deoxynivalenol ( don ) is a mycotoxin produced by fusarium spp . and a major contaminant of wheat and other grains in many countries around the world . pigs are the animal most sensitive to don , followed by rodents , dogs , cats , poultry , and ruminants . animals fed low to moderate doses of don show a reduction in feed consumption and lower weight gain ; higher doses induce vomiting , increased salivation , and malaise . don cytotoxicity is mainly caused by its inhibition of protein synthesis via binding to the ribosome . leukocytes are central targets of don , and high - dose don exposure promotes leukocyte apoptosis , which were confirmed in mouse and in vitrostudies . the hepatotoxic effect of don was reported in cell culture experiments with primary hepatocytes of rats , pigs   and humans , but apoptosis of hepatocytes induced by don has not been reported in animal experiments . in addition , in vivo pig studies on the hepatotoxic potential of don using naturally contaminated feedstuffs have produced inconsistent results . therefore , pure don administration experiments are needed for direct demonstration of the hepatotoxic effect of don , as naturally contaminated feedstuffs can contain several kinds of mycotoxins other than don . it was also reported that the effect of a pure don - spiked diet on pigs was milder than naturally contaminated feed containing the same level of don , suggesting the presence of additional known and/or unidentified toxic compounds in naturally contaminated grain . acute trichothecene poisoning in humans and animals has been characterized as a multisystem shock - like syndrome , with clinical signs that include dermal irritation , nausea , vomiting , diarrhea , hemorrhage , and hematological lesions such as leukopenia and anemia , some of which could be mediated by cytokines . trichothecene exposure affects the immune function by initiating a rapid and transient upregulation of proinflammatory cytokines including interleukin ( il)-1 , il-6 , tumor necrosis factor ( tnf)- , interferon ( ifn)- and il-2 in mice . cytokines play a critical role in the regulation of immune responses ; however , information about cytokine modulation by don in pigs is very limited and has only been reported after prolonged exposure to don . the aims of this study were : 1 . to demonstrate in vivo hepatotoxicity of don in pigs , compared to immunotoxicity , with special attention to apoptosis . 2 . to evaluate the effect of don on the modulation of cytokine genes in lymphoid organs of acutely exposed pigs nine healthy , 1-month - old lwd ( triple crossbreed of landrace , large white and duroc ) piglets ( 8~10 kg ) from an spf farm ( chiba prefecture , japan ) were used for the experiments . six pigs were intravenously injected with don ( wako , japan ) into the jugular vein at the concentration of 1 mg / kg body weight in 0.9% physiological saline ( total volume 10 ml ) , which corresponds to the concentration that previously induced apoptosis of hepatocytes in vitro . three don pigs were necropsied at 6 h post - injection ( pi ) and the remaining 3 don pigs and 3 control pigs at 24 h pi after euthanization ( exsanguinated under anesthesia ) . a complete necropsy was performed , and collected tissue samples were fixed in 10% phosphate - buffered formalin . the tissues were routinely embedded in paraffin , and 3~4 m thick sections stained with hematoxylin and eosin ( h&e ) were prepared for histopathological examination . all animal experiments were carried out under the approval of the animal ethics committee of the national institute of animal health . tdt - mediated dutp - biotin nick end - labeling ( tunel ) method was performed on the liver , spleen , mesenteric lymph node , thymus , and ileum using a commercial kit for tunel ( apoptag kit ; chemicon , usa ) according to the manufacturer 's instructions . for immunohistochemical examination , sections of the liver , spleen , mesenteric lymph node , thymus , and ileum were deparaffinized , and endogenous peroxidase activity was blocked by 3% h2o2 in methanol at room temperature for 30 min . proteolytic digestion for single - stranded dna ( ssdna ) detection was performed by 0.1% actinase e ( kaken pharmaceutical , japan)-pbs at 37 for 10 min . for detection of cleaved caspase-3 , sections were autoclaved at 120 for 10 min in 1 mm edta solution ( ph 8.0 ) . sections were incubated with anti - ssdna antibody ( dako japan , japan ) diluted 1 : 200 or with anti - cleaved caspase-3 antibody ( cell signaling technology , usa ) diluted 1 : 100 at 4 overnight . next , all sections were incubated with histofine simple stain max - po(r ) ( nichirei , japan ) at room temperature for 30 min , using diaminobenzidine as the chromogen for 5 min , then counterstained with hematoxylin . for the morphometrical analysis , the total number of tunel - positive cells in the thymus ( cortex ) , ileum ( peyer 's patches ) , and liver were counted in 5 fields each section displayed randomly on the monitor at high - power view . the mean values and standard deviations were calculated from three pigs each in the don-6 h , don-24 h and control groups , and assessed for statistical difference by dunnett 's method . small pieces of tissue samples were collected from the spleen , thymus and mesenteric lymph nodes at the necropsy and stored in rnalater ( ambion , usa ) at -20. total rna from the samples was isolated using trizol reagent ( invitrogen , usa ) according to the manufacturer 's manual . extracted rna was resuspended in 20 l ultra - pure water . expressions of il-1 , il-6 , il-18 and tnf- mrna were determined by semi - quantitative rt - pcr as described previously . briefly , 1 g of rna isolated from each organ was reverse transcribed using oligo ( dt)15 primers ( roche diagnostics , germany ) and an rna pcr kit ( amv ) ver.2.1 ( takara , japan ) , essentially according to the manufacturer 's protocol . the cdna equivalent to 25 ng total rna was subjected to pcr for il-1 , il-6 , il-18 , tnf- and -actin . electrophoresis band intensities of the pcr products were quantified using light capture type ae-6962 and a cs analyzer ( atto , japan ) . mean il-1 , il-6 , il-18 and tnf- mrna expression levels normalized against -actin levels from each tissue were presented in arbitrary units . mean values and standard deviations were calculated from three pigs each in the don- 6h pi , don-24h pi and control groups , and analyzed for statistical difference by dunnett 's method . nine healthy , 1-month - old lwd ( triple crossbreed of landrace , large white and duroc ) piglets ( 8~10 kg ) from an spf farm ( chiba prefecture , japan ) were used for the experiments . six pigs were intravenously injected with don ( wako , japan ) into the jugular vein at the concentration of 1 mg / kg body weight in 0.9% physiological saline ( total volume 10 ml ) , which corresponds to the concentration that previously induced apoptosis of hepatocytes in vitro . three don pigs were necropsied at 6 h post - injection ( pi ) and the remaining 3 don pigs and 3 control pigs at 24 h pi after euthanization ( exsanguinated under anesthesia ) . a complete necropsy was performed , and collected tissue samples were fixed in 10% phosphate - buffered formalin . the tissues were routinely embedded in paraffin , and 3~4 m thick sections stained with hematoxylin and eosin ( h&e ) were prepared for histopathological examination . all animal experiments were carried out under the approval of the animal ethics committee of the national institute of animal health . tdt - mediated dutp - biotin nick end - labeling ( tunel ) method was performed on the liver , spleen , mesenteric lymph node , thymus , and ileum using a commercial kit for tunel ( apoptag kit ; chemicon , usa ) according to the manufacturer 's instructions . for immunohistochemical examination , sections of the liver , spleen , mesenteric lymph node , thymus , and ileum were deparaffinized , and endogenous peroxidase activity was blocked by 3% h2o2 in methanol at room temperature for 30 min . proteolytic digestion for single - stranded dna ( ssdna ) detection was performed by 0.1% actinase e ( kaken pharmaceutical , japan)-pbs at 37 for 10 min . for detection of cleaved caspase-3 , sections were autoclaved at 120 for 10 min in 1 mm edta solution ( ph 8.0 ) . sections were incubated with anti - ssdna antibody ( dako japan , japan ) diluted 1 : 200 or with anti - cleaved caspase-3 antibody ( cell signaling technology , usa ) diluted 1 : 100 at 4 overnight . next , all sections were incubated with histofine simple stain max - po(r ) ( nichirei , japan ) at room temperature for 30 min , using diaminobenzidine as the chromogen for 5 min , then counterstained with hematoxylin . for the morphometrical analysis , the total number of tunel - positive cells in the thymus ( cortex ) , ileum ( peyer 's patches ) , and liver were counted in 5 fields each section displayed randomly on the monitor at high - power view . the mean values and standard deviations were calculated from three pigs each in the don-6 h , don-24 h and control groups , and assessed for statistical difference by dunnett 's method . small pieces of tissue samples were collected from the spleen , thymus and mesenteric lymph nodes at the necropsy and stored in rnalater ( ambion , usa ) at -20. total rna from the samples was isolated using trizol reagent ( invitrogen , usa ) according to the manufacturer 's manual . expressions of il-1 , il-6 , il-18 and tnf- mrna were determined by semi - quantitative rt - pcr as described previously . briefly , 1 g of rna isolated from each organ was reverse transcribed using oligo ( dt)15 primers ( roche diagnostics , germany ) and an rna pcr kit ( amv ) ver.2.1 ( takara , japan ) , essentially according to the manufacturer 's protocol . the cdna equivalent to 25 ng total rna was subjected to pcr for il-1 , il-6 , il-18 , tnf- and -actin . electrophoresis band intensities of the pcr products were quantified using light capture type ae-6962 and a cs analyzer ( atto , japan ) . mean il-1 , il-6 , il-18 and tnf- mrna expression levels normalized against -actin levels from each tissue were presented in arbitrary units . mean values and standard deviations were calculated from three pigs each in the don- 6h pi , don-24h pi and control groups , and analyzed for statistical difference by dunnett 's method . all of the don - injected pigs showed hypersalivation from approximately 5 min pi , vomiting from 10 min pi , diarrhea from around 1 h pi , and the pigs were depressed and anorectic . the salivation , vomiting , and diarrhea disappeared at around 3 h pi , but depression and anorexia continued . the pigs appeared to have recovered from clinical symptoms of the disease at 24 h pi . the common necropsy findings in don - injected pigs ( 6 and 24 h pi ) were mucosal hemorrhage from the fundus to the pylorus of the stomach . common findings in don - injected pigs were systemic apoptosis of lymphocytes in lymphoid tissues including the spleen ( data not shown ) , tonsil ( data not shown ) , lymph nodes ( data not shown ) , thymus , and peyer 's patches of the ileum . apoptotic lymphocytes had chromatin - condensed nuclei , often with chromatin margination to the nuclear membrane . apoptotic changes of lymphocytes were observed at both 6 and 24 h pi , but were more severe at 24 h pi ( fig . 1 ) . increased apoptosis was also seen in the bone marrow and infiltrated / resident lymphocytes in the epithelia , lamina propria , and submucosa of the trachea and gastrointestinal tract ( data not shown ) . in the liver , activation of kupffer cells and dilation of sinusoids were observed , together with apoptotic hepatocytes exfoliated from hepatic cords , apoptotic bodies of hepatocytes ( acidophilic bodies ) , and acidophilic material in the sinusoids in don - injected pigs ( fig . these lesions were conspicuous at 6 h pi , but were mild at 24 h pi . in addition to these findings , periacinar degeneration to necrosis was occasionally observed in the liver at 6 h ( 1/3 ) and 24 h pi ( 2/3 ) in don - injected pigs . the most commonly observed stomach lesions in don - injected pigs were hemorrhage and necrosis of the mucous membrane , principally in the fundic to pyloric area . the tunel method and immunohistochemical staining against ssdna and caspase-3 revealed apoptosis of lymphocytes in the spleen , mesenteric lymph nodes , thymus and ileum , and hepatocytes in the liver . a positive reaction was seen in the nuclei of apoptotic cells with the tunel method and anti - ssdna immunostaining , but in the cytoplasm with anti - caspase-3 immunostaining ( fig . there were significantly greater numbers of tunel - positive cells in the lymphoid tissues and the liver of don - injected pigs compared to the controls ( fig . the number of tunel - positive cells in the thymus and peyer 's patches of the ileum increased further at 24 h pi compared to those at 6 h pi ( figs . , the number of tunel - positive cells peaked in the liver at 6 h pi ( fig . a statistically significant increase of il-1 mrna at 6 h pi and decrease of il-18 mrna at 24 h pi were observed in the spleen by rt - pcr ( fig . 4a ) . increased expression of il-6 at 6 h pi was also observed without significant difference . the mrna expression for il-1 and il-6 increased significantly at 6 h pi in the thymus . a decrease of tnf- mrna was observed at 6 h pi in the mesenteric lymph nodes . until now , don - induced apoptosis of hepatocytes has only been reported in vitro . here , this phenomenon was confirmed to exist in vivo as well . although distortion of hepatic cords with degenerative to necrotic changes of individual hepatocytes were observed at 3 and 24 h following intraperitoneal administration of don ( 10 mg / kg body weight ) in rats , those findings could be related to apoptosis . the number of tunel - positive cells in piglet livers peaked at 6 h pi and was reduced at 24 h pi . this is in agreement with the study of ihara et al . , which showed that hepatocyte apoptosis in mice induced by t-2 toxin , a trichothecene mycotoxin , was observed at 2 h pi , but not at 12 h pi . these findings may reflect the fact that kupffer cells in the liver swiftly and efficiently remove apoptotic cells . immunohistochemical staining using anti - cleaved caspase-3 antibody is an easy and reliable method for the detection and qualification of apoptotic cells . don - induced apoptosis in porcine hepatocytes was demonstrated to occur through the caspase-3 activation pathway . our immunohistochemical results support that finding in the liver of the don - injected pig as well . it is speculated that p38 and erk act as molecular rheostats in don - induced apoptosis , that activation of both erk and p38 leads to the expression of immune genes and antiapoptotic pathways , and that only p38 activation initiates the apoptotic response involving caspase-3 activation . it was reported that don concentrations in liver tissue after intravenous administration ( 1 mg / kg body weight ) in pigs was 1,114.3  153.9 ng / g tissue at 0.33 h pi . although we did not determine the concentration of don in the organs , these observation suggested the don concentration of the liver may have reached the same level as in our previous in vitro study , in which we observed apoptosis of hepatocytes macroscopically at the concentration of 1 g / ml . induction of lymphocytic apoptosis by don has been previously reported in mice , but has not been clearly demonstrated in pigs . in this study , we confirmed that don induced apoptosis of lymphocytes in pigs . coppock et al .   reported mild lympholysis of the mesenteric lymph nodes in intravenously don - injected ( 0.5 mg / kg ) pigs , and lymphocytic depletion and atrophy of lymphoid organs were reported in long - term swine feeding experiments [ 6 - 8 ] . it was suggested that don can suppress host resistance to listeria , salmonella   and enteric reovirus   in mice . the same predisposition may be also a possible outcome in pigs exposed to a high level of don because extensive apoptosis of lymphocytes is considered to be a manifestation of immunosuppression . more detailed study is needed to elucidate the impact of don on the porcine immune system , as don can be either immunostimulatory or immunosuppressive in mice , depending on the dose , exposure frequency , and timing of functional immune assays . we also found hemorrhagic lesions in the fundic area of the stomach in don - injected pigs . " hemorrhagic gastritis " is often seen in pigs with acute septicemia , and the severe congestion of mucous membranes in this condition is attributed to venous infarction from endotoxemia . our findings could be associated with this condition , although venous thrombi were not conspicuous in h&e stain section of don - injected pigs . the transient elevation of il-1 and il-6 mrna at 6 h pi was in accordance with the previous mice studies . it was reported that mrna levels for il-1 , il-6 , tnf- , ifn- and il-2 increased rapidly in spleens of mice exposed orally to 5 or 25 mg / kg body weight don . no elevation of tnf- mrna expression was observed in the spleen of don - inoculated pigs in our study . detected tnf- mrna elevation at 3 h pi in mice . since tnf- is the most acutely responsive cytokine , the peak of this cytokine concentration in the serum was at 1 h pi in lipopolysaccharide ( lps)-inoculated pigs . our preliminary data indicates the elevation of tnf- mrna up to 2 times greater than control at 3 h pi ( data not shown ) . elevated cytokine mrna returned to the normal levels after 24 h pi in our study , in agreement with the results from don - exposed mice , showing that the maximum duration for the elevation of cytokine mrna was 4 to 8 h . in another experiment , mice were orally exposed to 12.5 mg / kg body weight don and splenic tnf- , il-1 and il-6 mrna were found to peak at 3 h pi and remained significantly elevated at 6 h pi , but not at 9 h pi . the mrna expression of ifn- , il-4 , il-6 and transforming growth factor- were not affected in the spleen and ileum of pigs fed a naturally don - contaminated diet ( 2.2 - 2.5 mg / kg ) for 9 weeks . the differences from our results are considered to be due to the exposure dose and duration . a high dose of don induce " shock - like death " in mice ; our results suggest that the " cytokine storm " which was observed in lps - inoculated pigs   may be induced in acutely don - exposed pigs as well . the decrease of il-18 mrna at 24 h pi and the decrease of tnf- at 6 h pi were observed only in the spleen and mesenteric lymph nodes , respectively . we did not determine serum cytokine levels in this study , but mrna expression in the spleen may reflect a systemic reaction , as the expression of cytokine mrna in the spleen has been reported to correlate well with the cytokine concentrations in the serum . the use of pigs for immunotoxicological studies is a chance to extend the knowledge gained from rodent animal models . one problem in our experiment was the rather high inter - individual variation of mrna expression of cytokines as observed by rt - pcr . an experimental design using line - bred miniature pigs may eliminate the influence of inter - individual variation . in this study , we showed the apoptosis of hepatocytes in vivo , indicating the hepatotoxic potential of don in pigs , as well as its immunotoxic effect on the modulation of proinflammatory cytokine genes in lymphoid organs with extensive apoptosis of lymphocytes induced by acute exposure to don .
six 1-month - old piglets were intravenously injected with deoxynivalenol ( don ) at the concentration of 1 mg / kg body weight , with three pigs each necropsied at 6 and 24 h post - injection ( pi ) for investigation of hepatotoxicity and immunotoxicity with special attention to apoptotic changes and cytokine mrna expression . 
 histopathological examination of the don - injected pigs revealed systemic apoptosis of lymphocytes in lymphoid tissues and hepatocytes . 
 apoptosis of lymphocytes and hepatocytes was confirmed by the tdt - mediated dutp - biotin nick end - labeling ( tunel ) method and immunohistochemical staining against single - stranded dna and cleaved caspase-3 . 
 the number of tunel - positive cells in the thymus and peyer 's patches of the ileum was increased at 24 h pi compared to 6 h pi , but the peak was at 6 h pi in the liver . 
 the mrna expression of interleukin ( il)-1 , il-6 , il-18 , and tumor necrosis factor ( tnf)- in the spleen , thymus and mesenteric lymph nodes were determined by semi - quantitative rt - pcr , and elevated expression of il-1 mrna at 6 h pi and a decrease of il-18 mrna at 24 h pi were observed in the spleen . il-1 and 
 il-6 mrna expressions increased significantly at 6 h pi in the thymus , but tnf- decreased at 6 h pi in the mesenteric lymph nodes . 
 these results show the apoptosis of hepatocytes suggesting the hepatotoxic potential of don , in addition to an immunotoxic effect on the modulation of proinflammatory cytokine genes in lymphoid organs with extensive apoptosis of lymphocytes induced by acute exposure to don in pigs .