Roquefortine Research Articles

Molecular and Biological Investigation of Isolated Marine Fungal Metabolites as Anticancer Agents: A Multi-Target Approach.

Cancer is the leading cause of death globally, with an increasing number of cases being annually reported. Nature-derived metabolites have been widely studied for their potential programmed necrosis, cytotoxicity, and anti-proliferation leading to enrichment for the modern medicine, particularly within the last couple of decades. At a more rapid pace, the concept of multi-target agents has evolved from being an innovative approach into a regular drug development procedure for hampering the multi-fashioned pathophysiology and high-resistance nature of cancer cells. With the advent of the Red Sea Penicillium chrysogenum strain S003-isolated indole-based alkaloids, we thoroughly investigated the molecular aspects for three major metabolites: meleagrin (MEL), roquefortine C (ROC), and isoroquefortine C (ISO) against three cancer-associated biological targets Cdc-25A, PTP-1B, and c-Met kinase. The study presented, for the first time, the detailed molecular insights and near-physiological affinity for these marine indole alkaloids against the assign targets through molecular docking-coupled all-atom dynamic simulation analysis. Findings highlighted the superiority of MEL's binding affinity/stability being quite in concordance with the in vitro anticancer activity profile conducted via sulforhodamine B bioassay on different cancerous cell lines reaching down to low micromolar or even nanomolar potencies. The advent of lengthy structural topologies via the metabolites' extended tetracyclic cores and aromatic imidazole arm permitted multi-pocket accommodation addressing the selectivity concerns. Additionally, the presence decorating polar functionalities on the core hydrophobic tetracyclic ring contributed compound's pharmacodynamic preferentiality. Introducing ionizable functionality with more lipophilic characters was highlighted to improve binding affinities which was also in concordance with the conducted drug-likeness/pharmacokinetic profiling for obtaining a balanced pharmacokinetic/dynamic profile. Our study adds to the knowledge regarding drug development and optimization of marine-isolated indole-based alkaloids for future iterative synthesis and pre-clinical investigations as multi-target anticancer agents.

Roquefortine C in blue-veined and soft-ripened Cheeses in the USA

ABSTRACT Certain fungi can produce secondary metabolites that are toxic, mycotoxins. Two groups of cheeses where fungi are used for ripening are the blue-veined cheeses (Penicillium roqueforti) and the “soft-ripened” cheeses (P. camemberti). An enzyme-linked immunosorbent assay was used to screen for the mycotoxin roquefortine C (ROQC) in 202 samples of cheeses sold in the United States. Of these 152 were blue-veined cheeses, 46 were soft-ripened cheeses and 4 were other varieties of mould-ripened cheeses. ROQC was not detected in any of the soft-ripened cheeses, at a limit of detection of 1.8 µg/kg. ROQC was found in 151 of 152 blue-veined cheeses. The maximum level found was 6,630 µg/kg (median 903 µg/kg, average of positives 1430 µg/kg, limit of quantitation 6.9 µg/kg). These levels are consistent with the levels found previously in blue-veined cheeses in the United Kingdom and Europe, which have generally been considered non-hazardous for human consumption.

Exploratory real-time kinetic analysis of the cytotoxicity induced by maize silage mycotoxins in a calf intestinal epithelial cell line

In a temperate climate, the mycotoxins deoxynivalenol (DON), nivalenol (NIV), enniatin B (ENN B), mycophenolic acid (MPA), roquefortine C (ROC) and zearalenone (ZEN) are often found in maize silage. Although rumen microbiota are able to degrade some mycotoxins (e.g. DON), others are known to stay mainly intact (e.g. ROC). In addition, mycotoxin degradation can be hampered by a low ruminal pH or decrease in rumen microbial activity. Hence, these mycotoxins can reach the small intestine and exert a cytotoxic effect on intestinal epithelial cells. In this study, a real-time kinetic analysis of the cytotoxicity of these six mycotoxins and some of their metabolites (α- and β-zearalenol, α- and β-ZOL) was performed in a calf small intestinal epithelial cell line (CIEB). Confluency as well as the cell death parameters apoptosis and necrosis were determined to evaluate the mycotoxin-induced cytotoxicity. A combination of Annexin-V green and Cytotox red staining was used to determine early and late apoptosis as well as necrosis. Six different concentrations were tested ranging from 0.78 to 12.5 μM. Compared to cells not exposed to mycotoxins, DON and NIV exert a fast toxic effect with DON being more toxic than NIV within the first hours of incubation, whereas the inverse was observed at 16 h of incubation. On the other hand, MPA and ZEN induced increased Annexin V green positive cells within several hours of incubation with higher toxicity over time. Increased Annexin V green and Cytotox red positive cells were seen for ROC only at the highest concentration tested. For ENN B, increased Annexin V green positive cells were observed only after 12 h and α- and β-ZOL did not show cytotoxic effects. Hence, mycotoxin exposure causes either severe (DON and NIV) or more limited (ZEN, ROC, MPA, and ENN B) risk of bovine intestinal epithelial damage.

Development and characterisation of a monoclonal antibody to detect the mycotoxin roquefortine C

ABSTRACT Roquefortine, also known as roquefortine C (ROQC) is a fungal secondary metabolite (mycotoxin) that is produced by some of the same Penicillia as the tremorgen penitrem-A (PEN-A). The two mycotoxins have been linked to sporadic cases of toxicosis in dogs, cattle, and humans, leading some to consider ROQC as a biomarker of PEN-A. Reported here are the development of a monoclonal antibody (mAb) and associated competitive enzyme-linked immunosorbent assay (ELISA) for the screening of ROQC in extracts of nuts (nut “milks”), and dog serum. The ELISA was sensitive for ROQC, with a level of 0.117 ng ml−1 inhibiting colour development by 50% (IC50), a limit of detection of 0.026 ng ml−1, and a dynamic range (IC20 to IC80) of 0.038 to 0.289 ng ml−1 in buffer. The assay was tolerant to significant levels of methanol. Recoveries from 4 types of nut milks spiked over the range of 0.25 to 2 ng ml−1 were in the range of 83.5% to 116%. A small survey of commercial nut “milks” and “creamers” indicated 4 of 35 samples contained ROQC at levels so low that they are unlikely to be significant to human health (<0.6 ng ml−1). The assay was also applied to canine serum. Recoveries from serum spiked over the range of 0.2 to 5 ng ml−1 ranged from 98.1% to 123%. The results suggest the ELISA can be applied to the screening of food products, such as nut extracts, as well as for the screening of serum from dogs suspected to be suffering from mycotoxin-induced tremors.

Evaluation of the Efficacy of Mycotoxin Modifiers and Mycotoxin Binders by Using an In Vitro Rumen Model as a First Screening Tool.

Ruminal microbiota of cattle are not able to detoxify all mycotoxins. In addition, detoxification can be hampered by adverse ruminal conditions (e.g., low ruminal pH). Hence, in the cattle husbandry, mycotoxin binders and modifiers could be used to prevent animal exposure to mycotoxins. In this study, an in vitro rumen model, including feed matrix, was established as first screening tool to test the efficacy of five products claiming to detoxify mycotoxins. The detoxifiers had different modes of action: (a) binding (three products); (b) enzymatic detoxification of zearalenone (ZEN; one product, ZenA); and (c) bacterial transformation of trichothecenes (one product, BBSH 797). For the mycotoxin binders, the binding to the mycotoxins enniatin B (ENN B), roquefortine C (ROQ-C), mycophenolic acid (MPA), deoxynivalenol (DON), nivalenol (NIV), and zearalenone (ZEN) were tested at a dose recommended by the manufacturers. The in vitro model demonstrated that all binders adsorbed ENN B to a certain extent, while only one of the binders also partially adsorbed ROQ-C. The binders did not change the concentrations of the other mycotoxins in the ruminal fluid. The enzyme ZenA detoxified ZEN very quickly and prevented the formation of the more toxic metabolite α-zearalenol (α-ZEL), both at normal (6.8) and low ruminal pH (5.8). The addition of BBSH 797 enhanced detoxification of DON and NIV, both at normal and low ruminal pH. The in vitro rumen model demonstrated that the addition of ZenA seems to be a very promising strategy to prevent estrogenic effects of ZEN contaminated feed, and BBSH 797 is efficient in the detoxification of trichothecenes.

In Vitro Rumen Simulations Show a Reduced Disappearance of Deoxynivalenol, Nivalenol and Enniatin B at Conditions of Rumen Acidosis and Lower Microbial Activity.

Ruminants are generally considered to be less susceptible to the effects of mycotoxins than monogastric animals as the rumen microbiota are capable of detoxifying some of these toxins. Despite this potential degradation, mycotoxin-associated subclinical health problems are seen in dairy cows. In this research, the disappearance of several mycotoxins was determined in an in vitro rumen model and the effect of realistic concentrations of those mycotoxins on fermentation was assessed by volatile fatty acid production. In addition, two hypotheses were tested: (1) a lower rumen pH leads to a decreased degradation of mycotoxins and (2) rumen fluid of lactating cows degrade mycotoxins better than rumen fluid of non-lactating cows. Maize silage was spiked with a mixture of deoxynivalenol (DON), nivalenol (NIV), enniatin B (ENN B), mycophenolic acid (MPA), roquefortine C (ROQ-C) and zearalenone (ZEN). Fresh rumen fluid of two lactating cows (L) and two non-lactating cows (N) was added to a buffer of normal pH (6.8) and low pH (5.8), leading to four combinations (L6.8, L5.8, N6.8, N5.8), which were added to the spiked maize substrate. In this study, mycotoxins had no effect on volatile fatty acid production. However, not all mycotoxins fully disappeared during incubation. ENN B and ROQ-C disappeared only partially, whereas MPA showed almost no disappearance. The disappearance of DON, NIV, and ENN B was hampered when pH was low, especially when the inoculum of non-lactating cows was used. For ZEN, a limited transformation of ZEN to α-ZEL and β-ZEL was observed, but only at pH 6.8. In conclusion, based on the type of mycotoxin and the ruminal conditions, mycotoxins can stay intact in the rumen.

Modelling Fungal Growth, Mycotoxin Production and Release in Grana Cheese.

No information is available in the literature about the influence of temperature (T) on Penicillium and Aspergillus spp. growth and mycotoxin production on cheese rinds. The aim of this work was to: (i) study fungal ecology on cheese in terms of T requirements, focusing on the partitioning of mycotoxins between the rind and mycelium; and (ii) validate predictive models previously developed by in vitro trials. Grana cheese rind blocks were inoculated with A. versicolor, P. crustosum, P. nordicum, P. roqueforti, and P. verrucosum, incubated at different T regimes (10–30 °C, step 5 °C) and after 14 days the production of mycotoxins (ochratoxin A (OTA); sterigmatocystin (STC); roquefortine C (ROQ-C), mycophenolic acid (MPA), Pr toxin (PR-Tox), citrinin (CIT), cyclopiazonic acid (CPA)) was quantified. All the fungi grew optimally around 15–25 °C and produced the expected mycotoxins (except MPA, Pr-Tox, and CIT). The majority of the mycotoxins produced remained in the mycelium (~90%) in three out of five fungal species (P. crustosum, P. nordicum, and P. roqueforti); the opposite occurred for A. versicolor and P. verrucosum with 71% and 58% of STC and OTA detected in cheese rind, respectively. Available predictive models fitted fungal growth on the cheese rind well, but validation was not possible for mycotoxins because they were produced in a very narrow T range.

Development of an UPLC-MS/MS Method for the Analysis of Mycotoxins in Rumen Fluid with and without Maize Silage Emphasizes the Importance of Using Matrix-Matched Calibration.

Ruminants are less susceptible to the effects of mycotoxins than monogastric animals as their rumen microbiota are claimed to degrade and/or deactivate at least some of these toxic compounds. However, the mycotoxin degradation is not well-known yet. For this, a sensitive, specific, and accurate analytical method is needed to determine mycotoxins in the rumen fluid. This study aims to develop and thoroughly validate an ultra-performance liquid chromatography tandem mass spectrometry method for the quantitative determination in the rumen fluid of some of the most relevant mycotoxins found in maize silage in Western Europe: deoxynivalenol (DON), nivalenol (NIV), zearalenone (ZEN), mycophenolic acid (MPA), roquefortine C (ROQ-C) and enniatin B (ENN B), as well as their metabolites deepoxy-deoxynivalenol (DOM-1), α-zearalenol (α-ZEL), β-zearalenol (β-ZEL), zearalanone (ZAN), α-zearalanol (α-ZAL) and β-zearalanol (β-ZAL). As feed is often present in the rumen fluid samples, the potential interaction of feed particles with the mycotoxin extraction and analysis was investigated. Extraction recovery and matrix effects were determined in the rumen fluid with and without maize silage. Differences in those parameters between rumen fluid alone and rumen fluid with maize silage highlight the importance of using matrix-matched calibration curves for the quantification of mycotoxins in rumen fluid samples. A cross-validation of the method with rumen fluid and maize silage demonstrates that this analytical method can be applied in research on rumen fluid samples to investigate the degradation of the reported mycotoxins by rumen microbiota if matrix-matched calibration is performed.

Modeling Growth and Toxin Production of Toxigenic Fungi Signaled in Cheese under Different Temperature and Water Activity Regimes.

The aim of this study was to investigate in vitro and model the effect of temperature (T) and water activity (aw) conditions on growth and toxin production by some toxigenic fungi signaled in cheese. Aspergillus versicolor, Penicillium camemberti, P. citrinum, P. crustosum, P. nalgiovense, P. nordicum, P. roqueforti, P. verrucosum were considered they were grown under different T (0–40 °C) and aw (0.78–0.99) regimes. The highest relative growth occurred around 25 °C; all the fungi were very susceptible to aw and 0.99 was optimal for almost all species (except for A. versicolor, awopt = 0.96). The highest toxin production occurred between 15 and 25 °C and 0.96–0.99 aw. Therefore, during grana cheese ripening, managed between 15 and 22 °C, ochratoxin A (OTA), penitrem A (PA), roquefortine-C (ROQ-C) and mycophenolic acid (MPA) are apparently at the highest production risk. Bete and logistic function described fungal growth under different T and aw regimes well, respectively. Bete function described also STC, PA, ROQ-C and OTA production as well as function of T. These models would be very useful as starting point to develop a mechanistic model to predict fungal growth and toxin production during cheese ripening and to help advising the most proper setting of environmental factors to minimize the contamination risk.

Unraveling the Tritrophic interactions between fruit host-pathogen-antagonist in the post-harvest environment

Fruits and vegetables post-harvest losses due to the attack of several fungal pathogens can reach levels of up to 50% depending on the commodity. Besides the economic losses, some of these fungi may represent a potential health risk because of mycotoxin production. To control post-harvest diseases of fruits, synthetic fungicides, when permitted, are the primary means used. However, the emergence of fungicide-resistant strains and the willingness to use safer and eco- friendly treatments, have generated interest in the development of alternative non-chemical methods to reduce post-harvest losses. Biological control using microbial antagonists has emerged as one of the most promising alternatives to fungicides, either alone or as part of an integrated pest management. The antagonistic activity of biocontrol agents has been widely demonstrated, however the mechanisms of action of most biocontrol agents of post-harvest diseases has not yet been fully understood which is important to know for a successful implementation of post-harvest biocontrol technology. Therefore a part of my PhD focused on this aspect; we have successfully cloned, expressed in P. pastoris and confirmed the involve- ment of a novel endochitinase gene MfChi and the protease gene ALP5 in the biocontrol activity of the antagonistic yeasts Metschnikowia fructicola AP47 and Aureobasidium pullulans PL5 successively. Our work proved that the alkaline serine protease ALP5 and the endochitinase MfChi could be developed as post-harvest treatments with antimicrobial activity for fruits undergoing a short shelf life to control Botrytis cinerea, Monilinia fructicola and Monilinia laxa on pome and stone fruits. Moreover, besides developing biocontrol agents as alternative approaches to chemicals to control the pathogens, we should also be familiar with the decaying agents, such as their nature and the molecular basis of the infection. Due to recent technological advances, sequencing has revolutionized biological research and has become the forefront of bio- logical experimentation in the last decade. Therefore, we have sequenced for the first time Penicillium griseofulvum PG3 which is associated with blue mould decay, the most important post-harvest disease of pome fruit worldwide. The fungal pathogen has the ability to simultaneously produce both detrimental and beneficial Secondary Metabolites (SM). Our work aimed to analyse some important SM clusters present in the studied strain in order to gain insight into SM synthesis in P. griseofulvum. Genome-wide analysis of PG3 genes revealed a complete putative gene cluster for patulin biosynthesis, and partial griseofulvin and roquefortine C clusters. Besides the bioinformatics analysis of these gene clusters, we quantified the SM production in vitro and during disease development on apple. Furthermore, the SM detected in infected apple were examined by studying the expression kinetics of their key genes under controlled conditions. In addition, we found additional SM clusters in PG3, including those potentially responsible for the synthesis of penicillin and cyclopiazonic acid. These findings provide relevant information to understand the molecular basis of SM biosynthesis in P. griseofulvum and this resource will allow further research directed to the overexpression or blocking of specific SM synthesis, to assess its potential in terms of biotechnological applications for beneficial SM, such as griseofulvin.

Individual and combined effects of roquefortine C and mycophenolic acid on human monocytic and intestinal cells

Roquefortine C (ROC) and mycophenolic acid (MPA) are secondary metabolites produced by various fungal species. It is known that both ROC and MPA may co-occur in raw materials or food. However, to date there is a lack of information regarding their toxicity. In this study, ROC and/or MPA cytotoxicity was evaluated on human intestinal (Caco-2) and monocytic cell cultures (THP-1 and CD14+). After 48 h single mycotoxin exposure, viability tests showed that monocytes (THP-1 and CD14+) were more sensitive to ROC (inhibitory concentration 50% (IC50)=55 and 45 μM, respectively) than to MPA (IC50&gt;780 μM). IC50 values determined from ROC and MPA mono-exposure experiments on Caco-2 cells were &gt;100 and &gt;780 μM, respectively. Caco-2 cell viability was significantly reduced after 48 h co-exposure at high ROC/MPA concentrations. A synergistic effect was observed at 10/78, 25/780 and 50/780 μM ROC/MPA concentrations, while an additive effect was seen at 100/780 μM. THP-1 apoptosis rate increased after 3 and/or 6 h single ROC (from 10 to 100 μM) and MPA (780 μM) exposures in a dose-dependent manner. Co-exposure to 100/780 μM of ROC/MPA led to an increase in the THP-1 apoptotic cell population. No apoptosis mechanism was observed on Caco-2 cells. This is the first time that combined ROC and MPA cytotoxic effects, as well as the associated mechanisms are investigated.

Influence of intraspecific variability and abiotic factors on mycotoxin production in Penicillium roqueforti

Penicillium roqueforti has the ability to produce secondary metabolites, including roquefortine C (ROQC) and mycophenolic acid (MPA). In a previous study, the presence of these mycotoxins, alone or in co-occurrence, has been reported in blue-veined cheese. A high variability of mycotoxin content has also been observed, although the majority of samples exhibited relatively low concentrations. The observed variability raises the question of the factors impacting ROQC and MPA production. In this context, the mycotoxigenic potential of 96 P. roqueforti strains (biotic factor) and the effect of some abiotic factors (pH, temperature, NaCl and O2 contents, and C/N ratio) on mycotoxin production were evaluated. A high intraspecific diversity, established via genotypic (RAPD) and phenotypic (FTIR) approaches, was observed. It was associated with mycotoxigenic potential variability and may thus explain part of the observed variability in mycotoxin content of blue-veined cheese. Moreover, a significant decrease of ROQC and MPA production was observed for conditions (temperature, C/N ratio, O2 and NaCl concentrations) encountered during cheese-making compared with optimal growth conditions. The results also highlighted that there was no significant effect of addition of ROQC amino-acid precursor on the production of both mycotoxins whereas a pH increase from 4.5 to 6.5 slightly reduced MPA but not ROQC production.

Occurrence of roquefortine C, mycophenolic acid and aflatoxin M1 mycotoxins in blue-veined cheeses

Penicillium roqueforti, a food and feed contaminant, is known for its potential to produce roquefortine C (ROQC) and mycophenolic acid (MPA) amongst other mycotoxins. In blue-veined cheeses, selected P. roqueforti ripening cultures are used for organoleptic development but little is known about mycotoxin occurrence in these products. In this study, aflatoxin M1 (AFM1), ROQC and MPA levels were determined in 86 blue-veined cheeses collected worldwide using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). AFM1 was absent in all samples while 97.7% and 37.2% of cheeses contained quantifiable ROQC and MPA levels, respectively. Overall, the analyzed cheeses contained a large range of mycotoxin concentrations with ROQC and MPA mean levels at 848 ± 1670 μg/kg and 841 ± 1271 μg/kg, respectively. Noteworthy, 75% of cheese samples contained less than 792 μg/kg ROQC and 705 μg/kg MPA.

Study of the effects of PR toxin, mycophenolic acid and roquefortine C on in vitro gas production parameters and their stability in the rumen environment

SUMMARYMoulds belonging to Penicillium section roqueforti are common contaminants of feedstuffs and produce several mycotoxins that can cause health hazards when ingested by farm animals. Among these, PR toxin (PR), mycophenolic acid (MY) and roquefortine C (RC) have been frequently detected in forages, particularly silages. The aims of the current trials were to study the effects of the presence of pure mycotoxins on in vitro rumen fermentation parameters and to assess their stability in the rumen environment. Two successive in vitro gas production experiments were carried out: a central composite design with four replications of central point (CCD) and a completely randomized design with a fully factorial arrangement of treatments (FFD). In CCD, the effects of PR, MY and RC concentrations in diluted rumen fluid (i.e. 0·01, 0·30, 1·01, 1·71 and 2·00 μg of each mycotoxin/ml) were tested. Gas volume produced after 48 h of incubation (Vf) decreased linearly as concentrations of RC and MY in diluted rumen fluid increased, with marginal effects similar for two mycotoxins, being respectively −14·6 and −13·4 ml/g organic matter (OM) for each 1·0 μg/ml of increment in mycotoxin concentration. Similarly, total volatile fatty acid (VFA) production decreased quadratically as concentrations of RC and MY increased, with marginal effects about two times higher for MY than RC, being −4·22 and −2·62 mmol/l for each 1·0 μg/ml of increment in mycotoxin concentration. With respect to maximum Vf (i.e. 410·6 ml/g OM) and VFA (98·06 mmol/l) values estimated by the model, decreases of 13·6 and 15·2% were obtained when incubating the highest RC and MY concentrations, respectively. The PR did not interfere with rumen fermentation pattern and it was not recovered after 48 h of incubation, whereas the stabilities of MY and RC in rumen fluid were similar and on average equal to about 50%. On the basis of CCD results, a second experiment (FFD) was carried out in which only effects of MY and RC concentrations (i.e. 0, 0·67, 1·33 and 2·00 μg of each mycotoxin/ml of diluted rumen fluid) were tested. Data from FFD showed Vf decreased linearly when concentrations of MY and RC increased, with marginal effect two-folds higher for MY than for RC (−11·1 ml/g OM and −6·7 ml/g OM, respectively). Similar marginal effects of MY and RC in decreasing VFA production were recorded: −2·38 and −2·86 mmol/l for each 1·0 μg/ml of increment in mycotoxin concentration, respectively. At the highest RC and MY tested concentrations, Vf and VFA decreased by 8·7 and 10·7%, respectively, over maximum estimated values. In FFD, the average amounts of MY and RC recovered in rumen fluid after 48 h of incubation were 79·0 and 40·6%, respectively. In conclusion, the MY and RC from standards interfered with rumen microorganisms at relatively low levels and were partially stable in the rumen environment after 48 h of incubation. These findings suggested that MY and RC could interfere with digestive processes and might represent a potential risk for ruminants fed diets containing feeds contaminated by mycotoxins produced by P. roqueforti.

Effect of a heterotrimeric G protein alpha subunit on conidia germination, stress response, and roquefortine C production in Penicillium roqueforti.

Heterotrimeric G protein signaling regulates many processes in fungi, such as development, pathogenicity, and secondary metabolite biosynthesis. For example, the Galpha subunit Pga1 from Penicillium chrysogenum regulates conidiation and secondary metabolite production in this fungus. The dominant activating allele, pga1G42R, encoding a constitutively active Pga1 Galpha subunit, was introduced in Penicillium roqueforti by transformation, resulting in a phenotype characterized by low sporulation and slow growth. In this work, the effect of the constitutively active Pga1G42R Galpha subunit on conidial germination, stress tolerance, and roquefortine C production of P. roqueforti was studied. Pga1G42R triggered germination in the absence of a carbon source, in addition to negatively regulating thermal and osmotic stress tolerance. The presence of the Pga1G42R Galpha subunit also had an important effect on roquefortine C biosynthesis, increasing production and maintaining high levels of the mycotoxin throughout a culture period of 30 days. Together, the results suggest that G protein-mediated signaling participates in the regulation of these three processes in P. roqueforti.

Contamination of Fresh and Ensiled Maize by Multiple Penicillium Mycotoxins

Toxins produced by Penicillium species are reported in maize silage and have been associated with health problems in cattle. Our objectives were to evaluate the prevalence and dynamics of patulin (PAT), mycophenolic acid (MPA), cyclopiazonic acid (CPA), and roquefortine C (ROC) in fresh and ensiled maize. To achieve these objectives we developed a high-performance liquid chromatography method coupled with mass spectrometry to detect all four toxins simultaneously in silage. In addition we collected weather data, information on agronomic practices, and silage fermentation characteristics for each study site. Silage was collected at harvest and after ensiling in 2001 and 2002 from 30 Pennsylvania dairies. The average concentration of toxins (range in parentheses) was: PAT 0.08 microg/g (0.01 to 1.21), MPA 0.16 microg/g (0.02 to 1.30), CPA 0.12 microg/g (0.02 to 1.43), and ROC 0.38 microg/g (0.01 to 5.71). ROC was the most frequently detected toxin (60%), followed by MPA (42%), CPA (37%), and PAT (23%). Of 120 samples tested, 15% contained no detectible levels of toxin, 25% were contaminated with one toxin, 32% with two, 18% with three, and 10% with all four toxins. All four mycotoxins were found in freshly harvested material, contradicting the belief that Penicillium toxin formation occurs exclusively during storage. We observed that weather conditions during specific growth stages of the crop affected the final concentration of toxins in freshly harvested maize. In ensiled material, PAT levels were affected by concentrations of propionic and isobutyric acids. Based on our data, Penicillium mycotoxins can form while the crop is in the field and after ensiling, suggesting that preventative measures should begin prior to ensiling.

Combined effects of selected Penicillium mycotoxins on in vitro proliferation of porcine lymphocytes

The in vitro effect of combinations of the Penicillium mycotoxins citrinin (CIT), cyclopiazonic acid (CPA), ochratoxin A (OTA), patulin (PAT), penicillic acid (PIA) and roquefortine C (RQC) on mitogen induced lymphocyte proliferation was determined using purified lymphocytes from six piglets. Dose-response curves for each mycotoxin and mycotoxin combinations were generated. The combined effects of toxin pairs based on IC20 were illustrated in isobole diagrams and statistically calculated. OTA and CIT elicited a synergistic effect. Four toxin pairs elicited additive effects, four pairs less-than-additive effects and six pairs independent effects. Thus, the majority of toxin pairs tested produced lower combined effects than an additive effect. The results indicate that the sum effect of all toxins is less than that from the summation of concentrations of the individual compounds, adjusted for differences in potencies.

The effects of the Penicillium mycotoxins citrinin, cyclopiazonic acid, ochratoxin A, patulin, penicillic acid, and roquefortine C on in vitro proliferation of porcine lymphocytes

The in vitro effect of each of the Penicillium mycotoxins citrinin (CIT), cyclopiazonic acid (CPA), ochratoxin A (OTA), patulin (PAT), penicillic acid (PIA) and roquefortine C (RQC) on mitogen induced lymphocyte proliferation was determined using purified lymphocytes from 6 piglets. Dose response curves for each mycotoxin were generated and the concentrations producing 50% inhibition of cell proliferation (IC(50)) were estimated. OTA and PAT were the most potent toxins with IC(50) of 1.3 and 1.2 micromol/l, respectively (0.52 and 0.18 mg/l, respectively). Based on molar concentrations, OTA was 15, 30, 40, and 65 times more potent as an inhibitor than PIA, CIT, CPA and RQC, respectively.