Journal of Toxicology and Environmental Health
ISSN: 0098-4108 (Print) (Online) Journal homepage: http://www.tandfonline.com/loi/uteh19
Investigations in the use of mice exposed to mycotoxins as a model for growing pigs Barbara A. Rotter , Roland G. Rotter , Brian K. Thompson & H. Locksley Trenholm To cite this article: Barbara A. Rotter , Roland G. Rotter , Brian K. Thompson & H. Locksley Trenholm (1992) Investigations in the use of mice exposed to mycotoxins as a model for growing pigs, Journal of Toxicology and Environmental Health, 37:2, 329-339, DOI: 10.1080/15287399209531673 To link to this article: http://dx.doi.org/10.1080/15287399209531673
Published online: 20 Oct 2009.
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INVESTIGATIONS IN THE USE OF MICE EXPOSED TO MYCOTOXINS AS A MODEL FOR GROWING PIGS Barbara A. Rotter, Roland G. Rotter Centre for Food and Animal Research, Agriculture Canada, Central Experimental Farm, Ottawa, Ontario, Canada
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Brian K. Thompson Research Program Service, Agriculture Canada, Central Experimental Farm, Ottawa, Ontario, Canada H. Locksley Trenholm Centre for Food and Animal Research, Agriculture Canada, Central Experimental Farm, Ottawa, Ontario, Canada
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 OONIkg diet) was more pronounced in a doss-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 × 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.
The authors wish to thank W. Bogie, S. Clarkin, L. A. Robinson, C. Mitchell, D. Fielder, S. Croteau, R. Doak, and staff for their capable assistance. The mycotoxins used were provided courtesy of J. D. Miller, Plant Research Centre, Agriculture Canada. This research was supported by a grant (Ontario Pork Industry Improvement Program) from the Ontario Ministry of Agriculture and Food (OMAF) and Ontario Producers Marketing Board (OPPMB) under the Ontario Pork Industry Improvement Program. Research Program Service contribution no. R104; Centre for Food and Animal Research contribution no. 2026. Present address for R. C. Rotter is Toxicological Evaluation Division, Bureau of Chemical Safety, Food Directorate, Health Protection Branch, Health and Welfare Canada, Ottawa, Ontario, K1A 0L2. Requests for reprints should be sent to Dr. B. A. Rotter, Centre for Food and Animal Research, Agriculture Canada, Ottawa, Ontario K1A 0C6, Canada.
329 Journal of Toxicology and Environmental Health, 37:329-339, 1992 Copyright © 1992 by Hemisphere Publishing Corporation
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There was a trend (p — .064) suggesting a possible interaction between the two toxins after 7 d of exposure. For animals consuming 4 mg T-2 toxin/kg diet, increasing the DON concentration from 4 to 8 mglkg diet did not result in further depression in weight gain. Only at 2 mg T-2 toxin did increasing the DON concentration from 4 to 8 significantly (p < .05) reduce weight gains further. Food conversion was depressed for both toxins after 7 d of exposure, but over the whole 2-wk period, this effect was only seen for T-2 toxin. Compared to growing pigs, mice display similar dose-dependent linear responses in feed consumption and weight gain due to presence of dietary DON and T-2 toxin. Further experiments are needed to establish responses of mice to a range of mycotoxins as a part of the mouse model evaluation.
INTRODUCTION Mold-contaminated feedstuffs often contain more than one mycotoxin or other secondary fungal metabolites. Each of the metabolites, in turn, potentially may have its own effect(s) on the biological systems of animals that ingest them. Recent studies at the Centre for Food and Animal Research (CFAR) have examined possible interactions between deoxynivalenol (vomitoxin, DON) and co-occurring metabolites of Fusarium graminaerum (Rotter et al., 1992), and DON and T-2 toxin in swine (Friend et al., 1992). Although certain combinations appeared to indicate possible interactions, the studies were hampered by severely limited quantities of the metabolites, thereby limiting the number of animals exposed (Rotter et al., 1992). Scarcity of pure metabolites generally precludes the use of larger animals such as swine in extensive toxicity trials. Mice and rats are often used because of their low cost, availability, uniformity, and the lesser amounts of compound needed to conduct toxicity tests. Studies with mice have examined the subacute effects of dietary DON (Forsell et al., 1986; Tryphonas et al., 1986) and T-2 toxin (Hayes and Schiefer, 1980, 1982). Forsell et al. (1986) observed a significant (p < .01) reduction in weight gain of B6C3F1 female mice fed diets containing 2 or more mg DON/kg diet over an 8-wk trial. Although feed refusal was observed sporadically at 2 mg DON/kg diet, food consumption decreased only on the 25 mg DON/kg diet after 6 d of exposure (p < .01). Dose-related depressions in weight gain were also more pronounced in juvenile (16.4 g) than in young adult (32.2 g) male mice after 14 or 28 d of ingesting 10 or 20 mg T-2 toxin/kg diet (Hayes and Schiefer, 1982). Mice and rats exposed to DON (Arnold et al., 1986) exhibit the same general effects, such as reduced weight gain and feed refusal, seen in swine (Friend et al., 1982, 1986a, 1986b). Feed intake in growing-finishing pigs decreased in a linear fashion as the DON level increased, but, even though weight gains differed significantly in the first week, the differences were not maintained over a 7-wk period (Friend et al., 1982). In a subsequent study, pigs exposed to a 4.2 mg DON/kg diet containing Fusarium-inoculated corn were more strongly affected in the first week
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and never recovered when compared to pigs fed a contaminated wheat diet containing 3.7 mg DON/kg. This suggested the presence of another toxic metabolite(s) (Friend et al., 1986a). Although differences in kinetic parameters (metabolism, absorption, elimination, etc.) exist between the pigs and mice, and extrapolation of data between species presumes certain conditions, the mouse may prove to be a satisfactory animal model for initial studies on mycotoxin interactions. The present research examined the effects of pure DON and T-2 toxin at dietary concentrations comparable to naturally occurring levels on growing female mice. This series of experiments is the first step in determining similarities between mice and pigs in response to selected toxins as a part of an overall evaluation of the mouse model for growing pigs in mycotoxin research. MATERIALS AND METHODS Animals Weanling 3-wk-old outbred female mice (ICR, weighing 15-17 g) were obtained from the CFAR Research Farm (original source: Charles River Canada, St-Constant, Quebec) and housed either 3 per cage (Experiment 1) or singly (Experiment 2 and 3) in transparent polypropylene cages (Maryland Plastics, Inc., Federalsburg, Md.). Each cage unit consisted of a stainless-steel wire lid, water bottle, individual feeder, and a layer of heattreated hardwood shavings. Water was provided ad libitum and changed every 3 d. Feeders (PFF-6D, Allentown Company Inc., Allentown, N.J.) containing fresh diet were changed every 3 d to minimize exposure of animals to feed contaminated with feces and urine. The mice were kept in an environmentally controlled room (22 ± 2°C) on a 12-h light/dark cycle. Diet The mice were fed ground or pelleted mouse chow 5015 (Purina Mills, St. Louis, Mo.). Chow was ground using a plate grinder (model 105A, Sprout-Waldron, Muncy, Pa.), and the specified amount of toxin was mixed into the diet. The pelleted form of the diet was prepared by mixing ground chow with water, then freeze-drying and cutting in into small blocks as outlined by Morrissey and Norred (1984). Sufficient quantities of feed were prepared for the whole experiment and stored at 4°C. Toxins Purified DON (99.1%) and T-2 toxin (99.1%) were provided by J. D. Miller, Plant Research Centre, Agriculture Canada, Ottawa, Ontario. Deoxynivalenol was prepared from the hydrolysis of 3-acetyldeoxynivalenol and purified from the media of large-scale Fusarium culmorum cultures following procedures outlined by Greenhalgh et al. (1986), while T-2 toxin
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was isolated and purified from Fusarium sporotrichioides (Greenhalgh et al., 1990).
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Experimental Design In Experiment 1,180 mice were randomly assigned to 6 dietary treatments (0, 4, 8, 12, 16, and 20 mg DON/kg diet), x 2 age groups using 5 replicates with 3 mice per cage (15 mice per treatment group). Mice allocated to the first group were fed experimental diets from d 1 (first exposure at 21 d of age). The second group was fed a regular chow pellet for the first 7 d, followed by experimental diets (first exposure at 28 d of age). Body weights were recorded on d 0, 2, 4, 7, and at 7-d intervals thereafter until wk 6 for group 1 (42 d) and 5 for group 2 (35 d). All diets were fed as pellets, but food consumption was not measured. In Experiment 2, 96 mice, housed singly, were randomly assigned to 4 dietary treatments (0, 8, 12, and 16 mg DON/kg diet) x 2 dietary regimens in a randomized block design with 12 replicates. The regimens consisted of two forms of diet, one prepared by mixing the toxin in the ground chow and the other by mixing the toxin in the ground chow, adding water, freeze-drying, and regrinding the product as outlined above. Body weight and food consumption were measured on d 0, 2, 4, 7, and 14. In Experiment 3,100 mice, housed singly, were assigned randomly to 10 dietary treatments fed as a mash in a randomized block design with 10 replicates. The treatments were as follows: control; 4, 8 mg DON/kg diet; 2, 4, 8 mg T-2 toxin/kg diet; and 4 + 2, 4 + 4, 8 + 2, and 8 + 4 mg DON + T2 toxin/kg diet. Body weight and food consumption were measured on d 0, 2, 4, 7, and 14. Analysis All diets were analyzed for DON (Trenholm et al., 1985) and T-2 toxin (Croteau et al., 1992) to confirm dietary concentrations and to ensure a uniform mix. Analysis of variance was applied to the weight gains and food consumption over specific periods. In order to look for trends with increasing toxin levels, the treatment sums of squares were partitioned into the portions due to linear regression and to deviations from the regression (Agriculture Canada Software no. SO22-RPS) as outlined by Snedecor and Cochran (1980). RESULTS Experiment 1 There were no differences (p > .05) in initial body weights among treatments for the two age groups at randomization (21 d of age). Figure 1a shows that the order of the treatments seen in the body weights established at d 2 of exposure to contaminated diets for group 1 per-
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100-f
3 g>
80
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T3 O CQ
50 (a)
O)
50
10
20
30
40
Time of exposure (days) FIGURE 1. Effects of various concentrations of DON (mg/kg diet: x •= 0, A - 4, • - 8, 0 - 12, o - 16, + - 20) on body weight over time, (a) Started at 21 d of age (SEM ranged from 0.73 at 0 d to 1.26 at 42 d). (b) Started at 28 d of age (SEM ranged from 0.84 at 0 d to 1.7 at 35 d).
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sisted at 6 wk. After 7 d of exposure to DON, differences in body weights were seen in mice fed the control and 8,12,16, and 20 mg DON/kg diets (p < .01). For mice exposed at an older age (28 d, Fig.ib), the differences narrowed by d 7, and by the end of the experiment only mice fed the 20 mg DON/kg diet clearly differed from those fed the other diets. The fact that treatment differences were larger in mice exposed at 21 d (group 1) compared to group 2 was reflected in the analysis of variance; the variance among diets after 7 d of exposure for body weights was twice as great for the younger mice (group 1, mean square = 190.34) as for the older mice (group 2, mean square = 91.62), even though the error mean squares were essentially the same (5.23 and 5.17, respectively). After 7 d of exposure, the percent decreases in body weight compared to controls for the early exposed mice (Fig. 1a) were 0.2, 2.8, 9.7,15.4, and 21.4 on 4, 8, 12, 16, and 20 mg DON/kg respectively, while the percent change in body weight for those exposed at 28 d of age (Figure 16) increased 1.1 and decreased 0.2,1.1, 5.6, and 12.1, respectively. Experiment 2 Differences in weight gains during the first week (Table 1) were significant between diet types (p < .01) and different DON concentrations (p < .001), but not between control diets. Although differences were not apparent in the second week, the overall response to DON was significant (p < .001). Food consumption was significantly affected by diet type and DON concentration throughout the experiment (p < .001). For both food consumption and weight gain, most of the differences between DON concentrations were accounted for by a linear trend (not shown) toward decreasing performance with increasing DON levels. After 7 d of exposure, diet type x DON interaction for food consumption was evident (p < .01), suggesting that the effect of DON differed with the type of diet. Although the advantage in food consumption of ingesting mash versus freeze-dried diets was consistent throughout the range of toxin levels used, it was notably greater for the 8 and 16 mg DON/kg diets. There was no difference in food conversion efficiency data (weight gain/ food consumed) between the two types of diet after 7 d of exposure for the controls or the highest toxin concentration (Table 1). However, differences between the two types of diet at various toxin concentrations were significant (p < .01) during the second week and overall (p = .049) (i.e., no difference between controls). Experiment 3 After 7 d on test, there were significant (p < .001) linear decreases in weight gain and food consumption as the DON and T-2 toxin concentrations increased (Table 2). The 4 mg DON + 2 mg T-2/kg diet caused a significant (p < .05) reduction in weight gain (0.78 g/d) compared to 4 mg DON/kg diet (0.96 g/d) or 2 mg T-2 toxin/kg diet (0.93 g/d) alone. When
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TABLE 1. Weight Cain and Food Consumption of Young Mice Fed Control or DON-Containing Diets as Freeze-Dried vs. Regular Mash (Experiment 2) Food consumption (g/d)a
Weight gain (g/d)
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DON (mg/kg diet)
Food conversion*1
Diet type
0-7
0-14
0-7
0-14
0-7
0-14
0
FDC Mash
0.85 0.87
0.55 0.56
3.57 3.94
3.62 3.84
0.24 0.22
0.15 0.15
8
FD Mash
0.73 0.84
0.50 0.56
3.39 4.03
3.31 3.99
0.22 0.21
0.15 0.14
12
FD Mash
0.57 0.64
0.48 0.46
3.22 3.55
3.32 3.62
0.18 0.18
0.14 0.13
16
FD Mash
0.47 0.64
0.39 0.46
2.70 3.59
2.79 3.57
0.18 0.18
0.14 0.13
SEM
0.05
0.03
0.09
0.08
0.01
0.007
***
NS ***
Probabilities Analysis of variance Diet type DON Diet type x DON
*• • **
NS ***
NS
NS
***
**•
• *
*•*
NS
*d
NS NS
Note. * , **, * * * Significant effects at p < .05, p < .01, and p < .001, respectively. NS, not significant. a Corrected for dry matter content. ^Weight gain/food consumed. c Freeze-dried. d p - .049.
the T-2 toxin concentration was 2 mg/kg diet and DON concentration increased from 4 to 8 mg/kg diet, a further depression in weight gain from 0.78 to 0.70 was observed. However, there was no difference in weight gain when T-2 toxin concentration was 4 mg/kg diet and DON concentration increased from 4 to 8 mg/kg diet (0.74 vs. 0.74). Food conversion was reduced after 7 d of exposure to both toxins, but the effect of T-2 toxin was more persistent throughout the experiment. DISCUSSION The overall purpose of this research was to determine the feasibility of using mice as a potential model for growing swine in mycotoxin studies. The present investigation examined the effects of dietary exposure of DON at 21 d versus 28 d of age, type of diet (freeze-dried vs. mash), and the effects of DON and T-2 toxin combinations on young mice. The severity of effects as measured by changes in body weight varied, depend-
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TABLE 2. Weight Gain and Food Consumption of Young Mice Fed Diets Containing DON and T-2 Toxin Alone or in Combination (Experiment 3)
Weight gain (g/d)
Food conversion8
(mg/kg diet)
T-2 toxin (mg/kg diet)
0-7
0-14
0-7
0-14
0-7
0-14
0 4 8
0 0 0
1.12 0.96 0.83
0.74 0.66 0.65
3.71 3.69 3.50
3.81 3.90 3.71
0.30 0.26 0.24
0.20 0.17 0.17
0 0 0
2 4 8
0.93 0.83 0.59
0.69 0.58 0.50
3.66 3.61 2.96
3.90 3.77 3.33
0.25 0.23 0.20
0.18 0.15 0.15
4 4 8 8
2 4 2 4
0.78 0.74 0.70 0.74
0.59 0.54 0.55 0.55
3.39 3.29 3.18 3.26
3.64 3.48 3.47 3.53
0.23 0.23 0.22 0.24
0.16 0.15 0.16 0.16
SEM
0.05
0.04
0.09
0.07
0.01
0.009
*
*** ***
***
** **#
NS **
NS
NS
*b
NS
NS
DON
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Food consumption (g/d)
Probabilities Analysis of variance DON T-2
* * •
DON X T-2
NS
• **
Note. *, • * , * * * Significant effects at p < .05, p < .01, and p < .001, respectively. NS, not significant. ^Weight gain/food consumed. b p - .036.
ing on whether the mice were first exposed to a contaminated diet at 21 or at 28 d of age. The former permitted exposure of the animal during the rapid growing period when the sensitivity to dietary toxin is much greater. Other studies used either a 7-d acclimatization involving inbred female mice-B6C3F1 (Forsell et al., 1986) or no acclimatization using male mice (Arnold et al., 1986; Tryphonas et al., 1986). In an 8-wk experiment, Forsell et al. (1986) determined that the rate of body weight gain was significantly reduced (p < .01) after 24 d for all mice consuming 2 mg DON/kg diet. In the current study, there was a dose-dependent linear decrease in weight gain and food consumption with increasing toxin concentration (Tables 1 and 2). The lowest detectable level that resulted in growth effects significantly different from the control after 7 d of exposure (p < .05) was 4 mg DON/kg diet (Table 2). However, the linear trends in weight gain and food consumption suggest that increasing levels of dietary DON had a subtle effect on performance without displaying signs of distress. Differences in sensitivity between the two studies may
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be attributable, in part, to differences in diet type—semipurified (Forsell et al., 1986) versus mouse chow (present study)—and/or sensitivity of the animals (inbred vs. outbred). Tryphonas et al. (1986) showed that male inbred Swiss Webster mice fed 2.0 and 4.0 mg DON/kg diet gained significantly less weight than the controls during the second week of exposure. During wk 4 and 5, the weight gain of treated groups followed a dose-response relationship (control > 1.00 > 2.00 > 4.00 mg DON/kg diet). Compared to the current data, where differences were only apparent at higher DON levels, gender or inbred versus outbred mice differences in sensitivity to DON may exist. Iverson et al. (1985) also noted that DON given orally was more toxic to male than female mice. A more detailed comparison between sexes and between strains in a single study is required to test this hypothesis. Differences in food consumption between diet type (freeze-dried vs. mash) and the diet type x DON interaction after 7 d of exposure likely reflected a difference in physical properties of the diet and how the toxin is influenced by these properties, rather than merely a difference in food utilization. However, a difference in food conversion was observed in a second week on DON-containing diets (p < .01). Morrissey and Norred (1984) found no differences in the average weight gain per rat per day and feed conversion between freeze-dried and dry meal for rats, but, these diets did not contain any toxin. While freeze-drying offers advantages over using a dry diet, the fact that it may influence the activity of a toxin must be taken into consideration when designing toxicological experiments. Although numerous studies on individual mycotoxins, such as DON and T-2 toxin, have been conducted, there is no information on interactions between them. The current study showed a potentiation of effects on food consumption when the two toxins were combined at 2 mg T-2 toxin and 4 mg DON/kg diet (3.39 g/d) compared to 2 mg T-2 toxin/kg diet and 8 mg DON/kg diet (3.18 g/d), confirming DON's involvement in feed refusal. In the presence of 4 mg T-2 toxin, increasing the DON concentration from 4 to 8 mg/kg diet did not cause further growth depression or reduction in food consumption. It seems that only the lower (2 mg/kg diet) concentration of T-2 toxin reinforced the effect of DON on food consumption. Based on these three experiments, mice respond in a similar manner to dietary DON and T-2 toxin as do growing pigs, and the degree of response is dose dependent. Friend et al. (1982) observed that feed intake in growing pigs decreased in a linear fashion as the concentration of DON increased (up to 1 mg/kg diet), but differences in weight gain established after the first week were not maintained over a 7-wk period. When younger animals (21 kg) were fed diets containing 0-3 mg DON/kg diet, differences in weight gain (p < .05) but not feed intake were observed.
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Trends toward reduced weight gain and feed consumption were also noted in pigs fed 0-3.2 mg T-2 toxin/kg diet (Friend et al., 1992). Responses in both mice and swine suggest that increasing levels of DON and T-2 toxin have subtle effects on performance, but it is difficult to identify early measurable signs of discomfort or stress. Although the minimum dietary levels required to reduce weight gain in growing pigs have been identified as 2 mg DON/kg diet (-0.15-0.20 mg/kg body weight/d) and 3 mg T-2 toxin/kg diet (—0.14 mg/kg body weight/d), respectively, subclinical effects can occur at lower levels. For mice, the minimum effective level (reduction in weight gain) for DON was 4 mg/kg diet (0.91 mg/kg body weight/d) and for T-2 toxin 2 mg/kg diet (0.46 mg/ kg body weight/d). While there seems to be a three- to fourfold difference in sensitivity to DON and T-2 toxin between growing pigs and mice, the values for pigs were estimated from experiments using naturally contaminated diets and for mice from experiments using pure toxins. Although differences in sensitivity exist, linear responses to increasing concentrations of toxins are more important criteria to use when making comparisons between growing mice and pigs. Further experiments are necessary to establish responses of mice to a range of mycotoxins with more defined end points as a part of the mouse model evaluation.
REFERENCES Arnold, D. L., McGuire, P. F., Nera, E. A., Karpinski, K. F., Bickis, H. G., Zawidzka, Z. Z., Fernie, S., and Vesonder, F. R. 1986. The toxicity of orally administered deoxynivalenol (vomitoxin) in rats and mice. Food Chem. Toxicol. 24:935-941. Croteau, S. M., Prelusky, D. B., and Trenholm, H. L. 1992. Analysis of trichothecene mycotoxins by gas chromatography with electron capture detection. J. Assoc. Offic. Anal. Chem. (submitted). Forsell, J. H., Witt, M. F., Tai, J.-H., Jensen, R., and Pestka, J. J. 1986. Effects of 8-week exposure of the B6C3F1 mouse to dietary deoxynivalenol (vomitoxin) and zearalenone. food Chem. Toxicol. 24:213-219. Friend, D. W., Trenholm, H. L, Elliot, J. I., Thompson, B. K., and Hartin, K. E. 1982. Effect of feeding vomitoxin-contaminated wheat to pigs. Can. J. Anim. Sci. 62:1211-1222. Friend, D. W., Trenholm, H. L., Thompson, B. K., Fiser, P. S., and Hartin, K. E. 1986a. Effect of feeding diets containing deoxynivalenol (vomitoxin)-contaminated wheat or corn on the feed consumption, weight gain, organ weight and sexual development of male and female pigs. Can. J. Anim. Sci. 66:765-775. Friend, D. W, Trenholm, H. L., Thompson, B. K., Prelusky, D. B., and Hartin, K. E. 1986b. Effect of deoxynivalenol (DON)-contaminated diet fed to growing-finishing pigs on their performance at market weight, nitrogen retention and DON excretion. Can. J. Anim. Sci. 66:1075-1085. Friend, D. W., Thompson, B. K., Trenholm, H. L., Hartin, K. E., Boermans, H. J., and Panich, P. L. 1992. Toxicity of T-2 toxin and its interaction with DON when fed to young pigs. Can. J. Anim. Sci., in press. Greenhalgh, R., Levandier, D., Adams. W., Miller, J. D., Blackwell, B. A., McAlees, A. J., and Taylor, A. 1986. Production and characterization of deoxynivalenol and other secondary metabolites of Fusarium culmorum (CMI 14764, HLX 1503). J. Agric. Food Chem. 34:98-102. Greenhalgh, R., Fielder, D. A., Blackwell, B. A., Miller, J. D., Charland, J.-P., and ApSimon, J. W. 1990. Some minor secondary metabolites of Fusarium sporotrichioides DAOM 165006. J. Agric. Food Chem. 38:1978-1984.
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Hayes, M. A., and Schiefer, H. B. 1980. Subacute toxicity of dietary T-2 toxin in mice: Influence of protein nutrition. Can. J. Comp. Med 44:219-228. Hayes, M. A., and Schiefer, H. B. 1982. Comparative toxicity of dietary T-2 toxin in rats and mice. J. Appl. Toxicol. 2:207-212. Iverson, F., Lok, E., and Nera, E. A. 1985. Pathological and biochemical effects of vomitoxin in rodents. Toxicologist 5:6 (abstr.). Morrissey, R. E., and Norred, W. P. 1984. An improved method of diet preparation for toxicological feeding experiments. Lab. Anim. 18:271-274. Rotter, R. G., Thompson, B. K., Trenholm, H. L., Prelusky, D. B., Hartin, K. E., and Miller, J. D. 1992. A preliminary examination of potential interactions between deoxynivalenol (DON) and other selected Fusarium metabolites in growing pigs. Can. J. Anim. Sci. 72:107-116. Snedecor, G. W., and Cochran, W. G. 1980. Statistical Methods, 7th ed., p. 507. Ames: Iowa State University Press. Tryphonas, H., Iverson, F., Ying, S., Nera, E. A., McGuire, P. F., O'Grady, L., Clayson, D. B., and Scott, P. M. 1986. Effects of deoxynivalenol (vomitoxin) on the humoral and cellular immunity of mice. Toxicol. Lett. 30:137-150. Trenholm, H. L., Warner, R. M., and Prelusky, D. B. 1985. Assessment of extraction procedures in the analysis of naturally contaminated grain products for deoxynivalenol (vomitoxin). J. Assoc. Offic. Anal. Chem. 68:645-649. Received February 6, 1992 Accepted April 21, 1992
ISSN: 0098-4108 (Print) (Online) Journal homepage: http://www.tandfonline.com/loi/uteh19
Investigations in the use of mice exposed to mycotoxins as a model for growing pigs Barbara A. Rotter , Roland G. Rotter , Brian K. Thompson & H. Locksley Trenholm To cite this article: Barbara A. Rotter , Roland G. Rotter , Brian K. Thompson & H. Locksley Trenholm (1992) Investigations in the use of mice exposed to mycotoxins as a model for growing pigs, Journal of Toxicology and Environmental Health, 37:2, 329-339, DOI: 10.1080/15287399209531673 To link to this article: http://dx.doi.org/10.1080/15287399209531673
Published online: 20 Oct 2009.
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Date: 07 November 2015, At: 18:47
INVESTIGATIONS IN THE USE OF MICE EXPOSED TO MYCOTOXINS AS A MODEL FOR GROWING PIGS Barbara A. Rotter, Roland G. Rotter Centre for Food and Animal Research, Agriculture Canada, Central Experimental Farm, Ottawa, Ontario, Canada
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Brian K. Thompson Research Program Service, Agriculture Canada, Central Experimental Farm, Ottawa, Ontario, Canada H. Locksley Trenholm Centre for Food and Animal Research, Agriculture Canada, Central Experimental Farm, Ottawa, Ontario, Canada
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 OONIkg diet) was more pronounced in a doss-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 × 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.
The authors wish to thank W. Bogie, S. Clarkin, L. A. Robinson, C. Mitchell, D. Fielder, S. Croteau, R. Doak, and staff for their capable assistance. The mycotoxins used were provided courtesy of J. D. Miller, Plant Research Centre, Agriculture Canada. This research was supported by a grant (Ontario Pork Industry Improvement Program) from the Ontario Ministry of Agriculture and Food (OMAF) and Ontario Producers Marketing Board (OPPMB) under the Ontario Pork Industry Improvement Program. Research Program Service contribution no. R104; Centre for Food and Animal Research contribution no. 2026. Present address for R. C. Rotter is Toxicological Evaluation Division, Bureau of Chemical Safety, Food Directorate, Health Protection Branch, Health and Welfare Canada, Ottawa, Ontario, K1A 0L2. Requests for reprints should be sent to Dr. B. A. Rotter, Centre for Food and Animal Research, Agriculture Canada, Ottawa, Ontario K1A 0C6, Canada.
329 Journal of Toxicology and Environmental Health, 37:329-339, 1992 Copyright © 1992 by Hemisphere Publishing Corporation
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There was a trend (p — .064) suggesting a possible interaction between the two toxins after 7 d of exposure. For animals consuming 4 mg T-2 toxin/kg diet, increasing the DON concentration from 4 to 8 mglkg diet did not result in further depression in weight gain. Only at 2 mg T-2 toxin did increasing the DON concentration from 4 to 8 significantly (p < .05) reduce weight gains further. Food conversion was depressed for both toxins after 7 d of exposure, but over the whole 2-wk period, this effect was only seen for T-2 toxin. Compared to growing pigs, mice display similar dose-dependent linear responses in feed consumption and weight gain due to presence of dietary DON and T-2 toxin. Further experiments are needed to establish responses of mice to a range of mycotoxins as a part of the mouse model evaluation.
INTRODUCTION Mold-contaminated feedstuffs often contain more than one mycotoxin or other secondary fungal metabolites. Each of the metabolites, in turn, potentially may have its own effect(s) on the biological systems of animals that ingest them. Recent studies at the Centre for Food and Animal Research (CFAR) have examined possible interactions between deoxynivalenol (vomitoxin, DON) and co-occurring metabolites of Fusarium graminaerum (Rotter et al., 1992), and DON and T-2 toxin in swine (Friend et al., 1992). Although certain combinations appeared to indicate possible interactions, the studies were hampered by severely limited quantities of the metabolites, thereby limiting the number of animals exposed (Rotter et al., 1992). Scarcity of pure metabolites generally precludes the use of larger animals such as swine in extensive toxicity trials. Mice and rats are often used because of their low cost, availability, uniformity, and the lesser amounts of compound needed to conduct toxicity tests. Studies with mice have examined the subacute effects of dietary DON (Forsell et al., 1986; Tryphonas et al., 1986) and T-2 toxin (Hayes and Schiefer, 1980, 1982). Forsell et al. (1986) observed a significant (p < .01) reduction in weight gain of B6C3F1 female mice fed diets containing 2 or more mg DON/kg diet over an 8-wk trial. Although feed refusal was observed sporadically at 2 mg DON/kg diet, food consumption decreased only on the 25 mg DON/kg diet after 6 d of exposure (p < .01). Dose-related depressions in weight gain were also more pronounced in juvenile (16.4 g) than in young adult (32.2 g) male mice after 14 or 28 d of ingesting 10 or 20 mg T-2 toxin/kg diet (Hayes and Schiefer, 1982). Mice and rats exposed to DON (Arnold et al., 1986) exhibit the same general effects, such as reduced weight gain and feed refusal, seen in swine (Friend et al., 1982, 1986a, 1986b). Feed intake in growing-finishing pigs decreased in a linear fashion as the DON level increased, but, even though weight gains differed significantly in the first week, the differences were not maintained over a 7-wk period (Friend et al., 1982). In a subsequent study, pigs exposed to a 4.2 mg DON/kg diet containing Fusarium-inoculated corn were more strongly affected in the first week
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and never recovered when compared to pigs fed a contaminated wheat diet containing 3.7 mg DON/kg. This suggested the presence of another toxic metabolite(s) (Friend et al., 1986a). Although differences in kinetic parameters (metabolism, absorption, elimination, etc.) exist between the pigs and mice, and extrapolation of data between species presumes certain conditions, the mouse may prove to be a satisfactory animal model for initial studies on mycotoxin interactions. The present research examined the effects of pure DON and T-2 toxin at dietary concentrations comparable to naturally occurring levels on growing female mice. This series of experiments is the first step in determining similarities between mice and pigs in response to selected toxins as a part of an overall evaluation of the mouse model for growing pigs in mycotoxin research. MATERIALS AND METHODS Animals Weanling 3-wk-old outbred female mice (ICR, weighing 15-17 g) were obtained from the CFAR Research Farm (original source: Charles River Canada, St-Constant, Quebec) and housed either 3 per cage (Experiment 1) or singly (Experiment 2 and 3) in transparent polypropylene cages (Maryland Plastics, Inc., Federalsburg, Md.). Each cage unit consisted of a stainless-steel wire lid, water bottle, individual feeder, and a layer of heattreated hardwood shavings. Water was provided ad libitum and changed every 3 d. Feeders (PFF-6D, Allentown Company Inc., Allentown, N.J.) containing fresh diet were changed every 3 d to minimize exposure of animals to feed contaminated with feces and urine. The mice were kept in an environmentally controlled room (22 ± 2°C) on a 12-h light/dark cycle. Diet The mice were fed ground or pelleted mouse chow 5015 (Purina Mills, St. Louis, Mo.). Chow was ground using a plate grinder (model 105A, Sprout-Waldron, Muncy, Pa.), and the specified amount of toxin was mixed into the diet. The pelleted form of the diet was prepared by mixing ground chow with water, then freeze-drying and cutting in into small blocks as outlined by Morrissey and Norred (1984). Sufficient quantities of feed were prepared for the whole experiment and stored at 4°C. Toxins Purified DON (99.1%) and T-2 toxin (99.1%) were provided by J. D. Miller, Plant Research Centre, Agriculture Canada, Ottawa, Ontario. Deoxynivalenol was prepared from the hydrolysis of 3-acetyldeoxynivalenol and purified from the media of large-scale Fusarium culmorum cultures following procedures outlined by Greenhalgh et al. (1986), while T-2 toxin
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was isolated and purified from Fusarium sporotrichioides (Greenhalgh et al., 1990).
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Experimental Design In Experiment 1,180 mice were randomly assigned to 6 dietary treatments (0, 4, 8, 12, 16, and 20 mg DON/kg diet), x 2 age groups using 5 replicates with 3 mice per cage (15 mice per treatment group). Mice allocated to the first group were fed experimental diets from d 1 (first exposure at 21 d of age). The second group was fed a regular chow pellet for the first 7 d, followed by experimental diets (first exposure at 28 d of age). Body weights were recorded on d 0, 2, 4, 7, and at 7-d intervals thereafter until wk 6 for group 1 (42 d) and 5 for group 2 (35 d). All diets were fed as pellets, but food consumption was not measured. In Experiment 2, 96 mice, housed singly, were randomly assigned to 4 dietary treatments (0, 8, 12, and 16 mg DON/kg diet) x 2 dietary regimens in a randomized block design with 12 replicates. The regimens consisted of two forms of diet, one prepared by mixing the toxin in the ground chow and the other by mixing the toxin in the ground chow, adding water, freeze-drying, and regrinding the product as outlined above. Body weight and food consumption were measured on d 0, 2, 4, 7, and 14. In Experiment 3,100 mice, housed singly, were assigned randomly to 10 dietary treatments fed as a mash in a randomized block design with 10 replicates. The treatments were as follows: control; 4, 8 mg DON/kg diet; 2, 4, 8 mg T-2 toxin/kg diet; and 4 + 2, 4 + 4, 8 + 2, and 8 + 4 mg DON + T2 toxin/kg diet. Body weight and food consumption were measured on d 0, 2, 4, 7, and 14. Analysis All diets were analyzed for DON (Trenholm et al., 1985) and T-2 toxin (Croteau et al., 1992) to confirm dietary concentrations and to ensure a uniform mix. Analysis of variance was applied to the weight gains and food consumption over specific periods. In order to look for trends with increasing toxin levels, the treatment sums of squares were partitioned into the portions due to linear regression and to deviations from the regression (Agriculture Canada Software no. SO22-RPS) as outlined by Snedecor and Cochran (1980). RESULTS Experiment 1 There were no differences (p > .05) in initial body weights among treatments for the two age groups at randomization (21 d of age). Figure 1a shows that the order of the treatments seen in the body weights established at d 2 of exposure to contaminated diets for group 1 per-
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100-f
3 g>
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T3 O CQ
50 (a)
O)
50
10
20
30
40
Time of exposure (days) FIGURE 1. Effects of various concentrations of DON (mg/kg diet: x •= 0, A - 4, • - 8, 0 - 12, o - 16, + - 20) on body weight over time, (a) Started at 21 d of age (SEM ranged from 0.73 at 0 d to 1.26 at 42 d). (b) Started at 28 d of age (SEM ranged from 0.84 at 0 d to 1.7 at 35 d).
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sisted at 6 wk. After 7 d of exposure to DON, differences in body weights were seen in mice fed the control and 8,12,16, and 20 mg DON/kg diets (p < .01). For mice exposed at an older age (28 d, Fig.ib), the differences narrowed by d 7, and by the end of the experiment only mice fed the 20 mg DON/kg diet clearly differed from those fed the other diets. The fact that treatment differences were larger in mice exposed at 21 d (group 1) compared to group 2 was reflected in the analysis of variance; the variance among diets after 7 d of exposure for body weights was twice as great for the younger mice (group 1, mean square = 190.34) as for the older mice (group 2, mean square = 91.62), even though the error mean squares were essentially the same (5.23 and 5.17, respectively). After 7 d of exposure, the percent decreases in body weight compared to controls for the early exposed mice (Fig. 1a) were 0.2, 2.8, 9.7,15.4, and 21.4 on 4, 8, 12, 16, and 20 mg DON/kg respectively, while the percent change in body weight for those exposed at 28 d of age (Figure 16) increased 1.1 and decreased 0.2,1.1, 5.6, and 12.1, respectively. Experiment 2 Differences in weight gains during the first week (Table 1) were significant between diet types (p < .01) and different DON concentrations (p < .001), but not between control diets. Although differences were not apparent in the second week, the overall response to DON was significant (p < .001). Food consumption was significantly affected by diet type and DON concentration throughout the experiment (p < .001). For both food consumption and weight gain, most of the differences between DON concentrations were accounted for by a linear trend (not shown) toward decreasing performance with increasing DON levels. After 7 d of exposure, diet type x DON interaction for food consumption was evident (p < .01), suggesting that the effect of DON differed with the type of diet. Although the advantage in food consumption of ingesting mash versus freeze-dried diets was consistent throughout the range of toxin levels used, it was notably greater for the 8 and 16 mg DON/kg diets. There was no difference in food conversion efficiency data (weight gain/ food consumed) between the two types of diet after 7 d of exposure for the controls or the highest toxin concentration (Table 1). However, differences between the two types of diet at various toxin concentrations were significant (p < .01) during the second week and overall (p = .049) (i.e., no difference between controls). Experiment 3 After 7 d on test, there were significant (p < .001) linear decreases in weight gain and food consumption as the DON and T-2 toxin concentrations increased (Table 2). The 4 mg DON + 2 mg T-2/kg diet caused a significant (p < .05) reduction in weight gain (0.78 g/d) compared to 4 mg DON/kg diet (0.96 g/d) or 2 mg T-2 toxin/kg diet (0.93 g/d) alone. When
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TABLE 1. Weight Cain and Food Consumption of Young Mice Fed Control or DON-Containing Diets as Freeze-Dried vs. Regular Mash (Experiment 2) Food consumption (g/d)a
Weight gain (g/d)
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DON (mg/kg diet)
Food conversion*1
Diet type
0-7
0-14
0-7
0-14
0-7
0-14
0
FDC Mash
0.85 0.87
0.55 0.56
3.57 3.94
3.62 3.84
0.24 0.22
0.15 0.15
8
FD Mash
0.73 0.84
0.50 0.56
3.39 4.03
3.31 3.99
0.22 0.21
0.15 0.14
12
FD Mash
0.57 0.64
0.48 0.46
3.22 3.55
3.32 3.62
0.18 0.18
0.14 0.13
16
FD Mash
0.47 0.64
0.39 0.46
2.70 3.59
2.79 3.57
0.18 0.18
0.14 0.13
SEM
0.05
0.03
0.09
0.08
0.01
0.007
***
NS ***
Probabilities Analysis of variance Diet type DON Diet type x DON
*• • **
NS ***
NS
NS
***
**•
• *
*•*
NS
*d
NS NS
Note. * , **, * * * Significant effects at p < .05, p < .01, and p < .001, respectively. NS, not significant. a Corrected for dry matter content. ^Weight gain/food consumed. c Freeze-dried. d p - .049.
the T-2 toxin concentration was 2 mg/kg diet and DON concentration increased from 4 to 8 mg/kg diet, a further depression in weight gain from 0.78 to 0.70 was observed. However, there was no difference in weight gain when T-2 toxin concentration was 4 mg/kg diet and DON concentration increased from 4 to 8 mg/kg diet (0.74 vs. 0.74). Food conversion was reduced after 7 d of exposure to both toxins, but the effect of T-2 toxin was more persistent throughout the experiment. DISCUSSION The overall purpose of this research was to determine the feasibility of using mice as a potential model for growing swine in mycotoxin studies. The present investigation examined the effects of dietary exposure of DON at 21 d versus 28 d of age, type of diet (freeze-dried vs. mash), and the effects of DON and T-2 toxin combinations on young mice. The severity of effects as measured by changes in body weight varied, depend-
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TABLE 2. Weight Gain and Food Consumption of Young Mice Fed Diets Containing DON and T-2 Toxin Alone or in Combination (Experiment 3)
Weight gain (g/d)
Food conversion8
(mg/kg diet)
T-2 toxin (mg/kg diet)
0-7
0-14
0-7
0-14
0-7
0-14
0 4 8
0 0 0
1.12 0.96 0.83
0.74 0.66 0.65
3.71 3.69 3.50
3.81 3.90 3.71
0.30 0.26 0.24
0.20 0.17 0.17
0 0 0
2 4 8
0.93 0.83 0.59
0.69 0.58 0.50
3.66 3.61 2.96
3.90 3.77 3.33
0.25 0.23 0.20
0.18 0.15 0.15
4 4 8 8
2 4 2 4
0.78 0.74 0.70 0.74
0.59 0.54 0.55 0.55
3.39 3.29 3.18 3.26
3.64 3.48 3.47 3.53
0.23 0.23 0.22 0.24
0.16 0.15 0.16 0.16
SEM
0.05
0.04
0.09
0.07
0.01
0.009
*
*** ***
***
** **#
NS **
NS
NS
*b
NS
NS
DON
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Food consumption (g/d)
Probabilities Analysis of variance DON T-2
* * •
DON X T-2
NS
• **
Note. *, • * , * * * Significant effects at p < .05, p < .01, and p < .001, respectively. NS, not significant. ^Weight gain/food consumed. b p - .036.
ing on whether the mice were first exposed to a contaminated diet at 21 or at 28 d of age. The former permitted exposure of the animal during the rapid growing period when the sensitivity to dietary toxin is much greater. Other studies used either a 7-d acclimatization involving inbred female mice-B6C3F1 (Forsell et al., 1986) or no acclimatization using male mice (Arnold et al., 1986; Tryphonas et al., 1986). In an 8-wk experiment, Forsell et al. (1986) determined that the rate of body weight gain was significantly reduced (p < .01) after 24 d for all mice consuming 2 mg DON/kg diet. In the current study, there was a dose-dependent linear decrease in weight gain and food consumption with increasing toxin concentration (Tables 1 and 2). The lowest detectable level that resulted in growth effects significantly different from the control after 7 d of exposure (p < .05) was 4 mg DON/kg diet (Table 2). However, the linear trends in weight gain and food consumption suggest that increasing levels of dietary DON had a subtle effect on performance without displaying signs of distress. Differences in sensitivity between the two studies may
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be attributable, in part, to differences in diet type—semipurified (Forsell et al., 1986) versus mouse chow (present study)—and/or sensitivity of the animals (inbred vs. outbred). Tryphonas et al. (1986) showed that male inbred Swiss Webster mice fed 2.0 and 4.0 mg DON/kg diet gained significantly less weight than the controls during the second week of exposure. During wk 4 and 5, the weight gain of treated groups followed a dose-response relationship (control > 1.00 > 2.00 > 4.00 mg DON/kg diet). Compared to the current data, where differences were only apparent at higher DON levels, gender or inbred versus outbred mice differences in sensitivity to DON may exist. Iverson et al. (1985) also noted that DON given orally was more toxic to male than female mice. A more detailed comparison between sexes and between strains in a single study is required to test this hypothesis. Differences in food consumption between diet type (freeze-dried vs. mash) and the diet type x DON interaction after 7 d of exposure likely reflected a difference in physical properties of the diet and how the toxin is influenced by these properties, rather than merely a difference in food utilization. However, a difference in food conversion was observed in a second week on DON-containing diets (p < .01). Morrissey and Norred (1984) found no differences in the average weight gain per rat per day and feed conversion between freeze-dried and dry meal for rats, but, these diets did not contain any toxin. While freeze-drying offers advantages over using a dry diet, the fact that it may influence the activity of a toxin must be taken into consideration when designing toxicological experiments. Although numerous studies on individual mycotoxins, such as DON and T-2 toxin, have been conducted, there is no information on interactions between them. The current study showed a potentiation of effects on food consumption when the two toxins were combined at 2 mg T-2 toxin and 4 mg DON/kg diet (3.39 g/d) compared to 2 mg T-2 toxin/kg diet and 8 mg DON/kg diet (3.18 g/d), confirming DON's involvement in feed refusal. In the presence of 4 mg T-2 toxin, increasing the DON concentration from 4 to 8 mg/kg diet did not cause further growth depression or reduction in food consumption. It seems that only the lower (2 mg/kg diet) concentration of T-2 toxin reinforced the effect of DON on food consumption. Based on these three experiments, mice respond in a similar manner to dietary DON and T-2 toxin as do growing pigs, and the degree of response is dose dependent. Friend et al. (1982) observed that feed intake in growing pigs decreased in a linear fashion as the concentration of DON increased (up to 1 mg/kg diet), but differences in weight gain established after the first week were not maintained over a 7-wk period. When younger animals (21 kg) were fed diets containing 0-3 mg DON/kg diet, differences in weight gain (p < .05) but not feed intake were observed.
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Trends toward reduced weight gain and feed consumption were also noted in pigs fed 0-3.2 mg T-2 toxin/kg diet (Friend et al., 1992). Responses in both mice and swine suggest that increasing levels of DON and T-2 toxin have subtle effects on performance, but it is difficult to identify early measurable signs of discomfort or stress. Although the minimum dietary levels required to reduce weight gain in growing pigs have been identified as 2 mg DON/kg diet (-0.15-0.20 mg/kg body weight/d) and 3 mg T-2 toxin/kg diet (—0.14 mg/kg body weight/d), respectively, subclinical effects can occur at lower levels. For mice, the minimum effective level (reduction in weight gain) for DON was 4 mg/kg diet (0.91 mg/kg body weight/d) and for T-2 toxin 2 mg/kg diet (0.46 mg/ kg body weight/d). While there seems to be a three- to fourfold difference in sensitivity to DON and T-2 toxin between growing pigs and mice, the values for pigs were estimated from experiments using naturally contaminated diets and for mice from experiments using pure toxins. Although differences in sensitivity exist, linear responses to increasing concentrations of toxins are more important criteria to use when making comparisons between growing mice and pigs. Further experiments are necessary to establish responses of mice to a range of mycotoxins with more defined end points as a part of the mouse model evaluation.
REFERENCES Arnold, D. L., McGuire, P. F., Nera, E. A., Karpinski, K. F., Bickis, H. G., Zawidzka, Z. Z., Fernie, S., and Vesonder, F. R. 1986. The toxicity of orally administered deoxynivalenol (vomitoxin) in rats and mice. Food Chem. Toxicol. 24:935-941. Croteau, S. M., Prelusky, D. B., and Trenholm, H. L. 1992. Analysis of trichothecene mycotoxins by gas chromatography with electron capture detection. J. Assoc. Offic. Anal. Chem. (submitted). Forsell, J. H., Witt, M. F., Tai, J.-H., Jensen, R., and Pestka, J. J. 1986. Effects of 8-week exposure of the B6C3F1 mouse to dietary deoxynivalenol (vomitoxin) and zearalenone. food Chem. Toxicol. 24:213-219. Friend, D. W., Trenholm, H. L, Elliot, J. I., Thompson, B. K., and Hartin, K. E. 1982. Effect of feeding vomitoxin-contaminated wheat to pigs. Can. J. Anim. Sci. 62:1211-1222. Friend, D. W., Trenholm, H. L., Thompson, B. K., Fiser, P. S., and Hartin, K. E. 1986a. Effect of feeding diets containing deoxynivalenol (vomitoxin)-contaminated wheat or corn on the feed consumption, weight gain, organ weight and sexual development of male and female pigs. Can. J. Anim. Sci. 66:765-775. Friend, D. W, Trenholm, H. L., Thompson, B. K., Prelusky, D. B., and Hartin, K. E. 1986b. Effect of deoxynivalenol (DON)-contaminated diet fed to growing-finishing pigs on their performance at market weight, nitrogen retention and DON excretion. Can. J. Anim. Sci. 66:1075-1085. Friend, D. W., Thompson, B. K., Trenholm, H. L., Hartin, K. E., Boermans, H. J., and Panich, P. L. 1992. Toxicity of T-2 toxin and its interaction with DON when fed to young pigs. Can. J. Anim. Sci., in press. Greenhalgh, R., Levandier, D., Adams. W., Miller, J. D., Blackwell, B. A., McAlees, A. J., and Taylor, A. 1986. Production and characterization of deoxynivalenol and other secondary metabolites of Fusarium culmorum (CMI 14764, HLX 1503). J. Agric. Food Chem. 34:98-102. Greenhalgh, R., Fielder, D. A., Blackwell, B. A., Miller, J. D., Charland, J.-P., and ApSimon, J. W. 1990. Some minor secondary metabolites of Fusarium sporotrichioides DAOM 165006. J. Agric. Food Chem. 38:1978-1984.
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Hayes, M. A., and Schiefer, H. B. 1980. Subacute toxicity of dietary T-2 toxin in mice: Influence of protein nutrition. Can. J. Comp. Med 44:219-228. Hayes, M. A., and Schiefer, H. B. 1982. Comparative toxicity of dietary T-2 toxin in rats and mice. J. Appl. Toxicol. 2:207-212. Iverson, F., Lok, E., and Nera, E. A. 1985. Pathological and biochemical effects of vomitoxin in rodents. Toxicologist 5:6 (abstr.). Morrissey, R. E., and Norred, W. P. 1984. An improved method of diet preparation for toxicological feeding experiments. Lab. Anim. 18:271-274. Rotter, R. G., Thompson, B. K., Trenholm, H. L., Prelusky, D. B., Hartin, K. E., and Miller, J. D. 1992. A preliminary examination of potential interactions between deoxynivalenol (DON) and other selected Fusarium metabolites in growing pigs. Can. J. Anim. Sci. 72:107-116. Snedecor, G. W., and Cochran, W. G. 1980. Statistical Methods, 7th ed., p. 507. Ames: Iowa State University Press. Tryphonas, H., Iverson, F., Ying, S., Nera, E. A., McGuire, P. F., O'Grady, L., Clayson, D. B., and Scott, P. M. 1986. Effects of deoxynivalenol (vomitoxin) on the humoral and cellular immunity of mice. Toxicol. Lett. 30:137-150. Trenholm, H. L., Warner, R. M., and Prelusky, D. B. 1985. Assessment of extraction procedures in the analysis of naturally contaminated grain products for deoxynivalenol (vomitoxin). J. Assoc. Offic. Anal. Chem. 68:645-649. Received February 6, 1992 Accepted April 21, 1992
