Penitrem A Research Articles

Development and application of a competitive enzyme immunoassay for the detection of penitrem A in fungal mycelium: Evidence for frequent occurrence of multiple indole-containing mycotoxins in mouldy foods

The present study aimed to develop a novel enzyme immunoassay (EIA) for the tremorgenic mycotoxin, penitrem A (PTA). This competitive EIA, based on rabbit anti-PTA polyclonal antibodies and a PTA-bovine serum albumin conjugate, yielded a mean standard curve detection limit of 220 ng/mL. The PTA-EIA, together with two EIAs for paxilline (PAX) and ergoline alkaloids, were used to study the toxin profiles in culture of fungi isolated from some naturally infected food sample materials, including dairy products, citrus fruits, and walnuts. PTA was produced by eight out of 27 mycelium extracts from malt extract agar and Sabouraud glucose chloramphenicol selective agar (2–200 μg/mL). Ten isolates from all types of food were also positive in the PAX-EIA (0.004–4 μg/mL). The EIA for ergoline alkaloids yielded positive results (0.0002–0.4 μg/mL) in isolates from dairy products and from walnuts, but not from citrus fruits. Control analyses of selected fungal extracts by HPLC-MS/MS for PTA and PAX qualitatively confirmed the EIA results, poor quantitative agreement could be attributed to the presence of penitrems other than PTA, and to PAX analogues, respectively. Selected PTA-positive fungal isolates were subjected to sequencing of the internal transcribed spacer region and the β-tubulin gene, and were identified as Penicillium polonicum and P. crustosum. In conclusion, Penicillium spp. capable to produce PTA, PAX, and ergoline alkaloids, either alone or in combination, appear to be quite common as contaminants on foods at the retail or production level. Some are capable to produce PTA in culture at high levels, which would have to be considered as toxic if present in food.

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.

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.

Astaxanthin and Docosahexaenoic Acid Reverse the Toxicity of the Maxi-K (BK) Channel Antagonist Mycotoxin Penitrem A.

Penitrem A (PA) is a food mycotoxin produced by several terrestrial and few marine Penicillium species. PA is a potent tremorgen through selective antagonism of the calcium-dependent potassium BK (Maxi-K) channels. Discovery of natural products that can prevent the toxic effects of PA is important for food safety. Astaxanthin (AST) is a marine natural xanthophyll carotenoid with documented antioxidant activity. Unlike other common antioxidants, AST can cross blood brain barriers (BBBs), inducing neuroprotective effects. Docosahexaenoic acid (DHA) is polyunsaturated ω-3 fatty acid naturally occurring in fish and algae. DHA is essential for normal neurological and cellular development. This study evaluated the protective activity of AST and DHA against PA-induced toxicity, in vitro on Schwann cells CRL-2765 and in vivo in the worm Caenorhbitidis elegans and Sprague Dawley rat models. PA inhibited the viability of Schwann cells, with an IC50 of 22.6 μM. Dose-dependent treatments with 10–100 μM DHA significantly reversed the PA toxicity at its IC50 dose, and improved the survival of Schwann cells to 70.5%–98.8%. Similarly, dose-dependent treatments with 10–20 μM AST reversed the PA toxicity at its IC50 dose and raised these cells’ survival to 61.7%–70.5%. BK channel inhibition in the nematode C. elegans is associated with abnormal reversal locomotion. DHA and AST counteracted the in vivo PA BK channel antagonistic activity in the C. elegans model. Rats fed a PA-contaminated diet showed high levels of glutamate (GLU), aspartate (ASP), and gamma amino butyric acid (GABA), with observed necrosis or absence of Purkinjie neurons, typical of PA-induced neurotoxicity. Dopamine (DA), serotonin (5-HT), and norepinephrine (NE) levels were abnormal, Nitric Oxide (NO) and Malondialdehyde (MDA) levels were significantly increased, and total antioxidant capacity (TAC) level in serum and brain homogenates was significantly decreased in PA-treated rats. DHA and AST treatments effectively counteracted the toxic effects of PA and normalized most biochemical parameters in rats. DHA and AST can be useful food additives to prevent and reverse PA food-induced toxicity.

Brain neurotoxicity of Penitrem A: electrophysiological, behavioral and histopathological study

The neurotoxicity of Penitrem A (PA) in rats was assessed against neurophysiological, behavioral and histopathological parameters. Animals were acutely given intracerebroventricular (22–45 mg) or intraperitoneal injections (0.5–1.5 mg/kg) of PA. A typical trembling syndrome associated with PA was always noted. Depending on the dose administered, animals may convulse and eventually die (1–1.5 mg/kg). PA-induced transient alterations of the EEG involving an increase in the frequency and voltage of electrical activity recorded from the cerebral cortex. Hippocampal activity was not modified and some pathologic activities may be recorded at the thalamus. Generally these EEG alterations disappeared at d 3 after the injection and the animals progressively recovered. However in the most severe cases, neuromotor disturbances were maintained at d 7 (rotarod test). Coronal sections of the brain at the striatal, thalamic, hippocampal and pons levels mainly revealed that PA was able to induce dose related injuries in the cerebellum with massive degeneration of Purkinje cells and a significant vacuolization within the molecular layer. The neurotoxic mechanism remains unclear. Action of the mycotoxin on the cerebello–thalamo-cortical tract is discussed.

Effects of penitrem A on learning in rats

Effects of penitrem A on learning in rats

Optimization of Yeast Bioassay for Trichothecene Mycotoxins

An improved disc diffusion type bioassay was developed for T-2 toxin (T-2), HT-2 toxin (HT-2), diacetoxyscirpenol (DAS), deoxynivalenol (DON), nivalenol (NIV), neosolaniol (NSL), fusarenon-X (FUS-X), trichothecin (TIN), roridin A (RDA) and verrucarin A (VCA) using the yeast, Kluyveromyces marxianus. Factors such as type of medium, agar volume per plate, preincubation time and temperature, incubation temperature, inoculum size and pH had variable, and in some cases a dramatic effect on the sensitivity of the assay. The effect of pH of the assay medium was particularly pronounced. The highest sensitivity was obtained when 6 ml per plate of a tryptic-soy-agar (TSA) medium (pH 7.5) containing 105 CFU inoculum per ml was incubated at 38°C for 18 h. All of the trichothecenes (TNN) were able to inhibit the growth of the yeast with the detection limit being 0.005, 0.01, 0.02, 0.02, 0.1, 0.5, 10, 10, 10 and 50 μg/disc for VCA, RDA, T-2, TTN, DAS, HT-2, DON, NSL, FUS-X and NIV, respectively. The detection limits for corn and wheat spiked with T-2 were 0.1 and 0.2 μg/g, respectively. Non-TNN mycotoxins that did not inhibit yeast at a concentration of 200 μg/disc were aflatoxin B1 (AFB1), ochratoxin A (OA), citrinin (CT), penicillic acid (PA), cyclopiazonic acid (CPA), penitrem-A (PTA) and zearalenone (ZEE). These results indicate that disc diffusion assay using K. marxianus under optimized conditions provides a sensitive method for the detection of low concentrations of several TNN.

Tremorgenic mycotoxins produced by strains of Penicillium spp. isolated from toxic Poa huecu parodi

Seventeen strains of Penicillium spp. have been isolated from Poa huecu Parodi from the Zapala zone, exhibiting toxicity to sheet. The following strains have been identified: P. crustosum, cyclopium, notatum, palitans, puberulum, verrucosum, viridicatum and Penicillium spp. The toxigenic capacity of the strains was studied after growing them under suitable conditions. Toxins produced were analysed by thin layer chromatography (TLC). Penitrem A (PA) and Penitrem B (PB) neurotoxins were identified and quantitated in twelve strains; verruculogen (VERR) and fumitremorgen B (FTB) being present in one of them. The effect of these mycotoxins was studied in mice. Neurological symptoms characteristic of the intoxication by tremorgenic toxins and similar to those observed in sheep suffering from 'huecu's disease' were observed. The possible role of these toxins as causative agents of 'huecu's disease' is discussed.

Neurobehavioral studies of tremorgenic mycotoxins verruculogen and penitrem A.

Verruculogen (V) and penitrem A (PA) represent a group of toxic secondary metabolites of mold contaminants of foodstuffs known as 'tremorgenic mycotoxins', which produce a unique neurotoxic syndrome characterized by sustained tremors, limb weakness, ataxia and convulsions. In the present study the intraperitoneal median tremogenic dose in mice for V was found to be 0.92 mg/kg and that for PA, 0.19 mg/kg. Behavioral and neurochemical parameters were assessed following acute exposure to varying neurotoxic and subneurotoxic doses of these mycotoxins. Measures of spontaneous motor activity (photoactometer) and exploratory reflex behaviors (open field) were markedly depressed by both V and PA. Notably, at dose levels of V or PA that were not accompanied by any overt signs of neurotoxicity, significant neuromotor depression was still observed. Acquisition of a conditioned avoidance shuttle response was disrupted, but only at a high neurotoxic dose level of V. Neurochemical analyses revealed no clear catecholaminergic or cholinergic involvement in the neurotoxic syndrome of eigher V or PA.

Effects of prenatal administration of penicillic acid and penitrem A to mice

METABOLITES of certainmolds growingon foodstuffs causetoxicdiseasesinman and animals;however,anunderstanding oftherelationship betweenthemold,thefoodandthe diseasehas onlynow beguntodevelop(NEWBERNE, 1974).Pencillia and Aspergillia are especially importantas contaminatingorganismsbecausecertainspeciescan grow and producetoxicmetabolites on agricultural commoditiesunderwidelydifferent conditions of moistureand temperature .Effectsof mycotoxinson biological systemsarenumerous; rangingfrom suddendeathtotumor formationfollowingingestion ofsmallquantities of toxinoveranextendedperiodoftime.The economicsignificance ofmycotoxicoses, though unknown,certainly ishigh.The presenceofmycotoxinsinfeedstuffs probablycauses,in additiontoacuteeffects, economiclossesby affecting suchimportantaspectsasweight gain,impairmentof resistance and immunitytodisease, and damage totheoffspring (PIER, 1973). A number offungithatgrowon standingcropsorstoredfeedarecapableofproducing toxicmetabolites .Two suchcompounds arepenicillic acidand penitremA .Penicillic acid, 3-methoxy-5-methyl-4-oxo-2,5-hexadienoic acid,first isolated by AISBERG andBLACK (1913), isa secondarymetaboliteproducedby speciesofPenicillium and Aspergillus (OXFORD etal.,1942).Penicillic acidistoxictomammals (HAYES etal.,1977),hasantibiotic activity (OXFORD, 1942;OXFORD etal.,1942),andhasbeenreportedtobecarcinogenic toratsand mice(DICKENS and JONES, 1961;DICKENS and JONES, 1965).Thismycotoxinoccursnaturallyinseveralagricultural products,includingmoldy tobacco,cornand peas(WILSON, 1976). Severalspecies ofPenicillium produceametabolite, penitremA (C87H 44NO,,Cl), capable ofcausingsustainedtremors,convulsionand evendeathinanimals(WILSON and HAYES, 1973). P.crustosunt and P.palitans, two tremorgenicspecies, havebeen implicatedin mycotoxicosesinvolvingcontaminatedfeedstuffs (WILSON etal.,1968;CIEGLER, 1969). Cows, sheep,mice,rats,chickens, rabbits, guineapigs,dogsand hamstersareamong the animalssusceptible toitsneurotoxic properties (CYSEWSKI etal.,1975;WILSON etal.,1972; WYATT etal.,1972;HAYES etal.,1976;HAYES etal.,1977).The neurotoxic actionofpenitremA inmiceandratsismostunusualbutremainsunexplained (WILSON etal.,1972).The effects ofselected drugson penitremA-inducedtremorsinmiceand ratssuggestthatthe toxinmay actcentrally byinhibition ofinhibitory interneurons (STERN, 1971).WILSON etal.

Production of Penitrem A and of an Unidentified Toxin by Penicillium lanoso-coeruleum Isolated from Weevil-Damaged Pecans

Production of Penitrem A and of an Unidentified Toxin by Penicillium lanoso-coeruleum Isolated from Weevil-Damaged Pecans