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P.F. Surai and J.E. Dvorska 93

93

Effects of mycotoxins on antioxidantsystems

A delicate balance between antioxidants andpro-oxidants in the body in general andspecifically in the cell is responsible forregulation of various metabolic pathwaysleading to maintenance of immuno-competence, growth and development andprotection against stress conditions associatedwith commercial poultry production (Surai andDvorska, 2001). This balance can be regulatedby dietary antioxidants, including vitamin E

(Surai et al., 1999), carotenoids (Surai andSpeake, 1998; Surai et al., 2001) andselenium (Se) (Surai, 2000). On the otherhand, nutritional stress factors have a negativeimpact on this antioxidant/pro-oxidantbalance. In this respect mycotoxins areconsidered to be among the most importantfeed-borne stress factors.

It is not clear at present whethermycotoxins stimulate lipid peroxidationdirectly by enhancing free radical productionor if the increased tissue susceptibility to lipidperoxidation is a result of a compromised

antioxidant system. It seems likely that bothprocesses are at work. In most cases lipidperoxidation in tissues caused by mycotoxinswas associated with decreased concentrationsof natural antioxidants. For example, in anexperiment with quail, levels of the primaryliver antioxidants ( tocopherol, tocopherol, carotenoids and ascorbic acid)were significantly decreased as a result of T-

2 toxin consumption (Dvorska and Surai,2001; Figure 1).

Similarly, the presence of T-2 toxin in thediet decreased the concentration of tocopherol in the chicken liver (Hoehler andMarquardt, 1996). T-2 toxin consistentlydepressed concentrations of vitamin E inchicken plasma (Coffin and Combs, 1981).Addition of micelle-promoting compounds(taurocholic, monoolein, and oleic acids)

alleviated depression in plasma vitamin E,indicating interference of T-2 toxin withmicelle formation during vitamin Eabsorption. Similarly, aflatoxin B

1(AFB

1) in

the feed interfered with the accumulation ofcarotenoids in chicken tissues (Schaeffer etal., 1988) inducing pale bird syndrome inbirds. In fact, AFB

1 caused a significant

depression of lutein in the toe web, liver,serum and mucosa (Schaeffer et al.,1988a).Pigment restoration was accomplished byfeeding the same diet supplemented withlutein (70 mg/kg). In young chickens AFB

1

reduced the lutein content of jejunal mucosaup to 35% while serum lutein was reducedup to 70% (Tyczkowski and Hamilton, 1987),suggesting that AFB

1 interfered with the

absorbtion, transport and deposition ofcarotenoids. More precisely, AFB

1impaired

lutein absorption in chickens (Tyczkowskiand Hamilton, 1987a). In similar fashion,ochratoxin A (OTA) was shown to affect

5

EFFECTS OF MYCOTOXINS ON ANTIOXIDANT STATUSAND IMMUNITY

1*Peter F. Surai and 2Julia. E. Dvorska1Avian Science Research Centre, Scottish Agricultural College, Auchincruive, Ayr,Scotland, UK and 2Sumy National Agrarian University, Sumy, Ukraine


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94 Effects of mycotoxins on antioxidant status and immunity

carotenoid assimilation in chickens. Again,depression in uptake of carotenoids byintestinal mucosa and depressed transport inserum were considered to be importantmechanisms of AFB

1 action on carotenoid

metabolism (Schaeffer et al.,1988; Huff andHamilton, 1975).

In general, malabsorption syndrome isconsidered a common result ofmycotoxicoses. For example, aflatoxicosis,ochratoxicosis and T-2 toxicosis wereproduced by feeding diets containing gradedconcentrations of the appropriate toxin tobroiler chickens from hatching until 3 weeksof age (Osborne et al., 1982). In thisexperiment AFB

1, even at levels lower than

needed for growth inhibition, produced amalabsorption syndrome characterized bysteatorrhea, hypocarotenoidemia, anddecreased concentrations of bile salts andpancreatic enzymes. T-2 toxin also producedmalabsorption, but at concentrations higherthan required to inhibit growth. Ochra-toxicosis produced mainly hypocarote-noidemia (Osborne et al., 1982). It ispostulated that the decreased level of vitaminA in the quail liver as a result of T-2 toxinconsumption (Dvorska and Surai, 2001) is also

a reflection of the decreased intestinalabsorption of fat soluble nutrients.

The presence of OTA in the dietsignificantly decreased the concentration oftocopherol in the chicken liver (Hoehlerand Marquardt, 1996). Furthermore, aflatoxin-treated barrows had decreased serumtocopherol and retinol concentrationscompared with control and pre-test values,and decreased tocopherol concentration incardiac tissue (Harvey et al., 1994).Aurofusarin decreased vitamin E andcarotenoid concentration in quail egg yolk(Dvorska et al., 2001a), in the liver, yolk sacmembrane and other tissues of newly hatchedquail (Dvorska et al.,2002; Dvorska et al.,2003; Dvorska and Surai, 2004). It isinteresting to note that the carotenoid andvitamin A concentrations in the quail liverwere also decreased when the maternal dietwas supplemented with aurofusarin.

A pro-oxidant effect of mycotoxins inmany cases could be mediated throughchanges in reduced glutathione (GSH)concentration. For example, Rizzo et al.(1994) demonstrated that T-2 toxin decreasedGSH in rat liver. Treatment of fasted micewith a single dose of T-2 toxin (1.8 or 2.8

0

5

10

15

20

25

Control T-2 T-2+Zeolite T-2+Mycosorb

-tocopherol -tocopherol

carotenoids ascorbate

g/g

tissue

Figure 1. Effect of T-2 toxin with and without supplemental toxin adsorbents on antioxidant concentrations in

quail liver (adapted from Dvorska and Surai, 2001).


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mg/kg BW) by oral gavage led to a markeddecrease in hepatic GSH (Atroshi et al.,1997).In male broiler chicks, hepatic GSHconcentration decreased after 7 days oftreatment (1.5 mg T-2 toxin/kg BW/day) (Lealet al.,1999). Acute exposure of mice to T-2toxin (4 mg/kg, s.c.) resulted in a progressivedecrease in hepatic GSH, reaching a minimum6-8 hrs after toxin administration (Fricke andJorge, 1991). Intraperitoneal administrationof AFB

1to rats (2 mg/kg) was also associated

with decreased GSH in the liver. In contrast,in 3-week-old male chickens daily aflatoxin

gavage (2 mg/kg BW, in corn oil) for 5 and10 days elevated hepatic GSH; and renal GSHwas elevated after 10 days (Beers et al.,1992a). Similarly, hepatic GSH increased 2and 8 hrs following a single AFB

1dose and

continued to increase through five daily dosesof AFB

1(Beers et al.,1992b).

There was GSH depletion in cultured rathepatocytes as a result of AFB

1toxicosis (Liu

et al.,1999). Similarly, consumption of OTAfor two weeks was associated with a depletionof GSH from the mouse liver (Atroshi et al.,2000). The mycotoxin patulin also decreased

GSH concentration in rat hepatocytes (Busbeeet al.,1999). Considering that glutathione isresponsible for the maintenance of redoxstatus of the cell (Sies, 1999) and thereforeparticipates in regulation of gene expression(Arrigo, 1999), changes in GSH status couldbe detrimental.

One of the most important mycotoxinactions is their effect on antioxidant enzymes.Depending on experimental conditions(species, doses, route and duration ofadministration, concentrations of otherantioxidants etc.), antioxidant enzyme

activities can be increased in response tooxidative stress or decreased by direct orindirect action of mycotoxins. For example,treatment of pig kidney cells with 50 mMfumonisin B

1 (FB

1) for 24 hrs significantly

decreased cellular GSH and increased theactivities of glutathione reductase (Kang andAlexander, 1996). The activities ofglutathione peroxidase (GSH-Px), catalase,

and Cu/Zn-superoxide dismutase (SOD) werenot changed by this treatment. Oraladministration of T-2 mycotoxin to rats (1.25mg/kg) for 5 days decreased the activity ofliver glutathione-S-transferase (Ahmed andRam, 1986). In contrast, feeding a single doseof T-2 toxin (2 mg/kg BW) to rats increasedactivities of GSH-shuttle enzymes includingGSH-Px, glutathione reductase and glucose-6-phosphate dehydrogenase (Suneja et al.,1989), probably reflecting an adaptiveresponse to oxidative stress. On the otherhand, when male rats were given a diet

deficient in vitamins C and E and Se and wereadministered orally a single dose ofdeoxynivalenol (DON) or T-2 toxin, there wasa significant decrease in activities of GSH-Px, catalase, SOD and glutathione reductase(Rizzo et al., 1994). Activity of GSH-Px inrat blood was decreased due to consumptionof AFB

1(Choi et al.,1995). Administration of

AFB1to rats (2 mg/kg intraperitoneally) caused

a significant decrease in the activities of SOD,catalase, GSH-Px, glutathione-S-transferaseand glutathione reductase in liver (Rastogi etal., 2001). A significant increase in SOD

activity occurred in the liver following AFB1exposure of the ducks (Barraud et al.,2001).

When considering detrimental effects ofmycotoxins on antioxidant systems it isnecessary to be aware that combinations ofseveral mycotoxins can be more toxic thanindividual mycotoxins, as was shown for acombination of T-2 toxin and OTA (Kubenaet al.,1989).

One of the most important targets formycotoxins is embryonic development. Sincechicken embryo tissues contain high levelsof PUFA, they are vulnerable to peroxidation

and oxidative stress caused by mycotoxinscould be lethal. As mentioned previously,aurofusarin increased late mortality of quailembryos (Dvorska et al.,2001a). Furthermore,contamination of the diet with T-2 toxinmarkedly decreased egg production andimpaired hatchability (Tobias et al.,1992).Confirmation of a possible association of thiseffect with oxidative stress came from data

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indicating that increased dietary vitamin Eduring the first week of the experimentsignificantly decreased the number of infertileeggs and significantly improved the hatchingpercentage (Tobias et al.,1992).

Increased lipid peroxidation as a

consequence of mycotoxicoses

As illustrated in Table 1, OTA has astimulating effect on lipid peroxidation. Inmost of cases, accumulation of thiobarbituricacid reactive substances (TBARS) was usedas a measurement of lipid peroxidation.Ethane exhalation, EPR-registered freeradicals, hydroxyl radical formation, single-

strand cleavage DNA, DNA adduct formationas well as LDH release were also used toconfirm pro-oxidant properties of OTA.Various in vitro and in vivo systems werealso used including liver microsomes,phospholipid vesicles, primary cell cultures,whole organ and whole body measurements.

T-2 toxin was also shown to have pro-oxidant properties (Table 2). Those propertieswere confirmed with rat, mouse and quailliver tissue and yeast. TBARS accumulationwas the method used in most of the studies,however, conjugate diene formation and DNA

fragmentation were also demonstrated. Effectof AFB

1 on lipid peroxidation has been

studied in rat liver and kidney as well as incultured hepatocytes and in anin vitromodel

Table 1. Stimulation of lipid peroxidation by ochratoxin A.

Mycotoxin Tissue Lipid peroxidation Protective effect Referencesmeasurement of antioxidants

OTA in vitro Rat liver TBARS - Rahimtula et al.,1988,microsomes Gautier et al.,2001; Khan et

al., 1989

OTA in feed Rats Ethane exhalation - Rahmitula et al.,1988OTA in feed Rats MDA in serum, - Meki and Hussein, 2001liver, kidney

OTA in vitro Phospholipid TBARS , oxygen - Omar et al., 1990vesicles uptake

OTA and its Bacillus brevis Free radical Vitamin E Hoehler et al.,1996analogs bacteria generation (EPR)

OTA in feed Chicken liver TBARS - Hoehler and Marquardt,1996; Hoehler et al.,1997

OTA in vitro Vero cells in TBARS SOD, catalase Baudrimont et al.,1997a;culture aspartame 1997b

OTA in diet Rat liver TBARS - Hoehler et al.,1997OTA in vitro A model oxid- OH, single- - Gillman et al.,1999

ation system strand cleavageDNA

OTA in vitro Astrocytes and TBARS, LDH - Belmadani et al.,1999neurons release

OTA in feed Mouse and rat DNA adduct Retinol, ascorbic Grosse et al.,1997kidney formation acid, vitamin E

OTA in feed Rats DNA adduct Aspartame Creppy et al.,1998formation

OTA in vitro Rat kidney TBARS - Gautier et al.,2001microsomes


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system (Table 3). Similar to the examplesabove, TBARS accumulation was substantiallyincreased as well as conjugate dieneproduction. At the same time GSHconcentration and activities of antioxidantenzymes significantly declined as a result ofAFB

1action.

Fumonisin B1 also stimulated lipid

peroxidation in rat liver, rat liver nucleifraction, primary rat hepatocytes, Vero cellsin culture and phosphatidyl choline (PC)bilayers. In those systems TBARSaccumulation and DNA strand breaks wereincreased (Table 4). DON increased TBARSformation in rat and mouse liver anddecreased GSH in rat brain and spleen. Thereare also data indicating pro-oxidant propertiesof zearalenone (Karagezyan et al., 1995;Ghedira-Chekir et al., 1999) and citrinin(Ribeiro et al.,1997). Aurofusarin has beenshown to decrease antioxidant defences andstimulate lipid peroxidation in quail egg andtissues of newly hatched quail (Dvorska etal.,2002; 2003; Dvorska and Surai, 2004).

It is clear from these data that mycotoxinsstrongly promote lipid peroxidation in variousin vitro and in vivosystems. This effect wasobvious no matter which measurement was

used to assess the process of lipidperoxidation.

Mycotoxins and apoptosis

The maintenance of tissue homeostasisinvolves the removal of superfluous anddamaged cells. This process is often referredto as programmed cell deathor apoptosis,since it is thought that cells activate anintrinsic death program contributing to theirown demise (Sastre et al., 1996). Severalprocesses, such as initiation of death signalsat the plasma membrane, expression of pro-apoptotic oncoproteins, activation of deathproteases, endonucleases etc., ultimatelycoalesce to a common irreversible execution

phase leading to cell demise. A balancebetween cell death and cell survival factorsplays a major role in the decision process asto whether a cell should live or die (Ray etal.,2000).

Apoptosis is distinguishable from necrosis.When cell death is induced by osmotic,physical or chemical damage, early disruptionof external and internal membranes takesplace with subsequent liberation of denatured

Table 2. Stimulation of lipid peroxidation by T-2 toxin.Mycotoxin Tissue Lipid peroxidation Protective effect References

measurement of antioxidants

T-2 in feed Rat liver TBARS Se, ascorbic Tsuchida et al.,1984;acid, vitamin E Suneja et al.,1989;

Schuster et al.,1987; Rizzoet al.,1994

T-2 in feed Rat liver nuclei TBARS - Ahmed and Ram, 1986T-2 in feed Mouse liver TBARS - Karppanen et al.,1989T-2 in feed Mouse MDA in liver after Vila et al.,2002

24-48 hrs aftersupplementation

T-2 in feed Mouse liver DNA CoQ10

and Atroshi et al.,1997

fragmentation vitamin ET-2 in feed Quail liver TBARS - Dvorska and Surai, 2001T-2 in feed Rat tissues Conjugate diene - Chang and Mar, 1988T-2 in feed Chicken Liver MDA Lycopene Leal et al.,1999T-2 in feed Chicken, duck, Liver MDA Mezes et al.,1999

goose

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proteins into the cellular space and inductionof an inflammatory response in the vicinityof the dying cell (Sastre et al., 1996). Incontrast, apoptosis is characterised by cellshrinkage, nuclear pyknosis, chromatincondensation, DNA cleavage into fragmentsof regular sizes and activation of proteasescalled caspases (Dare et al.,2001).

Reactive oxygen species (ROS) arethought to play a major role in apoptosis(Hockenbery et al.,1993; Ratan et al.,1994),being involved in both initiation andexecution of apoptosis (Herdener et al.,2000). GSH depletion increases thepercentage of apoptotic cells in a givenpopulation; and increased GSH concentrationis shown to decrease the percentage ofapoptosis in fibroblasts (Sastre et al.,1996).In fact, GSH depletion sensitises cells forintracellular induction of apoptosis (Zuckerand Bauer, 1997). Therefore, a decrease inGSH, or an increase in GSSG or perhaps a

change in the ratio of the two constitutes atrigger for apoptosis (Beaver and Waring,1995). For example ROS from mitochondriacan cause apoptosis after GSH depletion(Zucker et al.,1997). Therefore, apoptosis isinduced by oxidative damage either directlyfrom oxygen free radicals or hydrogenperoxide or from their generation in cells byinjurious agents. For example, hydrogenperoxide is considered a common mediatorfor the apoptosis induced by variousanticancer drugs (Simizu et al.,1998). In linewith those findings there are data showingprotective effects of catalase and SOD fromdifferent inducers of apoptosis (Sandstrom andButtke, 1993; Herdener et al.,2000). Indeed,intracellular induction of apoptosis dependson ROS production and can be efficientlyblocked by antioxidants (Langer et al.,1996;Schaefer et al.,1995).

As previously mentioned, in many casesmycotoxins decreased cellular GSH, which

Table 3. Stimulation of lipid peroxidation by aflatoxin.Mycotoxin Tissue Lipid peroxidation Protective effect References

measurement of antioxidants

In feed Rat liver TBARS , Se , vitamin E Shen et al.,1994conjugated dienes

In feed Rat liver MDA , NO melatonin - El-Gibaly et al.,2003loaded chitosan

In feed Rat liver MDA , NO melatonin Meki et al.,2001In feed Rat testis MDA Vitamin E Verma and Nair, 2001In vitro Cultured rat TBARS , LDH SOD, catalase Shen et al.,1995

hepatocytes release

In feed Rat liver TBARS Semecarpus Premalatha et al.,anacardiumnut extract 1997

In vitro Cultured TBARS ROS Silvia Liu et al.,1999primary rat formation miltorrhizahepatocytes extract

Intraperitoneally Rat liver TBARS Vitamin E, ternatin Souza et al.,1999In feed Rat liver GSH-Px Se, vitamin E Choi et al.,1995Intraperitoneally Rat liver and TBARS Picroliv Rastogi et al.,2001

kidney

Intraperitoneally Rat liver GSH, SOD, - Rastogi et al.,2001bCatalase, GSH-Px

In vitro Primary rat TBARS , GSH , - Yang et al.,2000hepatocytes ROS generation


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can trigger apoptosis. In general, T-2 toxinis the most potent apoptotic agent among

mycotoxins. However, there are also reportsindicating apoptosis caused by FB

1, OTA and

AFB1. For example, based on the DNA

fragmentation profile in gel electrophoresisand the morphological changes seen inelectron microscopy, the induction ofapoptotic nuclear changes by variousmycotoxins was investigated in HL-60human promyelotic leukemia cells (Ueno et

al.,1995). The results showed that T-2 toxin,nivalenol (NIV), DON, OTA, citrinin, AFB

1

and some other mycotoxins induced DNAfragmentation. Morphological evidence ofapoptosis was related to the toxicity of themycotoxins and more toxic NIV and DONresulted in more late stage apoptotic eventsthan FB

1. The results suggested that DNA

damage and apoptosis rather than plasmamembrane damage and necrosis may beresponsible for the observed cytotoxicity.

Table 4. Stimulation of lipid peroxidation by fumonisin, deoxynivalenol, aurofusarin and citrinin.

Mycotoxin Tissue Lipid peroxidation Protective effect Referencesmeasurement of antioxidants

Fumonisin Rat liver TBARS , DNA Catalase, Sahu et al.,1998nuclei strand breaks mannitol

Fumonisin B1

Primary rat TBARS Vitamin E Abel and Gelderblom, 1998hepatocytes

Fumonisin B1

Rat liver TBARS - Abel and Gelderblom,1998; Lemmer et al.,1999

Fumonisin B1

Phosphati- Rate of - Yin et al.,1998dyl choline peroxidation ,bilayers free radical

Formation ,Acceleration ofchain formation

Fumonisin B1

Vero cells TBARS - Abado-Becognee et al.,1998

Fumonisin B1

Glioma cells, TBARS , DNA - Mobio et al.,2003Mouse fragmentationfibroblasts

Fumonisin B1

Macrophage MDA - Ferrante et al.,2002cell line

DON in feed Rat liver TBARS Se, ascorbic Rizzo et al.,1994acid, vitamin E

DON + Mouse liver TBARS - Karppanen et al.,1989 3-AcDON

in feedAurofusarin Quail egg yolk TBARS - Dvorska et al.,2001b, in feed Dvorska, 2001

Aurofusarin Liver of newly TBARS - Dvorska et al.,2002 in feed hatched quail Dvorska and Surai, 2004

Zearalenone Rat liver TBARS - Karagezyan et al.,1995injected IV

Zearalenone Vero cells TBARS Vitamin E Ghedira-Chekir et al.,1999 in vitro

Citrinin in feed Rat liver TBARS - Ribeiro et al.,1997

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Fumonisin B1, T-2 toxin, fusarenon-X andDON could induce apoptosis of liver cells,kidney cells, gastrointestinal epithelial cellsand immunological cells as well as severalcell lines. The possible mechanisms ofapoptosis induction are not well understood.It seems likely that sphingol, p21 gene,protein kinase and intracellular Ca2+ levelmight be involved in the cellular apoptosisinduction by Fusariummycotoxins (Wang andZhang, 2000).

T-2 TOXIN

The rank order of the potency of trichothecenemycotoxins to induce internucleosomal DNAfragmentation was found to be T-2, satratoxinG, roridin A >> diacetoxyscirpenol >baccharin B-5 >> nivalenol, deoxynivalenol,3-acetyldeoxynivalenol, fusarenon-X, baccharinB-4 vehicle control (Nagase et al.,2001). Theauthors showed that T-2-induced apoptosisinvolved activation of caspase-3 throughcytosolic accumulation of cytochrome c alongwith caspase-9 activation. Both T-2 and HT-2

induced apoptosis after 24 hrs in HL-60 cells,with T-2 being somewhat more potent thanHT-2 and the apoptotic process was almostcompletely blocked in the presence of acaspase inhibitor (Holme et al., 2003).Development of apoptosis and changes inlymphocyte subsets were examined inthymus, mesenteric lymph nodes and Peyerspatches of mice up to 24 hrs after oralinoculation with T-2 toxin (10 mg/kg). Thedegree of lymphocyte apoptosis wasprominent in the thymus, moderate in thePeyers patches, and somewhat mild in the

mesenteric lymph nodes (Nagata et al.,2001). Cytocentrifugation and lightmicroscopy of leukocyte-enriched cellsamples from the pronephros (the primaryhematopoietic compartment in the fish)demonstrated T-2-related increases inapoptotic bodies and the apoptotic probesconfirmed apoptosis in fish (Gogal et al.,2000).

Female mice were treated orally with T-2toxin (10 mg/kg BW) and thymus and spleenwere examined to detect apoptotic changes(Shinozuka et al., 1997). The number ofapoptotic lymphocytes in the thymusincreased dramatically from 9 to 24 hrs aftertreatment and began to increase in the spleenat 12 hrs after treatment indicating thatthymus was more sensitive to apoptosis thanspleen. In another experiment, mice weregiven 5 mg/kg BW of T-2 toxin and werekilled 12 hrs later (Ihara et al.,1997). Massivecellular destruction was observed in the

thymus and spleen and electron microscopyrevealed the presence of apoptotic bodies.Similarly, in the liver of mice given 2.5 mg/kg T-2 toxin and killed 2 hrs later, theinduction of apoptotic cellular lesions wasobserved. Electron microscopic characteristicsof damaged lymphocytes were shrinkage ofthe cell body, nuclear chromatin condensationand fragmentation (Li et al.,1997). Of ninetrichothecene mycotoxins tested, T-2 toxinwas the most potent agent to induce apoptosisin the thymus in young female mice (Islamet al.,1998; 1998a). It is interesting that both

the acetyl group at the C-4 position and theisovaleryl or 3-hydroxyisovaleryl group atthe C-8 position of the T-2 toxin moleculeare important for inducing apoptotic changesin the thymus (Islam et al.,1998). T-2 toxinwas shown to induce apoptosis in thehematopoietic (bone marrow) tissues offemale mice (Shinozuka et al., 1998). Itshould be noted that the gastrointestinal tractis also sensitive to trichothecene-inducedapoptosis, since such changes were observedin the gastric mucosa, gastric glandularepithelium and intestinal crypt cell

epithelium (for review see Bondy and Pestka,2000). These changes could impair nutrientassimilation as well as decrease defencesagainst enteric pathogens and endotoxin.

To examine the effect of T-2 toxin on thedeveloping embryo, pregnant mice weredosed orally with T-2 toxin (3 mg/kg BW) at11 days of gestation (Ishigami et al.,1999).Ultrastructural changes characteristic of


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apoptosis were observed. For example, in somelayers of the central nervous system moderatepyknosis or karyorrhexis was seen. Using DNAfragmentation and fluorescence microscopyassays Yang et al. (2000a) demonstrated thatrank order of trichothecene-mediated apoptosisis similar to their cytotoxicity. Therefore,cytotoxicity of trichothecene mycotoxins ismediated through an apoptotic process. Inparticular, the embryotoxic effect of T-2 toxinon the mouse fetus is mediated throughapoptosis. T-2 toxin (2 mg/kg BW) was givenorally to pregnant mice at various gestational

days and the fetuses were examined 24 hrs later.T-2 toxin-induced apoptosis was detected. SinceT-2 toxin readily crosses the placenta, T-2 toxincould directly cause fetal apoptosis (Ishigamiet al.,2001).

It was shown that in human promyeloticcell line HL-60, T-2 toxin induced apoptosisat 10 ng/ml levels within 2-6 hrs (Ueno etal.,1995). Apoptosis in HL-60 cells inducedby T-2 toxin was dose dependent when cellswere treated with concentrations of 5-100 ng/ml for more than 2 hrs (Yoshino et al.,1996).The authors suggested that the Ca2+ signal

triggered by T-2 toxin is transduced by theactivation of endonuclease and protease, andultimately evokes apoptosis.

Molecular mechanisms of T-2 stimulationof apoptosis are not clear at present.However, recently it has been shown thattrichothecene mycotoxins trigger a ribotoxicstress response that activates the stress-activated kinases c-Jun N-terminal kinase and/or p38 mitogen-activated kinase and inducesapoptosis (Shifrin and Anderson, 1999). Suchactivation may signal cell survival or induceapoptosis in various cell types depending on

the conditions, e.g. length of signal.However, the pathway leading from theactivation of these kinases to the activationof caspases and apoptosis has not beenelucidated and needs further research.Albarenque et al. (2001) suggested that theelevation of TNF-mRNA expression mayplay an important role in T-2 toxin-inducedepidermal cell apoptosis. The induction of c-

fos and perhaps of c-jun mRNAs may beassociated with T-2 toxin-induced epidermalcell apoptosis (Albarenque et al.,2001).

FUMONISIN B1

The effects of FB1on apoptosis in various in

vitro and in vivo studies have receivedsubstantial attention over the last few years.For example, FB

1-treated keratinocytes

developed morphological features consistentwith apoptosis. In particular, they released

nucleosomal DNA fragments into themedium 2-3 days after exposure to 0.1 mMFB

1and showed increased DNA strand breaks

(Tolleson et al., 1996). FB1 induced a

significant and dose-related increase ofmodifications of DNA bases (8-OH-dG) andDNA fragmentation in both C6 glioma andmouse embryonic fibroblasts cells (Mobio etal.,2003). In the same study, apoptotic C6glioma cells were also observed after FB

1

incubation. Recently genes that inhibit FB1-

induced apoptosis in African green monkeykidney fibronlasts (CV-1 cells) and two mouse

embryo fibroblasts have been identified (Joneset al., 2001). Primary mouse embryofibroblasts underwent apoptosis following FB

1

treatment (Ciacci-Zanella et al.,1999). Theauthors also demonstrated that the tumournecrosis factor (TNF) pathway and caspasesplay an important role in FB

1-induced

apoptosis. Effects of exposure of humanfibroblasts to FB

1were investigated. After 72

hrs of treatment, FB1 (50 and 100 mM)

induced DNA damage, an enhancement ofcaspase-3-activity and cleavage of poly(ADP-ribose)polymerase compared to controls

(Galvano et al.,2002).The cytotoxicity and genotoxicity of FB

1

in rabbit kidney RK13 cells were examined.Exposure to FB

1caused a significant increase

in micronucleus frequency in a concentration-and in a time-dependent manner and anincreased number of the cells were dying bythe process of apoptosis (Rumora et al.,2002).The author suggested that nanomolar

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concentrations of FB1 induced apoptosis,which subsequently may proceed tosecondary necrosis. Treatment of monkeykidney cells (CV-1 cells) with FB

1led to cell

cycle arrest and apoptosis (Zhang et al.,2001). It was suggested that the ability ofFB

1 to alter gene expression and signal

transduction pathways may be necessary forits carcinogenic and toxic effects.

In general, FB1 induces apoptosis of

hepatocytes and of proximal tubule epithelialcells. More advanced lesions in both organswere characterised by simultaneous cell loss

(apoptosis and necrosis) and proliferation(Voss et al., 2001). A dose-dependentincrease in TNF-induced apoptosis wasobserved in porcine renal epithelial cellspretreated with FB

1. In particular, cells treated

with FB1 showed increased DNA

fragmentation and terminal uridine nucleotideend labeling in response to TNF-treatment(Johnson et al.,2003). In the same study, FB

1

also increased DNA synthesis and resultedin cell cycle arrest in the G2/M phase of thecell cycle.

FB1toxicity caused induction of cytokine

networks in liver with involvement of theTNF- signaling pathway (Bhandari andSharma, 2002). The authors suggested thatincreased expression of caspase 8 involvedin the TNF- signaling pathway maycontribute to the apoptosis, whereas IL-1Rainduction could contribute to the proliferatingeffects observed in FB

1 toxicity. Similarly,

FB1 caused an activation of the cytokine

network in liver, particularly the TNF-signaling pathway, suggesting its involvementin hepatotoxic mechanisms (Bhandari et al.,2002). Induction of apoptosis was suggested

to be a consequence of ceramide synthaseinhibition and disruption of sphingolipidmetabolism by FB

1(Dragan et al.,2001). It

seems likely that the elevated endogenoussphinganine acts as a contributing factor tothe fumonisin-induced cell death (Yu et al.,2001). Moreover recent results of Mobio etal . (2000) showed that cytotoxicconcentrations of FB

1 induce cellular cycle

arrest in phase G(2)/M in rat C6 glioma cells.Furthermore, under experimental conditions(9 or 18 mM FB

1in the medium) there was

an induced DNA fragmentation and ladderingand many apoptotic bodies in glioma cells.

As mentioned above, FB1 caused

morphological changes (i.e., cell shrinkage,membrane blebbing) and time-dependentincreases in DNA fragmentation characteristicfor apoptosis. For example, Kim et al. (2001)demonstrated that FB

1kills LLC-PK(1) kidney

cells by inducing apoptosis. This includedinitial disruption of sphingolipid metabolism

and accumulation of sphinganine (or ametabolite), which, in turn, inducedexpression of calmodulin. FB

1, but not AFB

1,

induced the apoptosis of swine alveolarmacrophages (AM) with evidence of DNAladdering and nuclear fragmentation.However, both FB

1 and AFB

1 exposure

induced the expression of apoptosis-relatedheat shock protein 72 in AM (Liu et al.,2002).

FB1has been shown to cause apoptosis in

a variety of cell types and tissues, but theapoptotic potential of other fumonisins and

fumonisin metabolites could substantiallyvary. For example, Seefelder et al. (2003)exposed human proximal tubule-derivedcells (IHKE cells) to FB

1, FB

2, FB

3, hydrolyzed

FB1 and N-palmitoyl-hydrolyzed FB

1 and

investigated caspase-3 activation, chromatincondensation and DNA fragmentation. Theresults demonstrated that all compounds ledto increased sphinganine levels in IHKE cells,but only FB

1was able to induce apoptosis. In

the short-term studies, increases in tissueconcentration of the ceramide synthase substratesphinganine and the sphinganine:sphingosine

ratio were correlated with apoptosis (Voss etal.,2002).

Although it has been demonstrated thatFB

1induces apoptosis in many cell lines, the

precise mechanism of apoptosis is not fullyunderstood. For example, FB

1-induced

apoptosis involves the activation of caspase3, which is associated with the repression ofprotein kinase C and possibly its downstream


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effectors, NF-B and TNF- (Gopee et al.,2003).After intravenous injection of male rats

with FB1(1.25 mg/kg), hepatic and renal GSH

concentrations were depressed andproliferation and apoptosis were observed inthe outer medulla of the kidney cell (Lim etal.,1996). When male mice were injectedwith FB

1subcutaneously at doses of 0, 0.25,

0.75, 2.25 and 6.75 mg/kg BW daily for 5days, a dose-dependent increase in apoptosisin liver and kidney was observed (Tsunodaet al., 1998). Similar observations were

described by Sharma et al. (1997). Male micewere injected subcutaneously with vehicleor 2.25 mg/kg/day of FB

1 for 5 days and

sampled 1 day after the last treatment. FB1

increased apoptotic cells in liver (Bhandariet al., 2002). Similarly, histopathologyrevealed the occurrence of apoptosis in theliver of rats exposed to FB

1 (Pozzi et al.,

2001).Multiple daily doses of FB

1after surgery

elevated the number of apoptotic hepatocytesin rats (Li et al.,2000). Including FB

1in the

diets of rats increased hepatocellular and renal

tubule epithelial cell apoptosis (Howard etal., 2001). It is interesting that female ratsdemonstrated more sensitivity than male ratsin the induction of hepatocellular apoptosisand mitosis. Mild toxic effects, includingapoptosis, proliferation of bile duct epithelialcells, and early signs of fibrosis were noticedin the liver of rats fed FB

1(25 mg/FB

1/kg diet;

Gelderblom et al.,2001). Six hours afteradministration of FB

1, marked morphologic

changes of rat hepatocytes included theappearance of small vacuoles along the marginof the cell membrane. Electron microscopic

analysis revealed margination of nuclearchromatin and swollen mitochondria withamorphous matrical deposit (Moon et al.,2000). The authors suggested that FB

1-induced

alteration of hepatocyte membrane lipidcomposition is an early key event in the FB

1

apoptotic effect. Therefore, the induction oftubular epithelial cell apoptosis was theprimary response of the kidneys to dietary

FB1. Apoptosis was present at all doses (99-484 ppm for 28 days) in the kidney of malerats and occurred in females at doses of 153-484 ppm FB

1(Tolleson et al.,1996). Wistar

rats were fed diets containing 0 (control) or100 ppm FB

1 for 12 weeks. Necrosis and

apoptosis of tubular epithelial cells in thekidney were observed and increased mitoticfigures and lymphocytic infiltrate in the smallintestine were found (Theumer et al.,2002).Increased hepatocellular apoptosis was alsodetected in the mice consuming dietscontaining 72 and 143 mmol/kg FM

1(Howard

et al.,2002).FB

1 can induce renal injury and organ

sphingolipid alterations in cattle. Forexample, renal lesions in FB

1-treated calves

consisted of vacuolar change, apoptosis,caryomegaly, and proliferation of proximalrenal tubular cells, as well as dilation ofproximal renal tubules, which containedcellular debris and protein (Mathur et al.,2001). Probably there is species-specificityin susceptibility to FB

1. For example, FB

1was

investigated in four groups of growing ducks,each receiving 0, 5, 15 or 45 mg/kg FB

1by

daily oral administration over 12 days. Nosign of apoptosis was present in the liver orin peripheral blood lymphocytes and onlymoderate oxidative damage was noted (Baillyet al.,2001).

The apoptosis-stimulating effect of FB1has

been clearly demonstrated; howevermolecular mechanisms of this stimulation arenot clear at present. Nevertheless, stimulationof lipid peroxidation by FB

1and decreased

antioxidant concentrations including GSH intissues could lead to changes in redox statusof the cell and trigger a cascade of apoptotic

changes.

OCHRATOXIN A

Lipid peroxidation due to OTA consumptionmay be implicated in DNA damage. When aspecially designed oxidation system was used,OTA was found to facilitate single-strand

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cleavage of supercoiled plasma DNA throughROS production (Gillman et al.,1999). Ingeneral, OTA is implicated in DNA-adductformation (Grosse et al., 1997). OTAtreatment of rats resulted in a 5-fold increasein the expression of the protein haemoxygenase-1 generated during oxidative stress(Gautier et al.,2001). Changes in redox statusof the cell due to the pro-oxidant action ofOTA could trigger cell apoptosis. Forexample, OTA at a dose of 20 mg/kg causednuclear changes characteristic of apoptosis inhamster kidney (HaK) and HeLa cells

(Seegers et al., 1994). Furthermore, theresults indicated that OTA might activatedifferent cellular processes involved in thedegradation of DNA in HaK and HeLa cells.Exposure of human proximal tubule-derivedcells to 30 nmol/L or more OTA led to DNAfragmentation and chromatin condensation(Schwerdt et al.,1999). It was concluded thatexposure to low OTA concentrations can leadto direct or indirect caspase-3 activation andsubsequently to apoptosis in cultured humanproximal tubule cells and in other renalepithelial cell lines of different origins.

Administration of OTA twice a week forone or two weeks resulted in apoptosis inthe liver of mice (Atroshi et al., 2000). Inparticular, the presence of intracellularapoptosis bodies was detected two weeksafter toxin treatment. Light microscopicexamination demonstrated the presence ofeosinophilic globules, often containingapoptotic bodies. They were found within thecytoplasm of intact hepatic cells. The numberof apoptotic bodies was further enhanced attwo weeks, resulting in an 8-fold increase inliver over the control values (Atroshi et al.,

2000). Wistar rats were treated with a lowdose (5 mM and 1 mg/kg, respectively) or ahigh dose (12.5 mM and 10 mg/kg,respectively) of OTA for 24 or 72 hrs. Markedtreatment-specific transcriptional changeswere detected for genes involved in DNAdamage response and apoptosis (Luhe et al.,2003). Because of OTA consumption, therewas a striking increase in the counts of

eosinophils and of apoptotic phagocytes inweaner pigs in a dose-dependent manner(Muller et al.,1999).

Nanomolar concentrations of OTApromote apoptosis in a cell-type specificfashion. For example, OTA, at noncytotoxicdoses, was able to detach collagen- andfibronectin-adherent cells from immobilizedsubstratum causing apoptosis as measured bycaspase-3 activation (Scibelli et al., 2003).Relatively low OTA concentrations (547.2,752.5 and 930.3 ng OTA/g kidney tissue)have activated apoptotic processes and

oxidative damage in kidney cells (Petrik etal.,2003). In human monocyte/macrophageline THP-1 metabolic activity, cellproliferation, cell membrane integrity, celldifferentiation, phagocytic behaviour, NOsynthesis and cell surface markers werelargely suppressed by OTA at concentrationsbetween 10 and 1000 ng/ml (Mulleret al.,2003). OTA greatly induced apoptosis inhuman myc-transfected cell line (Horvath etal.,2002).

Exposure to low OTA concentrations (5-30 nmol/L) can lead to time- and

concentration-dependent direct or indirectcaspase-3 activation and subsequently toapoptosis in cultured human proximal tubulecells and in other renal epithelial cell linesof different origins (Schwerdt et al.,1999).Additional evidence was provided indicatingthat OTA interacts in a cell type-specific waywith distinct members of the mitogen-activated protein kinase family providinginduction of apoptosis via the c-jun aminoterminal kinase pathway (Gekle et al.,2000).

OTHER MYCOTOXINS

When thein vitro effect of deoxynivalenol(DON) on apoptosis in specific T- and B-cellsubsets within thymus, spleen and Peyerspatch cultures was studied (Pestka et al.,1994) it was shown that depending onlymphocyte subset, tissue source andglucocorticoid induction, DON could either


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enhance or inhibit apoptosis. Combinedtreatment of mice with DON andlipopolysaccharide significantly increased theamount of apoptotic thymic and splenic tissue(Zhou et al., 2000). It was suggested thatinhibition of protein synthesis and inductionof apoptosis are the main mechanisms ofDON toxicity in intestinal cells (Maresca etal.,2002).

It seems likely that DON can interact withother inducers of apoptosis to increaseapoptotic action. As demonstrated by DNAfragmentation and flow cytometric analysis,

apoptosis in thymus, Peyers patches, andbone marrow was marked in mice 12 hrs afteradministering Escherichia coliLPS (0.1 mg/kg BW i.p.) concurrently with DON (12.5 mg/kg BW p.o.), whereas apoptosis in controlmice or mice treated with either toxin alonewas minimal (Islam et al.,2002). Therefore,lipopolysaccharide can interact with DON inmice to induce the glucocorticoid-drivenapoptotic loss of immature thymocytes andcytotoxic T-lymphocytes in thymus, mature-B-lymphocytes in Peyers patch, and pro/pre-B-lymphocytes and mature B-lymphocytes in

bone marrow in mice (Islam et al.,2003).Dead lymphocytes in the thymus, spleen

and Peyers patches showed ultrastructuralcharacteristics of apoptosis. DON andaflatoxin G

1 (AFG

1) could induce and

accelerate apoptosis in human peripheralblood lymphocytes. For example, a typicalsub-diploid apoptosis peak was demonstratedin lymphocytes treated with DON and AFG

1

(Wang et al.,1999). In particular, a significantdose response effect and time effectcorrelation was found between apoptosis ratesand mycotoxin concentrations and the treated

time.In an experiment with male rats, AFB

1was

administered i.p. or intratracheally at 2-8 mg/kg BW and animals were killed after 26 hrs(Raj et al.,2001). Dose-dependent inductionof micronuclei in bone marrow and lung cellswas observed. Furthermore, apoptotic bodieswere found in lung cells of rats given AFB

1,

which was correlated with micronucleiinduction. Clearly, AFB

1induced micronuclei

and apoptosis in lung and bone marrow cells.This stimulation could be associated with thestimulation of the phosphatidylinositol (PI)cycle and activation of PI kinase by AFB

1

(Pasupathy et al.,1999). Apoptotic rates inrat liver were significantly reduced whenmelatonin was co-administered with AFB

1(El-

Gibaly et al.,2003). Rats were treated withAFB

1(50 mg/kg BW) for 8 weeks. The levels

of caspase-3 activity in the AFB1group were

significantly higher than in controls and the

apoptosis was associated with degenerativeand necrotic changes in the hepatocytes(Meki et al.,2001a). In the study, caspase-3activity was positively correlated with MDAwhile negatively correlated with GSH, GSH-Px and GSH-reductase in rat livers treatedwith AFB

1. It is interesting that melatonin

treatment of rats could enhance hepaticantioxidant/detoxification system, whichconsequently reduced the apoptotic rate andthe necrobiotic changes in the liver.

It seems likely that apoptosis is theprincipal mechanism contributing to germ

cell depletion and testicular atrophy followingzearalenone (ZEA) exposure. In particular,Kim et al. (2003) demonstrated that a singledose of ZEA (5 mg/kg) induced testiculargerm cell apoptosis in a time-dependent andstage-specific pattern. In another study, ZEA(10, 20 and 40 mM) induced concentration-dependent DNA fragmentation in three celllines (Vero, Caco-2 and DOK) resulting inDNA laddering patterns on agarose gelelectrophoresis. This observation wasconsistent with apoptosis, which wasconfirmed by observations of formation of

apoptotic bodies. Furthermore, ZEA inducedcell cycle arrest in the three cell linescharacterised by an increase in the numberof cells in the G2/M phase of the cell cycle(Abid-Essefi et al.,2003). It is interesting thatvitamin E (25 mM) added simultaneously withZEA partially reduced DNA fragmentation andapoptotic body formation after 24 hrsincubation.

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Mycotoxins and the immune system

All animals protect themselves from invasionby microorganisms, parasites, fungi, viruses andany foreign molecules. This protective capacityrequires an effective immune system, which isan important determinant of animal health andwell being. In that sense, the remarkable abilityof the immune system to distinguish betweenself and non-self is a great achievement ofevolution. Commercial animal production isbased on balanced feed, providing nutrientrequirements and optimised environmental

conditions. However, it is very difficult to avoidvarious nutritional or environmental stresses,which are responsible for immunosuppressionand increased susceptibility to various diseasesand consequently decreased productive andreproductive performance of farm animals. Inthis respect mycotoxins are one of the mostimmunosupressive factors in animal diets.

There are two major types of immunefunction: natural and acquired immunity (Figure2). Natural immunity, called the innate immunesystem, includes physical barriers (e.g. skin,mucus layer of the gastrointestinal tract), specific

molecules such as agglutinins, precipitins, acutephase proteins and lysozyme, phagocyticdestruction of pathogens by macrophages andneutrophils, and lysing activity by lymphocytescalled natural killer (NK) cells.

Macrophages perform a range of functions,including phagocytosis of foreign particles,destruction of bacterial or tumor cells,secretion of prostaglandins and cytokines,and as a result regulating activity oflymphocytes and other macrophages(Qureshi, 1998). In fact, phagocytosis is themajor mechanism by which microbes are

removed from the body and is especiallyimportant for defence against extracellularmicrobes. As a result of stimulation (forexample by microbes) monocytesdifferentiate into macrophages that are morepowerful in mediating host defence (Klasing,1998a).

Macrophage activation and phagocytosisof foreign particles are regularly accompaniedby a respiratory burst, an increase in theproduction of reactive oxygen species (ROS),exerted by the enzyme complex NADPHoxidase. Therefore macrophages as well as

Immunity

Natural (innate) Aquired (adaptive, specific)

Physical

barriers Phagocytes

Specific

moleculeHumoral Cell-mediated

Macrophages Neutrophils

NK cells Mastcells

Dendritic

cells

ROS, RNS,Small peptides

Elcosanoids

Cytokines, Complement

Acute phase proteins

Kill pathogen

B-lymphocytes T-lymphocytes

Immunoglobulins

(antibodies)

Direct contact

with target cells

Pathogen removal and resistance

to pathogens (immunologic memory)

Inflammatory

response

Figure 2. General overview of the immune system.


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other phagocytic leukocytes (e.g.,neutrophils, monocytes and eosinophils) cansynthesize toxic oxygen metabolites such assuperoxide anion (O

2-), hydroxyl radical

(OH), singlet oxygen (1O2), hydrogen

peroxide (H2O

2), nitric oxide (NO) and

peroxinitrite (ONOO-) during the respiratoryburst (Zhao et al., 1998). For example, abacterium coming into contact with theplasma membrane is enclosed in a plasmamembrane vesicle containing NADPHoxidase, and exposed to an intensive flow ofsuperoxide radical (Giller and Sigler, 1995).

Superoxide radical can dismutate to H2O2,which penetrates the bacterium withproduction of hydroxyl radical, which isultimately deadly to biological molecules.In general, the production of ROS andreactive nitrogen species (RNS) is acharacteristic of both mammalian and avianmacrophages (Qureshi et al.,1998a).

In general ROS, RNS and eicosanoids (e.g.leukotrienes and prostaglandins) haverecently received substantial attention asmajor metabolites produced by macrophages(Dietert and Golemboski, 1998). Because of

this powerful weapon, macrophages bind,internalize, and degrade foreign antigens(e.g. bacteria) quite quickly. It takes only 15minutes for chicken macrophages to kill morethan 80% of the internalized Salmonella(Qureshi et al., 1998a). Therefore naturalimmunity works rapidly, and gives rise to theacute inflammatory response. Macrophagescontain various substances (includingenzymes producing free radicals and smallpeptides with antibiotic activity) involved inmicrobial killing. They also have receptorsfor chemoattractive factors released from

microbes. In addition to ROS, RNS andeicosanoids mentioned above, macrophagesalso synthesize and secrete a great numberof cellular communication molecules such ascytokines, including the pro-inflammatorycytokines interleukin 1 (IL-1), interleukin 6(IL-6), and TNF-. They also produce cytokineinhibitors, hormones and neurotransmitters(Klasing, 1998a), which regulate specific

immunity (initiating and directing theimmune and inflammatory responses) andmany other related physiological responses.Therefore macrophages are importantamplifiers of the immune response, both bycytokine production and by serving to presentparasite derived peptides to T-cells.

Acquired or specific immunity consists ofhumoral and cell-mediated immunity. Thereare two major types of lymphocytes, B-cellsand T-cells. Humoral immunity is mediatedby antibodies that are released by B-cells intothe bloodstream. The bursa of Fabricius is the

site of B-lymphocyte development anddifferentiation in birds. This immunity isbased on the production of immunoglobulins.They are responsible for recognition andelimination of specific antigens: they bindand remove from the host invading organisms/substances.

Cell-mediated immunity is based onspecific antigen recognition by thymus-derived T-lymphocytes. Due to this immunitycells infected with a foreign agent, forexample virus, are destroyed via directcontact between an activated T-cell and the

target (infected) cell (Qureshi et al.,1998).Cell-mediated immunity is responsible fordelayed-type hypersensitivity (DTH)reactions, foreign graft rejections, resistanceto many pathogenic microorganisms andtumor immunosurveillance (Wu andMaydani, 1998).

In birds, both T-cell and B-cell precursorsoriginate in the bone marrow. Actualdevelopment of T-cells takes place in thethymus and B-cells develop in the bursa ofFabricius in birds and in bone marrow ofmammals (Lahtala, 1998). Interactions

between T- and B-cells, as well as antigenpresenting cells, are responsible for thedevelopment of specific immunity. Thedefence mechanisms of specific immunity areinduced or stimulated by exposure to foreignsubstances, are specific for distinctmacromolecules and increase in magnitudewith each successive exposure to a particularmacromolecule (Miles and Calder, 1998). In

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comparison to natural immunity, specificimmunity takes longer to develop, but ishighly specific for antigens and has memory.These two parts of the immune system worktogether through direct cell contact andthrough interactions involving such chemicalmediators as cytokines and chemokines.Therefore, the animal immune systemrequires the co-operation of macrophages,bursa-derived B-lymphocytes and thymus-derived T-lymphocytes with various othertypes of cells.

The immune response involves cellular

proliferation (T-lymphocytes), enhancedprotein synthesis (including immunoglobulinsynthesis by B-lymphocytes and acute phaseprotein synthesis in the liver) andinflammatory mediator production.Physiological changes resulting fromstimulation of the immune system includefever, anorexia and loss of tissue components(Grimble, 1997).

When analysing data related toimmunomodulation careful attention must bepaid to methods used to assess immunologicalfunctions. For example, in vivomethods of

immune function assessment are based on twomain approaches: antibody response tovaccine or delayed-type hypersensitivity(DTH) reactions. In the first method,immunisation with appropriate antigens (viralor bacterial) can elicit serum antibodies. Thehaemagglutination (HA) assay measuresserum antibody concentration (titre) againstantigens, which are often sheep red bloodcells (SRBC). This assay provides informationabout humoral immunity (B-cellresponsiveness) and its association with cell-mediated immunity (T-cell co-operation). The

second (DTH) method is used to assess cell-mediated immune function.

In vitro indices of immune functioninclude (Wu and Meydani, 1998):

Lymphocyte proliferation assay. This assayprovides information about cell-mediatedimmune response and consists ofmeasuring the number of cells in culture

with or without addition of a stimulatoryagent (mitogen). In this assay, isolatedlymphocytes are incubated with mitogens,which activate division of either T-or B-lymphocytes. Various mitogens are usedin such assays, but most often they includeconcanavalin A (con A, a T-cell mitogen),phytohemagglutin (PHA, a T-cellmitogen), lipopolysaccharide (LPS, a B-cell mitogen) and pokeweed mitogen(PWM, T- and B-cell mitogen) (Hayek etal., 1996). Decreased proliferation mayindicate impaired cell-mediated

immunity. Cytokine production. T-cells produce a

range of protein mediators calledcytokines, which regulate cell activation,growth, differentiation, inflammation andimmunity.

Cytotoxicity assay. This assay assessesactivity of cytotoxic T-lymphocytes (agroup of T-cells that kill other cells byrecognising their cell-surface antigens)and NK cells (a group of non-T and non-Blymphocytes that kill virus-infected andtumour cells).

Flow cytometric analysis. This assayidentifies cells with different surfacemarkers. The results can be used forunderstanding the cellular basis ofimmune response.

Plaque-forming cell (PFC) test. This showsthe number of antibody-producing cells.

The impact of the avian immune system inmodern production cannot be overestimated.The banning of feed grade antibiotics in Europehas made immunocompetence the major factordetermining efficiency of poultry production.

Molecular immunology is developing veryquickly; and mechanisms and factors affectingavian immunocompetence have recentlyreceived substantial attention (McCorkle, 1998;Saif and Swayne, 1998) and nutritionalmodulation of resistance to infectious diseasesin poultry (Klasing, 1998) is a frontline for futureresearch. It must be emphasised that cellularintegrity is very important for receiving and


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responding to the messages needed to co-ordinate an immune response (Latshaw, 1991).Therefore, the antioxidant/pro-oxidant balanceof the host is a critical consideration in optimalimmune system function.

The clinical toxicologic syndromes causedby ingestion of mycotoxins have beencharacterized in domestic animals, poultryand laboratory animals and range from acutemortality to decreased performance andimmunosuppression. In fact, consumption ofcertain mycotoxins, at levels that do notcause overt clinical mycotoxicosis, suppresses

immune function and decrease resistance toinfectious disease (Corrier, 1991). The effectsof several mycotoxins on immune responseshave been investigated (Tables 5-9);however, most data were obtained withlaboratory animals. In some instances, farmanimals and cells derived from livestockspecies have been employed to evaluate theimmunotoxicity of mycotoxins (Sharma,1993; Bondy and Pestka, 2000).

Immunosupressive potency of variousmycotoxins differs substantially. Effects of DON,3-acetyldeoxynivalenol, fusarenon-X, T-2 toxin,

ZEA, zearalenol, -zearalenol and NIV onT- and B-cells were studied using a proliferationassay and antibody-dependent cellular cytotoxicNK cell activity on human peripheral bloodmononuclear cells (Berek et al., 2001). Themycotoxin concentrations used in theexperiments were comparable to those foundin normal human peripheral blood (0.2-1800ng/ml). Among the mycotoxins tested, T-2toxin, fusarenon-X, NIV and DON showed thehighest immunosuppressing effects in vitro. Inparticular, mycotoxin-induced immuno-suppression was related to depressed T- or

B-lymphocyte activity. Furthermore, they alsoinhibited NK cell activity (Berek et al.,2001).

As can be seem from Table 5,immunosuppression caused by AFB

1has been

demonstrated in various livestock species(e.g., chickens, turkeys, pigs and lambs) andalso in laboratory animals (mice and rats) andin various in vitro systems. Aflatoxin is animmunomodulating agent that acts primarily

on cell-mediated immunity and phagocyticcell function. Therefore, AFB

1 mainly

decreases lymphocyte activity and may affectmacrophages assisting lymphocyte function.For example, in macrophages exposed toAFB

1, NO production stimulated by LPS

significantly decreased (Moon et al.,1998).Furthermore, AFB

1 decreased pro-

inflammatory and increased anti-inflammatory cytokine mRNA expression inweanling piglets (Marin et al.,2002). AFB

1

can be transferred from chicken to the eggand further to the developing embryo.

Therefore, the progeny of hens consumingan AFB

1-contaminated diet may be

increasingly susceptible to disease owing tosuppression of humoral and cellularimmunity. For example, anti-Brucella abortusantibody production was compromised andROS production by macrophages from AFB

1

progeny decreased (Qureshi et al., 1998).Long-term immune depression of macrophage-mediated functions can occur followingembryonic exposure to AFB

1(Neldon-Ortiz and

Qureshi, 1992). Furthermore, depressedcomplement and interferon production could

also result from AFB1 exposure. Therefore,acquired immunity from vaccination programsmay be substantially suppressed; and in suchcases the signs of disease observed are thoseof the infectious process rather than those ofthe aflatoxin that predisposed the animal toinfection. Mixtures of aflatoxin with othermycotoxins can result in greatly augmentedbiological responses in terms of weight gaindepression, lethality, and immune reactivity(Pier, 1992).

The immunomodulatory effects ofochratoxins have also been considered for

many years and a summary is shown in Table6. OTA has been shown to affect mainlyhumoral immune function at the level ofantibody synthesis in chickens, rats and mice.However, number and phagocytic activity ofmacrophages were also decresed in growinggilts receiving OTA for 35 days (Harvey et al.,1992). IL production was also compromised. Ithas been demonstrated that exposure of purified

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Table 5. Effects of aflatoxin B1on immunity.

Species Dose Effects on immune system Reference

Broiler chicks 1 mg/kg, 7-49 days Titres to NK Shivachandra et al.,2003Broiler chicks 2.5 mg/kg diet for Peripheral T-lymphocyte counts , Celik et al.,2000

21 days Splenic plasma cell counts Broiler chicks 2.5 mg/kg diet for Percentage/mean phagocytic Ibrahim et al.,2000

21 days activity ; Immune responsemeasured by HI test

Broiler breeder 0.2; 1; 5 or In progeny, anti-Brucella abortus Qureshi et al.,1998 hens 10 mg/kg titres ; Phagocytosis of SRBC

and ROS production bymacrophages in 5 mg AFB

1/kg

Broiler chicks 5 mg/kg Secondary antibodies Okotie-Eboh et al.,1997against IBD

Broiler chicks 400 g/kg AFB1

Cell-mediated immunity Giambrone et al.,1985a+AFB

2for 5 weeks measured by DTH skin test ;

Humoral immunityunchanged;Acquired immunity to ND orfowl cholera vaccinationunchanged

Broiler chickens 2.5 g/g of feed Total complement activity Stewart et al.,1985for 42 days

Chickens 2.5 g/g diet from Cell-mediated immunity (graft- Giambrone et al.,1978hatching to 4 vs. host reaction) ; DTHweeks of age reactions to tuberculin ;

Humoral immunity (to rabbitRBC) unchanged; Serum IgG and

IgA; IgMunchangedChickens 0.3 mg/kg from Cell mediated immune response Kadian et al.,19880 to 6 weeks (DTH reaction ) suppressed at

30, 45 and 60 days of age

14-day-old 200 g/kg Cell-mediated immunity (DTH skin Giambrone et al.,1985b turkeys and test) ; Humoral immunity

unchanged; Acquired immunity toND or fowl cholera vaccinationunchanged

4-month-old 100-400 g/kg Lymphoblast transformation Quist et al.,2000 wild turkeys feed for 2 wk

Chick embryo 1.09-17.4 g/g Mitotic index B-cells; Mitotic Potchinsky and Bloom,embryo at day 18 index T-cells unchanged; Sister 1993of incubation chromatid exchanges in B-

lymphocytes ; Sister chromatidexchanges in T-lymphocytes

6-day chick 0.1, 0.5, and 1 Macrophages recruited in the Neldon-Ortiz and Qureshi, embryos g/per embryo peritoneal cavity after i.p. 1992

Sephadex elicitation ; Substrateadherence potential of peritonealexudate cells at 1 g AFB

1;

Macrophage phagocytic potential at 0.5-1.0 g AFB

1


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Table 5. Continued.Species Dose Effects on immune system Reference

6-day chick 0.1 g Incidence of sister chromatid Dietert et al.,1985 embryos exchanges in blood cells (5-fold);

Cell-mediated immunity (graft vshost and cutaneous basophilhypersensitivity reactions)

Weanling 140-280 g/kg, AFB1decreased proinflammatory Marin et al.,2002

piglets 4 wks (IL-1, TNF-) and increasedanti-inflammatory (IL-10) cytokinemRNA expression

Weaned pigs 140, or 280 g/kg, Skin thickness (PHA test) van Heugten et al.,1994for 3 wks linearly with dietary AFB

1

Pigs 300 or 500 mg/kg Humoral immune response Panangala et al.,1986of feed to Erysipelothrix rhusiopathiae,measured by ELISA unchanged;Proliferative responses to mitogensunchanged;Complement titers;Serum IgG and M values

Female lambs 2 mg/kg for 37 d Response to intradermal Fernandez et al.,2000injection of PHA

White-tailed 0.8 mg/kg for 8 Lymphocyte proliferation and Quist et al.,1997deer wks DTH reactionsunchangedWeanling rats 60, 300 and Cell mediated immunity, Raisuddin et al.,1993

600 g/kg BW measured by DTH responseassay, at the 300 and 600 gdose levels

Weanling rats 350 and Population and phagocytic Raisuddin et al.,1990700 g/kgBW capacity of macrophages

Rats and mice Aerosol inhalation Alveolar macrophage Jakab et al.,1994or intratracheal phagocytosis at 16.8 g/kginstillation to AFB

1with the effect persisting for~2 weeks

Mice 0.03, 0.145 or [3H]thymidine uptake in Reddy et al.,19870.70 mg/kg orally lymphocyte cultures; Synthesisevery other day for of DNA in mixed lymphocyte ;2 weeks Primary antibody production by

splenic cells from animalschallenged with SRBC; DTHreaction to hemocyanin

Murine 10 or 50 mM NO production stimulated Moon et al.,1998 peritoneal by LPS , mediated by the macrophages reduction of iNOS activity,

mRNA, and protein

Human 0.1-1 pg/ml Phagocytosis and microbicidal Cusumano et al.,1996 monocytes activity , Intrinsic antiviral

activity or superoxideproduction unchanged

Rat Kupffer 0.01 pg/ml Phagocytosis , intracellular Cusumano et al.,1995 cells in vitro killing of Candida albicans ,

intrinsic anti-Herpes virus activity

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Table 6. Effect of Ochratoxin A on immunity.Species Dose Effects on immune system Reference

Broiler chicks 2 mg/kg Vaccination titres to NK , Santin et al.,2002Mitotic cells in the bursa

Chicks 5 mg/kg Weight of lymphoid organs , Stoev et al.,2002Humoral immune response

Broiler chicks 130, 305 and HI titers to ND vaccination Stoev et al.,2000790 mg/kg

Broiler chicks Up to 4 mg/kg for Ig-containing cells in Dwivedi and Burns, 198420 days from hatch lymphoid organs ; Total Ig

levels at 2-4 ppmBroiler chickens 0.5-8 g/g from Total circulating lymphocytes ; Chang et al.,1979

day-old to 3 weeks Monocytes at 2.0 g/g;

of age Circulating heterophils unchangedSalmonella- 3.0 mg/kg PHA- and ConA-stimulated Elissalde et al.,1994 challenged blastogenesis ; S.typhimurium broiler chicks alone had no effect on the

variables measured

Growing gilts 2.5 mg/kg of feed Cutaneous basophil hyper- Harvey et al.,1992for 35 days sensitivity response to PHA ,

DTH to tuberculin , Stimulationindex for lymphoblastogenesis ,Il-2 production when lymphocyteswere stimulated with ConA ,Number and phagocytic activity ofmacrophages

Rats Gavage with 0, Thickening of the basement Dortant et al.,2001

0.07, 0.34 or membrane and reduction in1.68 mg OTA/kg splenic T-cell fraction. IgG atBW for 4 wks 0.34-1.68 mg/kg OTA

Rat Dams exposure to The proliferative response of Thuvander et al.,1996ba single dose of splenocytes to ConA in pupsOTA (0, 10, 50 or from dams given 10 or 50 g250 mg/kg BW) on OTA/kg BW; Proliferation ofday 11 of lactation thymocytes in response to

ConA in pups from damsexposed to 50 g OTA/kg BW

Mice Dams, OTA, In offspring on days 14 and 28 Thuvander et al.,1996200 g/kg before postpartum percentages of splenicand during gestation CD4+ and CD8+ cells

Mice Dams exposure to Proliferation of splenic and Thuvander et al.,1996a

OTA (500 g/kg BW) thymic lymphocytes in responseon day 16 of to mitogens in pups at 15gestation days of age ; percentages of

mature CD4+ cells andpercentages of immature, double-positive (CD4/CD8+) cells inthe exposed pups

Mice Dams exposure to Proliferative responsiveness of Thuvander et al.,1996aOTA (500 g/kg lymphocytes in the offspringBW) on day 10 when stimulated with B- or T-cellpostpartum mitogens 3 days after exposure


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human lymphocyte populations andsubpopulations to the toxin will abrogate the

cells ability to respond to activating stimuli invitro (Lea et al., 1989). Thus, both IL-2production and IL-2 receptor expression ofactivated T-lymphocytes were severelyimpaired. The results strongly suggest that thetoxin caused immunosuppression throughinterference with essential processes in cellmetabolism (Lea et al., 1989). In particular,OTA appears to suppress NK cell activity byinhibiting production of basal interferon. (Lusteret al.,1987).

It is important to note that subchronic oralexposure to OTA affects certain immune

functions in mice at exposure levels that maybe found in contaminated food products(Thuvander et al.,1995). In the mouse modelOTA had a non-selective suppressive effecton various immune reactions. They includeweight depression, lymphopenia, neutrophiliaand eosinophilia. Furthermore, antibody-producing cells, antibody titres in bloodserum and phagocytosis of E. coliby bloodphagocytes become suppressed. Immunizedanimals also showed a lower survival rateafter experimental infection with Pasteurellamultocidaas well as an increase in oxygen

radicals in blood cells (Muller et al.,1995).Similarly, in Salmonella-challenged broilerchicks PHA- and ConA-stimulatedblastogenesis was depressed as a result ofOTA consumption, while Salmonellaalonehad no effect on these parameters (Elissaldeet al.,1994).

In weaner pigs subtoxic amounts of OTAproduced immunomodulation in a dose-dependent mode (Muller et al., 1999). It

included increased counts of total leukocytesand neutrophils in the blood and reduced

lymphocyte levels. There was also asubstantial increase in the counts ofeosinophils and of apoptotic phagocytes.Reduced phagocytosis and reducedexpression of a swine-specific surface markeron lymphocytes were also observed.

It is interesting that the time of exposuresignificantly influences the immunotoxiceffects of OTA on the developing immunesystem in rodents (Thuvander et al.,1996a).Recent studies have examined immunefunction in the offspring of rats and miceexposed to OTA pre- and perinatally (Bondy

and Pestka, 2000). It has been shown thatthe decrease in proliferation and antibodyproduction in young animals resulted fromprenatal modulation of the immune system.In particular, prenatal exposure to relativelylow doses of OTA may induce immuno-suppression in the progeny. In contrast,short-term exposure of suckling pups to OTAvia the milk stimulated the proliferativeresponses of lymphocytes to polyclonalactivation.

Fumonisin toxicity has been characterizedrelatively recently in comparison to aflatoxin

and ochratoxin (Table 7), and fumonisin-induced immunotoxicity is an area of activeresearch (Bondy and Pestka, 2000). In fact,FB

1 has diverse effects on the immune

system, causing both stimulation andsuppression of responses to foreign antigens,and apparently inducing an antigenic responseto FB

1. For example, in chickens FB

1caused

a decrease in total immunoglobulins, in IgGand macrophage phagocytic activity (Qureshi

Table 6. Continued.


Mice 250 or 2600 mg/kg After 28 days, AB production Thuvander et al.,1995diet, 28 days and plaque-forming cells ;

Decrease in thymocyte cellcounts in the 250-g/kggroup; After 90 days, proportionof mature CD4+ or CD8+ cells

Mice 0.005 g/kg BW Immune response to SRBC Haubeck et al.,1981

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Table 7. Effects of Fumonisins on immunity.Species Dose Effects on immune system Reference

White Leghorn 6.1, 10.5, and Splenic, thymic, and liver Qureshi et al.,1995 chicks 42.7 mg/kg from weights ; Bursa of Fabricius

day 7 to 42 unchanged; Total Ig andIgG; Macrophage phagocyticactivity ; NK cell activityunchanged

Turkey poults 75 mg/kg feed and Primary immune response to Weibking et al.,1994AB

1, 200 g/kg SRBC (combination of FB

1and

feed, from day AFB1); PHA responseunchanged

1 to 21

Weaned pigs 1-100 mg/animal/d Titres unchanged; Lymphocyte Tornyos et al.,2003stimulation by PHA, ConA, LPSunchanged

Rat Injected at 7.5 or Thymus weight , Serum Bondy et al.,199510 mg/kg BW/day IgM; Circulating phagocyticfor 4 days cell numbers

Mice 50 (LD), FM1+ After challenge with T. cruzi, Dresden et al.,2002FM

2, 6 weeks NO production by MGafter

14 days

Mice Five daily s.c. Relative spleen and thymus Johnson and Sharma,injections of weights in F , No effect on organ 20012.25 mg/kg/d FB

1weights in M; Splenic cellularityand the basal rate of lymphocyteproliferation in F ; PHA-inducedT-lymphocyte and LPS-inducedB-lymphocyte proliferation in F ;Expression of IL-2 mRNA insplenocytes in F

Mice I.p. with SRBC, Number of plaque-forming cells Martinova et al.,1995at 5-100 g produced against SRBC

Mice I.p daily, at 1 to 4 to 12-fold increase in the Martinova et al.,199550 g number of of plaque-forming

cells after SRBC injection

Macrophage 1-10 M Membrane fluidity ; Membrane Ferrante et al.,2002 cell line peroxidative damage

Murine 1-100 M LPS-induced production of NO Meli et al.,2000 macrophage and PGE

2at 0.1-10 M

cell line

Murine 1, 10, and 100 M LPS-induced NO production Rotter and Oh, 1996 macrophage

cellRat splenic 1-100 g/ml NO production ; ConA-induced Dombrink-Kurtzman et al., macrophages proliferation of splenic cells in 2000 and lymphocytes the presence of NO synthase

inhibitor

et al.,1995). However, in turkey poults acombination of FB

1and AFB

1was responsible

for increased primary immune response toSRBC (Weibking et al., 1994). In weaned

pigs, FB1(up to 100 mg/animal/day) did not

affect immunity (Tornyos et al.,2003). Effectsof FB

1on immunocompetence of laboratory

animals also vary substantially (Table 7).


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Immunomodulatory properties of FB1probably depend on its effect on lipidmetabolism, antioxidant/pro-oxidant balanceand interactions with other factors. Forexample, FB

1 decreased CD3 receptor

expression on the surface of thymus cells invitroand in vivo, which is consistent withthe sharp decrease in the ceramide contentin this organ (Martynova et al.,1995).

Similar to FB1, the trichothecenes can both

suppress and stimulate immune function(Table 8). Most of the research on T-2 toxineffects on immunity was perfomed with

laboratory animals and there are only a fewpublications addressing immunomodulatingproperties of T-2 toxin in farm animals.Futhermore, the molecular basis of immunemodulation by mycotoxins remains mostlyunknown and continues to be an active areaof research (Bondy and Pestka, 2000). Itappears that trichothecenes are potentimmunosuppressive agents that directly affectimmune cells and modify immune responsesbecause of tissue damage elsewhere. Forexample, sheep and calves treated withFusarium T-2 toxin develop leukopenia and

decreased function of peripheral lymphocytes(Sharma, 1993). In fact, exposure ofexperimental animals and humans to T-2 toxinhas been shown to have a variety ofimmunosuppressive effects, including alteredparameters of humoral immunity. It is wellestablished that T-2 toxin is cytotoxic tolymphocytic cellsin vitro, however, limitedinformation is presently available regardingthe contribution of such a mechanism toimmunosuppression in vivo, or to potentialimmune cell targets. It seems likely thatlymphocyte progenitors, in contrast to

thymocytes, represent highly sensitive targetsof T-2 toxin exposure, responsible for thymicatrophy (Holladay et al.,1993). For example,subchronic T-2 toxin treatment of timed-pregnant B6C3F1 mice resulted in significantand selective depletion of fetal liver cellsexpressing low levels of surface CD44 andCD45 antigens, suggestive of possiblelymphoid progenitor cell sensitivity to this

agent (Holladay et al.,1995). The authorsshowed that the precursors of B-cells mightrepresent highly sensitive targets of T-2 toxinexposure. Impaired resistance to pathogenicmicroorganisms occurs after exposure to thetrichothecenes T-2 toxin and DON. This maypredispose food animals to infectious diseaseand could result in decreased productivity aswell as increased animal-to-humantransmission of pathogens such as Salmonellaand Listeria (Pestka and Bondy, 1990). Forexample, mice challenged with S.typhimuriumand then treated with T-2 toxin

every other day for 10 days had markedlylarger and more bacteria-related lesions in thespleen, kidney, and liver than animalschallenged with S. typhimuriumalone (Taiand Pestka, 1990). In great contrast, in miceexposed to T-2 toxin at 3 mg/kg BW bygavage, virulence of both E. coli and S.auerus decreased. Apparently there arespecies-specific features in sensitivity totrichothecenes. For example, recently it hasbeen shown that T-2 toxin (up to 1 ppm) inthe poult diet did not affect antibodyproduction stimulated by various antigens

(Sklan et al.,2003).Immunomodulating properties of DON

have been studied mainly with rodents (Table9). The evidence is quickly accumulating toshow that DON can be immunosuppressiveor immunostimulatory, depending upon thedose and duration of exposure. Whileimmunosuppression is probably related to theinhibition of translation, immunostimulationcan be a result of interference with variouscellular regulatory mechanisms (Rotter et al.,1996). For example, in vivoDON suppressesnormal immune response to pathogens and

simultaneously induces autoimmune-likeeffects. Characteristic effects of DON alsoinclude superinduction of cytokineproduction by T-helper cells (in vitro) andactivation of macrophages and T-cells toproduce proinflammatory cytokines. Althoughthese effects have been largely characterisedin the mouse, several investigations withDON suggest that immunotoxic effects are

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Table 8. Effects of T-2 toxin on immunity.


Young poults Up to 1 mg/kg for Antibody titres unchanged Sklan et al.,200332 days

7-week-old pigs 0.5, 1.0, 2.0 or Leukocyte count ; Proportion of Rafai et al.,19953.0 mg/kg diet for T-lymphocytes ; Antibody3 weeks formation ; Blastogenic

transformation of lymphocytes

9-10-week- Aerosol, 390 g/L Lymphocyte count , Neutrophil Pang et al.,1988 old pigs count ; ConA, PHA, and PWM

blastogenic responses and H1 titersto SRBC

9-10-week- 15 mg/kg Blastogenic responses to ConA, Pang et al.,1987 old pigs PHA and PWM at early (3 to

5 days) and late (20 to 28 days)postdosing intervals; HA titer to SRBCunchanged

Calves 0.6 mg/kg/day Lymphocyte responses to Buening et al.,1982mitogens ; Chemotaxic migrationof neutrophils

Calves 0.6 mg/kg per day IgM and IgA ; IgG Mann et al., 1982for 43 days

Monkeys Gastric intubation Leucocyte countsat the end of Jagadeesan et al.,1982at 100 g/kg BW/ wk 4; Bactericidal activity ofday for 4-5 wk neutrophils , T-cell number ;

Lymphocyte transformation ,

B-cell number ; IgG and IgMlevels

Rats 2 mg/kg In progeny, the cortex was Lafarge-Frayssinet et al.,atrophic while the medulla 1990was proliferative. In thespleen, both B- and T-cells wereimpaired and responsivenessto PHA and LPS decreased

Mice 3 mg/kg BW by Virulence of both E. coliand Cooray and Jonsson,gavage S. aureus 1990

Mice 1 mg/kg every Markedly larger and more Tai and Pestka, 1990 challenged other day for 10 d bacteria-related lesions in thewith spleen, kidney, and liver than S. typhimurium animals challenged with

S. typhimuriumaloneMice 20 ng Immune response to sheep RBC Holt and DeLoach,1988

Mice 1.5 or 3.0 ppm T-lymphocyte-dependent Schiefer et al.,1987in the diet for humoral immunity unaffected16 months

Mice exposed 4.0 mg/ kg of BW Growth of Listeria ; Mortality Corrier and Ziprin, to Listeria 1986


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Table 9. Effects of DON on immune system.


Growing pigs 0.6, 1.8 and Secondary antibody response Overnes et al.,19974.7 mg/kg to tetanus toxoid dose-

dependentlyYoung pigs 0.75- 3.00 mg/kg -globulin levels, and Rotter et al.,1994

diet for 28 days antibody titers to SRBCMice 5 or 25 mg/kg BW mRNAs for IL-1, IL-6, and Zhou et al.,1997

TNF-; the T-helper 1 cytokinesIFN-and IL-2; and the T helper2 cytokines IL-4 and IL-10

Mice 25 mg/kg for 8 or Serum IgA and microhematuria Dong and Pestka, 199324 ws index after 4 to 8 wks and

continued to increase with furtherexposure

Mice 25 ppm for 4 and Total IgA ; Total IgG and IgM Rasooly ands Pestka, 19928 wks

Mice 10-100 mg/kg in Serum levels of anti-SRBC Robbana-Barnat et al.,

the diet antibodies ; Stimulation of B- 1988and T-cells by mitogens

Weanling mice 0.5- 25.0 mg/kg Total circulating white blood Forsell et al.,1986over 8 wks cells ;PMN neutrophils ; Serum

IgM ; IgA Weanling mice 0.75, 2.5 or 7.5 Serum IgM to SRBC ; Plaque- Tryphonas et al.,1984

mg/kg BW by forming cell numbers gavage

Murine 0.1 ng/ml-1 g/ml Phagocytosis ; Microbiocidal Ayral et al.,1992 peritoneal activity ; Superoxide anion macrophages production at 1 ng/ml;

Phagosome-lysozome fusion at 100 ng/nl

Lymphocytes 0.005-100 ng/ml PHA-induced lymphocyte Miller and Atkinson, 1986 in vitro proliferation at 0.005- 0.5 ng

DON/ml; at 50-100 ng/mllymphocyte proliferation

Human 216 ng/ml 50% inhibition in cell Meky et al.,2001 lymphocytes proliferation

Murine 25-100 ng/ml Production of H2O

2after LPS Ji et al.,1998

macrophage stumulation , but at 250 ng/m; cell line NO production at 25-250 ng/ml

Table 8. Continued.


Mice 20 ppm for Spleen proliferative responses Friend et al.,19831- 4 weeks to ConA ;

Human 1 -5 ng/ml 50% inhibition in cell Meky et al.,2001 lymphocytes proliferation

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also likely in domestic animals (Rotter et al.,1996). Therefore, DON can selectively andconcurrently upregulate or downregulatecritical functions associated with activatedmacrophages. For example, a single DONexposure rapidly induces gene expression invivo for a wide range of cytokines withapparently complete recovery occurring after24 hrs. Elevated cytokine expression mayplay an important role in the patho-physiological effects of DON and othertrichothecenes (Zhou et al., 1997). Malesappeared more susceptible than female mice

to DON-induced IgA dysregulation and IgAnephropathy in terms of latency, thresholddose, and severity (Greene et al.,1994). It isinteresting that experimental dysregulationof IgA production and IgA nephropathypersisted up to four months after a discreteperiod of dietary DON exposure (Dong andPestka, 1993).

Mycotoxin-induced immunosuppression isrelated to both natural and adaptiveimmunity:

depressed T- or B-lymphocyte activity

depressed NK cell activity suppressed immunoglobulin and antibody

production reduced complement or interferon activity impaired macrophage functions

The sensitivity of the immune system tomycotoxin-induced immunosuppression arisesfrom the vulnerability of the continuallyproliferating and differentiating cells thatparticipate in immunomediated activities andregulate the complex communication networkbetween cellular and humoral components

(Corrier, 1991). In fact, high levels ofpolyunsaturated fatty acids in the immune cellsand presence of sensitive receptors on cellsurfaces make them an important target for freeradical attack (Surai, 2002). As mentionedabove, oxidative stress caused by mycotoxinconsumption could be responsible for breakingeffective communication between immunecells, which ultimately would misregulate the

immune system and result in immuno-suppression. Although the molecular basis formany of the specific immunosuppressive effectsof mycotoxins is presently unclear, inhibitionof DNA, RNA and protein synthesis via a varietyof different mechanisms appears to be directlyor indirectly responsible for theimmunosuppressive action of many mycotoxins(Corrier, 1991). Furthermore, detrimental effectson antioxidant defences, stimulation of lipidperoxidation and apoptosis and damage toimmune cell receptors appear to be involvedin the immunomodulation properties of

mycotoxins. Consequences of oxidative stressfor immunocompetence are shown in Figure3.

Protective effect of antioxidants

against mycotoxicoses

The wide range of mycotoxins that cancontaminate animal feed and their differingchemical compositions makes protectionagainst mycotoxin-related toxicity a difficult

task. There are various approaches to controlor combat mycotoxin problems. The simpleststrategy is based on the prevention ofmycotoxin formation in feeds by specialmanagement programmes including storageat low moisture levels and prevention ofgrain damage during processing (Dawson,2001). However, modern agronomictechnology cannot eliminate pre-harvestinfection of susceptible crops by fungi (Wood,1992). Therefore, this strategy can be onlypartially effective; and in countries withwarm and humid conditions, this strategy can

be quite costly.Other strategies based on microbial orthermal inactivation of toxins, physicalseparation of contaminated feedstuffs,irradiation, ammoniation and ozonedegradation have not been developed to anindustrial level because they are either time-consuming or comparatively expensive(Dawson, 2001a). In recent years, nutritionalmanipulation has been actively used to


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improve animal defence against mycotoxinsor to decrease the detrimental consequencesof mycotoxin consumption.

Since lipid peroxidation plays an importantrole in mycotoxin toxicity, a protective effectof antioxidants is expected (Galvano et al.,2001). Indeed, in several experiments withvarious animal species, protective effects ofantioxidants against the toxic effects ofmycotoxins were observed. For example,

vitamin E supplementation ameliorated the pro-oxidative effects of OTA in the chicken(Hoehler and Maraquardt, 1996) and mouse(Grosse et al.,1997). Trolox C, a water-solubleform of vitamin E, prevented the genotoxiceffects of OTA in E. coli (Malavielle et al.,1994). Protective effects of vitamin E were alsoobvious in aflatoxicosis (Shen et al.,1994; Choiet al.,1995; Souza et al.,1999; Verma andNair, 2001), DON or T-2 toxicoses (Segal etal.,1983; Tsuchida et al.,1984; Rizzo et al.,1994) in rats; T-2 toxicosis in mice in vivo(Atroshi et al.,1997) or in an in vitrosystem

chapter mycotoxine immunity- blue book 2005

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