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. Author manuscript; available in PMC: 2009 Jun 15.
Published in final edited form as: Toxicol Lett. 2008 Feb 23;178(2):83–87. doi: 10.1016/j.toxlet.2008.02.005

Immunochemical Assessment of Deoxynivalenol Tissue Distribution Following Oral Exposure in the Mouse

James J Pestka *,†,§,1, Zahidul Islam *, Chidozie J Amuzie §,
PMCID: PMC2696392  NIHMSID: NIHMS50788  PMID: 18395371

Abstract

Deoxynivalenol (DON or vomitoxin) is a trichothecene mycotoxin commonly found in cereal grains that adversely affects growth and immune function in experimental animals. A competitive enzyme-liked immunosorbent assay (ELISA) was used to monitor the kinetics of distribution and clearance of DON in tissues of young adult B6C3F1 male mice that were orally administered 25 mg/kg bw of the toxin. DON was detectable from 5 min to 24 h in plasma, liver, spleen and brain and from 5 min to 8 h in heart and kidney. The highest DON plasma concentrations were observed within 5 to 15 min (12 μg/ml) after dosing. There was rapid clearance following two-compartment kinetics (t1/2α=20.4 min, t1/2β=11.8 h) with 5 and 2% maximum plasma DON concentrations remaining after 8 and 24 h, respectively. DON distribution and clearance kinetics in other tissues were similar to that of plasma. At 5 min, DON concentrations in μg/g were 19.5±1.9 in liver, 7.6±0.5 in kidney, 7.3±0.8 in spleen, 6.8±0.9 in heart and 0.8±0.1 in the brain. DON recoveries in tissues by ELISA were comparable to a previous study that employed 3H-DON and 25 mg/kg bw DON dose. The ELISA was further applicable to the detection of DON in plasma of mice exposed to the toxin via diet. This approach provides a simple strategy that can be used to answer relevant questions in rodents of how dose, species, age, gender, genetic background and route/duration of exposure impact DON uptake and clearance.

Introduction

The trichothecene mycotoxins are group of more than 200 structurally related sesquiterpenoid metabolites produced by foodborne and environmental fungi that are characterized by the tetracyclic 12,13-epoxytrichothec-9-ene ring system (Grove, 2007). Toxicological effects associated with trichothecene mycotoxin poisoning in humans and animals include anorexia, gastroenteritis, emesis and hematological disorders (Pestka and Casale, 1990). The immune system is extremely sensitive to trichothecenes (Pestka et al., 2004). Exposure to low trichothecene doses induces rapid, transient upregulation of proinflammatory cytokines causing immune stimulation, whereas high doses of trichothecenes cause apoptosis in lymphoid tissues resulting in immunosuppression.

Deoxynivalenol (DON, vomitoxin) is a trichothecene commonly found in wheat, barley and corn that have been infected by the mold Fusarium graminearum (Placinta et al., 1999; Schothorst and van Egmond, 2004). The presence of ppm levels of DON in foods is of major human health concern worldwide (Pestka and Smolinski, 2005). All animal species evaluated to date are susceptible to DON according to the rank order of swine>mice>rats>poultry≈ ruminants (Rotter et al., 1996). Differences in metabolism, absorption, distribution, and elimination of DON among animal species have been suggested to account for this differential sensitivity (Pestka, 2007).

In addition to species sensitivity, critical questions still remain as to how age, gender, genetic background and route of exposure affect absorption distribution, metabolism and excretion of DON. Radiolabeled DON has been employed to investigate the toxicokinetics and tissue distribution of this mycotoxin in several animal species in several studies (Azcona-Olivera et al., 1995)(Rotter et al., 1996) (Lake et al., 1987; Lun et al., 1989; Meky et al., 2003; Prelusky et al., 1986; Prelusky et al., 1988). Limitations of such radioisotope approaches include (1) challenges associated with preparing these compounds with high specific activity, (2) unavailability of radiolabeled DON from commercial sources, (3) difficulties applying their use in large animal studies and (4) general safety issues related to handling and disposal of these materials. While conventional chemical analyses have also been employed to track DON absorption and distribution in tissues (Prelusky et al., 1985; Prelusky et al., 1990), these methods lack sensitivity, require large amounts of tissue and involve extensive sample clean-up.

Mycotoxin immunoassays are rapid, extremely sensitive, do not require extensive clean-up and typically do not require evaporation or concentration (Pestka,1988). Our laboratory was the first to describe DON-specific antibodies and an ELISA for this mycotoxin (Casale et al., 1988). Immunochemical assays are now widely employed for the analysis of DON in cereal grains (Schneider et al., 2004). ELISA might be similarly applicable to detecting DON in tissues of experimental animals exposed to this and other trichothecenes. The purpose of this study was to apply the ELISA to monitoring the kinetics of DON distribution and clearance in mouse plasma and tissues following a single oral exposure to the toxin. The results indicated that the ELISA was comparable to a previously described radioisotope approach for monitoring DON toxicokinetics, with both showing DON to be quickly distributed throughout the body and removed during sequential rapid clearance and slower terminal elimination phases.

Materials and Methods

Chemicals

All reagents were of reagent-grade quality or better and purchased from Sigma Chemical Co. (St. Louis, MO) unless otherwise described. For acute oral exposure studies DON was purchased from Sigma and used. For the feeding study, DON was produced in Fusarium graminearum R6576 cultures and purified by silica gel chromatography (Clifford et al., 2003). Purity of DON was verified by a single HPLC peak at 224 nm. Concentrated toxin solutions were handled in a fume hood. Labware that was contaminated with mycotoxin was detoxified by soaking for >1 h in 100 mL/L sodium hypochlorite.

Animals

Male B6C3F1 (C57B1/6J×C3H/HeJ) mice (7 wk) were obtained from Charles River (Portage, MI). Animal handling was conducted in accordance with recommendations established by the National Institutes of Health and were approved by the Michigan State University Institutional Animal Care and Use Committee. Mice were kept in environmentally protected transparent polypropylene cages with stainless steel wire tops and filter covers and acclimated for 1 wk prior to onset of different experimental treatments. Environmental conditions included 23–25°C, relative humidity of 45–55%, and a 12-h light:dark cycle. Mice were 3 housed per cage and provided food and water ad libitum.

Experimental design

For the acute exposure study, food and water were withdrawn from cages 1 h prior to DON administration. DON was administered at 25 mg/kg bw by oral gavage in 250 μl of endotoxin-free water. Food and water were restored immediately after gavaging. Ungavaged animals were used as controls. At 5, 15, 30 min and 1, 2, 4, 8, 24 h, mice were anesthetized and blood collected. Mice were then euthanized and liver, kidney, heart, spleen and brain removed. Plasma was isolated from blood and stored at -20°C. Organs were frozen at -80°C, pulverized using a mortar and pestle and then mixed with phosphate-buffered saline (PBS) at a 1:5 ratio. The tissue extract was centrifuged for 10 min at 14,000 × g. The resultant supernatant fraction was heated to 100°C for 5 min to inactivate enzymes and precipitate proteins. The heated extract was centrifuged for 10 min at 14,000 × g and the supernatant fraction subsequently used with appropriate dilution for ELISA.

For the subchronic feeding study, purified DON was added at 0, 2, 5, 10 and 20 mg/kg of powdered high fat AIN-93 G Purified Rodent Diet 101847 (Dyets Inc, Bethlehem, PA) as detailed previously (Pestka et al., 1989). Experimental diets were placed in special containers to minimize spillage. Cages were kept in class II ventilated cabinets for the duration of the experiment. Mice were fed for 4 wk and blood collected from the saphenous vein as described previously (Hem et al., 1998).

Measurement of DON by competitive direct ELISA

DON was analyzed using a commercial DON ELISA (Veratox for DON, Neogen Corp., Lansing, MI) which is based on a previously described direct competitive ELISA (Casale et al., 1988). The monoclonal antibody used in this assay binds to DON and 3-ADON but not to other trichothecenes. Since this assay was designed to analyze DON in grain and grain products, the manufacturer's protocol was adapted for use on plasma and organ tissue samples by preparing separate DON (Sigma) standards (4 to 1,000 ng/ml) in 10% (v/v) human plasma (Innovative Research Inc., Southfield, MI) or 1% bovine serum albumin (w/v) in PBS which functioned to prevent non-specific binding. Samples or standards (100 μl each) were combined with 100 μl of DON horseradish peroxidase conjugate solution in mixing wells provided by the manufacturer. A 100 μl aliquot of this mixture was transferred to antibody-coated wells and incubated for 5 min at 25°C. Wells were washed, 100 μl of K-blue Max TMB substrate (Neogen, Lansing, MI) added and the wells incubated for an additional 5 min at 25°C. The reaction was stopped with 100 μl of 2N sulfuric acid and absorbance read on a Molecular Devices ELISA reader (Menlo Park, CA) using 450 nm filter. DON concentrations were quantified from a standard curve using Molecular Devices Softmax software. DON recoveries of 90 percent or higher were observed in preliminary studies either in which heated liver extracts were spiked directly with the toxin at 12.5 to 250 ng/g tissue and analyzed or in which liver homogenates were spiked with the toxin at 10 to 250 ng/g tissue, heated and supernatants analyzed. Since this ELISA might detect DON as well as some of its metabolites, data were reported as DON equivalents per ml plasma or per g organ tissue.

Pharmacokinetic analysis

A two-compartment open model (Shargel et al., 2004) was employed to calculate toxicokinetic parameters. DON concentrations in plasma and tissue were fitted to biexponential expression to calculate clearance rates (Li et al., 1997).

Statistics

Data were analyzed using Sigma Stat for Windows (Jandel Scientific, San Rafael, CA). A Kruskal-Wallis One Way Analysis of Variance was performed. Data sets were significantly different when p<0.05.

Results

DON was rapidly absorbed into plasma following oral exposure to a 25 mg/kg bw dose. The highest concentrations, 12.1 and 11.5 μg/ml, were detectable at 5 and 15 min, respectively (Fig. 1). There was rapid clearance following two-compartment kinetics with half-lives of distribution (t1/2α) and clearance (t1/2β) being 20.4 min and 11.8 h, respectively.

Figure 1. Kinetics of DON distribution and clearance in plasma of the mouse after single oral exposure.

Figure 1

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Mice were orally administered with 25 mg/kg bw DON. Blood was collected at intervals and DON concentrations in plasma determined by ELISA. Data were plotted as mean DON equivalents ± SEM (n = 3).

DON distribution and clearance kinetics in liver (Fig. 2A) and kidney (Fig. 2B) similarly followed a two-compartment kinetic pattern. As observed for plasma, concentrations of DON peaked at 5 to 15 min in liver (19.6 and 12.1 μg/g, respectively) (Fig. 2A) and kidney (7.6 and 9.0 μg/g, respectively) (Fig. 2B). The t1/2α and t1/2β in liver were 22 min and 19.0 h, respectively, whereas the t1/2α and t1/2β for kidney were 47 min and 20.9 h, respectively.

Figure 2. Kinetics of DON distribution and clearance in liver and kidney of the mouse after single oral exposure.

Figure 2

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Mice were orally administered with 25 mg/kg bw DON and sacrificed at various time intervals. Liver (A) and kidney (B) were collected and DON concentrations determined by ELISA and data plotted as mean DON equivalents ± SEM (n = 3).

As seen in spleen and liver, the above tissues, the highest DON concentrations in spleen were observed at 5 min (7.3 μg/g) and 15 min (7.9 μg/g (Fig. 3A). The t1/2α and t1/2β were 29 min and 9.0 h, respectively. The highest DON concentrations were observed in the heart at 5 min (6.7 μg/g) and 15 min (6.8 μg/g) with t1/2α and t1/2β being 41 min and 12.3 h, respectively (Fig. 3B).

Figure 3. Kinetics of DON distribution and clearance in spleen and heart of the mouse after single oral exposure.

Figure 3

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Mice were orally administered with 25 mg/kg bw DON. Spleen (A) and heart (B) were collected and DON concentrations determined by ELISA and data plotted as mean DON equivalents ± SEM (n = 3).

Compared to other tissues, DON entered the brain much more slowly and peaked at much lower concentrations (0.7-1.0 μg/g), lasting from 5 min to 2 h (Fig. 4).

Figure 4. Kinetics of DON distribution and clearance in brain of the mouse after single oral exposure.

Figure 4

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Mice were orally administered with 25 mg/kg bw DON. Brain was analyzed for DON by ELISA and data plotted as mean DON equivalents ± SEM (n = 3).

The effects of subchronic dietary exposure to DON on plasma concentrations of this mycotoxin were also assessed in a 4 wk feeding study. Dose-dependent increases in plasma DON were observed in mice fed the toxin at 2, 5, 10 and 20 mg/kg diet with concentrations ranging from 20 to 100 ng/ml (Fig.5). All four of these doses reduced weight gain (data not shown) as has been reported previously for the B6C3F1 mouse (Forsell et al., 1986)

Figure 5. Dose-dependent effects of short-term DON consumption on DON plasma concentrations.

Figure 5

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Mice were fed various concentrations of DON for 4 wk and blood collected. Plasma was analyzed for DON by ELISA. Data plotted as mean DON equivalents ± SEM (n = 3).

Discussion

The results presented here suggested that DON uptake and distribution in the mouse could be monitored by ELISA. DON was rapidly taken up into tissues after exposure and maximum concentrations followed the rank order liver> plasma> kidney> spleen> heart> brain. DON was cleared initially at a rapid rate in all tissues except for brain and this was followed by a relatively slower rate of clearance. The data presented here were highly consistent with a previous [3H]-DON study conducted in the mouse by our laboratory (Azcona-Olivera et al., 1995). That prior study also reported two compartment kinetics with an initial rapid clearance rate and slower terminal elimination in plasma following exposure to DON doses of 5 mg/kg bw (t1/2α = 21.6 min and t1/2β =7.6 h, respectively) and 25 mg/kg (t1/2α = 33.6 min and t1/2β = 88.9 h, respectively). When DON uptake and clearance in plasma, liver and kidney observed here during the first 4 h were graphically compared to the aforementioned study at the 25 mg/kg bw dose, the results were found to be remarkably similar (Fig. 6). The ELISA was thus comparable to the radiolabel method for monitoring DON tissue concentrations and toxicokinetics in the mouse. Key advantages of this immunoassay approach are that (1) it was very sensitive and specific, (2) it employed a facile sample preparation protocol that did not require extensive clean-up and (3) it did not require evaporation or concentration.

Figure 6. Estimations by ELISA and radiolabel methods of DON distribution in plasma, liver and kidney after a single oral exposure are comparable.

Figure 6

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Data, determined as described in Figs. 1 and 2, were arithmetically plotted as mean DON equivalents (solid circles) and compared to data from a similar previous study (Azcona-Olivera et al. 1995) conducted with [3H]-DON and employing the identical DON dose and mouse strain (open circles).

The acute dose employed here, 25 mg/kg bw, is equivalent to 1/3 the LD50 previously reported for B6C3F1 female mice (Forsell et al., 1987). Although there were no obvious signs of toxicity at this dose, DON disposition and clearance kinetics seen here are highly consistent with the temporal patterns for biomarkers of stress-related responses DON rapidly induces the expression of the proinflammatory cytokines (IL-1β, IL-6, TNF-α) that potentially contribute to induction of anorexia and multiple immunologic effects (Azcona-Olivera et al., 1995; Zhou et al., 1997). Cytokine upregulation is mediated by mitogen-activated protein kinase (MAPK)-driven activation of transcription factors and enhancement of mRNA stability (Pestka et al., 2004). In mice, acute oral DON exposure (25 mg/kg bw), sequentially induces in the spleen: (1) phosphorylation of JNK 1/2, ERK 1/2 and p38 (15-30 min), (2) transcription factor activation (1-2 h), and (3) cytokine mRNA expression (1-4 h) (Zhou et al., 2003a). Thus, the time required to reach the highest DON concentrations in serum and tissues (5 to 15 min) correlates well with rapid stress-mediated gene expression.

It should be noted that in the subchronic feeding study, plasma DON levels increased in dose-dependent fashion with increasing concentrations of the toxin in diet. The DON concentration range of 2 to 20 mg/kg diet utilized here encompasses low and high levels that have previously been observed in human food products (Abouzied et al., 1991) and cause reduced weight gain in mice (Forsell et al.,1986). The DON concentrations found in plasma are also consistent with those having effects in leukocyte culture studies. For example, in cloned macrophages, DON at 100 ng/ml has previously been shown to rapidly induce MAPK kinase activation as well as cytokine gene expression (Zhou et al., 2003b; Zhou et al., 2005). Furthermore, we have observed that DON induces p38 activation in cultured human peripheral blood mononuclear cell cultures at concentrations as low as 25 ng/ml (Islam et al., 2006). Further investigation is warranted on how prolonged chronic consumption impacts plasma and tissue DON concentrations of how these effects might affect both kinase signaling and cytokine gene expression in immunocompetent cells.

DON has also been reported to have neuroendocrine effects (Rotter et al., 1996). It was therefore notable that this mycotoxin was detected in brain, albeit at peak concentrations that were approximately one-tenth those found in other organs. In a study of fate and distribution of the radiolabeled T-2 toxin, in guinea pigs, it was similarly observed that concentrations of this trichothecene in brain represented 10 to 25 percent that of other organs (Pace et al., 1985). Thus, while the blood brain barrier might impede uptake of trichothecenes, they apparently can still enter brain. Interestingly, DON's anorectic and emetic responses have been suggested to be mediated by the serotoninergic system based on increased levels of serotonin or its metabolites in DON-treated animals as well as the capacity of serotonin receptor antagonists to prevent DON-induced emesis (Prelusky et al., 1992; Prelusky, 1993; Prelusky and Trenholm, 1993). Clarification is needed on whether the neuroendocrine effects of DON result from direct interaction with the brain or, rather, are secondary effects of proinflammatory cytokines or other mediators evoked by the toxin in other tissues.

Numerous investigations have been conducted in several animal species to elucidate DON's tissue targets, mechanisms of action and threshold doses for adverse effects. These have been applied to establish tolerable daily intakes (TDIs) and regulatory limits in specific foods (Canady, 2001; Pieters et al., 2002). The potential for chemicals to differentially affect human subpopulations is an important consideration when determining TDIs and establishing regulatory standards (Falk-Filipsson et al., 2007). One way that subpopulations can differ in is their ability to distribute and clear a toxin, which will ultimately impact its concentration in a target organ and the magnitude of its pathophysiologic effects. The results presented here suggest that ELISA could be used to analyze for DON in animal plasma and tissues without extensive clean-up and that, in the mouse, the toxin was taken up into tissues throughout the body within a very short time. This approach provides a simple strategy that can be used to answer relevant questions of how dose, species, age, gender, genetic background and route/duration of exposure impact DON uptake and clearance in rodents.

Acknowledgments

This work was supported in part by the USDA, under a cooperative project with U.S. Wheat and Barley Scab Initiative. Any findings, opinions, conclusions or recommendations expressed in this publication are those of the authors and do not necessarily reflect the views of USDA. This project was also funded in part by Public Health Service Grant ES 3358 (JJP) from the National Institute for Environmental Health Sciences. We would like to thank Dr. Pavlina Yordanova, Sarah Godbehere and Mary Rosner for technical assistance.

Footnotes

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