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Aurora kinases are cell cycle regulated serine/threonine kinases that have been linked to cancer. Compound 1 was identified as a potent Aurora inhibitor but lacked oral bioavailability. Optimization of 1 led to the discovery of a series of fluoroamine and deuterated analogues, exemplified by compound 25, with an improved pharmacokinetic profile. We found that blocking oxidative metabolism at the benzylic position and decreasing the basicity of the amine are important to obtaining compounds with good biological profiles and oral bioavailability.

We report a series of potent imidazo[1,2-a]pyrazine-based Aurora kinase inhibitors. Optimization of the solvent accessible 8-position led to improvements in both oral bioavailability and off-target kinase inhibition. Compound 25 demonstrates anti-tumor activity in an A2780 ovarian tumor xenograft model., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

A recently developed sensitive indirect competitive enzyme-linked immunosorbent assay (ELISA) was applied to the determination of fumonisins in beer. Intra-assay and inter-assay recoveries averaged 98.7-102.8% at added fumonisin B1 (FB1) levels of 0.5-50 ng/ml beer, and coefficients of variation were 2.8-4.4 and 4.7-8.6%, respectively. Cross-reactivity of fumonisin B2 (FB2) compared with FB1 averaged 67% in beer. Two experiments were carried out to compare ELISA with liquid chromatography (LC) for determination of fumonisins in beer. In the first experiment, 19 samples (five previously known positive, nine other samples and five spiked samples) were passed through commercial immunoaffinity columns (ICs) and analysed by LC before conducting blind ELISA determinations on the extracts and beers directly. The known positive beers and extracts were used as blind duplicates. The second comparative experiment screened 46 beer samples by ELISA and then 22 positive and three of the negative samples were analysed by LC; the highest level found was 64.3 ng total fumonisins/ml measured by LC (24.7 ng/ml by ELISA). Regression analyses showed good correlation between ELISA and LC in the first experiment but low level interferences (equivalent to up to 5.35 ng fumonisin/ml) were observed by ELISA in the IC extracts. Five of nine beers negative by LC showed < 1 ng/ml ELISA responses on direct beer analysis. The second comparative experiment indicated underestimation by ELISA. However, there were two samples which tested positive by ELISA (0.2 ng/ml) but were found negative by LC (results close to the detection limits of both methods, which were 0.1 or 0.2 ng/ml by ELISA and 0.1-0.15 ng each fumonisin/ml by LC). There were no false negatives. It is concluded that ELISA has considerable value in rapid screening of beer directly for fumonisins.

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.

The mycotoxin deoxynivalenol (DON) is well documented to cause reduced feed consumption (FC) and weight gain (WG) rates in sensitive animal species. In the present study, the effect of DON on pigs when ingested as a DON-spiked diet was compared with toxin administered by a continuous-delivery osmotic pump that was implanted intraperitoneally (i.p.). The trial was conducted for a 7-day period. At 4 ppm dietary DON, overall FC and WG were reduced -14% and -8%, respectively, compared with control animals; at 9 ppm DON, values were -46% and -65% less, respectively. With i.p. infusion of te toxin, although FC was also reduced [-16% at 2 micrograms DON/hr (low dose) or -31% at 5 micrograms DON/hr (high dose)], WG rates were affected to a relatively less extent (-5% or -9%, respectively) than that noted with dietary DON. In pigs on dietary DON, both FC and WG were most affected during the initial 3-day period and showed significant recovery thereafter; however, in animals dosed by the i.p. pump, FC improved only moderately, and WG stayed relatively constant over the 7 days. The effect of dietary DON on WG appears to be influenced by more than just reduced feed intake, particularly at higher toxin concentrations.

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.

Fumonisins are carcinogenic to rats and are suspected human carcinogens. However, the mechanism(s) of carcinogenesis of fumonisn B1 (FB1) is poorly understood. Multiple signal transduction pathways such as protein kinase C (PKC) have been shown to play an important role in carcinogenesis. This study was undertaken to evaluate whether FB1 affects PKC activation. Similar to tumor-promoting phorbol ester, phorbol 12-myristate-13-acetate (PMA), PKC is also catalytically activated by FB1. Protein kinase C activity and its redistribution in response to FB1 were determined in rat cerebrocortical slices. Cytosolic and membranous PKC activities were determined by histone phosphorylation in the presence of [gamma-32P]ATP, phosphatidyl-L-serine, PMA, and Ca2+. Distribution of gamma PKC isozyme in the presence of FB1 was also assessed by immunoblotting using affinity purified anti-peptide antibodies. Similar to PMA, FB1 added in vitro to rat cerebrocortical slices facilitated PKC translocation from cytosol to membrane in a concentration-dependent manner. This FB1-induced PKC translocation was inhibited by incubation with the inactive 4 alpha-phorbol 12,13-didecanoate. The effects of FB1 and PMA were neither additive nor synergistic. In addition, PMA and FB1-induced PKC enzyme redistribution were inhibited by pretreating tissues with sphingosine. A concentration-related FB1 attenuation of specific phorbol dibutyrate, [3H]PDBu, binding was also observed when cortical membranes were incubated with either PMA or sphingosine. This is the first report of FB1-induced PKC translocation via a direct action on the diacylglycerol site that also binds phorbol esters. Because phorbol esters are well known tumor promoters, we provide a plausible cellular mechanism to explain the carcinogenicity of FB1.

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 10(3)-10(5) 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.

Trichothecene mycotoxins are a group of structurally similar fungal metabolites that are capable of producing a wide range of toxic effects. Deoxynivalenol (DON, vomitoxin), a trichothecene, is prevalent worldwide in crops used for food and feed production, including in Canada and the United States. Although DON is one of the least acutely toxic trichothecenes, it should be treated as an important food safety issue because it is a very common contaminant of grain. This review focuses on the ability of DON to induce toxicologic and immunotoxic effects in a variety of cell systems and animal species. At the cellular level, the main toxic effect is inhibition of protein synthesis via binding to the ribosome. In animals, moderate to low ingestion of toxin can cause a number of as yet poorly defined effects associated with reduced performance and immune function. The main overt effect at low dietary concentrations appears to be a reduction in food consumption (anorexia), while higher doses induce vomiting (emesis). DON is known to alter brain neurochemicals. The serotoninergic system appears to play a role in mediation of the feeding behavior and emetic response. Animals fed low to moderate doses are able to recover from initial weight losses, while higher doses induce more long-term changes in feeding behavior. At low dosages of DON, hematological, clinical, and immunological changes are also transitory and decrease as compensatory/adaptation mechanisms are established. Swine are more sensitive to DON than mice, poultry, and ruminants, in part because of differences in metabolism of DON, with males being more sensitive than females. The capacity of DON to alter normal immune function has been of particular interest. There is extensive evidence that DON can be immunosuppressive or immunostimulatory, depending upon the dose and duration of exposure. While immunosuppression can be explained by the inhibition of translation, immunostimulation can be related to interference with normal regulatory mechanisms. In vivo, DON suppresses normal immune response to pathogens and simultaneously induces autoimmune-like effects which are similar to human immunoglobulin A (IgA) nephropathy. Other effects include superinduction of cytokine production by T helper cells (in vitro) and activation of macrophages and T cells to produce a proinflammatory cytokine wave that is analogous to that found in lipopolysaccharide-induced shock (in vivo). To what extent the elevation of cytokines contributes to metabolic effects such as decreased feed intake remains to be established. Although these effects have been largely characterized in the mouse, several investigations with DON suggest that immunotoxic effects are also likely in domestic animals. Further toxicology studies and an assessment of the potential of DON to be an etiologic agent in human disease are warranted.

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.

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.

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.

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.

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.

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.

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.

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)

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.

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.

Deoxynivalenol (DON) is one of the major mycotoxins produced by Fusarium fungi, and is known to affect feeding behaviour in economically important animals. In this study the effect of low-level DON on cerebral spinal fluid (CSF) neurotransmitter concentrations, as a reflection of altered brain activity, was investigated in swine. Animals were given repetitive doses (6) at 30 min intervals, either intravenously (iv) (10 micrograms/kg) or intragastrically (ig) (30 micrograms/kg). Serial CSF samples were collected via a novel permanent indwelling catheter placed intracerebrally, and changes in catecholamine levels monitored. Results showed a rapid and sustained increase in 5-hydroxyindoleacetic acid (5HIAA) occurred following ig administration, remaining elevated for up to 20 hr post-dosing, and to a lesser extent after iv dosing, lasting up to 6 hr post-dosing. This was indicative of increased CNS serotoninergic activity and indicative to the theory that links elevated brain serotonin turnover to a decrease in feed intake. Also observed were alterations in dopamine (DA) metabolism. Following ig dosing, a delayed increase in homovanillyl alcohol (HVOL) suggested an enhancement of the dopaminergic response occurred as serotoninergic activity diminished. Following iv dosing, a rapid drop in homovanillic acid (HVA) levels alluded to a more direct toxic effect, possibly acting as an inhibitor of DA-metabolizing enzymes.

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.

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 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.

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.

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.

We devised a method for the atraumatic repeated collection of cerebrospinal fluid samples from conscious swine and sheep. Indwelling needles, with injection caps, were secured intracisternally and protected with plastic "crowns" attached to their skulls. The crowns permitted the animals freer movement with minimal risk of damage to the needles. With sheep, cerebrospinal fluid was withdrawn directly by puncturing the self-sealing injection caps attached to the hubs of the fixed needles. With the pigs, which are less amenable to handling, lengths of sterile polyethylene tubing inserted into the fixed needles enable collection without continuously disturbing the pigs. Serial samples were withdrawn from sheep (.10 to .30 ml) for up to 3 weeks with no problems, and from pigs (.05 to .15 ml) for 8 to 12 days, until the cannulae failed.

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.

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.

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.

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).

The guidelines in this report focus on prevention of mycotoxin contamination associated with plant and animal production in eastern Canada. The topics covered include a brief description of the Fusarium mycotoxins of particular concern in eastern Canada, the life cycle of Fusarium and recommendations for reducing fungal infection and mycotoxin production in grain crops and animal feed. The presence of complex mixtures of toxins, as well as research on detoxification and mycotoxin residues in animal food products are discussed. For those involved in advising producers and millers on occupational hazards associated with mycotoxins, guidelines on the safe handling of contaminated grain are also included. The review provides background information for scientists and technical personnel advising grain and animal producers on how to reduce mycotoxin problems associated with F. graminearum contamination of grain, especially wheat and corn. It may be necessary to revise the recommendations to suit different agricultural regions or different plant and animal production systems.

The transmission of radioactive residues of 14C-labelled deoxynivalenol (DON; vomitoxin) to eggs was investigated during prolonged administration of low levels of DON-contaminated feed to White Leghorn chickens. Laying hens were provided with a 5.5 ppm 14C-DON-spiked diet (.55 mg DON; .8.25 microCi bird/day) for a 65-day period, after which they received a clean, unadulterated diet for 21 days. Total residues (based on specific radioactivity) increased daily until the 8th day of 14C-DON exposure, when levels reached a plateau for several days, then decreased slowly thereafter. Maximum radioactivity measured was equivalent to 1.7 micrograms DON or metabolites per 60-g egg; the yolk, albumen, and shell membrane contributed 70, 29, and 1% of the total amount, respectively. By Day 30, levels had declined to 25% of peak levels (.40-micrograms DON equivalents/egg) and remained relatively constant until the spiked feed was removed at Day 65, at which time residues quickly declined to negligible values. These findings indicate that although very low concentrations of DON can be found in eggs under these feeding conditions, levels are so low that a potential health hazard to humans would likely be minimal.

The disposition of [14C]deoxynivalenol ([14C]DON) administered to hens as either a single oral dose or consumed in spiked feed over a 6-day period was determined by tracing the specific radioactivity of tissues and excreta. Following a single intubated dose (2.2 mg [14C]DON; 2.4 microCi/bird), the toxin was found to be poorly absorbed; peak plasma levels (2-2.5 hr post-treatment) accounted for less than 1% of the administered dose. Maximum tissue residues were measured at 3 hr in all tissues (liver, kidney, brain, heart, spleen, proventriculus, gizzard, small intestine) except for fat, muscle, and oviduct which occurred at 6 hr postdosing. Among the organs, the highest activities were measured in kidney, liver, and spleen; however, these levels were equal to less than 500 ng DON equivalents/g tissue, and declined quickly. Clearance of radioactivity from tissue had an average half-life of 16.83 +/- 8.2 hr (range 7.7-33.3 hr, depending on the tissue). Elimination of the labeled toxin in excreta occurred rapidly; recovery of radioactivity accounted for 78.6, 92.1, and 98.5% of the dose by 24, 48, and 72 hr, respectively. In continuously dosed birds fed 2.2 mg unlabeled DON for 6 days followed by 2.2 mg (1.5 microCi) [14C]DON for 6 days, accumulation of radioactivity in tissues did not occur. Maximum residual levels, which occurred in the kidneys, were only 60 ng DON equivalents/g. Estimated level of residues contained in the edible tissues amounted to only 13-16 micrograms DON/1.5 kg hen.

The pharmacokinetics of the trichothecene mycotoxin deoxynivalenol (DON) was investigated in swine following intravenous (0.30 mg, 0.35 microCi/kg) and intragastric (0.60 mg, 0.60 microCi/kg) administration of the 14C-labeled toxin. After iv dosing, plasma concentration data favored a three-compartment open model with half-life values for the rapid distribution (alpha), slower distribution (beta), and terminal elimination (gamma) phases of 5.8, 96.7, and 510.0 min, respectively. The apparent volume of distribution (V'd) was 1.34 liter/kg, the volume of the central compartment (Vc) was 0.166 liter/kg, and the plasma clearance was 1.81 ml/min/kg. DON was rapidly cleared essentially unchanged (greater than 95%), and was excreted primarily in urine (86-104%), with minor elimination in bile (3-5%). Following intragastric dosing DON was very rapidly absorbed, reaching near peak plasma levels within 15-30 min. Levels remained elevated (63-325 ng/ml) for approximately 9 hr, and began declining slowly (t1/2 beta = 7.1 hr) thereafter. The calculated systemic bioavailability (F) was between 48 and 65%, although urinary and biliary recoveries indicated marginally greater absorption actually occurred (54-85%). Overall, although DON was eliminated rapidly and completely within 24 hr following a single iv or intragastric dose, data suggest that residues may undergo temporary sequestration in a tissue depot.

In vitro metabolism of the aromatic ketone propiophenone and its nonaromatic isomer phenylacetone was studied using fortified 12 000 X g supernatants of liver homogenates from rat and rabbit. Reduction to the corresponding alcohols was the major metabolic route observed, although aliphatic C-hydroxylation and alcohol dehydrogenation also occurred. Marked differences were observed in the amounts of carbonyl reduction of the substrates, which was dependent on the species as well as the cofactor employed. Using rat liver preparation, phenylacetone was reduced to 1-phenyl-2-propanol much more efficiently with an NADH-fortified system than when NADPH was used whereas in rabbit, extensive reduction occurred in the presence of either cofactor. Reduction of propiophenone to 1-phenyl-1-propanol by rat liver preparation was slightly greater in the presence of NADH than with NADPH; the converse was observed in rabbit. Aliphatic hydroxylation of propiophenone to 2-hydroxy-1-phenyl-1-propanone was also a significant metabolic pathway in both species, with NADPH being the more efficient cofactor, but C-1 hydroxylation of phenylacetone to 1-hydroxy-1-phenyl-2-propanone occurred only to a minor extent. Small amounts of 1-phenyl-1,2-propanedione, as well as both erythro and threo isomers of 1-phenyl-1,2-propanediol, were also identified as metabolites in both species. Similar metabolic studies were carried out on the alcohols 1-phenyl-1-propanol and 1-phenyl-2-propanol and again the nature and quantities of metabolites isolated showed both species and cofactor dependencies.

A method is described for the analysis of zearalenone and its metabolites, alpha- and beta-zearalenol, in small volumes (0.5-2.0 ml) of biological fluids including milk, blood, plasma, urine and bile, using high-performance liquid chromatography with fluorescence detection. Isolation of the toxins from biological fluids was achieved using a series of pH-controlled solvent extractions. Detection limits for zearalenone and alpha-zearalenol were 1 ng/ml, and for beta-zearalenol ca. 5 ng/ml, both at a signal-to-noise ratio of 3. In bile, however, the detection was ca. five times less sensitive owing to interfering substances. Recoveries at low ng/ml concentrations were highest from urine (87-94%) and plasma (85-93%), slightly lower from whole blood (78-88%) and milk (75-84%), and lowest from bile (66-77%).

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.

The excretion profiles of deoxynivalenol (DON) and metabolites (DON glucuronide conjugate, 3 alpha, 7 alpha,15-trihydroxytrichothec-9,12-diene-8-one (DOM-1), and DOM-1 glucuronide conjugate) were defined in male sheep following either intravenous (iv) or oral administration of the toxin at levels of 0.5 and 5.0 mg DON/kg body wt, respectively. After iv dosing, urinary DON levels declined in a biphasic fashion with an average elimination half-life (terminal phase) of 1.2 hr, diminishing to baseline concentrations by 8 hr. Maximum urinary excretion rates for the two major metabolites identified (conjugated DON, conjugated DOM-1) occurred 0.5-1.5 hr after dosing, exhibiting elimination half-lives of 2.2 and 3.1 hr, respectively. Total recovery accounted for only about 66.5% of the dose: 63.0% in the urine (24.1% DON, 21.2% conjugated DON, 0.5% DOM-1, 17.2% conjugated DOM-1) and 3.5% in bile (made up almost completely of conjugated DOM-1). The peak biliary excretion rate for conjugated DOM-1 was found to occur within 1 hr postdosing, which rapidly declined to baseline levels by 5 hr. Following oral administration, urinary excretion rates of the major metabolites (DON, conjugated DON, conjugated DOM-1) reached maximum 6-9 hr post-treatment, and declined exponentially with t 1/2 values of 3.2, 4.0, and 5.0 hr, respectively. Urinary and biliary recovery of administered DON averaged approximately 7.1%: 7.0% in urine (2.1% DON, 3.6% conjugated DON, 0.06% DOM-1, 1.2% conjugated DOM-1) and 0.11% in bile (predominantly conjugated DOM-1).(ABSTRACT TRUNCATED AT 250 WORDS)

The stereospecificity of the metabolic reduction of arylalkylketones was investigated. The ketones, propiophenone (I), phenyl-acetone (III), and 1-phenyl-1,2-propanedione (V) were reduced in vitro and in vivo in rats and rabbits to the corresponding alcohols, 1-phenyl-1-propanol (II), 1-phenyl-2-propanol (IV), and 1-phenyl-1,2-propanediol (VIII), respectively. For the analysis, a capillary GLC method employing chiral derivatizing reagents for the resolution of these optically active alcohols was utilized. This study revealed that the metabolic reduction of each ketone produced the corresponding alcohol as a mixture of its enantiomers. With one exception, the mixtures obtained from all in vivo and in vitro reactions were shown to contain at least 70% of one isomer [S(-)-II, S(+)-IV, and erythro-VIII, respectively], with in vitro reduction showing the highest degree of stereospecificity (90-98%). The in vivo reduction of I by the rat was exceptional in that both optical isomers of II were recovered in equal proportions.

A specific analytical procedure for the quantitation of amphetamine (I), norephedrine (III), and their amphoteric metabolites, p-hydroxy-amphetamine (II) and p-hydroxynorephedrine (IV), in biological samples using electron-capture gas chromatography (GC-EC) is described. The procedure utilizes the ion-pairing reagent, bis(2-ethylhexyl)phosphoric acid, which frees the amines from most contaminants and permits the efficient extraction of the amphoteric compounds (as acetates) from the aqueous solution. Amines I and III and acetylated amines II and IV were perfluoroacetylated prior to GC-EC analysis. Metabolism of I, II, and III in the rat brain was studied. Results indicate that both in vivo and in vitro amines I and III are p-hydroxylated to II and IV, respectively, and II is beta-hydroxylated to give IV. Norephedrine (III) was not detected as a rat brain metabolite of amphetamine (I).

A comparison of 2 extraction solvent systems (acetonitrile-water, 21 + 4 and methanol-water, 1 + 1) and 3 mixing apparatus (high-speed blender, wrist-action shaker, and mechanical stirrer) was carried out for different extraction time periods. Methods were evaluated using uncontaminated corn spiked with pure deoxynivalenol (DON), field-inoculated (Fusarium graminearum) corn, and uncontaminated and naturally infected wheat in swine diets. After sample extraction, aliquots were passed through alumina-charcoal cleanup columns, evaporated to dryness, dissolved in 8% aqueous methanol, and injected onto the liquid chromatograph. Results confirm published reports of recoveries from DON-spiked samples; however, longer extraction times (less than or equal to 120 min) were required for naturally contaminated samples. Use of the high-speed blender resulted in faster extractions, but in our laboratory more samples could be more conveniently extracted simultaneously with the wrist-action shaker or mechanical stirrer. Less carryover (co-extraction) of interfering contaminants was observed when acetonitrile-water was used vs methanol-water. Results emphasize the importance of careful evaluation of extraction procedures with not only spiked samples but also naturally contaminated samples to establish extraction times required for maximum deoxynivalenol recoveries.

The optimization of a simple, sensitive procedure using a chick embryotoxicity screening test (CHEST) bioassay for detection of toxic compounds is presented. Dosing protocols of eggs, using several mycotoxins (aflatoxin B1, deoxynivalenol, T-2 toxin) and appropriate controls, were evaluated for embryonic sensitivity, overall practicality of the procedure, and consistency of results. It was found that both type of carrier solvent and volume injected could significantly affect overall embryonic mortality. The chick embryo was most sensitive to the effects of toxins and solvents after 1 or 2 days of incubation; a rapid decrease in response was observed as the age of the embryo at dosing increased. Following administration of the toxins just below the shell membrane by way of a small hole (less than 0.5 mm diameter) punched in the shell, a good dose-response (% mortality) could be obtained regardless of the site of injection (except directly into the yolk), although dosing via the air sac position resulted in a slightly better statistical outcome. Although some variations in calculated LD50 values were found among repeated assays, statistical analyses showed that the differences were not due to dosing protocol but to the variations in embryo sensitivities among batches of eggs. Thus, if standard reference toxins for comparison are run concurrently, the CHEST assay can prove to be a very satisfactory model, as well as having considerable flexibility to be adapted to the needs and resources of many laboratories.