fumonisin which belongs to the mycotoxins family was initially derived from the culture medium of fusarium verticillioides in south africa in 1988 ( 1 ) . fumonisin b1 ( fb1 ) , b2 and b3 are the most abundant types of fumonisins among which the b1 type can be found in maize , rice , sorghum , wheat and soybean ( 2 ) . fumonisins are absorbed through digestive system and affect organs at cellular level ( 3 ) . the biological effects of fumonisins have been studied in the past 20 years and have shown their toxic and carcinogenic side effects ( 4 , 5 ) . their most important role is contribution to sphingolipid synthesis , sphinganine and sphingosine aggregation and thus causing defective membrane duties ( 3 ) . they have also other cellular effects such as cellular growth regulation , differentiation , apoptosis and cell morphology changes ( 3 , 6 ) . on the other hand , repeated accumulation of free cellular sphingoid bases acts as cancer promoter and mutative factor ( 3 ) . a correlation between fumonisin contaminated maize ( > 100 mg / kg diet ) and the esophageal cancer has been found ( 7 ) . in vitro , fumonisins ( 1 to 10 g ) may activate apoptosis , inhibit proliferation and increase inflammatory cytokine production ( 8 , 9 ) . in vivo studies have also shown that fumonisins can cause intestinal villi atrophy and absorption disorder ( 10 ) . administration of fb1 in a dose of 8 to 10 mg / kg in pig s diet , changes cytokine profiles and reduces special antibody responses ( 10 , 11 ) . in addition , long - term consumption of fumonisin may also change intestinal cell morphology such as goblet cell reduction , cause atrophy and decrease mucin secretion ( 12 ) . previous studies have shown a strong correlation between gastric atrophy , and incidence of the gastric and esophageal cancers ( 13 - 16 ) . epidemiologic studies in some areas of south africa , japan , china and iran have shown the high incidence of the esophageal cancer with the fb1 contaminated maize high consumption compared to low risk regions of esophageal cancer that had a lower intake of the fb1 contaminated maize ( 13 , 17 - 20 ) . previous ecological studies similarly suggested positive correlation between fumonisin contamination of cereals and the risk of cancers ( 17 , 21 ) . some epidemiological studies have also shown a correlation between consumption of fb1 contaminated foods and the risk of esophageal cancer ( 7 , 18 ) . due to high incidence of esophageal cancer in developing countries including iran , and the existence of toxic fumonisin in their grains ( 20 ) , this hypothesis supports that fumonisin can act as a carcinogenic factor in high incidence rate of gastric and esophageal cancer in developing countries . thus , the objective of the present study is to detect apoptotic and proliferative activity of mouse gastric mucosa following administration of fb1 through histo - pathological and immunohistochemical methods . fumonisin b1 ( a gift by prof khosravi ar from tehran university , iran ) was mixed with rat chow purchased from pars animal feed co iran . first , the animal food was completely ground ( sk 100 comfort gubelisen , retsch , germany ) . fb1 was mixed with ground pellet for 30 min and then tap water was added to the mixture . the prepared dough was then shaped into strips and remade into pellet in a special pellet - making device . the pellets were finally put for 24 to 72 hr into an oven set at 75c to make it dry and ready to use . female mice ( 25 to 30 g ) were maintained in animal quarters under standardized conditions with a 12 hr light / dark cycle , 20 to 22c ambient temperature and 40 to 50% humidity with free access to rat chow and water . all experimental procedures were performed according to the guidelines of the animal and human ethical committee of tehran university of medical sciences . twenty - nine female mice divided into the treatment ( n=15 ) and control ( n=14 ) groups . animals in the treatment group received fb1 ( 150 mg / kg diet ) for 16 weeks ( 22 ) . mice were weekly weighed and euthanized at the end of 16 weeks . they were dissected and their stomachs were removed . in all animals , the body of stomach was sampled , fixed in 10% formaldehyde , passaged and embedded in paraffin . apoptotic changes were evaluated by immunohistochemistry using terminal deoxynucleotidyl transferase dutp nick - end labeling ( tunel ) , bcl2 and bax staining . for evaluation of proliferative activity of gastric mucosa ki-67 staining were used . gastric atrophy was allocated using grading criteria modeled on the updated sydney system ( 23 , 24 ) as follows : ( 0 ) no parietal cell loss ; ( i ) mild parietal cell loss , up to 25% ; ( ii ) moderate parietal cell loss , 25 to 75% and ( iii ) severe parietal cell loss , more than 75% . gastric glandular and mucosal heights were also measured using graticule linear method as previously described ( 25 ) . briefly , the sections were treated with 3% ( v / v ) h2o2 at room temperature , blocked with 10% ( v / v ) goat or rabbit serum ( nichirei , tokyo , japan ) , and incubated with a rabbit monoclonal antibody against ki-67 ( thermo fisher scientific inc . then , the sections were incubated with biotinylated goat anti - rabbit igg ( nichirei ) . the sections were incubated with a solution of streptavidin - conjugated horseradish peroxidase ( nichirei ) according to the manufacturer s recommen - dations . the sections were counterstained with hematoxylin before dehydration and mounting ( 26 ) . in each animal , mitotic indexes were measured in 10 to 15 gastric glands in transverse sections . at first , the ki-67 positive cells were counted and divided into ki-67 positive and non - positive cells . the obtained figure was then multiplied by 100 and reported . in situ detection of cells with dna strand breaks was performed in formalin - fixed , paraffin - embedded tissue sections by tunel according to the manufacturer s instructions ( roche , germany ) . paraffin sections of 5 m thick were deparaffinized , and endogenous peroxidase was blocked by immersing the sections in phosphate - buffered saline ( pbs ) containing 0.5% h2o2 for 15 min . for proteins nuclei stripping , tissue sections were incubated with proteinase k ( roche , germany ) ( 20 g / ml ) for 10 min . the sections were incubated with a mixture of terminal deoxynucleotidyl transferase ( tdt ) and reaction buffer containing digoxigenin - labeled dutp . hematoxylin staining was performed followed by incubation with anti - digoxigenin antibody conjugated with peroxidase . positive cells containing the fragmented nuclear chromatin which is a characteristic of apoptosis have brown - stained nuclei . in each animal , 2 to 3 slides and in each slide , 5 microscopic fields were studied . tunel - positive cell number in each field was divided by apoptotic + non apoptotic cells and expressed as percentage ( 27 ) . immunohistochemistry was carried out on 5 m tissue sections from formalin - fixed paraffin blocks using the avidin - biotin immunoperoxidase method . sections were stained by monoclonal mouse / rabbit bcl-2 , bax oncoprotein ( dako corporation , usa ) using a k0673 lsab2 detection system ( dako corporation , usa ) according to the manufacturer s instructions . briefly , the paraffin sections were deparaffinized with xylene and rehydrated through a series of descending graded ethanol solutions . endogenous peroxidase activity was blocked by incubation for 15 min in 0.3% h2o2 buffer . to unmask the epitopes of bcl-2 and bax , a microwave processing pretreatment was carried out in a citrate buffer , ph = 6.0 for 10 min . a biotinylated secondary antibody and an avidin - biotin complex with horseradish peroxidase were applied , followed by addition of the chromogen . finally , slides were counterstained with hematoxylin and observed with a light microscope . in each animal , 2 to 3 slides and in each slide , 5 microscopic fields were studied . bcl2 and bax positive cell number in each field were divided by apoptotic+non apoptotic cells and expressed as percentage ( 28 ) . student s t - test was used to find the differences between tunel , bax - and - bcl-2 labeled positive cell number and atrophy . fumonisin b1 ( a gift by prof khosravi ar from tehran university , iran ) was mixed with rat chow purchased from pars animal feed co iran . first , the animal food was completely ground ( sk 100 comfort gubelisen , retsch , germany ) . fb1 was mixed with ground pellet for 30 min and then tap water was added to the mixture . the prepared dough was then shaped into strips and remade into pellet in a special pellet - making device . the pellets were finally put for 24 to 72 hr into an oven set at 75c to make it dry and ready to use . female mice ( 25 to 30 g ) were maintained in animal quarters under standardized conditions with a 12 hr light / dark cycle , 20 to 22c ambient temperature and 40 to 50% humidity with free access to rat chow and water . all experimental procedures were performed according to the guidelines of the animal and human ethical committee of tehran university of medical sciences . twenty - nine female mice divided into the treatment ( n=15 ) and control ( n=14 ) groups . animals in the treatment group received fb1 ( 150 mg / kg diet ) for 16 weeks ( 22 ) . mice were weekly weighed and euthanized at the end of 16 weeks . they were dissected and their stomachs were removed . in all animals , the body of stomach was sampled , fixed in 10% formaldehyde , passaged and embedded in paraffin . apoptotic changes were evaluated by immunohistochemistry using terminal deoxynucleotidyl transferase dutp nick - end labeling ( tunel ) , bcl2 and bax staining . for evaluation of proliferative activity of gastric mucosa ki-67 staining were used . gastric atrophy was allocated using grading criteria modeled on the updated sydney system ( 23 , 24 ) as follows : ( 0 ) no parietal cell loss ; ( i ) mild parietal cell loss , up to 25% ; ( ii ) moderate parietal cell loss , 25 to 75% and ( iii ) severe parietal cell loss , more than 75% . gastric glandular and mucosal heights were also measured using graticule linear method as previously described ( 25 ) . briefly , the sections were treated with 3% ( v / v ) h2o2 at room temperature , blocked with 10% ( v / v ) goat or rabbit serum ( nichirei , tokyo , japan ) , and incubated with a rabbit monoclonal antibody against ki-67 ( thermo fisher scientific inc . , san jose , ca ) diluted 1:200 . then , the sections were incubated with biotinylated goat anti - rabbit igg ( nichirei ) . the sections were incubated with a solution of streptavidin - conjugated horseradish peroxidase ( nichirei ) according to the manufacturer s recommen - dations . the sections were counterstained with hematoxylin before dehydration and mounting ( 26 ) . in each animal , mitotic indexes were measured in 10 to 15 gastric glands in transverse sections . at first , the ki-67 positive cells were counted and divided into ki-67 positive and non - positive cells . in situ detection of cells with dna strand breaks was performed in formalin - fixed , paraffin - embedded tissue sections by tunel according to the manufacturer s instructions ( roche , germany ) . paraffin sections of 5 m thick were deparaffinized , and endogenous peroxidase was blocked by immersing the sections in phosphate - buffered saline ( pbs ) containing 0.5% h2o2 for 15 min . for proteins nuclei stripping , tissue sections were incubated with proteinase k ( roche , germany ) ( 20 g / ml ) for 10 min . the sections were incubated with a mixture of terminal deoxynucleotidyl transferase ( tdt ) and reaction buffer containing digoxigenin - labeled dutp . hematoxylin staining was performed followed by incubation with anti - digoxigenin antibody conjugated with peroxidase . positive cells containing the fragmented nuclear chromatin which is a characteristic of apoptosis have brown - stained nuclei . in each animal , 2 to 3 slides and in each slide , 5 microscopic fields were studied . tunel - positive cell number in each field was divided by apoptotic + non apoptotic cells and expressed as percentage ( 27 ) . immunohistochemistry was carried out on 5 m tissue sections from formalin - fixed paraffin blocks using the avidin - biotin immunoperoxidase method . sections were stained by monoclonal mouse / rabbit bcl-2 , bax oncoprotein ( dako corporation , usa ) using a k0673 lsab2 detection system ( dako corporation , usa ) according to the manufacturer s instructions . briefly , the paraffin sections were deparaffinized with xylene and rehydrated through a series of descending graded ethanol solutions . endogenous peroxidase activity was blocked by incubation for 15 min in 0.3% h2o2 buffer . to unmask the epitopes of bcl-2 and bax , a microwave processing pretreatment was carried out in a citrate buffer , ph = 6.0 for 10 min . a biotinylated secondary antibody and an avidin - biotin complex with horseradish peroxidase were applied , followed by addition of the chromogen . finally , slides were counterstained with hematoxylin and observed with a light microscope . in each animal , 2 to 3 slides and in each slide bcl2 and bax positive cell number in each field were divided by apoptotic+non apoptotic cells and expressed as percentage ( 28 ) . student s t - test was used to find the differences between tunel , bax - and - bcl-2 labeled positive cell number and atrophy . out of 30 mice , one died due to unknown reason three weeks after starting nutrition in control group . both in treated and control groups , the weight has been increased but not significantly . body weight rose steadily to reach a plateau after about 8 weeks in all mice , but showed a slight fall in the treated group in the last few weeks . there were no significantly differences in food intake ( g / day ) in the control and treatment groups . fumonisin b1 fed animals demonstrated no evidence of weight loss , and no gross organ changes were observed at necropsy . no behavioral changes were observed in the mice during the course of administration , or in the ensuing follow - up period . microscopic findings of the gastric mucosa in the treated animals revealed a mild to moderate gastric tissue atrophy ( p<0.05 ) . parietal cells significantly decreased in the treatment group ( 120.3 ) compared with the control ( 241.3 ) ( p<0.05 ) ( figures 1 and 2 ) . in addition , treatment with fb1 for 16 weeks , significantly reduced both gastric mucosa height ( 345.0068.04  vs. 512.8586.93  ) and mitotic index in gastric glands ( 43.2216.67 % vs. 72.6710.50 % ) ( p<0.05 ) ( table 1 ) . in both groups , the mitotic cells observing in basal part of gastric glands , indicated stem cells activity ( figure 2 ) . light photomicrograph of mice gastric gland ( a ) in control group , normal parietal cells present in the mucosa and ( b ) in treatment group ; a few parietal cells present in the final microscopy ( h & e , 400 ) light photomicrograph from mice gastric mucosa . note to reduce mitotic index and height in gastric mucosa ( gm ) in fumonisin treated animal ( b & b ) in compared to control ( a & a ) . bar : 50 . magnifications in a & b : 100  and in a & b : 400  scatter tunel positive cells were observed in controls among the gastric glands ( figure 3 ) . the percentage of tunel - positive cells in fb1 treated group were 1.560.21 vs. 0.560.11 , and it was significantly higher than controls ( table 1 ) . bar : 50 . magnifications in a & b : 200  and in a & b : 400  in control animals , the bcl-2 positive cells were mostly observed in surface and neck mucosal cells of the gastric glands . immunoreactivity with bcl-2 in control group was 8.900.33 , which was higher ( p<0.05 ) than the same cells in treated group ( figure 4 and table 1 ) . however , bax immunoreactivity in gastric mucosa was statistically higher in the fb1 group than the control ( p<0.05 ) ( figure 5 ) . treatment with fb1 caused a decrease in bcl-2/bax ratio compared to the control group ( p<0.05 ) ( table 1 ) . bar : 50 . magnifications in a & b : 200  and in a & b : 400  light photomicrograph from mice gastric mucosa . bar : 50 . magnifications in a & b : 200  and in a & b : 400 out of 30 mice , one died due to unknown reason three weeks after starting nutrition in control group . both in treated and control groups , the weight has been increased but not significantly . body weight rose steadily to reach a plateau after about 8 weeks in all mice , but showed a slight fall in the treated group in the last few weeks . there were no significantly differences in food intake ( g / day ) in the control and treatment groups . fumonisin b1 fed animals demonstrated no evidence of weight loss , and no gross organ changes were observed at necropsy . no behavioral changes were observed in the mice during the course of administration , or in the ensuing follow - up period . microscopic findings of the gastric mucosa in the treated animals revealed a mild to moderate gastric tissue atrophy ( p<0.05 ) . parietal cells significantly decreased in the treatment group ( 120.3 ) compared with the control ( 241.3 ) ( p<0.05 ) ( figures 1 and 2 ) . in addition , treatment with fb1 for 16 weeks , significantly reduced both gastric mucosa height ( 345.0068.04  vs. 512.8586.93  ) and mitotic index in gastric glands ( 43.2216.67 % vs. 72.6710.50 % ) ( p<0.05 ) ( table 1 ) . in both groups , the mitotic cells observing in basal part of gastric glands , indicated stem cells activity ( figure 2 ) . light photomicrograph of mice gastric gland ( a ) in control group , normal parietal cells present in the mucosa and ( b ) in treatment group ; a few parietal cells present in the final microscopy ( h & e , 400 ) light photomicrograph from mice gastric mucosa . note to reduce mitotic index and height in gastric mucosa ( gm ) in fumonisin treated animal ( b & b ) in compared to control ( a & a ) . bar : 50 . magnifications in a & b : 100  and in a & b : 400  scatter tunel positive cells were observed in controls among the gastric glands ( figure 3 ) . the percentage of tunel - positive cells in fb1 treated group were 1.560.21 vs. 0.560.11 , and it was significantly higher than controls ( table 1 ) . bar : 50 . magnifications in a & b : 200  and in a & b : 400  in control animals , the bcl-2 positive cells were mostly observed in surface and neck mucosal cells of the gastric glands . immunoreactivity with bcl-2 in control group was 8.900.33 , which was higher ( p<0.05 ) than the same cells in treated group ( figure 4 and table 1 ) . however , bax immunoreactivity in gastric mucosa was statistically higher in the fb1 group than the control ( p<0.05 ) ( figure 5 ) . treatment with fb1 caused a decrease in bcl-2/bax ratio compared to the control group ( p<0.05 ) ( table 1 ) . bar : 50 . magnifications in a & b : 200  and in a & b : 400  light photomicrograph from mice gastric mucosa . bar : 50 . magnifications in a & b : 200  and in a & b : 400 in this study , fb1 significantly reduced parietal cell number together with gastric body glandular cell height and caused pronounced atrophy . this study showed that administration of fumonisin b1 for 16 consecutive weeks in mouse induces atrophy in gastric mucosa partly through increasing apoptosis and decreasing proliferative activity of the cells . effects of fumonisin b1 on apoptosis and proliferative activity of cells in mouse gastric mucosa data are presented as mean  sem . fb1 : fumonisin b1 previous studies have shown that apoptosis is the early effect of fumonisin on target organs ( 29 , 30 ) . fb1 may be considered as the first clear example of a non - genotoxic substance that can give rise to a tumor through different ways including apoptotic necrosis and atrophy ( 30 ) . there are two possibilities ; one is that the dead cells are not replaced and atrophy occurs , and the other is alteration in cell regeneration or proliferative activity of gastric basal cells . in previous study , we showed the changes of cell viability in a dose dependent manner of fb1 . this toxin caused a decrease in the viability of gastric and intestinal cells ( 31 , 32 ) . in the present study , we showed that orally administration of fb1 for 16 weeks can induce apoptotic markers including increasing of bax and fragmentation of dna mouse gastric mucosa/ , and can also alter balance between bcl-2 and bax proteins in gastric epithelial cells . previous studies have suggested a correlation between gastric atrophy , apoptosis and alterations in the balance of bcl-2 and bax expression ( 33 , 34 ) . similarly , an increased bax expression and apoptosis but decreased bcl-2 expression is shown in chronic atrophic gastritis ( 33 , 34 ) . bcl-2 is an antiapoptotic gene family member ; while bax is a member of proapoptotic genes ( 35 ) . thus , these results suggest that an altered bcl-2/bax ratio may account for the atrophy - associated apoptosis in these mice models . on the other hand , fb1 has been identified as an inhibitor of ceramide enzyme , leading to significant alterations in sphingolipid metabolism , which is a key enzyme in sphingolipid metabolism , and various sphingolipids production involved in apoptosis ( 6 , 36 ) . conceivably , chain of events linking fb1-induced ceramide synthase inhibition , disrupted sphingolipid metabolism and apoptosis induction can be theoretically drawn , raising the issue of apoptosis role in gastric atrophy . on the other hand , previous studies have shown a strong relationship between the gastric atrophy and the onset of gastric and esophagus cancers ( 13 - 16 ) . in addition , there is strong board of evidence proving that nitrosamines are esophageal cancer initiators , and mycotoxins such as fb1 can play a key role in the development of esophageal cancer ( 37 ) . nitrosamines from stomach origin can reach the mucus of esophagus through the gastric - esophageal blood vessel networks . then , they can be converted to different active carcinogenic substances by p450 cytochrome isoforms existing in the mucus of esophagus ( 39 ) . therefore , endogenous nitrosamines produced by mycotoxins may etiologically play a role in esophageal and gastric cancer occurrence . however , further studies are required to understand the possible roles of fb1 in the signaling pathways . this study showed that the administration of fb1 for 16 consecutive weeks in mice can induce gastric mucosal atrophy partly through increasing apoptosis and decreasing proliferative activity of the cells .
objective(s):fumonisins are a group of toxic and carcinogenic mycotoxins , which contaminate the grains and their products . 
 the aim of this study was to examine the apoptotic and proliferative activity of mouse gastric mucosa following administration of fumonisin b1 ( fb1).materials and methods : twenty - nine female mice divided into treatment ( n=15 ) and control ( n=14 ) groups . 
 the treatment group received fb1 ( 150 mg / kg diet ) for 16 weeks . 
 the gastric atrophy was allocated using grading criteria modeled on the updated sydney system . 
 immunohistochemistry studies were performed for evaluation of apoptosis and proliferative activity in gastric mucosa.results:mild to moderate gastric atrophy were observed in microscopic findings of the gastric mucosa in treated animals ( p<0.05 ) . 
 number of parietal cells significantly decreased in the treatment group in comparison with the control ( p<0.05 ) . 
 treatment with fb1 for 16 weeks significantly reduced both gastric mucosa height and mitotic index in the gastric glands ( p<0.05 ) . 
 tunel- and bax - labeled positive cell numbers significantly increased in the fb1-treated group compared to the control ( p<0.05 ) . 
 in addition , proliferative activity of gastric glands in the treated group was significantly lower than the control ( p<0.05).conclusion : oral administration of fb1 caused atrophy in gastric mucosa both via increasing of apoptosis and suppressing the mitotic activity of these cells .