Effect of the appetite stimulant cyproheptadine on deoxynivalenol-induced reductions in feed consumption and weight gain in the mouse.

Deoxynivalenol (DON, vomitoxin), a Fusarium mycotoxin, is suspected of inducing its anorectic/feed refusal activity through a serotoninergic (5HT) mechanism, possible via 5HT2-receptors. In this study the efficiency of cyproheptadine (CYP), a serotonin antagonist and known appetite stimulant, to attenuate the adverse effect of DON was investigated in mice. CYP was administered in the feed for two days before animals began receiving the DON, which was also added to the feed. Both agents were administered concurrently thereafter for a 12-day period. Dosing levels included various combinations of the two compounds, ranging from 0-16 ppm DON and 0-20 ppm CYP.

Disposition of 14C-derived residues in tissues of pigs fed radiolabelled fumonisin B1.

The uptake and distribution of radioactive material-derived residues were determined in tissues of growing pigs consuming 14C-labelled fumonisin B1 (FB1) in the diet. Animals were fed 3.0 mg (3.0 microCi) 14C-FB1/kg feed from days 1-12, followed by 2.0 mg (2.0 microCi) 14C-FB1/kg feed during days 13-24, followed by a 9-day withdrawal period where pigs received clean feed only. Of the tissues analysed, residues were found to accumulate only in liver and kidney. Radioactivity was detected at the first sampling time (day 3), and continued to increase until the 14C-toxin was removed from the diet. Peak tissue levels (dpm/g tissue +/- SD, N = 2) in liver and kidney were 347 +/- 28 and 146 +/- 14, respectively, on day 24, which were equivalent to about 160 and 65 ng FB1 and/or metabolites per g tissue, respectively. Once pigs were placed on clean feed, tissue levels declined rapidly; down to approximately 35% of peak levels after 3 days, and only marginally above detection limits (approximately 25 dpm/g) after 9 days. Delayed recovery of the radioactive material consumed indicated a persistence within the body of FB1-derived residues, which could be eliminated only upon removal of the contaminated diet.

Response of growing swine to dietary exposure to pure fumonisin B1 during an eight-week period: growth and clinical parameters.

Consumption of corn or corn-based products contaminated with Fusarium moniliforme/fumonisins has been associated with a variety of animal and human diseases and is a major food/feed safety issue. This study focused on the clinical toxicity and performance parameters in growing swing exposed to low to moderate levels of pure fumonisin B1 (FB.) for 8 weeks. Male (castrated) and female pigs were fed diets containing 0,0.1,1.0, and 10 mg FB1/kg diet (ppm). Weight gains and feed consumption were measured weekly. Blood samples were collected throughout the study, and various clinical and hematological parameters were measured. Because fumonisins are potent inhibitors of sphingolipid biosynthesis, sphinganine and sphingosine concentrations were determined in the liver, lung, and kidney. Organ weights and carcass quality were measured at the end of the trial. In general, male pigs were more adversely affected by FB1 in the diet than females. The average daily gain for males decreased by 8% for pigs fed 1.0 ppm and by 11% at 10.0 ppm, when compared to the control (0 ppm). Males fed 0.1 ppm showed an erratic growth pattern during the first 5 weeks of the experiment. Feed consumption for the same animals was somewhat higher than that of the controls during each of the first 4 weeks but thereafter was 6-7% lower each week as compared to controls. Female pigs fed FB1-diets showed a general enhancement of feed consumption until week 4. Among clinical chemistry parameters, cholesterol increased in males for the 1.0 and 10.0 ppm diets as compared to controls after 2 weeks, while the levels in both sexes were elevated for the 1.0 ppm diet only by the end of the experiment. Serum liver enzyme concentrations were altered during week 2 only. Changes were observed in the weight of the pancreas and adrenals for male pigs fed FB1 diets as compared to controls. The free sphinganine to free sphingosine ratio (biomarker of exposure in FB1-consuming animals) increased in all three organs for the 10 ppm diet, regardless of sex. The study indicated that FB1 can cause different effects at each dose level, at concentrations as low as 0.1 ppm (showing erratic growth) followed by a reduced growth and biochemical abnormalities in blood (1.0 ppm) and sphingolipid alterations in tissues (10.0 ppm). Some of these effects occurred below the exposure level that caused alteration in sphingolipid metabolism.

Biological fate of fumonisin B1 in food-producing animals.

The presence of mycotoxins in grains and feedstuffs causes not only animal health problems, but also a valid concern about the transmission of potentially toxic residues into animal-derived products intended for human consumption. In a series of studies at Agriculture and Agri-Food Canada, we investigated the biological fate of fumonisin B1 (FB1) in several food-producing animals (grower pigs, laying hens, dairy cattle), as well as monitored various parameters for evidence of toxicity in these species. In several experiments involving either single-dose protocols (iv, po) or longer-term feeding trials, the pharmacokinetic profiles of FB1 (purity > 95%) in these species were determined, including tissue accumulation and transmission of residues. Toxicological (and economical) implications such as performance (feed consumption, growth), productivity, and carcass quality were also measured when appropriate.

Economic losses and decontamination.

Mycotoxin contamination of crops may cause economic losses at all levels of food and feed production including crop and animal production, and crop distribution and processing. The national economy would be affected adversely by losses incurred by crop and livestock producers and the multiplier effect this has on other industries as a result of the reduced spending power of producers. Costs of chemical analyses, quality control and regulatory programs, research and development, extension services, law suits, and the cost of human illnesses must all be borne by the national economy. The value of the losses encountered depends on grain, animal, and animal product prices, interest rates, degree of contamination, and other economic variables. Even during favourable seasons it is likely that millions of dollars are lost as a result of the contamination of crops with mycotoxins. Many compounds and treatments have been tested in order to reduce mycotoxin concentrations in food and feed or to alleviate their adverse effects on animals. Some of these treatments show promising prospects for commercial application, while others have had commercial applications already. However, until reliable, cost-effective, commercially applicable methods are more widely available, problems associated with mycotoxin contamination and the economic losses resulting, will continue to be seen in food and agriculture industries.

Pilot study on the plasma pharmacokinetics of fumonisin B1 in cows following a single dose by oral gavage or intravenous administration.

The pharmacokinetic fate of the mycotoxin fumonisin B1 (FB1) was investigated using 4 Holstein cows. Two animals each were administered FB1 intravenously (0.05 or 0.20 mg kg-1) and by oral gavage (1.0 or 5.0 mg kg-1). Blood samples were collected at specific time intervals over 12 hr postdosing, then daily for 13 more days, and analyzed for FB1, the hydroxylated aminopental metabolite, and their conjugates. Following intravenous dosing, the plasma-concentration profile of FB1 underwent a very rapid biexponential decrease, with toxin concentrations falling below detectable levels by 120 min postdosing. No known metabolites were detected in plasma. The similarity in pharmacokinetic parameters between the low- and high-dose animals suggests that FB1 distribution and elimination from blood was not dose-dependent at these levels of toxin administration. Following oral administration of the toxin, no FB1 or known metabolites could be found in the plasma, indicating no or very limited bioavailability in ruminants. The effects of FB1 on plasma-free sphinganine (Sa) and free sphingosine (So) concentrations were also determined. Following oral gavage at either dose, no effects on plasma sphingolipid concentration or Sa/So ratio were noted beyond typical daily variations. At the low intravenous dose (0.05 mg kg-1), changes in Sa or So concentrations were also not apparent. However, following intravenous administration at the higher dose (0.20 mg kg-1), the plasma Sa/So ratio was increased marginally in the one dosed cow, due essentially to a transient increase in Sa concentrations, which rose by approximately 60-65% over average predose levels; So levels remained relatively constant.

Determination of fumonisins in milk.

Fumonisin B1 (FB1) and fumonisin B2 (FB2) were determined in milk by liquid chromatography (LC) following immunoaffinity column cleanup. Recoveries from milk spiked with 5-50 ng each fumonisin/ml averaged 79-109%. The aminopentol hydrolysis product of FB1 (AP1) was determined by LC after cleanup on a C18 solid phase phase extraction column; mean recoveries were 69-83% at spiking levels of 50-100 ng AP1/ml milk. Detection limits were of the order 3-7 ng/ml for FB1 and FB2, and 20-25 ng/ml for AP1. A stability study showed no losses of FB1 and FB2 in milk under conditions of freezing, refrigeration and boiling. A transmission study using four cows dosed with pure FB1 either orally (1.0 and 5.0 mg FB1/kg b.w.) or by i.v. injection (0.05 and 0.20 mg FB1/kg b.w.) showed no detectable residues of FB1 or AP1 in the milk, with or without hydrolytic treatment with beta-glucuronidase/sulfatase to liberate any conjugates.

Influence of low-level exposure to Fusarium mycotoxins on selected immunological and hematological parameters in young swine.

The effects of low dietary concentrations of Fusarium mycotoxins (deoxynivalenol (DON), 15-acetyl-DON, and zearalenone) on growth, immunological, and hematological parameters were determined in young pigs during a 28-day feeding experiment. Clean and naturally contaminated corn were incorporated into basal diets formulated to contain 0.00, 0.75, 1.50, and 3.00 mg DON/kg diet. A pair-fed control animal was used for comparison with each animal receiving the highest level of contamination (diet 4). Skin temperature, measured during the first week of the experiment, decreased linearly as the dietary mycotoxin concentration increased. Several other linear effects were observed: depressed feed intake throughout the experiment, reduction in thyroid size (absolute/relative), and changes in the appearance of the esophageal region of the stomach (thicker and higher degree of folding with increasing toxin concentration). Serum T4 (thyroxine) levels increased quadratically after 7 and 28 days of exposure compared to control animals. This change coincided with an increase in albumin levels, a decrease in alpha-globulin levels, and an overall increase in albumin/globulin ratio as the level of contamination increased. After immunization with sheep red blood cells (SRBC), animals fed contaminated diets showed a delayed response in peak titers. At the end of the experiment an increase in the segmented neutrophil count was observed. The following observations were made for animals consuming diet 4 as compared to the pair-fed controls: lower skin temperature, better feed efficiency, more corrugated stomachs, reduced alpha-globulin levels, and lower antibody titers to SRBC.(ABSTRACT TRUNCATED AT 250 WORDS)

Pharmacokinetic fate of 14C-labelled fumonisin B1 in swine.

The pharmacokinetics of the mycotoxin fumonisn B1 (FB1) were investigated in pigs. Animals were administered 14C-FB1 intravenously (IV; 0.25 microCi, 0.40 mg/kg) or intragastrically (IG; 0.35 microCi, 0.50 mg/kg); separate groups of pigs underwent bile cannulation prior to dosing (groups IV/B and IG/B, respectively). Blood, urine, faeces, (and bile), were collected at specific time intervals over 72 hr, and assayed for specific activity. Following IV dosing, plasma concentration-time profiles were triexponential, with the following mean values: t1/2 alpha, 2.2 min; t1/2 beta, 10.5 min; t1/2 gamma, 182 min; apparent volume distribution (Vd gamma), 2.4 l kg-1; plasma clearance, 9.1 ml min-1 kg-1. After 3 days, clearance of FB1-derived radioactivity from the body had slowed to trace levels; total recoveries in urine and faeces were 21.2% and 58.3%, respectively. In bile-interrupted pigs (IV/B) the absence of the slow terminal elimination phase (gamma) suggested FB1 underwent enterohepatic circulation. Biliary recovery was 70.8% of the IV-dose. Radioactivity remaining in tissues after 72 hr amounted to 19.8% and 11.9% of the dose given to IV and IV/B pigs, respectively; highest activities were measured in liver and kidney equivalent to 1,076 and 486 ng FB1 and/or metabolites per g tissue, respectively. Based on plasma and excretion data, systemic bioavailability following IG dosing was estimated to be a very limited 3-6%. Tissue residue levels following IG dosing were 10-20-fold less than IV dosing.

Pharmacokinetic fate and pathological effects of 14C-fumonisin B1 in laying hens.

Presence of fumonisin B1 (FB1), a major metabolite of Fusarium moniliforme, in corn is of great concern to both human and animal health because of its wide range of toxicity. The pharmacokinetics of FB1 was studied in laying hens following oral and intravenous administration of 14C-labelled FB1. After iv dosing (2.0 mg = 23.68 kBq/kg bw) plasma radioactivity underwent a very rapid bi-exponential decline (t1/2 alpha = 2.5 +/- 0.3 min; t1/2 beta = 48.8 +/- 11.2 min) with negligible levels measured after 4-6 hr. Mean value for the apparent volume of distribution at steady state (Vdss) was 18.27 ml/kg, apparent volume of central compartment (Vd beta) was 82.20 ml/kg and plasma clearance was 1.18 ml/min/kg. At 24 hr post-dosing only trace residues were present in liver, kidney, and cecum. When dosed by the oral route (2.0 mg = 47.36 kBq/kg bw), systemic absorption of fumonisin appeared to be poor (F = 0.71 +/- 0.5%) with peak plasma concentrations of only 40-145 dpm/ml (equivalent to 28-103 ng FB1 and/or metabolites per ml) between 1.5 and 2.5 hr. At 24 hr post-dosing only trace amounts were present in crop, liver, kidney, small intestine, and cecum. In both orally and iv dosed birds almost all (97.7 +/- 3.73%) of the radioactivity was recovered in excreta by the end of the 24 hr experiment period and no residues were found in eggs laid during the 24 hr post-dosing period.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of low-level dietary deoxynivalenol on haematological and clinical parameters of the pig.

The subacute toxic effects of dietary deoxynivalenol (DON) were examined in grower pigs during a 32 day feeding period. DON was incorporated into the feed at 0, 1, and 3 mg/kg, added as either the purified toxin (P) or as naturally contaminated corn (N). Growth performance and blood biochemical and haematological parameters were monitored throughout the study. At the higher toxin levels (diets 3P, 3N) significantly lower feed consumption and body weight gains were evident soon after the start of feeding, but while weight gains of pigs fed the pure DON diet (3P) recovered after several days, values for pigs fed the naturally contaminated diet (3N) remained depressed over the course of the study. It is possible that these observations reflected the presence of other unidentified toxic compounds in the naturally contaminated grain. Generally, blood chemistry parameters of pigs fed the contaminated diets were not different from controls, with the exception of alpha-globulin and possibly cortisol in animals receiving diets 3N or 3P. Data suggested that the effect of DON on the alpha-globulin fraction may have been independent of the feed refusal syndrome associated with this toxin. Alterations in several haematological measurements were noted to occur sporadically with the 3 ppm diets, including higher RBC count, haematocrit and platelet level, however these effects could not be separated from the influence of decreased feed intake and were of limited value in diagnosing the effects of low level dietary DON on swine.

Influence of level of deoxynivalenol in the diet of dairy cows on feed intake, milk production, and its composition.

Eighteen primiparous Holstein cows were used in a 10-wk lactation study, preceded by a 2-wk covariate period, to determine the effect of concentration of deoxynivalenol in the diet on cow performance and transfer of deoxynivalenol and its metabolite, deepoxydeoxynivalenol, to milk. Diets were formulated to contain deoxynivalenol at 0, 6, and 12 mg/kg of concentrate DM, and daily intake of deoxynivalenol was .59, 42, and 104 mg, respectively. Increasing deoxynivalenol in the diet did not affect intake of concentrate or forage. Total milk output was not affected; however, milk fat responded quadratically; cows given deoxynivalenol at 6 mg/kg of concentrate DM had the lowest milk fat content and fat output. Overall energetic efficiency was not influenced because reduced energy output in milk was compensated by increased BW gains. No transfer of deoxynivalenol or deepoxydeoxynivalenol to milk was observed; concentrations were below detectable limits (1 microgram/ml) using HPLC-mass spectroscopy. We concluded that diets containing deoxynivalenol up to 6 mg/kg of dietary DM did not reduce feed intake of cows in this study and that deoxynivalenol or deepoxydeoxynivalenol was not transferred to milk. Further studies are required to confirm the apparent lack of effect of deoxynivalenol on milk production.

Biliary excretion and enterohepatic cycling of zearalenone in immature pigs.

The disposition of the estrogenic mycotoxin, zearalenone (ZEN) in female, 10- to 14-week-old Yorkshire pigs was investigated. Pigs were administered [3H]ZEN intravenously (IV; n = 4; 5 mg/kg; 15 microCi/kg), orally (n = 4; 10 mg/kg; 30 microCi/kg), or intravenously with bile removal (IVB; n = 2; 5 mg/kg; 15 microCi/kg). Plasma, urine, feces, and bile (IVB pigs only) were serially collected and analyzed for radioactivity. Metabolite profiles were determined in plasma and bile by HPLC. The biological half-life of total plasma radioactivity in IV and orally dosed pigs (86.6 hr) was much larger than that of IVB animals (3.34 hr). Metabolite profiles of plasma concentration vs time demonstrated secondary peaks in concentration during the terminal elimination phase in IV and orally dosed pigs. In IVB pigs these peaks were absent, relative metabolite profiles were altered, and ZEN and metabolites were no longer detectable after 16 hr post-dosing. Biliary recovery of radioactivity, principally as glucuronide conjugates, was extensive (45.61 +/- 4.7%) in IVB pigs and significantly greater (p < 0.05) than that of fecal recovery of radioactivity in IV (6.56 +/- 0.78) or orally dosed (21.74 +/- 1.56%) pigs. Intraduodenal administration of bile containing [3H]ZEN and metabolites resulted in recovery of 64.56 +/- 4.89% of the dose in bile, 20.78 +/- 3.94% in urine, and the presence of glucuronide conjugates of ZEN and alpha-zearalenol (ZEL) in portal and jugular plasma. Differences in metabolite profiles between administered bile and sampled plasma suggest that the intestinal mucosa was active in reducing ZEN to ZEL and conjugating these metabolites with glucuronic acid. These studies provide evidence for extensive biliary secretion and enterohepatic cycling of ZEN and metabolites in pigs.

The efficacy of various classes of anti-emetics in preventing deoxynivalenol-induced vomiting in swine.

The Fusarium mycotoxin deoxynivalenol (DON) is a potent emetic agent. While the basic mechanisms which invoke and mediate emesis are still poorly understood, various neurotransmitters appear to be involved. The action of these transmitters can be blocked by various receptor-specific antagonists. The current study investigated the efficacy of several classes of receptor antagonists to block the emetic effect of DON. Following anti-emetic pretreatment, pigs were administered the toxin (i.v., 80 micrograms/kg, or oral, 300 micrograms/kg) and the onset of emesis was monitored. Certain specific serotonin (5HT3)-receptor antagonists (ICS 205-930, BRL 43694 A) were found to efficaciously prevent DON-induced vomiting. These observations support the hypothesis that serotonin plays an important role in chemically induced emesis. Also moderately effective, but requiring high doses, were the 5HT2-receptor antagonists, cyproheptadine and sulpiride. A variety of compounds possessing strong anticholinergic activity were also efficacious. These, however, apparently act directly at the emetic center and thus are capable of preventing emesis regardless of the cause, including chemically induced vomiting. Non-effective were the antihistaminic and antidopaminergic anti-emetics; except, those which also possessed considerable anticholinergic activity, and i.v. administered chlorpromazine which has been speculated to block specific receptors found in the brain's chemoreceptor trigger zone (CTZ) reportedly involved in initiating emesis.

Investigations in the use of mice exposed to mycotoxins as a model for growing pigs.

A series of experiments was conducted to determine the feasibility of using mice to screen for possible dietary mycotoxin interactions before testing them with swine. Selected mycotoxins, deoxynivalenol (DON) and T-2 toxin, were fed to young mice, alone and in combination. The severity of effects on body weights caused by DON (0-20 mg DON/kg diet) was more pronounced in a dose-related manner when the animals were exposed to contaminated diets starting at 21 d of age than at 28 d (Experiment 1) as reflected in the analysis of variance. The relative variance among diets after 7 d was twice as great for the younger than for the older mice. In both age groups, the weight gain response was linear, similar to that seen in growing swine. In Experiment 2, a significant (p < .05) diet type x DON interaction for food consumption evident after 7 d, indicated that the effect of DON depended on the type of diet (freeze-dried vs. regular mash). There was no difference in food efficiency between diet type, but a strong dose-dependent effect due to DON was observed. When DON and T-2 toxin were fed together to young mice, a significant (p < .001) linear decrease in weight gain and food consumption was observed after 7 d on the contaminated diet as the toxin concentration increased.

Deltamethrin residues in milk and tissues of lactating dairy cows.

Lactating dairy cows were fed deltamethrin (2 or 10 mg kg-1 feed) for 28 consecutive days and deltamethrin residues measured in milk and tissues. Deltamethrin residues were higher relative to dose administered. The order of relative concentrations of deltamethrin in tissues, measured 1, 4, and 9 days after the last dose was: renal fat greater than subcutaneous fat greater than forequarter muscle greater than hindquarter muscle greater than liver greater than kidney. Depletion of deltamethrin residues in milk was very rapid indicating the half-life of the insectide of about 1 day. Trace amounts of deltamethrin metabolites 3-(2,2-dibromovinyl)-2,2-dimethylcyclopropane carboxylic acid (less than 0.0235 ppm) and 3-phenoxybenzoic acid (less than 0.034 ppm) were also detected in milk and tissues of treated cows.

Effect of deoxynivalenol on neurotransmitters in discrete regions of swine brain.

The effect of deoxynivalenol (DON, vomitoxin) on brain amine levels was investigated in swine. DON, a trichothecene mycotoxin, causes suppression of feed intake (anorexia) in susceptible species. Following acute administration of DON to pigs (0.25 mg/kg, IV), concentrations of endogenous catecholamines norepinephrine (NE), dopamine (DA), 3,4-dihydroxyphenyl-acetic acid (DOPAC), and homovanillic acid (HVA), and the indoleamines, 5-hydroxytryptamine (5HT, serotonin) and 5-hydroxyindoleacetic acid (5HIAA) were determined in five brain regions, periodically during the 24 h post-dosing. Analysis was carried out by high performance liquid chromatography, using electrochemical detection. Effects of DON in the swine brain were transmitter, time and region-specific. It was observed that levels of the major transmitters (NE, DA and 5HT) were statistically different from controls in the hypothalamus (Hypo), frontal cortex (FCX) and cerebellum (Cb) up to 8 h post-dosing. Overall, DON administration elevated NE and depressed DA concentrations in these regions, and levels of 5HT which increased initially in Hypo (1 h), had dropped significantly below controls in both Hypo and FCX at 8 h. These alterations, however, were not indicative of known neurochemical changes associated with chemical-induced anorexia. Instead, this data suggested that the neurochemical effects of acute DON exposure might be due to peripheral toxicological events (i.e., vomiting), which overwhelmed its more subtle feed refusal activity.

Evaluation of potential interactions involving trichothecene mycotoxins using the chick embryotoxicity bioassay.

The Chick Embryotoxicity Screening Test (CHEST) was used to examine possible interactions between different trichothecenes. Combinations of deoxynivalenol (DON), 15-acetyldeoxynivalenol (15-ADON) and HT-2 toxin were tested at various concentrations and the effects were determined based on percent mortality. The combined toxicity of any two trichothecenes was found to be additive. Discrepancies between observed and expected mortality values seldom differed by more than 10%, well within sampling error for the binomial distribution. Several limitations of the CHEST assay are discussed.

Distribution of deoxynivalenol in cerebral spinal fluid following administration to swine and sheep.

Deoxynivalenol (DON) is one of the major mycotoxins produced by Fusarium fungi. In evaluating DON as a potent CNS (emetic, anorexic) agent, its cerebral spinal fluid (CSF) and plasma pharmacokinetics were studied in pigs, a species very sensitive to the effects of DON, and sheep, a more tolerant animal. After intravenous administration, DON was detected very rapidly (less than 2.5 min) in the CSF of both species, but whereas peak levels (t-max) occurred at 5-10 min in sheep, in swine it was 30-60 min. It would appear that the very rapid and extensive tissue distribution of DON in swine (Vd gamma = 1.13 1 kg-1) may be slowing the rate of diffusion of the toxin into the CSF compared to sheep (Vd beta = 0.19 1 kg-1) where the toxin is confined essentially to the extracellular compartment. Area under curve calculations indicate approximately 2 1/2 times the amount of toxin eventually reaches the pig CSF compared to sheep CSF. A good relationship between blood-CSF DON levels was apparent in both species, although limitations in detection methods made it impossible to resolve a slow terminal phase (gamma) in swine CSF which was evident in the plasma profile after iv administration. Following oral administration of DON to pigs, a close correlation between plasma and CSF DON levels was observed. The toxin could be detected in CSF for up to 20 hr post-dosing.