The effect of deoxynivalenol on serotoninergic neurotransmitter levels in pig blood 1

Abstract

Deoxynivalenol (DON) produces two characteristic toxicological effects, decreased feed consumption (anorexia) and emesis. Both effects have been linked to increased central (CNS) serotoninergic activity. Although there has also been some indication of a peripheral involvement, the role of blood pools of serotonin and related compounds in mediating DON toxicity is not well defined. In this study, the effect of DON on plasma concentrations of serotonin (5‐hydroxytryptamine, 5HT), 5HIAA (5‐hydroxyindoleacetic acid) and tryptophan (TRP), as a reflection of an induced peripheral serotoninergic system, was investigated in swine.

Typical values for the plasma concentrations of 5HT, 5HIAA, and TRP were established in pigs. Following administration of DON, either intragastrically or intravenously, concentration changes in these substances were measured over an eight hour period. The effect of low and high toxin doses were also compared.

Analyses showed no effect on plasma levels of the compounds of interest, even at sufficient toxin doses to invoke emesis in the test animals. Any variation over the course of the study remained within acceptable control limits. These results indicated no peripheral effect by DON which could account for the increased serotoninergic activity associated with altered feeding behaviour or emesis.     

Plasma pharmacokinetics of the mycotoxin deoxynivalenol following oral and intravenous administration to sheep

The pharmacokinetics of deoxynivalenol (DON) were studied in sheep after administrating intravenous and oral doses (0.5 and 5.0 mg/kg, respectively). The plasma concentrations were measured using an electron-capture gas chromatographic method. After iv administration DON plasma levels were found to decrease biexponentially, showing a rapid distribution phase (t 1/2 alpha = 12-23 min), followed by a slower elimination phase (t 1/2 beta = 57-78 min). Only trace levels of DON could be detected in plasma 7 hr post-dosing. Further pharmacokinetic data suggest that DON was confined mainly to extracellular fluid, and did not appear to undergo any significant binding or uptake by tissue. After oral dosing, DON was quickly absorbed (t-max 4.0-5.3 hr), but had a systemic bioavailability of only 7.5%; due in part to its rapid and efficient metabolism by rumen microorganisms. Half-life of elimination (t 1/2 beta) was 100-125 min following oral administration, and depending on the animal, required 20-30 hr to be cleared from the system. The metabolic formation of the glucuronide conjugate after iv and oral administration of DON appeared to occur quite efficiently (iv, 21%; oral, 75%), and its elimination half-lives (iv, 150-200 min; oral 6.1-7.1 hr) were considerably longer than that of the parent toxin. Detection in plasma of the de-epoxide metabolite, DOM-1, accounted for only a minor portion of the dose after either dosing regimen (iv, less than 2.0%; oral, less than 0.3%), occurring predominantly as the glucuronide conjugate.     

Nontransmission of deoxynivalenol (vomitoxin) to milk following oral administration to dairy cows

The absorption of deoxynivalenol (DON; vomitoxin), a trichothecene mycotoxin produced by Fusarium species, was studied in the dairy cow. Serum and milk DON levels were quantitated following a single oral dose of 920 mg DON to each of two lactating cows of similar weight. Maximum blood levels for the two animals following DON administration were 200 and 90 ng/ml serum, occurring at times 4.7 and 3.5 hr, respectively. By 24 hr after dosing only trace levels (less than 2 ng/ml) were still detectable. DON in its conjugated form accounted for 24-46% of the total levels present in serum. Free and conjugated DON were also present in cow's milk, but only extremely low amounts (less than 4 ng/ml) were detected. Detection of DON was carried out utilizing Sep-Pak C18 extraction cartridges for isolation, with additional purification of the sample achieved by passing the extract through a short charcoal/alumina column. The extract was then reacted with N-heptafluorobutyrylimidazole prior to quantitation of the resulting DON-tris-heptafluorobutyrate derivative by combined gas chromatography-quadrupole mass spectrometry, using multiple selected ion monitoring. Detection limits were as low as 1 ng/ml (1 ppb).     

Minimal transmission of zearalenone to milk of dairy cows

Milk and plasma levels of zearalenone (ZEN), alpha-zearalenol (alpha-ZEL), beta-zearalenol (beta-ZEL) and conjugated metabolites were determined after feeding lactating cows with ZEN. In those instances where ZEN and alpha- and beta-ZEL were detected in milk or plasma, they occurred only as conjugates hydrolysable by treatment with a mixture of beta-glucuronidase and aryl sulfatase. With studies where 50 or 165 mg was fed daily to three cows for 21 day periods, neither dosage showed the presence of ZEN or metabolites in either milk or plasma (detection limits: milk, 0.5 ng/ml, ZEN, alpha-ZEL; 1.5 ng/ml, beta-ZEL; plasma, 2-3 times higher). A dose of 544.5 mg zearalenone per day given to a single cow for 21 days yielded maximum concentrations of only 2.5 ng ZEN/ml and 3.0 ng alpha-ZEL/ml in the milk. In plasma, up to 3 ng ZEN/ml could be detected during the initial 4 days of treatment. At a dose of 1.8 g of zearalenone given over a one day feeding period, maximum milk levels of 4.0 ng ZEN/ml, 1.5 ng alpha-ZEL/ml, and 4.1 ng beta-ZEL/ml were observed during the initial 2 days; corresponding maximum levels after a one day dose of 6.0 g zearalenone were 6.1, 4.0 and 6.6 ng/ml milk on days 2-3. In plasma, peak ZEN concentrations (9 and 13 ng/ml at the lower and higher one-day doses, respectively) occurred 12 hr after initial dosing, and declined to negligible levels by days 5-7. Neither alpha- nor beta-ZEL were detected in plasma. Since measurable levels required very high oral doses of ZEN, milk would not normally pose a human health hazard as a result of feeding rations containing ZEN to lactating dairy cows.     

The role of tryptophan in don‐induced feed rejection

Abstract

Deoxynivalenol (DON, vomitoxin) commonly produced by Fusarium fungi can alter feeding behaviour of pigs and other farm animals. The effects of dietary tryptophan (TRP, precursor of brain amine serotonin) in combination with DON were examined in mice to determine if TRP can modulate DON toxicity. Results indicated that brain TRP can be influenced by dietary TRP, but no evidence of TRP potentiating DON toxicity was observed. Higher TRP levels likely induced amino acid imbalance leading to weight gain suppression.     

A study on the effect of deoxynivalenol on serotonin receptor binding in fig brain membranes

Abstract

Central serotoninergic (5‐hydroxytryptamine, 5HT) pathways are believed to be involved in the mechanisms of anorexia and/or emesis evoked by the trichothecene mycotoxin deoxynivalenol (DON). Using an in vitro membrane receptor binding assay, the competitive potency of DON was investigated against several radioactive ligands that have a high affinity for selective 5HT‐receptor subgroups. Receptor site densities and displacement profiles in twelve selected regions of pig brain were investigated. Overall, DON possessed only minimal efficacy to competently block any of the 5HT‐ligands tested. IC₅₀ values (50% inhibitory concentration) of at least 5 mM DON were required to inhibit binding, and in certain regions concentrations of 100 mM were ineffective. In comparison, several standard 5HT‐antagonists showed 10³‐10⁵ times greater capability than DON to displace binding of these ligands. Because these results indicated DON possesses only weak affinity for the 5HT‐receptor subtypes investigated here, this suggested that in vivo, unless relatively high concentrations of the toxin are present, its pharmacological effects may be mediated by mechanisms other than a functional interaction with serotoninergic receptors at the central level.     

Determination of fumonisins in milk

Abstract

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 C₁₈solid 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 FB, or AP₁ in the milk, with or without hydroiytic treatment with β‐glucuronidase/sulfatase to liberate any conjugates.     

Distribution of deoxynivalenol in cerebral spinal fluid following administration to swine and sheep 1

Abstract

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 (<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 (Vdγ = 1.13 1 kg^‐1) may be slowing the rate of diffusion of the toxin into the CSF compared to sheep (Vd_β = 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 (γ) 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.     

Metabolic fate and elimination in milk, urine and bile of deoxynivalenol following administration to lactating sheep

Using a combination of radioisotopic counting and chromatographic detection techniques, the kinetics and metabolic fate of deoxynivalenol (DON) in plasma, urine and bile were studied in lactating sheep, as was the transmission of residues to milk. Following intravenous administration, the plasma clearance of 14C-DON-derived radioactivity was rapid and followed a tri-phasic decay curve comprised of a bi-exponential decrease in DON (rapid distribution phase, t1/2 alpha = 16.2 min; slower elimination phase t1/2 beta = 66.5 min) and the formation and elimination (t1/2 beta = 188.0 min) of its major plasma metabolite, DON-glucuronide conjugate, which accounted for 13% of the plasma radioactivity levels. DON was rapidly cleared from the body by metabolism to 7 possible metabolites, which were excreted essentially in the urine (91%) and to a lesser extent in the bile (6%). Most (67%) of the recovered radioactivity was in the form of the glucuronide conjugates of DON (54%) and the de-epoxide metabolite, DOM-1 (13%). Excretion of unmetabolized DON accounted for 11%. The remaining recovered dose (18%) comprised of minor amounts of DOM-1 (6%), DON-sulfate conjugate (2%) and 3 unidentified radioactive components (10%). Studies on the presence of DON-derived residues in milk indicated that, relative to the dose, only trace amounts were transmitted following either oral or iv administration of the toxin.     

Effect of the appetite stimulant cyproheptadine on deoxynivalenol ‐ induced reductions in feed consumption and weight gain in the mouse 1

Abstract

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.

Results showed that CYP could effectively offset the reduction in feed intake caused by dietary DON, but only when dose levels were optimized (CYP appeared to have a narrow effective dose range, which was also dependent on the DON concentration). In fact, optimum CYP doses were able to enhance feed intake in DON‐fed mice above control levels, indicating that more than just a direct block of the toxin's effect was occurring. Conversely though, the effect of CYP on weight gain (WG) was ambiguous. At the lower CYP doses (≤ 5 ppm), alone or in combination with the lowest DON level tested (4 ppm), a modest positive effect was noted, but at the higher DON concentrations (≥ 8 ppm) the overall WG generally remained depressed, and tended to be reduced further by increasing doses of CYP.

These results provide additional support that serotoninergic mechanisms probably play a role in mediating DON‐induced reduction in feed intake. Where and how DON actually initiates its anorectic effect, however, has not been resolved.