Reviewing the Analytical Methodologies to Determine the Occurrence of Citrinin and its Major Metabolite, Dihydrocitrinone, in Human Biological Fluids.

Until now, the available data regarding citrinin (CIT) levels in food and the consumption of contaminated foods are insufficient to allow a reliable estimate of intake. Therefore, biomonitoring configuring analysis of parent compound and/or metabolites in biological fluids, such as urine or blood, is being increasingly applied in the assessment of human exposure to CIT and its metabolite, dihydrocitrinone (DH-CIT). Most studies report urinary levels lower for the parent compound when compared with DH-CIT. A high variability either in the mean levels or in the inter-individual ratios of CIT/DH-CIT between the reported studies has been found. Levels of DH-CIT in urine were reported as being comprised between three to seventeen times higher than the parent mycotoxin. In order to comply with this objective, sensitive analytical methodologies for determining biomarkers of exposure are required. Recent development of powerful analytical techniques, namely liquid chromatography coupled to mass spectrometry (LC-MS/MS) and ultra-high-performance liquid chromatography (UHPLC-MS/MS) have facilitated biomonitoring studies, mainly in urine samples. In the present work, evidence on human exposure to CIT through its occurrence and its metabolite, in biological fluids, urine and blood/plasma, in different countries, is reviewed. The analytical methodologies usually employed to evaluate trace quantities of these two molecules, are also presented. In this sense, relevant data on sampling (size and pre-treatment), extraction, cleanup and detection and quantification techniques and respective chromatographic conditions, as well as the analytical performance, are evidenced.

Comparative metabolomics reveals defence-related modification of citrinin by Penicillium citrinum within a synthetic Penicillium-Pseudomonas community.

Co-occurring microorganisms have been proved to influence the performance of each other by metabolic means in nature. Here we generated a synthetic fungal-bacterial community comprising Penicillium citrinum and Pseudomonas aeruginosa employing the previously described membrane-separated co-culture device. By applying a newly designed molecular networking routine, new citrinin-related metabolites induced by the fungal-bacterial cross-talk were unveiled in trace amounts. A mechanically cycled co-culture setup with external pumping forces accelerating the chemically interspecies communication was then developed to boost the production of cross-talk-induced metabolites. Multivariate data analysis combined with molecular networking revealed the accumulation of a pair of co-culture-induced molecules whose productions were positively correlated to the exchange rate in the new co-cultures, facilitating the discovery of the previously undescribed antibiotic citrinolide with a novel skeleton. This highly oxidized citrinin adduct showed significantly enhanced antibiotic property against the partner strain P. aeruginosa than its precursor citrinin, suggesting a role in the microbial competition. Thus, we propose competitive-advantage-oriented structural modification driven by microbial defence response mechanism in the interspecies cross-talk might be a promising approach in the search for novel antibiotics. Besides, this study highlights the utility of MS-based metabolomics as an effective tool in the direct biochemical analysis of the community metabolism.

Citrinin Monomer and Dimer Derivatives with Antibacterial and Cytotoxic Activities Isolated from the Deep Sea-Derived Fungus Penicillium citrinum NLG-S01-P1.

Two previously unreported citrinin dimer derivatives, penicitol D (1) and 1-epi-citrinin H1 (2), were isolated from the culture of a deep sea-derived fungus Penicillium citrinum NLG-S01-P1, together with 11 biogenetic related compounds (3⁻13). A plausible biogenetic pathway for compounds 2⁻4 was proposed. Their structures, including absolute configurations, were established through analysis of extensive spectroscopic data and time-dependent density functional theory (TD-DFT) ECD calculations. Compounds 1 and 2 showed antibacterial activities against methicillin-resistant Staphylococcus aureus (MRSA). Compounds 5 and 10 displayed relatively stronger activities than the other compounds against Vibrio vulnificus and Vibrio campbellii. Compound 1 showed the most potent cytotoxic activity towards the HeLa cell.

Interaction of Dihydrocitrinone with Native and Chemically Modified Cyclodextrins.

Citrinin (CIT) is a nephrotoxic mycotoxin produced by Aspergillus, Penicillium, and Monascus genera. It appears as a contaminant in grains, fruits, and spices. After oral exposure to CIT, its major urinary metabolite, dihydrocitrinone (DHC) is formed, which can be detected in human urine and blood samples. Cyclodextrins (CDs) are ring-shaped molecules built up from glucose units. CDs can form host-guest type complexes with several compounds, including mycotoxins. In this study, the complex formation of DHC with native and chemically modified beta- and gamma-cyclodextrins was tested at a wide pH range, employing steady-state fluorescence spectroscopic and modeling studies. The weakly acidic environment favors the formation of DHC-CD complexes. Among the CDs tested, the quaternary-ammonium-γ-cyclodextrin (QAGCD) formed the most stable complexes with DHC. However, the quaternary-ammonium-ß-cyclodextrin (QABCD) induced the strongest enhancement in the fluorescence signal of DHC. Our results show that some of the chemically modified CDs are able to form stable complexes with DHC (logK = 3.2-3.4) and the complex formation can produce even a 20-fold increase in the fluorescence signal of DHC. Considering the above-listed observations, CD technology may be a promising tool to increase the sensitivity of the fluorescence detection of DHC.

Two new compounds from a marine-derived Penicillium griseofulvum T21-03.

A new phenolic acid compound, 46-dimethylcurvulinic acid (1) and a new citrinin monomer derivative penicitrinol P (2) were isolated from marine-derived Penicillium griseofulvum T21-03. The structures of 1 and 2 were elucidated on the basis of spectroscopic data.

Urinary biomarkers of ochratoxin A and citrinin exposure in two Bangladeshi cohorts: follow-up study on regional and seasonal influences.

Biomonitoring studies can provide valuable insights into human mycotoxin exposure, especially when food contaminant data are scarce or unavailable as in Bangladesh. First biomonitoring data in Bangladeshi adults indicated exposure to the nephrotoxic mycotoxins ochratoxin A (OTA) and citrinin (CIT). This led us to conduct a follow-up study with analysis of urinary biomarkers for both CIT and OTA to investigate regional and seasonal influences on mycotoxin exposure in two Bangladeshi cohorts. In total, 164 urines were collected (n = 69 in summer, n = 95 in winter) from residents of a rural and an urban area, among which there were 62 participants enrolled in both sampling periods. Most urines had detectable biomarker levels (OTA, CIT and its metabolite dihydrocitrinone, HO-CIT), with more or less pronounced differences with regard to season and region. In both cohorts, OTA was found at a mean level of 0.06 ± 0.10 ng/mL urine (range 0.01-0.55 ng/mL) in summer and a mean of 0.19 ± 0.38 ng/mL (range 0.01-1.75 ng/mL) in winter season. A season difference was significant in the rural cohort, but not in the urban cohort, and slightly higher mean OTA levels in the rural compared to the urban cohort were only observed in winter urines. CIT biomarkers showed more pronounced variations, with a CIT mean of 0.10 ± 0.17 ng/mL (range 0.02-1.22 ng/mL) and HO-CIT mean of 0.42 ± 0.98 ng/mL (range 0.02-5.39 ng/mL) in summer, and CIT mean of 0.59 ± 0.98 ng/mL (range 0.05-5.03 ng/mL) and HO-CIT mean of 3.18 ± 8.49 ng/mL (range 0.02-46.44 ng/mL) in winter urines of both cohorts. In both seasons, total CIT biomarker concentrations were significantly higher in the rural cohort than in the urban cohort. A provisional daily intake for CIT was calculated and exceeded a preliminary value set by EFSA (0.2 µg/kg/d) in 10 and 24 % of participants in summer and winter, respectively. No significant correlations were found between urinary biomarker levels and intake of certain types of food, except for a positive trend for higher rice consumption. Our results in the Bangladeshi population indicate frequent co-exposure to nephrotoxic mycotoxin food contaminants that vary by season and region.

Penicitols A-C and penixanacid A from the mangrove-derived Penicillium chrysogenum HDN11-24.

Three new citrinin analogues, penicitols A-C (1-3), and one new xanthone derivative, penixanacid A (4), together with four known biogenetically related compounds (5-8), were discovered from the extract of a mangrove-derived fungus, Penicillium chrysogenum HND11-24. The structures of penicitols A-C and penixanacid A were established through analysis of extensive spectroscopic data. Their cytotoxic activity against HeLa, BEL-7402, HEK-293, HCT-116, and A549 cell lines was evaluated.

Citrifelins A and B, Citrinin Adducts with a Tetracyclic Framework from Cocultures of Marine-Derived Isolates of Penicillium citrinum and Beauveria felina.

Citrifelins A (1) and B (2), two citrinin adducts possessing a unique tetracyclic framework, were characterized from a coculture of marine-derived fungal isolates of Penicillium citrinum and Beauveria felina. Neither fungus produced these compounds when cultured alone under the same conditions. The structures of these adducts were elucidated on the basis of spectroscopic analysis, and the absolute configurations were assigned on the basis of TDDFT-ECD calculations. A hypothesis that adducts 1 and 2 might be derived from a citrinin derivative through a non-pericyclic Michael reaction is proposed. Compounds 1, 2, and 5 showed inhibitory activities against several human and aquatic pathogens.

Fast and sensitive LC-MS/MS method measuring human mycotoxin exposure using biomarkers in urine.

A direct, fast and sensitive LC-MS/MS method was developed to measure biomarkers for mycotoxin exposure in human urine. In total, 32 biomarkers were quantitatively or semi-quantitatively measured in 32 urine samples of Belgian volunteers using two injections. All urine samples contained deoxynivalenol-15-glucuronide, the major detoxification metabolite of deoxynivalenol, in the ng/mL range. Also deoxynivalenol-3-glucuronide and de-epoxy-deoxynivalenol-glucuronide were present in, respectively, 90 and 25% of the samples, while deoxynivalenol was detected in 60% of the samples, in lower concentrations. Deoxynivalenol glucuronides were the major biomarkers for deoxynivalenol exposure. Ochratoxin A was detected in 70% of the samples in pg/mL. Citrinin and/or dihydrocitrinone were detected in 90% of the samples, also in concentrations of pg/mL. The presence of ochratoxin A and citrinin was confirmed by a second method using sample cleanup by immunoaffinity columns, followed by LC-MS/MS. Our data show that humans are much more exposed to citrinin than realized before and suggest further work on citrinin exposure in relation with ochratoxin A exposure, as both mycotoxins are nephrotoxic.

Occurrence of the mycotoxin citrinin and its metabolite dihydrocitrinone in urines of German adults.

As data on food contamination with the mycotoxin citrinin (CIT) are scarce, a recently developed method for biomarker analysis (Blaszkewicz et al. in Arch Toxicol 87:1087-1094, 2013) was applied to investigate CIT exposure of German adults. CIT and its human metabolite dihydrocitrinone (HO-CIT) were determined in urine samples from a group of 50 healthy adults (n = 27 females and n = 23 males). After cleanup by immunoaffinity (CitriTest®) columns, extracts were analyzed by LC-MS/MS. The mycotoxin and its major metabolite HO-CIT were detected in 82 and 84 % of all urine samples, at concentrations ranging from 0.02 (limit of detection, LOD) to 0.08 ng/mL for CIT, and 0.05 (LOD) to 0.51 ng/mL for HO-CIT. Median urine analyte levels in the cohort were 0.03 (CIT) and 0.06 ng/mL (OH-CIT) or adjusted to creatinine 20.2 ng/g crea (CIT) and 60.9 ng/g crea (HO-CIT), respectively. Except for higher urinary CIT levels in males, differences between subgroups were not significant. This first biomarker analysis indicates widespread and variable exposure to CIT in German adults, and conversion of ingested mycotoxin to its less toxic metabolite HO-CIT, which may serve as biomarker of exposure in addition to the parent compound.

A novel citrinin derivative from the marine-source fungus Penicillium citrinum.

A novel citrinin derivative, penicitrinol L (1), along with two known analogues, penidicitrinin B (2) and pennicitrinone A (3) were isolated from the marine-source fungus Penicillium citrinum. The structure of the new compound was elucidated by spectroscopic methods including one and two-dimensional NMR as well as high-resolution mass spectrometric analysis. Furthermore, compound 1 showed modest cytotoxic activity against HL-60 cell line and compound 3 showed weak cytotoxic activity against A375 cell line.

Toxicity of the mycotoxin citrinin and its metabolite dihydrocitrinone and of mixtures of citrinin and ochratoxin A in vitro.

Citrinin (CIT) and ochratoxin A (OTA) are mycotoxins produced by several species of the genera Aspergillus, Penicillium and Monascus. Both can be present as contaminants in various food commodities and in animal feed. The occurrence and toxicity of OTA and human exposure have been intensively studied, but for CIT such data are scarce by comparison. Recently, dihydrocitrinone (DH-CIT) was detected as main metabolite of CIT in human urine, and co-occurrence of CIT and OTA was shown in human blood plasma (Blaszkewicz et al. in Arch Toxicol 87:1087-1094, 2013). In light of these new findings, we have now investigated the toxicity of the metabolite DH-CIT in comparison with CIT and analysed the effects of mixtures of CIT and OTA in vitro. The cytotoxic potency of DH-CIT (IC50 of 320/200 µM) was distinctly lower compared with CIT (IC50 of 70/62 µM) after treatment of V79 cells for 24 and 48 h. Whereas CIT induced a concentration-dependent increase in micronucleus frequencies at concentrations ≥30 µM, DH-CIT showed no genotoxic effect up to 300 µM. Thus, conversion of CIT to DH-CIT in humans can be regarded as a detoxification step. Mixtures of CIT and OTA exerted additive effects in cytotoxicity assays. The effect of CIT and OTA mixtures on induction of micronuclei varied dependent on the used concentrations between additive for low µM concentrations and more-than-additive for high µM concentrations. Effects on cell cycle were mostly triggered by OTA when both mycotoxins were used in combination. The implications of our and related in vitro studies are discussed with respect to in vivo concentrations of CIT and OTA, which are found in animals and in humans.

The marine fungal metabolite, dicitrinone B, induces A375 cell apoptosis through the ROS-related caspase pathway.

Dicitrinone B, a rare carbon-bridged citrinin dimer, was isolated from the marine-derived fungus, Penicillium citrinum. It was reported to have antitumor effects on tumor cells previously; however, the details of the mechanism remain unclear. In this study, we found that dicitrinone B inhibited the proliferation of multiple tumor types. Among them, the human malignant melanoma cell, A375, was confirmed to be the most sensitive. Morphologic evaluation, cell cycle arrest and apoptosis rate analysis results showed that dicitrinone B significantly induced A375 cell apoptosis. Subsequent observation of reactive oxygen species (ROS) accumulation and mitochondrial membrane potential (MMP) reduction revealed that the apoptosis induced by dicitrinone B may be triggered by over-producing ROS. Further studies indicated that the apoptosis was associated with both intrinsic and extrinsic apoptosis pathways under the regulation of Bcl-2 family proteins. Caspase-9, caspase-8 and caspase-3 were activated during the process, leading to PARP cleavage. The pan-caspase inhibitor, Z-VAD-FMK, could reverse dicitrinone B-induced apoptosis, suggesting that it is a caspase-dependent pathway. Our data for the first time showed that dicitrinone B inhibits the proliferation of tumor cells by inducing cell apoptosis. Moreover, compared with the first-line chemotherapy drug, 5-fluorouracil (5-Fu), dicitrinone B showed much more potent anticancer efficacy, suggesting that it might serve as a potential antitumor agent.

Methods for analysis of citrinin in human blood and urine.

Citrinin (CIT), produced by several Penicillium, Aspergillus, and Monascus species, has been detected as contaminant in feeds, grains, and other food commodities. CIT can co-occur with ochratoxin A (OTA), a mycotoxin also known for its nephrotoxicity, and this raises concern regarding possible combined effects. But, in contrast to OTA, data on CIT contamination in foods for human consumption are scarce, and CIT biomonitoring has not been conducted so far due a lack of suitable methods for human specimen. Thus, it was the aim of the present study to develop sensitive methods for the analysis of CIT in human blood and urine to investigate human exposure. To this end, we assessed different methods of sample preparation and instrumental analysis for these matrices. Clean-up of blood plasma by protein precipitation followed by LC-MS/MS-based analysis allowed robust detection of CIT (LOD 0.07 ng/mL, LOQ 0.15 ng/mL). For urine, sample clean-up by an immunoaffinity column (CitriTest(®)) proved to be clearly superior to SPE with RP(18) material for subsequent analysis by LC-MS/MS. For CIT and its metabolite dihydrocitrinone (HO-CIT), the LOD and LOQ determined by external calibration curves in matrix were 0.02 and 0.05 ng/mL for CIT, and those for HO-CIT were 0.05 and 0.1 ng/mL urine. The newly developed method was applied in a small pilot study: CIT was present in all plasma samples from 8 German adults, at concentrations ranging from 0.11 to 0.26 ng/mL. The molar (nM) concentrations of CIT are similar to those measured for OTA in these samples as a result of dietary mycotoxin intake. CIT was detected in 8/10 urines (from 4 adults and 6 infants) in a range of 0.16-0.79 ng/mL, and HO-CIT was present in 5/10 samples at similar concentrations. Thus, CIT is excreted in urine as parent compound and also as metabolite. These first results in humans point to the need for further studies on CIT exposure.

Four new citrinin derivatives from a marine-derived Penicillium sp. fungal strain.

Four new citrinin derivatives, including two citrinin dimers and two citrinin monomer derivatives, were isolated and identified from a marine-derived fungal strain Penicillium sp. ML226 along with six known related compounds. Their structures were elucidated by spectroscopic and chemical methods. The new compounds showed modest cytotoxic activity against HepG-2 cell line and weak antimicrobial activity against Staphylococcus aureus.

The marine natural product, dicitrinone B, induces apoptosis through autophagy blockade in breast cancer.

Being a highly conserved catabolic process, autophagy is induced by various forms of cellular stress, and its modulation has considerable potential as a cancer therapeutic approach. In the present study, it was demonstrated that dicitrinone B (DB), a rare carbon­bridged citrinin dimer, may exert anticancer effects by blocking autophagy at a late stage, without disrupting lysosomal function in MCF7 breast cancer and MDA­MB­231 triple­negative breast cancer cells. Furthermore, it was discovered that DB significantly enhanced intracellular reactive oxygen species (ROS) production and that the removal of ROS was followed by the attenuation of autophagy inhibition. In addition, DB exerted notable inhibitory effects on the proliferation and promoting effects on the apoptosis of MCF7 and MDA­MB­231 cells. In combination with conventional chemotherapeutic drugs, DB exhibited a further enhanced synergistic effect than when used as a single agent. Overall, the data of the present study demonstrate that DB may prove to be a promising autophagy inhibitor with anticancer activity against breast cancer.

Citrinin derivatives from the marine-derived fungus Penicillium citrinum.

Three new citrinin derivatives, penicitrinols C, D, and E (1-3), along with two known compounds, citrinin (4) and decarboxydihydrocitrinone (5), were isolated from Penicillium citrinum. Their structures were determined by spectroscopic methods and X-ray diffraction analysis. Compounds 1 and 3 demonstrated weak cytotoxicity against the HL-60 cell line.

Efflux at the Blood-Brain Barrier Reduces the Cerebral Exposure to Ochratoxin A, Ochratoxin α, Citrinin and Dihydrocitrinone.

Recent studies have implied that environmental toxins, such as mycotoxins, are risk factors for neurodegenerative diseases. To act directly as neurotoxins, mycotoxins need to penetrate or affect the integrity of the blood-brain barrier, which protects the mammalian brain from potentially harmful substances. As common food and feed contaminants of fungal origin, the interest in the potential neurotoxicity of ochratoxin A, citrinin and their metabolites has recently increased. Primary porcine brain capillary endothelial cells were used to investigate cytotoxic or barrier-weakening effects of ochratoxin A, ochratoxin α, citrinin and dihydrocitrinone. The transfer and transport properties of the mycotoxins across the barrier formed by porcine brain capillary endothelial cell monolayers were analysed using HPLC-MS/MS. High levels of Ochratoxin A caused cytotoxic and barrier-weakening effects, whereas ochratoxin α, citrinin and dihydrocitrinone showed no adverse effects up to 10 µM. Likely due to efflux transporter proteins, the transfer to the brain compartment was much slower than expected from their high lipophilicity. Due to their slow transfer across the blood-brain barrier, cerebral exposure of ochratoxin A, ochratoxin α, citrinin and dihydrocitrinone is low and neurotoxicity is likely to play a subordinate role in their toxicity at common physiological concentrations.

New citrinin derivatives from the deep-sea-derived fungus Cladosporium sp. SCSIO z015.

During the course of our search for novel bioactive compounds from marine fungi, four new citrinin derivatives, cladosporins A-D (1-4) were isolated from a culture broth of the deep-sea-derived fungus Cladosporium sp. SCSIO z015. Their complete structural assignments were elucidated by the extensive spectroscopic investigation. The absolute configurations of 1-3 were established by quantum chemical calculations of the electronic circular dichroism (ECD) spectra. Compounds 1-4 showed weak toxicity towards brine shrine naupalii with LC50 values of 72.0, 81.7, 49.9 and 81.4 µM, respectively. And 4 also showed significant antioxidant activity against ɑ,α-diphenyl-picrylhydrazyl (DPPH) radicals with an IC50 value of 16.4 µM.

[Secondary metabolites of mangrove endophytic fungus SK5 in the South China Sea].

OBJECTIVE: To study the secondary metabolites of mangrove endophytic fungus SK5. METHODS: The compounds were isolated by chromatographic technique. Their structures were identified by comprehensive physico-chemical properties and spectroscopic methods. RESULTS: Five compounds were isolated and identified as 2,8-dihydroxy-3,4-dihydronaphthalen-1(2H)-one(1), Sclerotinin A (2), dihydrocitrinone(3), 4-hydroxybenzaldehyde (4) and 3-hydroxy-2-methylbenzoic acid (5). CONCLUSION: Compound 1 is isolated from nature for the first time.