A Study of Carry-Over and Histopathological Effects after Chronic Dietary Intake of Citrinin in Pigs, Broiler Chickens and Laying Hens.

Citrinin (CIT) is a polyketide mycotoxin occurring in a variety of food and feedstuff, among which cereal grains are the most important contaminated source. Pigs and poultry are important livestock animals frequently exposed to mycotoxins, including CIT. Concerns are rising related to the toxic, and especially the potential nephrotoxic, properties of CIT. The purpose of this study was to clarify the histopathological effects on kidneys, liver, jejunum and duodenum of pigs, broiler chickens and laying hens receiving CIT contaminated feed. During 3 weeks, pigs (n = 16) were exposed to feed containing 1 mg CIT/kg feed or to control feed (n = 4), while 2 groups of broiler chickens and laying hens (n = 8 per group) received 0.1 mg CIT/kg feed (lower dose group) and 3 or 3.5 mg CIT/kg feed (higher dose group), respectively, or control feed (n = 4). CIT concentrations were quantified in plasma, kidneys, liver, muscle and eggs using a validated ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method. Kidneys, liver, duodenum and jejunum were evaluated histologically using light microscopy, while the kidneys were further examined using transmission electron microscopy (TEM). Histopathology did not reveal major abnormalities at the given contamination levels. However, a significant increase of swollen and degenerated mitochondria in renal cortical cells from all test groups were observed (p < 0.05). These observations could be related to oxidative stress, which is the major mechanism of CIT toxicity. Residues of CIT were detected in all collected tissues, except for muscle and egg white from layers in the lowest dose group, and egg white from layers in the highest dose group. CIT concentrations in plasma ranged between 0.1 (laying hens in lower dose group) and 20.8 ng/mL (pigs). In tissues, CIT concentrations ranged from 0.6 (muscle) to 20.3 µg/kg (liver) in pigs, while concentrations in chickens ranged from 0.1 (muscle) to 70.2 µg/kg (liver). Carry-over ratios from feed to edible tissues were between 0.1 and 2% in pigs, and between 0.1 and 6.9% in chickens, suggesting a low contribution of pig and poultry tissue-derived products towards the total dietary CIT intake for humans.

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.

Comprehensive toxicokinetic analysis reveals major interspecies differences in absorption, distribution and elimination of citrinin in pigs and broiler chickens.

A comprehensive toxicokinetic analysis of citrinin (CIT) revealed interspecies differences for all toxicokinetic parameters and in absolute oral bioavailability. Oral bioavailability for CIT was complete for broilers (113-131%), while ranging from 37 to 44% in pigs. CIT was more rapidly absorbed in pigs (Tmax = 0.92 h) compared to broiler chickens (Tmax = 7.33 h). The elimination of CIT was slower in pigs (T1/2el = 26.81 h after intravenous (IV) administration) compared to chickens (T1/2el = 1.97 h after IV administration), due to the striking difference in clearance (Cliv=9.87 mL/h/kg for pigs versus Cliv = 863.09 mL/h/kg for broilers). Also, the volume of distribution differed significantly between pigs (Vd = 0.30 L/kg after IV administration) and chickens (Vd = 2.46 L/kg after IV administration). However, plasma protein binding did not differ statistically significant (91-98%). It is imperative to further investigate biotransformation and elimination pathways in different species, including humans.

LC-MS/MS methodology for simultaneous determination of patulin and citrinin in urine and plasma applied to a pilot study in colorectal cancer patients.

Biomarker-driven research has been proposed as a successful method to assess the exposure of individuals to xenobiotics, including mycotoxins, through estimation of their metabolites in biological fluids. A methodology to determine patulin (PAT) and citrinin (CIT) in human urine and plasma using liquid chromatography coupled to tandem mass spectrometry was developed and validated in the present study. Selectivity/specificity, linearity, limit of detection and quantification, apparent recovery, intraday- and interday-precision and measurement uncertainty were investigated for validation purposes. Finally, the method was used to analyze human urine (n = 100) and plasma (n = 100) case-control samples, where 50 samples originated from colorectal cancer patients and 50 from age/sex-matched controls. This case-control study revealed that PAT was not detected in urine samples, however occurred in 25% of the analysed plasma samples with an average concentration of 11.62 ± 6.67 ng/mL in the positive samples. CIT was found in urine samples (74%) and plasma samples (36%) with average concentrations in the positive samples of 0.45 ± 0.24 ng/mL and 0.49 ± 0.2 ng/mL respectively. No statistically significant difference of PAT and CIT concentration among colorectal cancer and control patients (p > 0.05) was observed.

Citrinin biomarkers: a review of recent data and application to human exposure assessment.

The mycotoxin citrinin (CIT) deserves attention due to its known toxic effects in mammalian species and a widespread occurrence in food commodities, often along with ochratoxin A, another nephrotoxic mycotoxin. Human exposure, a key element in assessing risks related to these food contaminants, depends upon mycotoxin levels in food and on food consumption. Yet, data available for CIT levels in food are insufficient for reliable intake estimates. Now biomonitoring, i.e., analysis of parent compound and/or metabolites in human specimen (blood, urine, breast milk), is increasingly used to investigate mycotoxin exposure. Biomonitoring requires sensitive methods for determining biomarkers of exposure, combined with kinetic data to conclude on the absorbed internal dose in an individual. Recent advances in LC-MS/MS-based analytical techniques have facilitated biomonitoring studies on the occurrence of CIT biomarkers in body fluids, mainly in urine samples. This review compiles evidence on human exposure to CIT in different countries, on CIT kinetics in humans, and on biomarker-based CIT intake estimates. Human CIT exposures are discussed in light of an intake value defined as 'level of no concern for nephrotoxicity' by the European Food Safety Agency, and some uncertainties in the toxicological data base. Further studies on CIT, including biomarker-based studies are warranted along with regular food surveys for this mycotoxin to protect consumers against undesirable health effects.

Analyses of biomarkers of exposure to nephrotoxic mycotoxins in a cohort of patients with renal tumours.

The Czech Republic occupies the first place in the world in the frequency of renal and other urinary tract tumours, but their aetiology is unknown. To explore whether carcinogenic and nephrotoxic mycotoxins may contribute to kidney diseases in the Czech population, biomarkers of ochratoxin A (OTA) and citrinin (CIT) exposure were determined in biological specimens from a cohort of 50 patients with malignant renal tumours. Biomarker analyses in blood and urine samples used validated targeted methods for measuring OTA and CIT plus dihydrocitrinone (DH-CIT) after enrichment of analytes by specific immunoaffinity clean-up. OTA and CIT plus its metabolite DH-CIT were frequently detected in patient urine samples (OTA 62%; CIT 91%; DH-CIT 100%). The concentration ranges in urine were 1-27.8 ng/L for OTA, 2-87 ng/L for CIT and 2-160 ng/L for DH-CIT. The analyses of blood samples revealed also a frequent co-occurrence of OTA and CIT, in the ranges of 40-870 ng/L serum for OTA and 21-182 ng/L plasma for CIT. This first analysis of biomarkers in blood and urine samples of Czech patients revealed no major differences in comparison with published data for the general healthy Czech and European populations. Nonetheless, a frequent co-occurrence of CIT and OTA biomarkers in patient samples may be of interest with regard to potential interactions with other risk factors for renal disease.

Preliminary data on citrinin kinetics in humans and their use to estimate citrinin exposure based on biomarkers.

Citrinin (CIT), a fungal metabolite causing nephrotoxicity, has a tolerable daily intake (TDI) value of 0.2µg/kg bw. Contamination of food with CIT is not sufficiently known to allow dietary exposure assessment. Urinary biomonitoring data are available from cohorts of several countries. However, kinetic information is lacking for CIT, hampering the use of urinary biomonitoring data to estimate the daily intake. We have investigated the kinetics of CIT after oral intake in two human volunteers on two occasions. Urinary excretion showed that ingested CIT undergoes conversion to dihydro-citrinone (DH-CIT) which is then excreted in the urine along with parent compound. The cumulative urinary excretion within 24h was between 32.9% and 70.8% (median 40.2%) of the sum of CIT and DH-CIT ('total CIT'). The median half-life in urine was 6.7h for CIT and 8.9h for DH-CIT. The median half-life in plasma accounted to 9.4h. The daily urinary excretion for 'total CIT' served to estimate a provisional daily CIT intake using published urine biomarker data in several cohorts. European cohorts had an exposure well below the TDI whereas in Bangladesh the exposure in one cohort exceeded the TDI.

Blood plasma biomarkers of citrinin and ochratoxin A exposure in young adults in Bangladesh.

Citrinin (CIT) and Ochratoxin A (OTA) are nephrotoxic mycotoxins which can co-occur in food commodities, resulting in internal exposure. Studies in many countries reported on the presence of OTA in human blood; however, such biomonitoring data for CIT is still scarce. This study was conducted to characterize both CIT and OTA biomarker levels in plasma of volunteers since food analysis data are insufficient to assess human exposure in Bangladesh. In total 104 blood samples were collected from university students in 2013 (sampling 1: n = 64, midsummer) and 2014 (sampling 2: n = 40, end winter) for analysis of CIT and OTA and their metabolites HO-CIT and OTα by LC-MS/MS and HPLC-FD techniques, respectively. CIT and HO-CIT were detected in 90% (max 2.70 ng/mL) and 85% (max 1.44 ng/mL) of all samples. Mean levels in sampling 2 (CIT 0.47 ng/mL; HO-CIT 0.40 ng/mL) were higher than in sampling 1 (0.25 ng/mL; 0.37 ng/mL) indicative of variable CIT exposure. OTA was present in all (max 6.63 ng/mL) and OTα in 98% (max 0.99 ng/mL) of the samples. In sampling 1, mean OTA (0.85 ng/mL) was higher than in sampling 2 (0.51 ng/mL); the reverse situation was found for OTα mean levels. The calculated dietary OTA intake among the students (mean 9.9; max 91.7 ng/kg bw/week) was lower than the tolerable weekly intake for this mycotoxin (120 ng/kg bw/week) set by EFSA. But frequent co-exposure to CIT should be considered, and the results of this study indicate the necessity to identify major sources of CIT and OTA intake in the Bangladeshi population.

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.

Determination of citrinin in barley by indirect and direct enzyme immunoassay.

Polyclonal antibodies against the mycotoxin citrinin were raised in rabbits after immunization with citrinin conjugated to keyhole limpet hemocyanin. The antibodies were used in a competitive indirect enzyme immunoassay (EIA) with citrinin coupled to glucose oxidase as the solid-phase antigen for coating microtiter plates. Detection limits of this indirect EIA for citrinin ranged from 0.4 to 0.8 ng/mL for buffer solutions. Recoveries of citrinin added to ground barley at 100-2000 ng/g ranged from 105 to 112%, with coefficients of variation between 4.5 and 12%. A direct competitive EIA also was established, with citrinin coupled to horseradish peroxidase as the labeled antigen. Detection limits of this direct EIA for citrinin ranged from 2 to 4 ng/mL for buffer solutions. Recoveries of citrinin added to ground barley at 500-2000 ng/g ranged from 108 to 111%, with coefficients of variation between 8.4 and 26.9%. In naturally contaminated barley samples assayed with the indirect EIA, optimum extraction of citrinin was obtained in 30 min, and only one extraction was necessary to recover 72-76% of the analyte.

Subacute toxicity testing of ochratoxin A and citrinin in swine.

Fourteen pigs were fed ochratoxin A and citrinin through a stomach tube at daily doses of 0.02 and 0.01 mg/kg body mass for 57 days. These toxin doses correspond to the average toxin contamination level of feeds in Central Europe. The clinical status of the pigs was monitored and clinical laboratory, haematological and mycotoxin-analytical examinations were performed throughout the trial. At the end of the experiment gross and histopathological examinations were carried out. The results of ochratoxin A and citrinin determination in the blood, obtained by high-performance liquid chromatography (HPLC), are important from the food hygienic point of view. The sensitivity of the method was 2 and 10 ng/ml for ochratoxin A and citrinin, respectively. The recovery rate of the mycotoxins was above 60%.

Isolation and identification of dihydrocitrinone, a urinary metabolite of citrinin in rats.

Dihydrocitrinone, 3,4-dihydro-6,8-dihydroxy-3,4,5-trimethylisocoumarin-7-carboxylic acid, was isolated and identified as a urinary metabolite after oral administration of citrinin to rats. Male and female Osborne-Mendel rats received 30 mg citrinin/kg body weight by oral intubation. The metabolite dihydrocitrinone was present in urine collected at 0-2, 2-4, 4-6, 6-8, and 8-24 h after treatment. Only unchanged citrinin was found in blood collected 24 h after administration of the compound. The metabolite had a blue fluorescence and the same Rf on thin-layer chromatography, the same retention time on reverse-phase high-pressure liquid chromatography, and the same mass spectrum as an authentic sample of dihydrocitrinone.

High-performance liquid chromatographic analysis of the mycotoxin citrinin and its application to biological fluids.

Citrinin is a toxic metabolite produced by several species of Penicillium and Aspergillus. Citrinin is nephrotoxic and has been implicated in disease outbreaks in animals and humans. Citrinin was resolved in a sharp peak by reversed-phase high-performance liquid chromatography on a small-article (10 micrometers) column by elution in 4.25 min with a phosphoric acid (0.25 N)-acetonitrile-2-propanol solvent (55:35:10). Detection was by ultraviolet absorbance at 340 nm. The relationship between peak height and area and quantity injected was linear over a range of 2--50 ng at 340 nm and 5--200 ng at 365 nm. Retention time and peak area were highly reproducible. As little as 2--5 ng citrinin was detectable. Complete recovery of citrinin from plasma samples containing known quantities of [14C]citrinin was obtained over a range of 5--40 micrograms/ml by treatment of the plasma with 1 N hydrochloric acid followed by extraction with ethyl acetate. The method provides for the direct analysis of citrinin in urine and bile without prior extraction.

Distribution and excretion of [14C]citrinin in rats.

The distribution and excretion of radioactivity from [14C]citrinin (3 mg/kg, i.v) was determined in male rats. At 0.5 h after administration maximum values of 14.7% and 5.6% of total radioactivity were observed in the liver and kidneys, respectively, and by 6 h decreased to 7.5% in the liver and 4.7% in the kidney. Plasma concentration of 14C decreased from 9.2% at 0.5 h to 4.7% at 6.0 h. 2 plasma elimination rates were observed, with half-lives of 2.6 and 14.9 h, respectively. Approximately 80% of the administered 14C activity was excreted in feces and urine by 24 h after administration. A second group of rats was pretreated with 50 mg/kg of citrinin, i.p., 4 days prior to administration of 3 mg/kg [14C]citrinin, i.v. 30% of the pretreated animals died and the remaining animals were divided into 2 groups on day 4 after pretreatment; rats which were "nephrotoxic" and rats which had "recovered" from the initial insult of citrinin. Proteinuria and glucosuria as well as enhanced urine output were observed in "nephrotoxic" rats 4 days after pretreatment. 24 h after [14C]citrinin, only 13% of 14C activity was detected in the urine of "nephrotoxic" rats. The plasma disappearance curve had 2 elimination rates, with half-lives of 0.6 and 14.1 h. "Nephrotoxic" rats retained 7.5% of the administered radioactivity in the liver compared to 1.3% in the "recovered" rats 24 h after the tracer dose and 47% of the radioactivity was either excreted in feces or in the colon contents after 72 h compared to 17.5% in "recovered" rats. Extraction of urine samples from "nephrotoxic" and "recovered" rats with chloroform suggested increased water soluble metabolites of citrinin in the urine from "nephrotoxic" rats. These data also suggested that in normal rats the kidneys are the major route of elimination of citrinin and its metabolite(s) while in rats rendered nephrotoxic by citrinin pretreatment, elimination is more dependent on hepatic excretion.