key: cord-0005045-j29m4y33
authors: Williams, Phletus P.
title: Effects of T-2 mycotoxin on gastrointestinal tissues: A Review ofin vivo andin vitro models
date: 1989
journal: Arch Environ Contam Toxicol
DOI: 10.1007/bf01062362
sha: 1b4b7716394cd9498863630c0e7fa742c0cfaa0a
doc_id: 5045
cord_uid: j29m4y33

T-2 mycotoxin, a trichothecene, is the principal toxic component ofFusarium sp. Agricultural products and food are frequently contaminated with this toxin. Various animal models have been used to determine its metabolic fate, rate of excretion, and distribution. A modulation effect on cell-mediated immunity and alterations in gastrointestinal propulsion have been demonstrated. The toxin has been shown to produce some similar pathologic alterations in various animal species studied. The consistent alteration appears to mainly affect mitotic cells of the gastrointestinal tract and the lymphoid system. A host of bioassay systems are now being used as alternative methods to the use of animals for testing of the mycotoxin. These tests may accurately assess and define the role of the subject-toxin interactions following consumption of T-2 mycotoxin contaminated food sources. T-2 mycotoxin, as observed above within vivo andin vitro models, promotes a chemically-induced change in structure and function of affected gastrointestinal cells from a transient and reversible aberration in a single enzymatic reaction to cell death. Regardless of the end point measured, the toxic response brought about in cells appears to involve the interactions of virtually all subcellular processes—membrane transport and permeability, chemical metabolism, DNA function, and energy production/expenditure—as cells attempt to maintain their functional integrity while disposing of the toxicant. The variation in the quality of the toxic response with dose suggests that more cellular processes are perturbed as the chemical dose is increased.

Trichothecenes are sequiterpenoid mycotoxins produced by fungal strains of the genera Cephalo-of Agriculture, Agricultural Research Services, Ames, Iowa 50010, sporium, Fusarium, Myrothecium, Stachybotrys, Trichoderma, Trichothecium, and Verticimonosporium. They are chemically related by the tetracyclic 12, 13-epoxytrichothec-9-ene skeleton (Ueno 1983; Bamburg and Strong 1971) . Currently, more than 45 kinds of derivatives have been detected from the metabolites of various species of fungi. These compounds have been isolated and identified, and shown to possess cytotoxic and phytotoxic activities (Ueno 1984) . Some of these compounds have been reported as associated with human and animal mycotoxicoses (Lutsky et al. 1978; Schoental et al. 1979) .

T-2 mycotoxin (413,15-diacetoxy-3a-hydroxy-8a[(3-methylbutyryl)oxy]-12,13-epoxytrichotec-9ene) is one of the most potent of the trichothecene mycotoxins. It is the principal biologically active fungal metabolite produced by Fusarium sp. The mycotoxin causes a well-documented toxicosis in a variety of animals after both experimental and natural exposures. The toxicosis, which is associated with cellular injury in multiple organ systems, causes an assortment of clinical signs. This toxin and its metabolites have been reported as possible constitutents of "yellow rain," a chemical biological warfare agent exploited in South East Asia (Holden 1982; Mirocha et al. 1982 Mirocha et al. , 1983 Robert and Rosen 1982; Robinson 1982) . Because T-2 mycotoxin is of apparent commercial importance to the chemical industry, there is a need for obtaining additional experimental toxicological information. Presently there is a substantial amount of information available in the literature on the occurrence and identification of T-2 mycotoxin in food residues. However, there is a lack of information on T-2 mycotoxin specially addressing gastrointestinal complications and pathology. An attempt is made here to consolidate such information while describing in vivo and in vitro models. Efforts were Toxin is also referred to as fusariotoxin T-2, insariotoxin, and T-2 toxin made to eliminate reported field cases where only circumstantial evidence implicated T-2 mycotoxin consumption.

T-2 mycotoxin is found as a contaminant of various agricultural products (Gareis et al. 1985; Szathmary 1983; Vesonder 1983) . General information concerning properties of the mycotoxin are given in Table 1 . It has been found in corn, barley, and mixed feeds in the U.S. and Canada at concentrations as high as 25 ppm (Vesonder 1983) . In vivo studies on the fate of T-2 mycotoxin in laboratory animals, poultry, and livestock have demonstrated that the parent compound is rapidly cleared from body fluids and tissues (Chi et al. 1978; Matsumoto et al. 1978; Pace et al. 1985; . T-2 mycotoxin, and other trichothecenes, in pigs and chickens are rapidly metabolized to various compounds and eliminated primarily through the bile into the gastrointestinal tract, and excreta (Bauer et al. 1985; Visconti and Mirocha 1985; Yoshizawa et al. 1981) , and secondarily in vomitus (Bauer et al. 1985) . T-2 mycotoxin is metabolized 375 to several hydrolyzed products, including HT-2 mycotoxin and T-2 tetraol, and hydroxylated compounds like 3'-hydroxy HT-2 toxin (TC-3) and 3'hydroxy T-2 toxin (TC-1) (Matsumoto et al. 1978; Visconti and Mirocha 1985; Yoshizawa et al. 1982) . In bile, T-2 mycotoxin metabolites have been identified as TC-3, 3'-hydroxy-7-hydroxy HT-2 mycotoxin (TC-6), and the glucuronide form of T-2 triol (Gareis et at. 1986; Roush et al. 1985) . Also deepoxidation products such as 3'-hydroxy-depoxy HT-mycotoxin, 3'-hydroxy-depoxy T-2 triol, and deepoxy T-2 tetraol have been isolated from excreta of rats intubated intragastrically with TC-3 and T-2 tetraol (Yoshizawa et aI. t985) . It has been suggested that T-2 mycotoxin is degraded by microorganisms of the large intestine of rats by first deacetylation to HT-2 toxin followed by the reduction of 12,13-epoxy group to deepoxy HT-2 mycotoxin (Conrady-Lorch et aL 1986; Ishii et al. 1986 ).

Man and domestic animals can be seriously affected by trichothecene mycotoxins (Ciegler 1979; Hayes 1980a Hayes , 1980b Hsu et al. 1972; Joffe 1971; Petrie et aI. 1977; Yagen and Joffe 1976) . A disease, alimentary toxic aleukia, which reportedly killed thousands in the Soviet Union (Joffe 1971) , was apparently caused by eating bread made from wheat infested with Fusarium. "Moldy corn disease" of domestic animals is caused by ingestion of corn infested with fungi producing T-2 mycotoxin (Hsu et al. 1972; Petrie et al. 1977) . Intoxication with T-2 rnycotoxin results in weight loss due to diarrhea and emesis, and inflammations with hematological changes and destruction of bone marrow (Bamburg and Strong 1971; Hayes and Schiefer 1980b; Marasas et al. 1969) . Low concentrations of the toxin fed to livestock and poultry have been reported to cause diarrhea and feed refusal; perioral, pharyngeal, and intestinal irritations; some hemorrhaging and lowered immunity; and infertility (Hoerr et al. 1982; Hsu et al. 1972; Palyusik and Koplik-Kovacs 1975; Rafai and Tuboly 1982; Speers et al. 1977; Weaver et al. 1977) . The toxin caused inhibition of rat liver mitochondrial electron transfer (Pace 1983) , and lipid peroxidation (Tsuchida et aI. 1984) .

Lethal dose (LD) values for T-2 mycotoxin administered by various routes to vertebrates are shown in Table 2 . The acute LDso obtained varied between 1.0 and 14 mg-kg-a, there being little difference between the various routes in any given species (Fairhurst et al. 1987) . In guinea pigs, the inhaled LDs0 of T-2 corresponding to an LCts0 1986; Weaver et al. 1978a (lethal concentration x time) of 5749 mg min m -3 is between 3.25 and 4.33 mg-kg -1. This is about twice the subcutaneous LDs0 of the same sample of T-2 mycotoxin used by Marrs et al. (1986) . One explanation for this would be that approximately 50% of the inhaled dose of aerosol was retained. The oral LD50 observed by De Nicola et al. (1978) was very similar to the inhaled LD50 observed by Marrs et al. (1986) .

Administering T-2 mycotoxin to the skin of mice has been reported to cause histological changes in the duodenum . These duodenal changes might be caused by either a generalized radiomimetric effect and/or by bilary excretion of the biologically active toxicant (Marrs et al. 1986 ). Cell damage is reported to result from the inhibitory effects of the toxin on protein and DNA synthesis (Ueno et al. 1973 , Agrelo and Schoental 1980 , Rosenstein and Lafarge-Frayssinet 1983 . Exposure of rats to T-2 mycotoxin resulted in an intestinal decrease in protein and an increase in RNA concentrations (Suneja et al. 1983 (Suneja et al. , 1984 . The decrease in protein might indicate either a decrease in synthesis or an increase in degradation. The increase in RNA content has not been explained.

In in vitro studies, the cytotoxic effects of T-2 mycotoxin in eukaryotic cells are correlated with inhibition of protein, by blockage of polypeptide chain initiation (Cannon et al. 1976; Cundliffe et al. 1974) , and DNA syntheses (Di Ninno et al. 1985; Mclaughlin et al. 1977; Melmed et al. 1985; Ueno et al. 1973) . T-2 mycotoxin markedly inhibits growth of human carcinoma and mouse leukemia cells (Perlman et al. 1969) , with cell differentiation asso-ciated with cytotoxicity of the toxin (Samara et al. 1987) .

Tritium-labeled T-2 mycotoxin administered intramuscularly to guinea pigs was observed to be distributed in all tissues within 30 rain (Pace et al. 1985) . The concentration of radiolabeled toxin rapidly declined, with no measurable long-term accumulation. In general, the early time (12 to 24 hr) distribution patterns of the guinea pig seem to parallel the distributions reported in chickens (Chi et al. 1978; Yoshizawa et al. 1980 ) and pigs .

Tritium-labeled T-2 mycotoxin and its metabolites rapidly distribute to tissues of orally dosed mice with maximum levels reached within 30 min, declining thereafter to non-detectable levels by 72 hr (Matsumoto et al. 1978) . Radiolabeled products were eliminated (feces to urine, 3:1) over a 72-hr time period.

The time-dependent progression of peak radioactivity from guinea pig bile (12 hr) to large intestine (24 hr) to feces (4 days) suggests that metabolites undergo enterohepatic circulation (Pace et al. 1985) . The slow elimination of radioactivity from the intestine might account for the reported histopathological lesions in the gastrointestinal tract of rodents (Brennecke and Neufeld 1982) . These studies suggest that enteric absorbants, such as charcoal, may be of some benefit in the treatment of T-2 mycotoxin intoxication (Pace et al. 1985) . Clays such as smectite are able to protect mice against T-2 mycotoxin-induced disturbances of gastrointestinal transit (Fioramonti et al. 1987) .

Following intravenous administration of tritiumlabelled T-2 mycotoxin to swine, the radioactivity accumulated in the greatest amounts in the ileum, followed by the jejunum and duodenum, stomach and large intestine (Corley et al. 1986 ). The distribution of radioactivity in the gastrointestinal tract is assumed to be primarily from the bile, with some contribution from blood flow to areas of the intestine (Corley et al. 1985) .

The effect of varying dietary levels of zinc has been evaluated in mice fed T-2 mycotoxin (Chanin et al. 1984) . Zinc retention in stomach and liver was enhanced by T-2 mycotoxin, whereas lungs and small intestines showed interactive effects. The dependence of the T-2 mycotoxin absorptive effect on zinc status is especially interesting. The mechanisms of this interaction could be the result of differences in such areas as transit time, transport inhibition, mucosal surface area, and gut permeability. T-2 mycotoxin appears to interfere with zinc homeostatis at the level of gastrointestinal tissues by enhancing zinc absorption in zinc-replete mice and decreasing zinc absorption in zinc-deficient mice. Alternatively, it did not appear to influence the rate of zinc excretion from the body tbtlowing absorption. A possible mechanism for this interaction is the formation of a zinc-T-2 mycotoxin complex which could upset the homeostatic mechanisms of the gastrointestinal tract for zinc. The gastrointestinal tract is probably the major organ involved in the control of zinc status (Beach et al. 1980; Cousins 1982; Fraker et al. 1977 Fraker et al. , 1982 Miranda et at. 1982) but any implication of a zinc-T-2 mycotoxin complex upsetting this control is entirely speculative and is not supported by any evidence in the literature (Chanin et al. 1984 ).

T-2 mycotoxin is immunosuppressive and depresses T-and B-lymphocyte mitogen responses (Buening et al. 1982; Friend et aI. 1983; Rafai and Tuboly 1982) , prolongs skin graft rejection time (Rosenstein et al. 1979) , and is cytotoxic to lymphocytes and rapidly dividing cells (Hayes et al. 1980a; Lafarge-Frayssinet et al. 1981; Saito et al. 1969) . Recently, it has been reported that mice pretreated with T-2 mycotoxin had alterations of their cell-mediated immunity. The treated animals show an increased resistance to listeriosis that apparently is associated with increased migration/activation of macrophage effector cells (Corrier et at. 1987; Zipr]n et al. 1987a Zipr]n et al. , 1987b . Administration of polymyxin E to these mice markedly reduced the gramnegative intestinal microflora but did not eliminate the toxin-induced resistance to listerosis. Enhancement of selective aspects of host immunity has been reported to accompany depletion of subpopulations of short-lived suppressor T-lymphocytes following treatment with T-2 mycotoxin (Masuko et al. 1977) . The depletion of a subpopulation of short-lived lymphocytes that exert a suppressive influence on macrophage activation is one mechanism whereby T-2 mycotoxin may concurrently induce lymphoid necrosis, modulate macrophagelymphocyte interaction, and enhance macrophage phagocytosis (Corrier et al. 1987) . The toxin has been reported to inhibit chemotaxis, phagocytosis, and to generate chemiluminescence in granulocytes. The toxin apparently does not affect the morphology of cells, but may cause perturbation of cell membranes (Niyo et al. 1988a , Gyongassy-Issa and Khachatourians 1984 , Yarom et al. 1984a , 1984b .

Among the physiological disturbances induced by T-2 mycotoxin administration, the toxin stimulates gastrointestinal propulsion that affects the Fate of digesta transit and, apparently, the absorption of nutrients (Sarr et al. 1980) . Shortly after orally dosing mice with the toxin (concentration one-tenth the LD50 daily for 4 days; Ueno t984), an increase in gastrointestinal propulsion was observed for 4 days.

The mechanisms involved in the stimulation of gastrointestinal propulsion by T-2 mycotoxin are unknown but a speculative hypothesis involving prostaglandins may be postulated because T-2 nqycotoxin has been found to increase the release of prostaglandins and related eicosanoids within the brain (Shohami and Feuerstein 1986) . Prostaglandins are known to act peripherally to accelerate gastric emptying and small intestine transit (Ruvcart and Rush 1984) , and to stimulate intestinal motility (Fargeas et al. 1984) . Disturbances of gastrointestinal motility by T-2 mycotoxin may be in part due to changes in mesenteric blood flow (Lundeen et al. 1986; Siren and Feuerstein 1986) . Another trichothecene, fusarenon-X, has been found to increase small intestinal propulsion and to inhibit spontaneous peristalsis (Matsuoka et al. 1979) . This finding has been attributed to an increase in intestinal fluid secretion evidenced by a leakage of plasma contents into the intestinal lumen (Matsuoka and Kubota 1981) . A similar mechanism may be postulated to explain the increase in small intestinal propulsion observed following T-2 mycotoxin administration to mice (Fioramonti et al. 1987) .

Predominant clinical features of subchronic trichothecene intoxication in humans (Goodwin et al. 1978) and animals (Wyatt et al. 1973 ) include signs of impaired central nervous system (CNS) function, such as retarded reflexes and general depression with coma (Martin et al. 1986 ). In animals, dietary and intragastrically administered T-2 mycotoxin produces loss of righting reflex, and meningeal vaso-congestion (Schoental et al. 1979; Wyatt et al. 1973 ). Whether or not the CNS represents a primary or secondary site of T-2 toxicant action is not known. It is generally acknowledged that T-2 mycotoxin affects multiple tissue target sites with predominant lesions occurring in the gastrointestinal, lymphoid, and cardiovascular systems (Bamburg 1983; Ueno 1977) . The toxin impairs regulatory aspects of neuronal nucleic acid metabolism at the transcriptional-translational level (Doebler et al. 1984; Yanagihara 1974) , and in this regard seems to exert direct action(s) on the CNS (Martin et al. 1986 ). Trichothecenes, in general, appear to target mitotic cells of the intestinal tract and lymphoid tissues (Otokawa 1983) .

Experimental administration of either culture preparations of Fusarium or purified T-2 mycotoxin, by various routes, produces the clinical picture of intoxication in rabbits (Gentry and Cooper 1981) , cats (Lutsky et al. 1978) , rats, and monkeys (Rukimini et al. 1980; Schoental and Joffe 1974; Schoental et al. 1979; Wilson et al. 1982) . Intoxication consists of a multisystem syndrome that is often rapidly fatal and dominated by hemorrhagic diathesis, leucopenia, and sepsis (Yarom et al. 1984b) . The cytotoxic effects of trichothecenes are correlated with their ability to inhibit protein and DNA synthesis in eukaryotic cells (Di Ninno et aI. 1985; McLaughlin et al. 1977; Melmed et al. 1985) , and to affect cell membrane functions (Samara et al. 1987) , inhibit platelet aggregation (Yarom et al. 1984a) , induce hemolysis of human red blood cells (Segal et al. 1983) , and inhibit phagocytosis and chemotaxis in polymorphonuclear ceils (Niyo et al. 1988a , Yarom et al. 1984b .

The T-2 mycotoxin LDs0 values for laboratory animals are shown in Table 2 . In rats, mice and guinea pigs the toxin values fell between 1 and 14 mg.kg -~. The mouse was less sensitive than either the rat or guinea pig (Fairhurst et al. 1987) . In rats, the toxin caused mucosal ulcerations, disruption of the lamina propria, and lymphocytolysis throughout Peyer's patches in follicular and parafollicular areas. Nuclear debris was frequently present. In the small intestine dead and dying lymphoid cells were seen in the lamina propria. The most severely affected segment seemed to be the duodenum. Disruption of duodenal Peyer's patches by the toxin may be significant in that this may represent interference in buildup of local antigen-specific immune responses (Enders et al. 1987) . The colon was practically unaffected by such changes but nuclear debris was seen in the gastric mucosa (Fairhurst et al. 1987) .

In mice, 10 ppm of T-2 mycotoxin in the diet is capable of inducing stomatitis, dermatitis, gastric. mucosal hyperplasia, necrosis of the intestinal mucosa (Hayes 1979) , and necrosis of splenic foUicules. After about 3 weeks on a T-2 mycotoxin diet, mice are apparently able to overcome the suppression of the hematopoietic system, and erythroid hyperplasia occurs in the spleen and bone marrow . A zone-specific necrosis of the adrenal cortex has also been seen in females but not males (Thurman et al. 1986 ). In mice, both lymphoid protein and DNA synthesis are inhibited by T-2 mycotoxin (Rosenstein and Lafarge-Frayssinet 1983) . Gastric emptying and small intestinal transit were significantly accelerated after T-2 mycotoxin administration to mice (Fioramonti et al. 1987) .

Mice given trichothecenes experimentally either by injection or by ingestion had pyknosis and karyorrhexis of the cryptal epithelial cells of the small intestine along with a marked edema and swelling of the intestinal villi with inflammatory cells infiltrating the lamina propria. The intestinal mucosa became necrotic and ulcerated. Necrosis of lymphoid cells in the intestinal lymphoid nodules was also reported. The toxin produced cellular necrosis in the thymus, especially in the cortical area, and in the germinal centers of the spleen. Bone marrow necrosis mimicked that seen with radiation damage, and the hematopoietic ceils in these areas were pyknotic and karyorrhexic (Carlton and Szezech 1978; Ueno 1983) .

Severe transmural intestinal necrosis was limited to mice given a single lethal dose of diacetoxyscir-penol (DAS) and may be the cause of death. Septicemia or endotoxemia or both may have resulted and caused the shock-like state of the animals before death (Conner et al. 1986 ). The lowest dose of DAS that produced overt morphologic injury to the intestinal epithelium was 10 mg/kg, given intraperitoneally. At this dosage, damage was limited to multifocal necrosis of crypt epithelium. Thus, the intestine was slightly less sensitive to DAS than are the lymphohematopoietic organs. When the toxin was administered (10 mg/kg) to mice via either gastric gavage or intraperitoneal injection, multifocal necrosis of intestinal epithelium occurred that was attributable to causing death. Guinea pigs exposed to T-2 mycotoxin at an inhalation dose of 4,424 to 6,510 mg min m -3, over an exposure time ranging from 22.5 to 75 min, showed petechial hemorrhages of the gastric mucosa. Animals from the highest dose had small mucosal hemorrhages in the jejunum, ileum and colon (Marrs et al. 1986 ). Microscopically, the small intestine was observed to have dead and dying lymphoid cells throughout the lamina propria, together with macrophages containing nuclear debris. Foci of nuclear debris were found in and around the columnar epithelium and at the bases of the crypts. Small zones of mucosal necrosis and ulceration were observed at the bases of the crypts in severely affected sections. All other organs examined were histologically normal but changes such as lymphocytolysis were seen in the lymphoid tissue of the lungs and adjacent to the pancreas and stomach in the decedents (De Nicola et al. 1978; Marrs et al. 1986 ). Changes in the lymphoid system have been shown to occur in many species other than the guinea-pig. These species include mice (Hayes et al. 1980) , chickens (Bitay et al. 1981) , cats (Lutsky and Mor 1981) and non-human primates (Jagadeesen et aI. 1982). Guinea pigs intubated with a single LD5o (1.0-2.0 mg/kg) of T-2 mycotoxin have hyperemia of the stomach mucosa and uterus (Mirocha 1983) .

Rabbits responded like other laboratory species with dermal trichothecene application producing intense irritation and necrosis of the epidermis and adnexal structures (Carlton and Szezech 1978) . Rabbits given 0.5 mg T-2 mycotoxin/kg/day developed leucopenia, and showed lowered concentrations of serum alkaline phosphatase and serum sorbitol dehydrogenase, and a lowered antibody response to AspergiIlus fumigatus (Niyo et al. 1988b) . Ingestion of the toxin caused lymphocyte necrosis and/or lymphoid depletion in ileal Peyer's patches and mesenteric/jejunal lymph nodes. Gastric mucosal hyperemia, hemorrhage, and superfi-cial mucosat necrosis were observed in the rabbits (Niyo et at. 1988b) .

The domestic cat appears to be highly susceptible to the radiomimetic effects of the trichothecene toxins. With this species, T-2 mycotoxin administered at 0.1-0.2 mg/kg causes major gross clinical signs in cats such as emesis, vomiting, diarrhea, anorexia, ataxia of the hind legs, discharge from the eyes, and ejection of hemorrhagic fluid (Cole and Cox, 1981; Sato et al. t975)~ Consecutive administration of toxin at sublethal dosages caused a marked decrease in leucocytes. Necropsy showed extensive cellular damage in the bone marrow, intestine, spleen, and lymph nodes. Also evident were meningeal hemorrhaging of the brain, bleeding in the lungs, and vacuolic degeneration of renal tubules (Sato et aI. 1975) .

Birds T-2 mycotoxin-induced feed refusal has been observed with pigeons (Kotsonis et aI. 1975) , similar to reports of trichothecene-induced food refusal observed in chickens (Kotsonis et al. 1975) , swine (Weaver et al. 1978a) , and cattle (Weaver et al. 1980) . In pigeons, T-2 mycotoxin caused vomiting (Fairhurst et al. 1987; Kotsonis et al. 1975; Lutsky et al. 1978; Rukmini et at. 1980; Sato et aI. 1975) . The onset and duration of the vomiting were doserelated. Vomiting began as early as 10 min postexposure and persisted for as long as 4 hr. Damage to the gastrointestinal mucosa and nausea probably promotes the onset of the vomiting phenomenon (Fairhurst et al. 1987; Ueno 1977) .

Chickens appear to be relatively resistant to the effects of T-2 mycotoxin, when the concentration in the feed does not exceed 5 ppm. Broilers fed toxin at 4-16 ppm for three weeks duration developed oral inflammatory lesions infected with bacteria. These oral bilateral necrotic lesions have also been observed in turkey pours, and are indicative of feed contaminated with trichothecenes. The trichothecenes, in general, appear to be detrimental to the immune system of birds, causing them to be more susceptible to infectious agents. The toxins cause a decrease in the cellularity of the bursa of Fabricius and general necrosis of other lymphoid sites. Turkeys are more susceptible to dietary T-2 mycotoxin than are chickens (Richard et al. 1978) . Turkey poults develop chronic oral lesions when fed T-2 mycotoxin for an extended period of time. T-2 mycotoxin, and other trichothecenes, do not seem to alter the clotting mechanism of poultry, however, leukopenia and anemia have been associated with chronic toxicoses (Mirocha 1983) .

When T-2 mycotoxin is present in the diet (50 ppm) of bovine species, for at least 15 days, there is no affect on target organs. The toxin apparently only causes congestion and edema to the gastrointestinal tract. The caustic nature of the toxin is neutralized and/or degraded in the rumen. However, the mycotoxin must remain intact for a period of time as it is secreted in the milk of treated-lactating animals (Weaver et al. 1977) . In calves, orally given 0.32-0.46 mg/kg of T-2 mycotoxin, ulcerations develop in the abomasum and rumen, and an acute enteric response with bloody feces occurs (Pier et al. 1976; Weaver et al. 1977) . The calves become inappetent, dehydrated and a slight to severe weight loss was observed respective to the toxin dose level. Clinicopathologic changes were restricted to the higher toxin dosages with increased prothrombin times and increased levels of serum glutamic oxalacetic transaminase (Pier et al. 1976) . No apparent reduction in leukocyte count or bone marrow alterations were observed in toxin-treated calves. In cattle, intramuscularly injected with T-2 mycotoxin to circumvent the rumen, petechial hemorrhages of the gastrointestinal tract have been observed (Mirocha 1983) .

Feeding cattle a T-2 mycotoxin-contaminated diet has been reported to cause intestinal necrosis that seems to be associated with altered release of lysosomal enzymes (Kosuri et al. 1970; Saito et al. 1969 ). However, this observation has not been seen with rats fed a diet containing T-2 mycotoxin (1.5 mg/kg body weight for 4 days; Suneja et al. 1984) . These rats showed no altered release of either acid phosphatase or acid ribonuclease from lysosomes. With other toxins, such as aflatoxin B and luteoskyrin, they have been shown to alter the integrity of rat lysosomal membranes in vivo and in vitro (Pokrovskii et al. 1972 (Pokrovskii et al. , 1974 Tung et al. 1970) .

Administration of lethal doses of T-2 mycotoxin, intravascularly to swine, caused congestion and hemorrhage in the stomach, small intestines (except the duodenum), and the large intestine (Beasly 1986; Weaver et al. 1978c) . By either intravenous or subcutaneous route in swine, the toxin apparently causes necrosis of epithelial and crypt cells of the jejunum and ~leum (Marrs et al. 1986; Pang et al. 1987a Pang et al. , 1987b Pang et al. , 1987c Weaver et al. 1978a) . When sows are fed a diet containing T-2 mycotoxin (12 ppm), during breeding and gestation intervals, the sows in some cases show congestion and edema of the gastrointestinal mucosa and a decrease in reproductive efficiency (Kurtz 1981) .

When T-2 mycotoxin is given parenterally it produces abortion in the pregnant sow but no histopathologic lesions are detected in the placenta or the fetuses (Weaver et al. 1978b (Weaver et al. , 1978d . T-2 mycotoxin fed in the diet at the rate of 1, 2, 4, and 8 ppm to young swine, for eight weeks, promotes the occurrence of only small erosions of the oral cavity. Diacetoxyscirpenol (DAS) on the other hand when fed to swine, at similar concentrations to T-2 mycotoxin, promotes ulcerations and proliferative lesions in the buccal mucosa and proliferative lesions on the lingual surfaces of the oral cavity (Weaver et al. 1978b (Weaver et al. , 1987d .

The parenteral administration of T-2 mycotoxin (LDs0, 1.21 mg/kg) and DAS (LDs0 0.376 mg/kg) produces diarrhea and emesis, posterior paresis, and acute death. With injection of these toxins, some of the pigs were believed to die from endotoxic shock while others prolonged and showed acute necrosis in the germinal centers of the lymph nodes and spleen along with moderate necrosis of the hematopoietic elements of the bone marrow (Weaver et al. 1977 (Weaver et al. , 1978a (Weaver et al. , 1981 . The lesions produced by DAS were similar to those of T-2 mycotoxin but DAS produced much more pyknosis and karyorrhexis of lymphoid cells. Feed containing DAS (4 ppm) was refused by Swine (Weaver et al. 1978b, 198I) . Feed containing T-2 mycotoxin concentrations greater than 12 ppm was refused by swine (Kurtz, 1981) . Also, topical exposure of swine to a sublethal dose of T-2 mycotoxin, 15 mg/kg, can cause significant systemic effects on parameters such as body weight gain, rectal temperature, hematology, serum biochemistry, and cellular immune response (Pang et al. 1987b ).

In terms of mycotoxin identification, a bioassay system for demonstrating toxic effects is the ultimate tool in classification. They are necessary analytical adjuncts to chemical and other techniques, and serve as alternative methods to the use of animals for testing. They are necessary for determining the precise nature of the toxins' toxic properties (Acosta et al. 1985; Dagani 1983; Grisham and Smith 1984) ). Bioassay systems serve as confirmatory evidence for the deleterious properties of (1987) Reference Source: f Reference Source: Suneja et al. (1984) . T-2 mycotoxin was given daily, by oral garage for four days g Reference Source: Veseley et al. (1982) newly identified fungal metabolites and aid in the risk assessment of possible hazardous effects to animal populations. It is within this framework that bioassay systems are helpful in the diagnosis of mycotoxicoses. Examples of in vitro bioassay systems used for testing of T-2 mycotoxin are shown in Table 3 .

Trichothecenes have been found to be potent inducers of terminal differentiation using human leukemic cells as a bioassay system (Table 3 ). The lipophilic compounds T-2 mycotoxin, HT-2 mycotoxin, DAS, and acetyl T-2 mycotoxin have been reported to be effective in the range of 2-10 ng/ml; while the less lipophilic T-2 triol and scirpentriol are effective at higher concentrations (50-100 ng/ml). Also 9,10-epoxy T-2 mycotoxin and 9,10-dihydro T-2 mycotoxin, compounds missing the 9,10 double bond, were effective at 50-I00 ng/ml~ Roridin A, a macrocyclic trichothecene, has been found to be the most effective, inducing differentiation at concentrations as low as 0.3 ng/ml (Samara et al. 1987) . The mechanism of induction of differentiation by these trichothecenes is apparently unknown.

The precise nature of the toxic action of 12,13epoxytrichothecenes is still uncertaim T-2 mycotoxin as shown in Table 3 is toxic to mammalian cells in culture (Bodon and Zoldag 1974; Hsia et al. 1983) , and has been reported to inhibit protein synthesis in animat cells in vitro (Cannon et al. 1976; Carter and Cannon 1977; McLaughlin et al. 1977; Ueno et al. 1973) , and to inhibit DNA synthesis in Ehrlich ascites tumour cells without affecting RNA synthesis (Ueno and Fukushima 1968) . The primary mechanism of trichothecene-induced cytotoxicity appears to be due to the ability of the toxin to bind to the 60 S subunit of the ribosomes causing the interference of the initiation of protein synthesis (McLaughlin et at. 1977) .

The effect of T-2 mycotoxin given by oral gavage on the intestinal transport system was examined by measuring the uptake of glucose and tryptophan us~ing everted jejunal sacs (Table 3) . Feeding of T-2 mycotoxin markedly decreased the uptake of glucose and tryptophan with respective differences over controls of: glucose, 549 p,g absorbed/brig; and tryptophan, 279 ~g absorbed/hr/g (Suneja et al. 1984 ). In addition, toxin-treated and non-treated intestinal segments were used as sources of mucosal layers for determining brush border sucrase and lactase activities. The toxin-treated segments showed lowered sucrase activity from 60 to 16 Ixg glucose liberated/10 mirdmg protein; and lactase activity from 14 to 10 txg glucose liberated/10 min/mg protein. The toxin also inhibited (Na+-K+)-ATPase of the small intestinal mucosa from 2.6 to 2.1 txmoles phosphorus liberated/10 rain/rag protein. (Suneja et al. 1984) . With impairment of (Na +-K+)-ATPase there would be, presumably, a lack of maintenance of the Na § gradient across the intestinal epithelial cell, resulting in disruption of absorption of sugars and amino acids (Skou 1965) . Apparently, the toxin does not interfere with intestinal alkaline phosphatase activity, an enzyme that is known to be localized in the microvillus membrane (Eichholz 1967) . The toxin also does not alter the release of the lysosomal enzymes, acid phosphatase and acid ribonuclease (Suneja et al. 1984) .

In studies with rat intestinal mucosal homogenates, T-2 mycotoxin appears to interfer with incorporation of 14C-leucine into protein, by about 15%. Intestinal mucosal protein content was reduced from 107 to 86 mg/g, indicating a significant loss of protein (Suneja et al. 1983) .

The above results on glucose and tryptophan uptake and brush border enzymes suggest that exposure to T-2 mycotoxin induces changes in the functioning of the brush border membrane in the small intestine of rats (Suneja et al. 1984) . The inhibition of the transport proteins by T-2 mycotoxin feeding could be due to their decreased synthesis or to direct interaction with the reactive site (epoxy group) of T-2 mycotoxin. Alkaline phosphatase, localized in the microvillus membrane, was not altered by T-2 mycotoxin suggesting that the toxin is more specific to the transport proteins (Eichholz 1967). Ueno and Matsumoto (1975) showed inhibition of SH-enzymes by prior incubation with T-2 mycotoxin in vitro, that could be prevented by dithiothreitol, suggesting a probable binding of epoxytricothecenes with thiol residues of SH-enzyme proteins. The inhibition of sucrase, lactase and (Na+-K+)-ATPase may be due to the interaction of the reactive site of T-2 mycotoxin with the SHgroup of enzyme proteins, with a concommitant reduction in glucose and tryptophan uptake. et aI. 1985) . Incorporation of this DNA precursor was observed as a gradual and continuous process, indicative of DNA synthesis associated with transformation of primarily epithelial crypt cells within the explants (Browning and Trier 1969; Eastwood and Trier 1973) . When similar ring explants were incubated with T-2 mycotoxin a decrease in uptake of radioactivity (about 90%) was observed. This decrease in radio-labeled thymidine incorporation to DNA by T-2 mycotoxin may not be due to direct inhibition of the appropriate polymerase or the accessory proteins necessary for replication, or damage to the DNA itself. T-2 mycotoxin may inhibit thymidine transport into the cells or damage membranes concomitant with cytotoxicity that indirectly inhibits the uptake of exogenous radio-labeled thymidine (Williams, unpublished data) . Chemicals that interfere with cell metabolism are able to induce quantitative as well as qualitative alterations of nucleotide pools (Bianchi 1982) .

T-2 mycotoxin incubated with the ring explants showed cytotoxicity effects to the gastrointestinal mucosa within 1 hr of T-2 mycotoxin exposure ( Table 3) . Necrosis of crypt epithelial cells, nuclear debris in the lamina propria, and loss of muscular retention were observed over a 48-hr incubation period. The most severely affected explants were the duodenum, followed by the jejunum, and ileum . When a proteinaceous toxin (Pasteurella multocida, PM) was compared to T-2 mycotoxin in explants, exposed to a pseudorabies virus (swine pathogen, Gustafson 1986), the following results were obtained. The cultures were observed over a four day incubation period to be metabolically active synthesizing DNA and protein, showed mitotic figures with morphologic integrity retained, and showed cellular changes with virus-toxin. The PM toxin was not caustic to the intestinal mucosa and did not impair but enhanced pseudorabies virus replication, causing epithelial necrosis. The T-2 mycotoxin was caustic to the intestinal mucosa resulting in loss of epithelial cells, causing a loss of pseudorabies virus replication . 

Sensitivity of cultured human and mouse fibroblasts to trichothecenes

An in vitro approach to the study of target organ toxicity of drugs and chemicals

Synthesis of DNA in human fibroblast treated with T-2 toxin and HT-2 (the trichothecene metabolites of Fusarium species) and the species of hydroxyurea

Biological and biochemical actions of trichothecene mycotoxins

13-Epoxy-trichothecenes

Comparative studies on the metabolism of diacetoxyscirpenol and zearalenone in pigs

Impaired immunologic ontogeny in postnatal zinc deprivation

Pharmacokinetics of the trichothecene mycotoxin T-2 toxin, in swine and cattle

Nucleotide pool unbalance induced in cultured cells by treatments with different chemicals

A case of T-2 mycotoxicosis in broiler chicks

Cytotoxicity studies on T2 fusariotoxin

Pathologic effects and LDs0 doses of T2 toxin in rats by intramuscular, subcutaneous, and intraperitoneal routes of administration

Organ culture of mucosal biopsies of human small intestine

The effect of T-2 toxin on the bovine immune system: cellular factors

Prevention by ribosome bound nascent polyphenylalanine chains of the functional interaction of T-2 toxin with its receptor site

Mycotoxicoses in laboratory animals

Structural requirement for the inhibitory action of 12,13 epoxytrichothecenes on protein synthesis in eukaryotes

Biointeraction of dietary T-2 toxin and zinc in mice

I978) Excretion and tissue distribution of radioactivity from tritium labeled T-2 toxin in chicks

Ciegler A (1979) Tricbothecenes, occurrence and toxicoses

Handbook of Toxic Fungal MetabNites

Toxicity of angnidine in mice

Investigations on the intestinal metabolism of T-2 toxin a major trichothecene mycotoxin. 27th Spring Mtg German Pharmacological Soc Naunyn-SchmJedeberg's

Glucuronide conjugates of T-2 toxin and metabolites in swine bile and urine

Disposition of T-2 toxin, a trichothecene mycotoxin, in intravascularly dosed swine

Modulation of cell-mediated resistance to listeriosis in mice given T-2 toxin

Mechanism of zinc absorption. In: Prasad AS (ed) Current topics in nutrition and disease

Mechanism of inhibition of eukaryotic protein synthesis by trichothecene group (fungal toxins)

Alternative methods could cut animal use in toxicity tests

T2 toxin mycotoxicosis in the guinea-pig

In vitro toxicity of T-2 toxin in mouse lymphoid cells

Soman toxication in hypo~a acclimated rats: Alterations in brain neuronal RNA and survival

Epithelia[ cell renewai in cultured rectal biopsies in ulcerative colitis

Eichholz A (t967) Structural and functional organization of the brush border of intestinal epithelial ceils. III. Enzymic activities and chemical composition of various fractions of tris-disrupted brush borders

Role of Peyer's patch in the intestinal immune response to cholera toxin in entericatty immunized rats

Acute toxicity of T-2 toxin in rats, mice, guinea pigs and pigeons. II. Variations in surface architecture found by scanning electron microscopy

Prostaglandins E2: A neuromodulator in the central control of gastrointestinal motility and feeding behavior by catcitenin

Comparative electron-microscopic features of normal, hyperplastic and adenomatous human colonic epithelium

Action of T-2 toxin on gastrointestinal transit in mice protective effect of an argillaceous compound

Effect of zinc deficiency on the immune response of the young adult A/J mouse

Delayed type hypersensitivity in zinc deficient adult mice: impairment and restoration of responsivity to dinitrofluorobenzene

The effect of T-2 toxin on acute herpes simplex virus type-1 infection in mice

Fusarien-toxine in Futtermittein. Nachweis und Vorkommen von Trichothecenen

Identification of glucuronide metabolites of T-2 toxin and diacetoxyscirpenol in the bile of isolated perfused rat liver

Effect of Fusarium T-2 toxin on hematological and biochemical parameters in rabbit

Changes induced by T-2 toxin in the erythrocyte membrane

Heller-Bettinger I, Hoogstraten B (1978) Phase I evaluation of aguidine (diacetoxyscirpenol

Predictive and mechanistic evaluation of toxic responses in mammalian cell culture systems

Diseases of Swine

A toxin from Pasteurella multocida serogroup D enhances swine herpesvirus 1 replication/lethality in vitro and in vivo

Mycotoxins: a review of biological effects and their role in human diseases

Morphological and toxicological studies on experimental T-2 mycotoxicosis

Subacute toxicity of dietary T2 toxin in mice: Morphological and hematological effects

Subacute toxicity of dietary T-2 toxin in mice: Influence of protein nutrition

Mycotoxicosis caused by either T-2 toxin or diacetoxyscirpenol in the diet of broiler chickens

Unequivocal evidence of Soviet toxin use

Proliferative and cytotoxic effects of Fusarium T2 toxin on cultured human fetal esophagus

Ribelin WE (1972) Identification of T-2 toxin in mouldy corn associated with lethal toxicosis in cattle

A proposal for deepoxydation and reepoxydation cycle of epoxy-trichothecene mycotoxin T-2 toxin

Immune studies with T2 toxin: Effects of feeding and withdrawal in monkeys

Alimentary toxic aleukia

Organ culture of rabbit small intestine: prolonged in vitro steady state proteinsynthesis and secretion and secretory IgA secretion

Penetration of {3H}T-2 toxin through excised human and guinea-pig skin during exposure to {3H}T-2 toxin absorbed to corn dust

Response of cattle to mycotoxins of Fusarium tricinctum, isolated from corn and fescue

Feed refusal factors in pure cultures of Fusarium roseum "graminareum

Comparative pathologic changes in trichothecene toxicosis

Current Veterinary Therapy-Food Animal Practice

Induction of DNA single-strand breaks by T-2 toxin, a trichothecene metabolite of Fusarium

Systemic distribution of blood flow during T-2 toxin induced shock in swine

Alimentary toxic aleukia (Septic angina, Endemic Panmyelotoxicosis. Alimentary hemorrhagic aleukia)

The role ofT-2 toxin in experimental alimentary toxic aleukia: A toxicity study in cats

Toxic effects on trout, rats and mice of T-2 toxin produced by the fungus Fusarium tricinctum (Cd) Synd ed Hans

Acute toxicity of T2 mycotoxin to the guinea pig by inhalation and subcutaneous routes

Scanning cytophotometric analysis of brain neuronal nuclear chromatin changes in acute T-2 toxin-treated rats

The enhancing effect of T-2 toxin on delayed hypersensitivity in mice

Toxicological approaches to the metabolites of fusaria. XII. Fate and distribution of T-2 toxin in mice

Mycotoxins in Human and Animal Health

Differential inhibition by T-2 toxin of total protein, DNA and isoprenoid synthesis in the culture macrophage cell line J 774

Protective action of zinc aganst pyrrolizidine alkaloid-induced hepatotoxicity in rats

Effect of trichothecene mycotoxins on farm animals

Analysis for Fusarium toxins in various samples implicated in biological warfare in Southeast Asia

Occurrence of trichothecenes in samples from Southeast Asia associated with "yellow rain

Effects ofT-2 mycotoxin ingestion on phagocytosis of Aspergillus fumigatus Fresenius conidia by rabbit alveolar macrophages and on hematologic, serum biochemical and pathologic changes in rabbits

The toxicological evaluation of the mycotoxins T-2 and T-2 tetraol using normal human fibroblasts in vitro

Immunological disorders

Effect of T2 mycotoxin on rat liver mitochondria electron transport system

Fate and distribution of tritium-labeled T-2 mycotoxin in guinea pigs

Effect on laying geese of feeds containing the fusariotoxins T2 and F2

The toxicity of T-2 toxin in swine following topical application. II. Effects on hematology, serum biochemistry and immune response

Experimental T-2 toxicosis in swine III. Morphologic changes following intravascular administration of T-2 toxin

The toxicity of T-2 toxin in swine following topical application. I. Clinical signs, pathology and residue concentrations

Differential agar-diffusion bioassay for cytotoxic substances

The identification of T-2 toxin and its association with haemorrhagic syndrome in cattle

Experimental mycotoxicoses in calves with aflatoxin, ochratoxin, rubratoxin and T-2 toxin

Effect of aflatoxin on rat liver lysosomes

Action of the mycotoxin of Fusarium sporotrichieUa v sporotrichioides on lysosomal membranes

Effect of T-2 toxin on adrenoco~ical function and immune response in growing pigs

Isolation and propagation of a human enteric coronavirus

Comparison of effects of dietary T-2 toxin on growth, immunogenic organs, antibody formation and pathologic changes in turkeys and chickens

Presence of four Fusarium mycotoxins and synthetic material in "yellow rain

Chemical warfare: Some events of the past year and their implications

Transmission of T-2 toxin into bovine and porcine milk

Distribution of tritiumqabeled T-2 toxin in swine

Inhibitory effect of Fusarium T-2 toxin on lymphoid DNA and protein synthesis

Immunosuppressive activity of Fusarium toxins: effects of antibody synthesis and skin grafts of crude extracts, T-2 toxin and diacetoxyscirpenol

Trichothecene metabolism studies: Isolation and structure determination of 15-acetyl-3a-(l'b-D-gtucopyranosiduronyl)-scirpen-3,4b,15-trioh

Effect of feeding T-2 toxin to rats and monkeys

The effects of PGF2a, PGE2 and 16,16 dimethyl PGE2 on gastric emptying and small intestinal transit in rat

Radiomimetic biological properties of the new scirpene metabolites of Fusarium nivale

Induction of differention in human myeloid leukemic cells by T-2 toxin and other trichothecenes

Canine jejunal absorption and transit during interdigestive and digestive motor stats

Toxicological approaches to toxic metabolites of Fusaria. VII. Acute and subacute toxicities of T-2 toxin in cats

Systemic effects of topical application of T2-toxin in mice

Pathology of acute 3-acetyldeoxynivalenol toxicity in mice

Lesions induced in rodents by extracts from cultures of Fusarium poae and F sporotrichioides

Cardiovascular lesions and various tumors found in rats given T-2 toxin, a trichothecene metabolite of Fusarium

Concerning the natural transplantation of maternal leukocytes in milk

Trichothecene induced hemolysis. The hemolytic activity of T-2 toxin

T-2 toxemia and brain prostaglandins

Effect of T-2 toxin on regional blood flow and vascular resistance in the conscious rat

Enzymatic basis for active transport of Na + and K § across cell membrane

Effects on laying hens of feeding corn invaded by two species of Fusarium and pure T-2 mycotoxin

Effects of feeding T-2 toxin on RNA, DNA and protein contents of liver and intestinal mucosa of rats

Trichothecene toxicoses and natural occurrence in Hungary

The target organella of trichothecenes in rodents and poultry. In: Ueno Y (ed) Trichothecenes: Chemical, Biological and Toxicological Aspects. Kodansha LTD

Ottbein ES eds 10th Ed

Adrenal cortical necrosis caused by T-2 mycotoxicosis in female but not male mice

Elevation of thiobarbituric acid values in the rat liver intoxicated by T2 toxin

Effects of aflatoxin on some marker enzymes of lysosomes

Toxicological features of T-2 toxin and related trichothecenes

Inhibition of protein and DNA synthesis in Ehrlich ascites tumour by Nivalenol, a toxic principle of Fusarium nivale on growing rice

Inactivation of some thiol-enzymes by trichothecene mycotoxins from Fusarium species

Comparative toxicology of trichothecene mycotoxins: Inhibition of protein synthesis in animal cells

Nineteen mycotoxins tested on chicken embryos

Natural occurrence in North America

Identification of various T-2 toxin metabolites in chicken excreta and tissues

The cell kinetics of the adaptation of the human esophagus to organ culture

The effect of Fusariurn toxins on food-producing animals

Acute and chronic toxicity of T-2 mycotoxin in swine

Diacetoxyscirpenol toxicity in pigs

Acute toxicity of the mycotosin diacetoxyscirpenol in swine

Effect of T-2 toxin on porcine reproduction

Mycotoxin-induced abortions in swine

The failure of purified T-2 mycotoxin to produce hemorrhaging in dairy cattle

Preparation and long-term cultivation of porcine tracheal and lung organ cultures by alternate exposure to gaseous and liquid medium phases

Swine intestinal explants as a model for the study of viruses and microbial toxins

Toxin enhanced porcine herpesvirus 1 replication in respiratory/gastrointestinal epithelial cells

Organ culture of piglet intestinal explants: Growth of viruses, incorporation or methyl-3H-thymidine, and uptake of fluorescein isothiocyanate-labelled colostral cells

Blood pressure changes and cardiovascular lesions found in rats given T-2 toxin, a trichothecene secondary metabolite of certain Fusarium microfungi

Neural disturbances in chickens caused by dietary T-2 toxin

Screening of toxic isolates of Fusarium poae and F sporotrichioides involved in causing alimentary toxic aleukia

Cerebral anoxia: Effect on transcription and translation

The effects of T-2 toxin on human platelets

T-2 toxin effect on rat aorta: Cellular changes in vivo and growth of smooth muscle cells in vitro

T-2 toxin effect on bacterial infection and leukocyte functions

Metabolic fate of T-2 toxin in a lactating cow

In vivo metabolism of T-2 toxin, a trichothecene mycotoxin

3'-hydroxy T-2 and 3'-hydroxy HT-2 toxins: New metabolites of T-2 toxin, a trichothecene mycotoxin, in animals

T-2 metabolites in the excreta of broiler chickens administered 3H-labeled T-2 toxin

LPS escape from T-2 mycotoxin damaged intestine is insufficient to account for T-2 toxin enhanced resistance to listeriosis

Acknowledgment. The author thanks Drs. John L. Richard, W.Michael Peden, and Loren H. Peterson for their suggestions during the preparation of this manuscript.

Ring explants of porcine duodenum, jejunum, and ileum have been reported to take up methyl-3H thymidine over a four-day incubation period (Williams