds were determined through comprehensive spectroscopic analyses, primarily using NMR and HRESIMS data. The stereochemistry was mainly confirmed by ECD calculations, and the configurations of C-7' in compounds 4 and 5 were determined using 13C NMR calculations. Compounds 4-5 and 8 showed antibacterial activity against five strains, with minimum inhibitory concentration values ranging from 7.8 to 125 µM. Compounds 4 and 7 exhibited inhibitions against three plant pathogenic fungi, with IC50 values ranging from 66.6 to 152.1 µM. Additionally, a putative biosynthetic pathway for compounds 1-5 derived from citrinin was proposed.

Study of cytotoxicity in neuroblastoma cell line exposed to patulin and citrinin.

Mycotoxins can be found in food and feed storage as well as in several kinds of foodstuff and are capable of harming mammals and some of them even in small doses. This study investigated on the undifferentiated neuronal cell line SH-SY5Y the effects of two mycotoxins: patulin (PAT) and citrinin (CTN), which are predominantly produced by fungi species Penicillium and Aspergillus. Here, the individual and combined cytotoxicity of PAT and CTN was investigated using the cytotoxic assay MTT. Our findings indicate that after 24 h of treatment, the IC50 value for PAT is 2.01 µM, which decreases at 1.5 µM after 48 h. In contrast, CTN did not attain an IC50 value at the tested concentration. Therefore, we found PAT to be the more toxic compared to CTN. However, the combined treatment suggests an additive toxic effect. With 2,7-dichlorodihydrofluorescin diacetate (DCFH-DA) DCFH-DA assay, ROS generation was demonstrated after CTN treatment, but PAT showed only small changes. The mixture presented a very constant behavior over time. Finally, the median-effect/combination index (CI-) isobologram equation demonstrated an additive effect after 24 h, but an antagonistic effect after 48 h for the interaction of the two mycotoxins.

Occurrence, Risk Implications, Prevention and Control of CIT in Monascus Cheese: A Review.

Monascus is a filamentous fungus that has been used in the food and pharmaceutical industries. When used as an auxiliary fermenting agent in the manufacturing of cheese, Monascus cheese is obtained. Citrinin (CIT) is a well-known hepatorenal toxin produced by Monascus that can harm the kidneys structurally and functionally and is frequently found in foods. However, CIT contamination in Monascus cheese is exacerbated by the metabolic ability of Monascus to product CIT, which is not lost during fermentation, and by the threat of contamination by Penicillium spp. that may be introduced during production and processing. Considering the safety of consumption and subsequent industrial development, the CIT contamination of Monascus cheese products needs to be addressed. This review aimed to examine its occurrence in Monascus cheese, risk implications, traditional control strategies, and new research advances in prevention and control to guide the application of biotechnology in the control of CIT contamination, providing more possibilities for the application of Monascus in the cheese industry.

Insight into selenium biofortification and the selenite metabolic mechanism of Monascus ruber M7.

Monascus species are functional fermentation fungi with great potential for selenium (Se) supplementation. This study investigated the effects of Se bio-fortification on the growth, morphology, and biosynthesis of Monascus ruber M7. The results demonstrated a significant increase in the yield of orange and red Monascus pigments (MPs) in red yeast rice (RYR) by 38.52% and 36.57%, respectively, under 20 µg/mL of selenite pressure. Meanwhile, the production of citrinin (CIT), a mycotoxin, decreased from 244.47 µg/g to 175.01 µg/g. Transcriptome analysis revealed significant upregulation of twelve genes involved in MPs biosynthesis, specifically MpigE, MpigF, and MpigN, and downregulation of four genes (mrr3, mrr4, mrr7, and mrr8) associated with CIT biosynthesis. Additionally, three genes encoding cysteine synthase cysK (Log2FC = 1.6), methionine synthase metH (Log2FC = 2.2), and methionyl-tRNA synthetase metG (Log2FC = 1.8) in selenocompound metabolism showed significantly upregulated. These findings provide insights into Se biotransformation and metabolism in filamentous fungi.

Development and validation of an analytical method for determination of citrinin in red rice and red yeast rice-based food supplements by ultra-high performance liquid chromatography tandem mass spectrometry.

Citrinin is a hepato-nephrotoxic mycotoxin produced by fungal species. The Monascus purpureus fungus plays a crucial role in the fermentation of red rice to produce red yeast rice-based food supplements, which represent the primary source of human exposure to citrinin. In this study, a simple and sensitive analytical method was successfully developed and validated for the citrinin determination in these products. The extraction process involved a QuEChERS (Quick, Easy, Cheap, Effective, Rugged, and Safe) step and citrinin determination by ultra high-performance liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS/MS). The proposed method provided satisfactory linearity, percentage of recovery from 82 to 104% with relative standard deviations (RSD) lower than 14%, and limits of detection and quantification of 0.07 µg/Kg and 0.24 µg/kg, respectively. Among the 14 samples analyzed, citrinin was found in two red rice samples (0.24 and 0.46 µg/kg) and in six food supplements (from 0.44 to 87 µg/kg).

Action of the fungal compound citrinin, a bioherbicide candidate, on photosystem II.

BACKGROUND: Bioherbicides are becoming more attractive as safe weed control tools towards sustainable agriculture. Natural products constitute an important source chemicals and chemical leads for discovery and development of novel pesticide target sites. Citrinin is a bioactive compound produced by fungi of the genera Penicillium and Aspergillus. However, its physiological-biochemical mechanism as a phytotoxin remains unclear. RESULTS: Citrinin causes visible leaf lesions on Ageratina adenophora similar to those produced by the commercial herbicide bromoxynil. Phytotoxicity bioassay tests using 24 plant species confirmed that citrinin has a broad activity spectrum and therefore has potential as a bioherbicide. Based on chlorophyll fluorescence studies, citrinin mainly blocks PSII electron flow beyond plastoquinone QA at the acceptor side, resulting in the inactivation of PSII reaction centers. Furthermore, molecular modeling of citrinin docking to the A. adenophora D1 protein suggests that it binds to the plastoquinone QB site by a hydrogen bond between the O1 hydroxy oxygen atom of citrinin and the histidine 215 of the D1 protein, the same way as classical phenolic PSII herbicides do. Finally, 32 new citrinin derivatives were designed and sorted according to free energies on the basis of the molecular model of an interaction between the citrinin molecule and the D1 protein. Five of the modeled compounds had much higher ligand binding affinity within the D1 protein compared with lead compound citrinin. CONCLUSION: Citrinin is a novel natural PSII inhibitor that has the potential to be developed into a bioherbicide or utilized as a lead compound for discovery of new derivatives with high herbicidal potency. © 2023 Society of Chemical Industry.

Research on pear residue dietary fiber and Monascus pigments extracted through liquid fermentation.

Pear residue, a byproduct of pear juice extraction, is rich in soluble sugar, vitamins, minerals, and cellulose. This study utilized Monascus anka in liquid fermentation to extract dietary fiber (DF) from pear residue, and the structural and functional characteristics of the DF were analyzed. Soluble DF (SDF) content was increased from 7.9/100 g to 12.6 g/100 g, with a reduction of average particle size from 532.4 to 383.0 nm by fermenting with M. anka. Scanning electron microscopy and infrared spectroscopic analysis revealed more porous and looser structures in Monascus pear residue DF (MPDF). Water-, oil-holding, and swelling capacities of MPDF were also enhanced. UV-visible spectral analysis showed that the yield of yellow pigment in Monascus pear residue fermentation broth (MPFB) was slightly higher than that in the Monascus blank control fermentation broth. The citrinin content in MPFB and M. anka seed broth was 0.90 and 0.98 ug/mL, respectively. Therefore, liquid fermentation with M. anka improved the structural and functional properties of MPDF, suggesting its potential as a functional ingredient in food.

Citrinin Provoke DNA Damage and Cell-Cycle Arrest Related to Chk2 and FANCD2 Checkpoint Proteins in Hepatocellular and Adenocarcinoma Cell Lines.

Citrinin (CIT), a polyketide mycotoxin produced by Penicillium, Aspergillus, and Monascus species, is a contaminant that has been found in various food commodities and was also detected in house dust. Several studies showed that CIT can impair the kidney, liver, heart, immune, and reproductive systems in animals by mechanisms so far not completely elucidated. In this study, we investigated the CIT mode of action on two human tumor cell lines, HepG2 (hepatocellular carcinoma) and A549 (lung adenocarcinoma). Cytotoxic concentrations were determined using an MTT proliferation assay. The genotoxic effect of sub-IC50 concentrations was investigated using the alkaline comet assay and the impact on the cell cycle using flow cytometry. Additionally, the CIT effect on the total amount and phosphorylation of two cell-cycle-checkpoint proteins, the serine/threonine kinase Chk2 and Fanconi anemia (FA) group D2 (FANCD2), was determined by the cell-based ELISA. The data were analyzed using GraphPad Prism statistical software. The CIT IC50 for HepG2 was 107.3 µM, and for A549, it was >250 µM. The results showed that sensitivity to CIT is cell-type dependent and that CIT in sub-IC50 and near IC50 induces significant DNA damage and cell-cycle arrest in the G2/M phase, which is related to the increase in total and phosphorylated Chk2 and FANCD2 checkpoint proteins in HepG2 and A549 cells.

Supressão da resposta imunitária humoral causada pela citrinina / Humoral immunosupression caused by citrinin

Avaliou-se o efeito imunotóxico causado por exposição a baixas doses de citrinina (2,5mg kg-1) em camundongos albinos expostos à micotoxina antes (n=15), durante (n=15) e após (n=15) a imunização com antígeno inerte, representado por eritrócitos de carneiro - sheep red blood cells (SRBC). Quinze camundongos foram usados como controle (não intoxicados). Sete dias após o tratamento, os animais foram sangrados e os títulos de anticorpos anti-SRBC e de complemento foram determinados. A citrinina diminuiu os títulos de anticorpos primários em todos os grupos intoxicados. A intoxicação antes e após a imunização provocou diminuição em 87,5 por cento nos títulos médios de anticorpos específicos. A exposição simultânea à imunização gerou diminuição de 75 por cento. Houve acentuada redução nos níveis de complemento circulante, detectada nos animais previamente intoxicados (93,8 por cento), ou intoxicados juntamente com a imunização (87,5 por cento).

Effect of citrinin and in association with aflatoxin B1 on the infectivity and proliferation of Toxoplasma gondii in vitro

Macrophages exposed to 10 mug/mL citrinin (CTR) or 0.01 mug CTR mixed with 0.04 mug aflatoxin B1 (AFB1) for a period of 2 h at 37ºC, were infected with 10(6) Toxoplasma gondii tachyzoites/muL. The parasites were treated with mycotoxins (2 h at 37ºC) before being added to the macrophage culture. The number of tachyzoites was quantified 2, 24, 48, 72 and 96 h after infection. During the first 2 hours, 59 percent infectivity was observed in the control. After exposure to CTR or the mixture of toxins (CTR-AFB1), macrophages were infected with 77.5 percent and 75 percent of the inoculated tachyzoites, respectively. Similarly, 72.3 percent of the cells were infected when cultured together with previously treated parasites. The treatment with CTR-AFB1 gave rise to 2.9 times more tachyzoites than the control at 72 h. An increased number of parasites was recovered from macrophages exposed to CTR after 96 h, and to CTR-AFB1 after 72 h of culture; The number of tachyzoites recovered from the supernatant was 1.94 and 2.06 times higher, respectively, than in the control (5 x 10(5) ± 0.054 /mL).

Citrininotoxinogenicity of Penicillium spp, isolated from decaying apples

A study on the occurrence of citrinin and citrinin production ability of Penicillium spp. isolated from bactericidal effect.decaying apples collected from households in Croatia was carried out. Among 100 samples of apples, 37 strains of Penicillium spp. were found, including P. expansum, P. roqueforti, P. implicatum and P. purpurogenum. Citrinin production in liquid yeast medium by 11 strains of P. expansum varied in a range of 0.07 to 9.00 mg.kg-1. Citrinin was isolated from 19 per center of apple samples in range of 0.05 to 0.24 mg.kg-1. Antimicrobial activity of isolated citrinin, evaluated through tests on Bacillus subtilis, presented inhibitory zones varying from 5 mm to 1 cm. Minimal inhibitory concentrations (MIC) were 0.0072 µg.mL-1 for bacteriostatic effect, and 0.0144 µg.mL-1 for bactericidaleefect.

Supressäo da resposta imunitária humoral causada por citrinina efeito imunotóxico da citrinina / Humoral immunoresponse discontinuance caused by citrinin

O presente trabalho foi realizado com a finalidade de avaliar o efeito imunotóxico causado por exposiçäo a baixas doses de citrinina (2,5mg.Kg(-1)). Para tanto, lotes de cinco camundongos albinos foram expostos à micotoxina antes, durante e após imunizaçäo com antígeno inerte (eritrócitos de carneiro - SRBC). Sete dias após tratamento, os animais foram sangrados e os títulos de anticorpos anti-SRBC e de complemento foram determinados, em relaçäo a um grupo controle näo intoxicado. Observou-se que a citrinina causou diminuiçäo nos títulos de anticorpos primários de todos os grupos de animais intoxicados. A intoxicaçäo antes e após à imunizaçäo provocou uma diminuiçäo nos títulos médios de anticorpos específicos equivalente a 87,5 por cento. A exposiçäo simultânea a imunizaçäo gerou uma diminuiçäo de 75 por cento. Houve, também, uma marcante reduçäo nos níveis de complemento circulante, detectada nos animais previamente intoxicados (93,75 por cento), ou intoxicados juntamente com a imunizaçäo (87,5 por cento). Os efeitos relativos da citrinina sobre populaçöes linfocitárias e sobre os processos inflamatórios e de apresentaçäo de antígenos também foram discutidos.

Introducción al tema de micotoxinas y micotoxicosis / Introduction to mycotoxins and mycotoxicoses

Se presenta un panorama general sobre las principales micotoxinas y sus efectos sobre la salud humana y animal

Ocratoxina A, aflatoxina B1 e citrina como agentes moduladores da produçäo de anticorpos em aves e mamíferos / Ochratoxin A, aflatoxin B1 and citrinin aas modulators agents of antibodies production in poultry and mammalian

O presente trabalho foi desenvolvido com o objetivo de demonstrar os efeitos causados pela associaçäo entre ocratoxina A, aflatoxina B1 e citrinina, sobre a resposta imunitária adaptativa de aves e mamíferos, a fim de determinar se estas micotoxinas poderiam levar à modulaçäo do sistema imune. A toxicidade relativa de cada micotoxina, bem como de suas associaçöes, foi determinada em células esplênicas de galinhas e camundongos, onde se observou a secreçäo primária de anticorpos (IgM) contra eritrócitos de carneiro, após quatro horas de exposiçäo às micotoxinas. Os ensaios foram realizados na presença e na ausência de soro heterólogo proveniente de animais adultos normais, a fim de determinar possíveis interaçöes entre o complemento presente no soro, com a açäo tóxica das micotoxinas. Observou-se uma notável depressäo imunológica nos grupos tratados com a associaçäo entre citrina e aflatoxina B1, porém, na presença de soro heterólogo, houve uma imunoestimulaçäo. Os resultados sugerem uma possível interferência de fatores inespecíficos, os quais estäo presentes no soro proveniente de animais adultos normais, cuja atividade poderia amenizar ou até mesmo neutralizar a açäo imunomodulatória das micotoxinas, sugerindo a possibilidade de imunoterapia.

Deficiências imunitárias por micotoxinas em camundongos C57BL/6, infectados com Toxoplasma gondii / Immunologic deficiencies for mycotoxins in mice C57BL/6, infected with Toxoplasma gondii

A adição de 0,04 mg/ml de Aflatoxina B1, 0,04mg/ml de Ocratoxina A, 0,01mg/ml de Citrinina e 0,05 mg/ml de Fumonisina B1, em combinações, sobre macrófagos murinos alteraram o número de placas hemolíticas contra eritrócitos de carneiro. A presença de soro heterólogo minimizou os efeitos imunosupressivos das micotoxinas. Macrófagos expostos às diferentes micotoxinas antes da infecção por T. gondii, permitiram investigar a infectividade relativa do protozoário sobre células intoxicadas. Todos os experimentos utilizando micotoxinas resultaram em um aumento da infecção por T. gondii.