Subacute Toxicity of Dietary T-2 Toxin in Mice:Influence of Protein Nutrition

M.A. Hayes and H. B. Schiefer*

ABSTRACT

The subacute toxic effects of dietaryT-2 toxin (20 ppm) incorporated in semi-purified diets of 8%, 12% or 16% protein,were examined in young Swiss mice afterone, two, three and four weeks. DietaryT-2 toxin caused substantial reductionsin growth and food consumption, thedegrees of which were greatest in mice fedthe diets of reduced protein content. T-2toxin consistently caused similar degreesof nonregenerative anemia, lymphopenia,thymic atrophy and gastric hyperkera-tosis irrespective of the dietary proteinlevel. However, erythroid hypoplasia wastemporary in mice fed T-2 toxin in the16%-protein diet such that erythroidprecursors regenerated in splenic andbone marrow and were hyperplastic afterfour weeks. Liver to body weight ratiosof mice fed T-2 toxin in the 16%-and12%-protein diets increased during thefour week trial in comparison to controlmice fed at a similar rate. These observa-tions indicated that suppression of ery-thropoiesis in mice by dietary T-2 toxinwas temporary and that the interval be-fore regeneration was prolonged by dietsof reduced protein content.

RESUME

Cette experience consistait 'a verifier,chez des jeunes souris suisses, au boutd'une, deux, trois et quatre semaines, leseffets toxiques subaigus de la presencede 20 ppm de toxine T-2 dans des dietessemi-purifiees qui contenaient respective-ment 8%, 12% et 16% de proteines. Cettetoxine entraina des reductions substan-

*Department of Veterinary Pathology, West-ern College of Veterinary Medicine, Univer-sity of Saskatchewan, Saskatoon, Saskat-chewan S7N OWO.

Submitted May 30, 1979.

tielles de la croissance et de la consom-mation alimentaire, lesquelles s'avererentplus marquees chez les souris dont ladiete contenait le plus faible pourcentagede proteines; elle provoqua aussi cons-tamment un certain degre d'ane'mie nonregeneratrice, de lymphopenie, d'atrophiethymique et d'hyperkeratose stomacale,independamment du pourcentage de pro-teines de la diete. L'hypoplasie erythroidese reve'la cependant transitoire chez lessouris, lorsqu'on incorporait la toxine T-2a la diete qui contenait 16% de proteines,de telle sorte que les precurseurs ery-throides regenererent dans la pulpe sple-nique et la moelle osseuse, au bout dequatre semaines. Le rapport entre lepoids du foie et le poids total des sourisqui recevaient la toxine T-2 dans lesdietes qui contenaient 12% et 16% deproteines, augmenta au cours des quatresemaines que dura l'experience, compara-tivement aux souris temoins. Les resultatsde cette experience demontrerent que lasuppression de l'hemopoiese, attribuablea l'ingestion de toxine T-2 par des souris,etait temporaire et que l'intervalle quiprecedait la regeneration se trouva pro-longe lorsque la diete contenait moinsde proteines.

INTRODUCTION

Trichothecene mycotoxins have been con-sidered to be one of the most importantgroups of mycotoxins in the temperateclimatic zones of Asia, Europe and NorthAmerica (24). This view has arisen fromthe knowledge that trichothecenes may beproduced by Fusarium and other speciesof fungi growing on cereal products undercold, moist conditions (24). Trichothecenes,especially T-2 toxin, have been associatedwith lethal mycotoxicoses such as ali-mentary toxic aleukia of man in whichsuppression of hematopoiesis is a majortoxic effect (9, 11, 24). More recently, T-2toxin has been implicated in mycotoxicosesof poultry (5, 26) and swine (5) charac-

Can. J. comp. Med. 44: 219-228 (April 1980) 219

terized by poor growth, food refusal, vomi-tion, perioral dermatitis and inflammationof the upper alimentary tract. In thesemycotoxicoses, effects on hematopoiesishave not been reported (5, 26), nor havethey been experimentally reproduced inanimals fed T-2 toxin in the diet (3, 25).Repeated doses of T-2 toxin (11, 20, 24)or other trichothecenes (22) will suppresshematopoiesis in some species, especiallycats (11, 20). However, because tricho-thecene mycotoxins inhibit voluntary foodconsumption (24), effects produced bynondietary exposure may not resembleeffects produced under natural exposure.Mice fed T-2 toxin for six weeks in a

balanced, semipurified diet containing 16%protein developed hematopoietic suppres-sion manifested as nonregenerative anemia,as described previously (7). During conti-nuous exposure, hypoplastic hematopoietictissues regenerated after several weeks ofsuppression, but lymphoid tissues remainedhypoplastic. Because optimally formulateddiets containing T-2 toxin were used instudies in which depression of hemato-poiesis was not observed (3, 25), we alsoexamined the influence of suboptimalprotein nutrition on the subacute toxicityof dietary T-2 toxin in mice to determineif low protein diets would enhance thetoxicity of T-2 toxin to the hematopoietictissues. Outbreaks of alimentary toxicaleukia have been much less severe inhunman populations consuming moldy grainin balanced diets (9), and variation innutritional composition and quality of thediet may modify biological detoxificationof many toxicants (13) including myco-toxins such as aflatoxin (14). This reportdescribes the influences of the dietaryprotein level on subacute dietary T-2 toxi-cosis of young Swiss mice.

MATERIALS AND METHODS

ANIMALS AND CARE

Male weanling outbred Swiss micewere housed in screen-top cages on softwood shavings in a room maintained at210C with 12 hours of fluorescent lightingper day and were managed according to

'Canadian Breeding Farms and Laboratories,St. Constant-Laprairie, Quebec.

the guidelines of the Canadian Council onAnimal Care. Mice were supplied with tapwater ad lib and with semipurified diets(Table I) at three protein levels (8%,12% and 16% as casein and gelatin). Thediets were prepared weekly as a moistgelatin bound cake, stored under refrigera-tion and freshly supplied each day inoverhead feeding racks.

EXPERIMENTAL DESIGN

A total of 192 mice weighing 16.0 ± 1.2g were randomly assigned to groups offour per cage. For each of the three dietaryprotein levels, mice were arranged inexperimental groups according to thedesign in Table II. The principal groups(A), of 20 mice each, were supplied ad libwith the semipurified diets containing

TABLE I. Composition of Semipurified MouseDieta

Gramper

Basic Diet Ingredients kilogram

c-cellulose 50.0gelatin 20.01-methionine-supplemented caseinb 140.00carbohydrate mixtured 590.00fat mixturee 100.0vitamin mixturef 50.0mineral mixtureg 50.0

Composition of prepared diet Proportionsbasic diet 100.0 gwater 70.0 mLethanol ( i T-2 toxin 10 mg/mL) 0.2 mLaModified from John and Bell. Part of the proteincontent was provided as gelatin (2%). Ethoxyquinand tetracycline were deleted. Iron content wasincreased by 50% and menadione increased by100%bVitamin-free casein, 96.9%; 1-methionine, 3.1%-Proportions shown are for a protein level of 16%(approx). This level is varied by interchanging1-methionine-supplemented casein with carbohy-drate mixturedCoMstarch, 50%; dextrose, 30%; sucrose, 20%eLard, 60%; sunflower oil, 40%fVitamin mixture components (g/kg): thiamin HCL,1.0; riboflavin, 2.0; d-Ca pantothenate, 1.0; nico-tinic acid, 0.5; a-biotin, 0.02; pyridoxine HCL,0.1; folic acid, 0.05; vitamin B12, 0.0005; meso-inositol, 0.04; choline chloride, 80; retinyl palmitate(Vitamin A, 500,000 IU/g), 0.2; cholecalciferol(Vitamin D3, 4 x 107 IU/g) 0.0001; dl-cc-tocopherolacetate (Vitamin E, 1,000 IU/g), 2.8; menadione,8.0; sunflower oil, 110.0; cornstarch, 794.3gMineral mixture components (g/kg): NaCl(iodized), 40.0; CaHPO4.2H20, 687.0; KHCO3,136.0; MgO, 18.0; MnSO4.H20, 3.0; FeSO4H2O,4.5; CuSO4.5H20, 1.0; ZnO, 1.0; cornstarch, 109.5

220

TABLE II. Experimental Design Used for Examination of the Influence of Dietary Proteinon Subacute Toxicity of Dietary T-2 Toxin (20 ppm) in Young Swiss Mice

Number of mice examined

Groupa Treatment Day 0 Day 7 Day 14 Day 21 Day 28

A Semipurified diets 4 x 3b 4 x 3 4 x 3 4 x 3containing T-2 toxin(20 ppm)

B Semipurified control 4 x 3 4 x 3 4 x 3 4 x 3diets - restricted intake

C As for group A for three 4 x 3weeks, then as for group B

D Semipurified control diets 8 x 3- ad lib

E Pelleted natural- 12 8ingredient mouse diete

aGroups A, B, C and D were repeated for each of the three dietary protein levels, 8%, 12%O and 16%bFour mice from each of the three dietscDepartment of Animal Science, University of Saskatchewan

purified crystalline T-2 toxin2 at a rateof 20 ug/g of dry diet (20 ppm). Matchingcontrol groups (B) of 20 mice were sup-plied with toxin-free diets at a restrictedrate such that cages of control mice weregiven an amount of diet similar to thatconsumed by a corresponding cage of micefed the toxic diet.The effect of withdrawal of the toxin

was examined in groups of four mice thatwere removed from the respective toxicdiets after three weeks and fed the cor-responding control diet for one week atthe rate at which the toxic diet had beenconsumed during the week before transfer.Groups D each contained eight mice whichwere supplied with the control diets adlib. Group E consisted of 20 mice, 12 ofwhich were examined on day 0, and eightwhich were fed ad lib for four weeks ona pelleted, natural-ingredient, balancedlaboratory mouse ration containing 18.6%crude protein.The experiment was conducted over a

four week period, with mice being examinedby necropsy according to the schedule inTable II. Mice remaining in groups A andB for the 8%- and 12%- protein dietsafter four weeks were discarded. Mice onthe 16%-protein level in this design werealso used for the six week morphologicaland hematological study described in theprevious paper (10).

EXPERIMENTAL OBSERVATIONS

spected at least once daily and, at weeklyintervals, were examined individually andweighed. The daily food consumption wasmeasured for all groups (except E) andaverage weekly food consumption rates formice in each cage were determined. Atweekly intervals, according to the schedulein Table II, mice were selected for hema-tological and necropsy examination.

Packed cell volume (PCV), erythrocytecount (RBC), hemoglobin concentration(Hb), mean corpuscular volume (MCV),mean corpuscular hemoglobin (MCH),mean corpuscular hemoglobin concentration(MCHC) and total leukocyte count (WBC)of peripheral blood were determined3 fromblood from the tail. Blood smears stainedwith Wright's-Giemsa were used for mor-phological evaluation and for differentialleukocyte counts, and blood was stainedwith methylene blue for determination ofreticulocyte counts. At necropsy, freshweights of liver, stomach, spleen, thymusand testes were determined and imprintsmears of tibial bone marrow were stainedwith Wright's-Giemsa. Various tissueswere selected for histological examination,as described in the previous paper (7).

ANALYSIS OF DATA

Continuous variables from groups A andB in this four week study were analysedin a three-way analysis of variance withtoxin, dietary protein and time as factors.

All mice in the experiment were in-

2Makor Chemicals, Jerusalem, Israel.3Coulter S, Coulter Electronics Inc., Hialeah,Florida.

221

The influence of diet on significant toxiceffects was assessed by a two-way analysisof variance of data from the toxin-treatedgroups. Intergroup comparisons between28-day means of groups A, B, C and D,for each dietary protein level were madeusing one-way analyses of variance andStudent-Newman-Keuls' multiple rangetests. Selected comparisons between meansof toxin-treated groups and the corres-ponding control groups were made by two-tailed Student's t tests. The 95%-probabi-lity level was used to determine significanceof factor effects and differences betweenmeans. All analyses and computations wereas described by Nie et al (15).

RESULTS

Similar hematological and morphologicalchanges were observed in all mice fed T-2toxin. Hematological and morphologicalchanges, including nonregenerative anemia,neutrophilia, generalized lymphoid deple-tion, atrophy of hematopoietic tissue inbone marrow and spleen, perioral derma-titis, hyperplasia with ulceration of thesquamous gastric mucosa and hepatomegalyhave been described in the previous paper(7). The present report deals with effectsof T-2 toxin on growth and feed consump-tion and with the influence of dietaryprotein on the various toxic effects.

Significant diet-related differences ingrowth rates of mice consuming T-2 toxinoccurred (Fig. 1), but a similar influenceof dietary protein level on growth occurredin the restricted-intake (B) and in thead lib fed control groups (D). Diet con-sumption rates were lower in mice fed T-2toxin in the two low protein diets, butthis difference was related to differencesin body weight, because consumption oftoxic diets per gram of body weight didnot differ significantly (p = 0.79) amongthe three diet groups (Table III). T-2toxin substantially reduced food consump-tion by mice; ad lib fed controls consumedabout two to three times as much as miceon toxic diets (Table III). Mice discardedtoxic diets into their bedding, especiallyduring the first two weeks.No deaths occurred in mice fed T-2 toxin

in the 16%-protein diet, nor in any ofthe control groups, but four mice fed T-2toxin in the diets of reduced protein

Days

Fig. 1. Growth curves of mice fed semipurifieddiets of 16% protein (squares), 12% protein(triangles), 8% protein (circles) or a naturalingredient diet of 18.5% protein (diamonds).Points are means of all mice on the respectivediets at each time.

content died during the experiment (TableIV). Of these, one on the 12%-proteindiet died on day 7 with intestinal hemor-rhage, perioral dermatitis and stomatitis.Two died (days 19 and 25) from the effectsof severe perioral dermatitis, whereas oneother which died was extremely anemic(day 24).Mice fed T-2 toxin developed dry ruffled

fur and some developed exudative derma-titis around the mouth, or less frequentlyon the feet. Many developed dry, scaleyskin on the tail, and pallor of the ears,eyes and muzzle was evident in someduring the later stages of the trial. Theseeffects occurred less frequently in micefed T-2 toxin in the 16%-protein diet(Table IV).The dietary protein level did not signi-

ficantly influence (p<0.05) the degree ofanemia (Fig. 2), reticulocytopenia (Fig.3), nor lymphopenia (Fig. 4). However,two mice on the 12%-protein diet becamemuch more severely anemic than othersin the same group (see Fig. 2). T-2 toxinalso caused eosinopenia, neutrophilia,thymic atrophy and gastric hyperkeratosisin all groups. The severity and rate ofdevelopment of these effects was notinfluenced by the dietary protein level andwere similar to those described for the16%-protein diet in the previous paper(7). Liver weights, both absolutely (p =

222

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TABLE III. Mean Rates of Diet Consumption of Mice Fed Semipurified Diets Containing T-2Toxin (20 ppm) and Different Levels of Protein

Diet Group Week 1 Week 2 Week 3 Week 4

A 11.3 ± 0.1a 8.3 z 1.8 9.1 i 0.9 13.1 ± 3.2(20ppm T-2) 1.4 0.1b 1.0 ± 0.3 1.2 + 0.2 1.5 + 0.3

8%protein B 15.2 i 0.5 11.5 + 0.6 13.6 ± 0.8 11.9 + 0.7

(restricted control) 2.4 i 0.0 1.9 ± 0.0 2.0 ± 0.0 1.9 ± 0.0

D 22.7 ± 1.7 18.5 i 0.3 14.3 i 0.1 13.7 i 0.4(ad lib control) 4.2 + 0.1 4.5 ± 0.0 4.2 ± 0.1 4.1 + 0.2

A 10.8 ± 1.4 10.4 ± 1.1 12.9 i 0.6 11.5 i 3.7(20ppm T-2) 1.4 ± 0.2 1.5 i 0.2 1.5 i 0.2 1.3 ± 0.4

12%protein B 12.3 i 0.3 12.2 i 0.6 11.9 i 0.6 11.7 ± 0.5

(restricted control) 2.4 ±4 0.1 1.9 i 0.0 2.0 i 0.0 2.0 : 0.0

D 18.0 ± 0.4 15.7 ± 1.1 12.0 i 0.1 12.2 i 0.5(ad lib control) 4.0 ± 0.0 4.4 ± 0.1 3.9 i 0.0 4.0 i 0.1

A 13.4 ± 0.8 10.1 i 2.0 13.9 ± 1.3 12.4 ± 1.2(20ppm T-2) 1.9 i 0.1 1.4 i 0.3 2.0 + 0.2 1.9 i 0.3

16%protein B 13.3

±0.3 10.8 ± 0.2 12.0 i 1.1 11.1 ± 0.1

(restricted control) 2.5 ± 0.1 2.0 i 0.1 2.0 ± 0.0 2.0 i 0.0

D 17.0 0.1 15.3 0.8 12.6 1.0 10.1 0.6(ad lib control) 4.1 ± 0.1 4.4 4 0.2 4.2 i 0.4 3.5 i 0.3

aMean consumption ( ± SEM) expressed as g/100g body weight/daybMean consumption ( ± SEM) expressed as g/mouse/dayOver the entire experiment, diet consumption rates of groups A and B were not significantly different(P = 0.79)

TABLE IV. Toxic Effects Observed in Mice Fed T-2 Toxin (20 ppm) in Semipurified Diets ofDifferent Protein Levels

Effects Day 8% protein diet 12% protein diet 16% protein diet

Mortality 1/15a (day 19) 1/24 (day 7) none1/6 (day 25) 1/6 (day 24)

Perioral dermatitis 14 5/20 4/19 1/2021 9/14 11/15 4/16

Scaliness of tail skin 14 5/20 6/19 2/2021 11/14 10/15 10/16

aValues are ratios of number atfected to number of mice remaining on the respective toxic diet

0.01) and as percentages of body weight(p = 0.04), were significantly greater inmice fed the two highest dietary proteinlevels (Fig. 5). Organ to body weightratios of testes, thymus, stomach andspleen of mice consuming T-2 toxin didnot differ significantly among groupsconsuming the different toxic diets.

Several quantitative and qualitative dif-ferences in the characteristic lesions in-duced by T-2 toxin were apparent amongthe groups fed different levels of protein.Mice fed T-2 toxin in each of the threediets initially developed atrophy of lym-phoid, granulopoietic and erythropoieticpopulations in the spleen. By two weeks,

myeloid and megakaryocyte precursorsreappeared in the splenic red pulp andsubsequently proliferated. The degree ofproliferation was more pronounced in miceon the 16%-protein diet and resulted insplenomegaly by day 28 in three mice inthis group (Fig. 6). Erythroid populationswere evident amid proliferating undiffe-rentiated cells in the splenic red pulp ofone of these mice, whereas granulopoiesiswas observed in all four.

Erythropoiesis in the spleen and bonemarrow of mice fed T-2 toxin rapidlydiminished during the first 14 days ineach of the three diet groups. Numbersof metarubricytes and polychromatophilic

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Fig. 2. Packed cell volumes of mice fed T-2toxin (20 ppm) (A) in comparison with re-stricted-intake control groups (B). For com-parison, 28-day values of mice after toxinwithdrawal (C), of ad lib-fed controls (D)and of natural-ingredient diet-fed controls(E) are shown. Asterisks indicate meanssignificantly different from control group B.All points are means (+ SEM) of differentgroups of four mice. Wide deviations at 14and 21 days for group A on the 12%-proteindiet are due to severe blood loss by one mousein each group.

erythrocytes in tibial bone marrow smearswere almost totally depleted. Erythropoie-sis resumed sooner and more frequentlyin mice consuming T-2 toxin in the 16%-protein diet (Table V).The dietary protein level did not in-

fluence the pattern of recovery that oc-curred in mice transferred to the toxin-free diets (groups C). Erythropoiesis inspleen and bone marrow, and lymphopoiesisin the thymus, lymph nodes, spleen and

Dietary protein level- %

- 12%16%

T- 2|wdraown(Gr.C)

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Fig. 3. Reticulocyte counts of young mice feddietary T-2 toxin (20 ppm) (solid symbols;group A), in comparison with counts frommice fed the control diets at the same rate(open symbols; group B). The effect of with-drawal of the toxin from the diet on day 21is shown (group C). All points are means ofdifferent groups of four mice.

intestinal lamina propria had resumed inall mice within seven days. No differencesor abnormalities in regenerating erythro-poiesis and lymphopoiesis were evidentamong mice on the different protein levels,but the degree of activity was variableamong individuals in each of the threediet groups.

DISCUSSION

The severity of the characteristic toxiceffects of dietary T-2 toxin at 20 ppm onthe gastric mucosa, lymphoid organs andbone marrow, and the degree of theresulting hematological changes, were gene-rally unrelated to the dietary protein level.Dietary T-2 toxin was slightly more harm-ful in diets containing 8% or 12% proteinbecause low mortality occurred in micefed T-2 toxin in these diets, but not inmice fed the toxic 16%-protein diet. Fur-thermore, mice fed T-2 toxin in the 16%-protein diet had higher growth rates thanthose consuming T-2 toxin in diets of

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Fig. 4. Lymphocyte counts of young mice feddietary T-2 toxin (20 ppm) (A), in compa-rison with restricted-intake control groups(B), ad lib-fed control groups (D) and miceon natural-ingredient diet (E). Mice in groupC were removed from the toxic diets on day21. Asterisks indicate means significantlydifferent from control group B. All pointsare means (+ SEM) of different groups offour mice.

reduced protein content. However, thesedifferences between the dietary groups

were small compared with the overalleffects.One toxic effect observed only in mice

fed T-2 toxin in diets of reduced proteincontent was intestinal hemorrhage. Fatalhemorrhage into the intestine was observedin only one mouse of the 60 fed toxic diets.In three others, hemorrhage probably oc-

curred because erythrocyte counts rapidlydecreased without hemosiderosis or icterus

Fig. 5. Liver weight to body weight ratios ofyoung mice fed dietary T-2 toxin (20 ppm)(A), in comparison with mice fed controldiets at the same rate (B). Means at 28 daysfor groups C (toxin withdrawn on day 21),ad lib-fed controls (D) and natural-ingredientdiet-fed controls (E) are shown. Asterisksindicate means significantly different fromcontrol group B. All points are means (+SEM)of different groups of four mice.

being evident, but in these mice neitherthe site of hemorrhage nor the patho-genesis was determined. Direct injury tothe intestinal mucosa by T-2 toxin mayhave been involved. Alternatively, infec-tious enteritis may have developed dueto depletion of lymphoid tissues by T-2toxin, but there was no histological evidencethat this may have occurred. Mice withevidence of previous hemorrhage hadhypoplastic bone marrow and reducednumbers of platelets on smears, but manyother mice that were similarly affecteddid not develop intestinal hemorrhage.

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DaysFig. 6. Splenic weight to body weight ratiosof young mice fed dietary T-2 toxin (20 ppm)(A), in comparison with mice fed controldiets at the same rate (B). Values at 28 daysfor groups C (toxin withdrawn on day 21),D (ad lib-fed controls) and E (natural-ingre-dient diet-fed controls) are indicated. Aste-risks indicate means significantly differentfrom control group B. All points are means(± SEM) of different groups of four mice.

TABLE V. Occurrence of Erythroid Regenera-tion in Tibial Bone Marrow in Mice Fed T-2Toxin (20 ppm) in Semipurified Diets of Dif-ferent Levels of Protein

Dietary protein levelPeriod on T-2 toxin 8% 12% 16%

7 days 0/4a 0/4 0/414 days 0/4 0/4 1/421 days 0/4 1/4 1/428 days 1/4 0/4 3/4

Values are ratios of number of mice exhibitingerythroid regeneration per group of four mice

Intestinal hemorrhage, associated withprolonged prothrombin times, has beenreported in calves given 30 daily intra-rumenal doses of T-2 toxin (18) and inone steer given 65 daily intramusculardoses of T-2 toxin (6). The pathogenesisof such hemorrhage is unknown. In someof the calves, hemorrhagic enteritis oc-curred before prothrombin times wereincreased, so direct injury to the aliment-ary mucosa by T-2 toxin may have beeninvolved (18). Hemorrhagic syndromes ofundetermined pathogenesis have beenreported in cattle fed rations contaminatedby T-2 toxin (8).

Several observations in this study indi-cated that the dietary protein level influ-enced the rate of spontaneous recoveryfrom toxic inhibition of erythropoiesis andmnyelopoiesis. During the trial, all miceconsuming T-2 toxin developed erythroidhypoplasia during the first 14 days, butsubsequently immature myeloid and ery-throid cells appeared in the spleen andbone marrow of many mice, as describedpreviously (7) The onset of recovery oferythropoiesis, indicated by the appearanceof maturing rubricytes, occurred mostfrequently in mice fed T-2 toxin in the16%-protein diet (Table V). Furthermore,during exposure to dietary T-2 toxin,undifferentiated hematopoietic cells re-generated in the splenic red pulp ingreatest numbers in mice fed the 16%-protein diet. Thus, splenomegaly occurredafter four weeks in these mice, while thespleens of mice fed the other toxic dietsremained small.The mechanism by which mice overcame

suppression of hematopoiesis has not beendetermined. Because hematopoiesis resumedin mice that were continuously consumingT-2 toxin in the diet, either the hemato-poietic cells became less sensitive to inhi-bition of proliferation by T-2 toxin, orthe amount of the active form of T-2toxin in the susceptible hematopoietictissues decreased. In this study, hepaticenlargement occurred in mice fed T-2toxin in both the 12% and 16%-proteindiets, and was correlated with the occur-rence of regeneration of hematopoietictissues. Hematopoiesis may have resumedbecause of an acquired competence of theliver to biotransform T-2 toxin into ametabolite that was less toxic to thehematopoietic precursors. Diets low inprotein are known to reduce production ofhepatic microsomal mixed function oxidases

226

(2) which are responsible for most bio-transformations of xenobiotics (17). Manyxenobiotics, such as DDT (23) and pheno-barbitone (21) cause hepatomegaly duringstimulation of iricrosomal enzyme activity,and the degree of DDT-induced hepato-megaly is much lower in rats consumingdiets deficient in protein (1). Mice inthe present study were housed on soft-wood shavings, which may contain agentscapable of inducing hepatic microsomalenzymes (21) so the recovery may havebeen influenced by bedding. However,further experiments with mice housed insuspension cages have shown similardevelopment of hepatomegaly and hemato-poietic regeneration during exposure todietary T-2 toxin (unpublished). Themetabolic fate of T-2 toxin during sub-acute exposure is not known. AlthoughT-2 toxin is deacetylated to HT-2 toxinby human, bovine and rat hepatic micro-somal enzymes (4, 24), HT-2 toxin is onlyslightly less acutely toxic than T-2 toxin(4, 24). However, the metabolic fate andsubacute toxic effects of HT-2 toxin havenot been examined.The degree of hyperplasia of the gastric

mucosa, as determined by gastric weight,was not diet-related, and progressivelyincreased throughout the trial period.This lesion is likely caused by directirritation of the gastric mucosa by T-2toxin, which is extremely irritant to theskin and mucous membranes (12). Sucha direct effect is unlikely to be lessenedby hepatic metabolism of T-2 toxin. Similarhyperplasia has been observed in miceand rats consuming diets containing 15ppm of T-2 toxin for up to 52 weeks (16).The ability of mice to overcome hemato-

poietic suppression by dietary T-2 toxindepended on the diet, suggesting thatdietary T-2 toxin might also impair hema-topoiesis of other animals under somenutritional conditions. Trichothecenes inadequate diets caused reduced growth,reduced food intake, and sometimes irrita-tion of the upper alimentary tract inpoultry (3, 26) and pigs (5, 25), but didnot cause hematological abnormalities. Themechanisms by which these species avoidthe suppressive effects of T-2 toxin, andthe influences of suboptimal nutrition onsuch resistance, have not been investigated.

Concerns have been expressed thattrichothecene mycotoxins may be hazardousto other livestock and man because thesetoxins have been associated with lethal

mycotoxicoses such as alimentary toxicaleukia of man (9), stachybotryotoxicosisof horses and cattle (19) and moldy cornpoisoning of cattle (8). In these diseases,toxic hematopoietic suppression is probablythe underlying problem, leading to criticaldepletion of granulocytes or platelets, andresulting in either bacterial sepsis orhemorrhagic diathesis respectively. Out-breaks of alimentary toxic aleukia havebeen reported to be more severe whenmoldy diets were of inferior nutritionalquality (9). Thus, evaluation of the hazardsdue to foodborne trichothecene mycotoxinsappears dependent on knowledge of theinfluence of nutrition and other factorson the toxicity of these mycotoxins tohernatopoietic cells. In addition to nutri-tional influences, other factors could affecttoxicity, including age, and other myco-toxins, particularly any which might becapable of interfering with hepatic meta-bolism of trichothecenes.

Although it is as yet unknown whetherthe murine responses to T-2 toxin resemblethose of other species, mice are usefulexperimental animals for evaluation offactors influencing the toxicity of tricho-thecenes. Dietary conditions that decreaseor potentiate systemic toxicity of tricho-thecenes could be identified in mice,thereby minimizing the number of expen-sive and potentially hazardous feedingtrials needed to determine the toxicity oftrichothecenes to various livestock.

ACKNOWLEDGMENTS

We are grateful to Mrs. G. Green, Mr.E. Bueckert and Mr. D. Olexson for experttechnical assistance, and to Mrs. CarolKettles for typing the manuscript.

This work was supported by the NaturalSciences and Engineering Research Councilof Canada, and is based upon a thesissubmitted by the senior author in partialfulfillment for a Ph.D. degree, Universityof Saskatchewan.During this study M.A. Hayes was a

Fellow of the Medical Research Councilof Canada.

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