Research Center for Veterinary Toxicology (CEPTOX), Department of Pathology, School of Veterinary Medicine andZootechny, University of Sao Paulo, Sao Paulo, Brazil

Effect of Senna occidentalis Seeds on Immunity in Broiler Chickens

I. M. Hueza1, A. O. Latorre

1, P. C. F. Raspantini1, L. E. R. Raspantini

1, D. P. Mariano-Souza1, J. L. Guerra

1

and S. L. Gorniak1,2

Address of authors: 1Research Center for Veterinary Toxicology (CEPTOX), Department of Pathology, School of VeterinaryMedicine and Zootechny, University of Sao Paulo, CEP 05508-900, Brazil; 2Corresponding author: Tel.: +55 11 30917693;fax: +55 11 30917829; E-mail: gorniak@usp.br

With 4 figures and 3 tables Received for publication July 5, 2006

Summary

This study investigated possible immunotoxic effects of Sennaoccidentalis (So) seeds incorporated in broiler chicken rations

at different concentrations (0.0%, 0.25%, 0.50% and 0.75%),for 28 or 42 days. We evaluated innate immune function(macrophage activities of spreading, phagocytosis, peroxide

and nitric oxide production) and acquired immune function(humoral and cellular immune responses), as well as lymphoidorgan weights and pathology. There was enhanced macroph-age activity, as hydrogen peroxide production increased

(P < 0.05) in cells of birds given 0.75% So, but there were noother pro-inflammatory effects. Birds receiving 0.75% of So inration for 42 days gained less weight (P < 0.01), and showed

a decrease in relative weight of the bursa of Fabricius(P < 0.05) and spleen (P < 0.01). In addition, morphologicalchanges were also noted in these lymphoid organs, with de-

pletion of lymphoid cells on the spleen and bursa of Fabricius,resulting in lower relative weight of both lymphoid organs. Noimpairment of humoral immune response against Newcastledisease and in cellular immune response after a phytohae-

magglutinin challenge was found. It is probable that mito-chondrial damage and related apoptosis may be responsiblefor the enhanced peroxide production and the reduced relative

weight of the bursa of Fabricius and spleen.

Introduction

Senna occidentalis (Syn. Cassia occidentalis) is a weed of theCaesalpinoideae family found ubiquitously in tropical andsubtropical regions of the world, and is a contaminant of

maize, and also soybean, sorghum, wheat and other cerealcrops (Lal and Gupta, 1973). Although most harvested cerealsare mechanically cleaned and selected before being processed,

if present Senna occidentalis (So) seeds always contaminate thefinal product, including animal rations, because of theirsimilarity in size and density to crop grains. For this reasonmany studies have been conducted in order to control the

contamination of the crops with these seeds; nevertheless, sofar the systems used for the management of this weed havebeen expensive and not completely effective (Teen et al., 1980;

Boyette et al., 1993; Keeton et al., 1996).The main toxic principles of So seed have been identified as

dianthrone (Haraguchi et al., 1996), an anthraquinone-derived

compound in So seeds that promotes the characteristic

mitochondrial myopathy produced by this plant (Graziano

et al., 1983; Calore et al., 1997, 1999, 2000; Haraguchi et al.,1998a,b; Tasaka et al., 2000). Furthermore, So toxicity inchickens has been proposed to impair mitochondrial function,resulting in swelling, loss of mitochondrial matrix, fragmented

mitochondrial cristae and glycogen depletion (O’Hara andPierce, 1974; Calore et al., 1997).Skeletal muscle degeneration is the predominant lesion

found in the majority of animals intoxicated with So seed inboth natural and experimental intoxication, as cattle (Dolahiteand Henson, 1965), swine (Colvin et al., 1986), horses (Irigo-

yen et al., 1991), sheep (Dolahite and Henson, 1965), rabbits(Tasaka et al., 2000), fowl (Simpson et al., 1971), goats(Suliman et al., 1982) and rats (Calore et al., 2000). The mainconsequence of So intoxication in broiler chickens is degen-

erative myopathy of myocardial muscle (Dolahite and Henson,1965; Barros et al., 1990; Calore et al., 1997).Moreover, studies conducted by Haraguchi et al. (2003)

with low concentrations of So seeds mixed in broiler chickrations showed that these birds were very sensitive to the toxiceffects of dianthrone. In addition, studies conducted in broiler

chicks treated with the seed coat of So also showed asignificant alteration in the relative weight of lymphoid organs,mainly in the bursa of Fabricius, and their histological

structure (Silva et al., 2003). Taken together, these studiesindicate that So toxicity might impact the immune system ofchickens.Thus, the purpose of the present study was to evaluate the

possible immunotoxic effects of low concentrations of So seedsincorporated in broiler chicken on both innate and acquiredimmune responses.

Materials and Methods

Plant material

Ripe So seeds were collected from plants grown at the

Biological Institute of Sao Paulo. So was identified atthe species level at the Maria Eneida Fidalgo Herbarium ofthe Botanical Institute of Sao Paulo, Sao Paulo state, Brazil.

The voucher herbarium specimen was deposited in thisInstitute under number SP-363817.After harvesting, the seeds were first frozen in liquid

nitrogen and then immediately ground and incorporated into

chicken rations at different concentrations.

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J. Vet. Med. A 54, 179–185 (2007)

� 2007 The Authors

Journal compilation � 2007 Blackwell Verlag, Berlin

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Experimental chicks and experimental design

All broiler chicks employed in the present study were housed inelectrically heated battery cages fitted with a feeder andwaterer. Food and water were provided ad libitum, except to

pair-fed (PF) group, as described later. All animal procedureswere approved by and in accordance with the ethical principlesfor animal research adopted by the Bioethics Committee of the

School of Veterinary Medicine and Zootechny, University ofSao Paulo, Brazil.To evaluate innate immunity, 60 1-day-old Hubbard com-

mercial broiler chicks were wing-banded, weighed and ran-domly assigned to four groups of 15 each, including onecontrol and three experimental groups treated for 28 days witha commercial ration mixed with 0.25%, 0.50% and 0.75% of

So seeds. At the end of the experimental period, peritonealmacrophages were harvested to determine macrophage activity[spreading, phagocytosis, and release of hydrogen peroxide

(H2O2) and nitric oxide (NO)]. Lymphoid organs werecollected to evaluate their relative weights and pathology(thymus, spleen and bursa of Fabricius).

A second group of 120 1-day-old Hubbard commercialbroiler chicks was wing-banded, weighed and randomlyassigned to five treatment groups with six replicates of four

chicks in each. The treated groups were one control, one PF,that received an amount of diet equivalent to that consumedby their So-treated partners, in order to determine if abnormalimmune responses are due to the direct effect of the plant or if

they result from dietary deficiencies, and three experimentalgroups treated for 42 days with 0.25%, 0.50% and 0.75% ofthe diet with So seeds, respectively, to evaluate acquired

immunity (i.e. humoral and cellular immune responses).Chicks from PF group received an amount of food equivalentto that consumed daily by birds from the 0.75% group. Thus,

food intake was identical in both the PF and the 0.75%groups.To access humoral immune responses, 16 chicks were chosen

at random from each group and were vaccinated with

Newcastle disease virus (NDV) vaccine and serum sampleswere collected before and after vaccination to determine serumtiters to NDV. Lymphoid organs (thymus, spleen and bursa of

Fabricius) from 12 birds of each group were also collected toevaluate their relative weights and pathology.Cellular immune response was tested using a standard

protocol for avian species (Smits et al., 1999), in that eightbroiler chicks from each treatment group received an intra-dermal injection of phytohaemagglutinin (PHA) at the third

and fourth inter-digital space 1 day before the end of experi-mental trial.

Macrophage activity

Forty-eight hours before being humanely sacrificed in CO2

chamber, birds received an intraperitoneal injectionof 5.0 ml/kg

of Sephadex solution (3%) in order to promote peritonealmacrophage activation. Themethods used to studymacrophagespreading and phagocytosis were based on those described by

Rabinovitch and De Stefano (1973) with some modifications.Briefly, peritoneal exudate cells were obtained by lavagewith cold RPMI1640 medium of the peritoneal cavity 48 h

after injection of Sephadex. The cells were adjusted to2 · 106 cells/ml ofRPMI1640.To studymacrophage spreading,

200 ll of each cell suspension was prepared in duplicate onglass slide monolayer that were kept in a multiwell (six wells)tissue culture plate (Corning Costar, Corning, NY, USA).

Within 20 min, the wells were washed several times with coldphosphate-buffer saline (PBS) (4�C);RPMI-1640-supplementedmedium was added to each well. The culture plates were

incubated for 60 min at 40�C in a humidified atmosphere of 5%CO2.After incubation, thewells were rinsedwith cold (4�C)PBSand the adherent cells fixed with 0.5% glutaraldehyde for

10 min. These cells were then counted with a phase con-trast microscope (Nikon, Inc., Melville, NY, USA) at 40·magnification. Using an ocular grid, 200 macrophages werescored as either round or spread. An index of macrophage

spreading (SI) was then calculated for each monolayer of eachwell: SI ¼ number of spreading macrophages · 100/200 adher-ent cells counted; hence, SI ¼ %of spreading macrophages.

Macrophage phagocytosis was evaluated using the samemethod as described above. One mg of zymozan-A solution(5.0 mg/ml) was added to each well 1 h before the 60 min

incubation period (40�C). Using the same microscope, and thesame objective and methods described above, an index ofphagocytosis (PI) was then obtained: PI ¼ number of macr-

ophages with phagocytic activity · 100/200 adherent cellscounted; hence, PI ¼ %of macrophages with zymosan parti-cles phagocytized. The mean of four counts obtained from thetwo slides of each bird was used to express the SI or PI index.

Spontaneous and phorbol myristate-acetate solution(PMA)-induced H2O2 release by macrophages was measuredby the method of Russo et al. (1989). Briefly, the peritoneal

cells adjusted to 2 · 106 cell/ml were centrifuged for 10 minand resuspended in 1 ml of phenol red solution (PRS,containing 140 mm NaCl, 10 mm potassium–phosphate buffer,

pH 7.0, 0.5 mm dextrose, 0.28 mm phenol red and 8.5 U/mlHRPO) for H2O2 detection. The cell suspensions were addedto 96-well flat-bottom microplates and incubated in a humid-ified 5% CO2 atmosphere at 40�C for 1 h. Subsequently, the

wells containing PRS received 10 ll of 1 n NaOH to stop thereaction. Hydrogen peroxide-dependent phenol red oxidationwas read on a Labsystem microplate reader (Model EX

Multiscan; Thermo Fischer Scientific Inc., Waltham, MA,USA) using test wavelength of 620 nm. The same procedurewas employed to determine H2O2 release after stimulation with

PMA, with 10 ll of 10 ng/ml PMA added to each well beforeincubation. Concentrations were calculated according to astandard curve prepared by serial dilutions of H2O2. Sponta-

neous and PMA-induced H2O2 production experiments wererepeated four times for each bird in each group, and the meanvalue of the four counts was used for H2O2 determination.

Nitric oxide production was indirectly measured by assess-

ment of the nitrite concentration, the stable end product of NOsynthase-generated reactive nitrogen intermediates. Nitriteconcentration was determined using a colorimetric assay

(Green et al., 1982). In brief, 150 ll of the cultured macroph-age supernatants were incubated with 300 ll of a 1:1 mixtureof 1% sulphanilamide in 30% acetic acid and 0.1% N-1-

naphtyl-ethylenediamine dihydrochloride in 60% acetic acid atroom temperature. After 5 min of incubation in the dark,absorbance was read at 540 nm. Concentrations were calcu-lated according to a standard curve prepared by serial

dilutions of sodium nitrite. Nitric oxide production experi-ments were repeated four times for each bird in each group,

180 I. M. Hueza et al.

and the mean value of the four counts was used for NOdetermination.

Body weight changes and feed intake

The body weight and the feed intake of chicks for each

replicate were recorded at weekly intervals. Feed conversionratio was calculated on the basis of feed:gain for each replicate.

Humoral immune response

Twenty-eight-day-old birds received the NDV vaccine (Hitch-ner B1) by the occulo-nasal route according to the recommen-

dation of the manufacturer (Biovet, Brazil). Blood samplecollection was performed before vaccination and at weeklyintervals from the main brachial vein. A commercial enzyme-

linked immunosorbent assay (ELISA) test kit (FlockcheckTM

IDEXX Laboratories Inc., Westbrook, ME, USA) was used totest serum for antibodies against NDV. Chicken serum

samples were diluted 1:500 and incubated in 96-well microtiterplates containing NDV antigen. The ELISA was performedaccording to the manufacturers� recommendations.

Cellular immune response

To evaluate the T-cell-mediated immunity the toe thicknesses

of both left and right foot at the third and fourth inter-digitalspace were measured by micrometer. Immediately after thosemeasurements, 0.1 ml of PHA (10 mg/ml in PBS) and 0.1 ml

of PBS were injected into right and left feet (acting as control)respectively. A stimulation index was calculated by subtractingthe skin thickness of the first measurement from the second

and the values of the left foot (control) from the right foot(Cheng and Lamont, 1988).

Pathology

On the last day of each experiment, all birds were killed andnecropsied, and representative samples of brain, thymus,

heart, skeletal muscle, liver, spleen, kidney and bursa ofFabricius were fixed in 10% neutral buffered formalin. Thesamples were processed, embedded in paraffin, sectioned at

5 lm thickness and stained with haematoxylin and eosin (HE).

Statistical analysis

Data were analysed by one-way analysis of variance (anova);significant F-tests were followed by post hoc analysis usingDunnett’s test to compare control and treated groups at

P < 0.05. Data are presented as mean ± SEM.

Results

Macrophage activity

The spreading and phagocytic activity of peritoneal macroph-ages from treated birds were not affected by So seeds at anylevel in the ration when compared with control birds (data not

shown). On the other hand, the administration of 0.75% Soseeds for 28 days increased the spontaneous and the PMA-induced H2O2 release by peritoneal macrophages, in a dose-

response fashion when compared with those cells from control

animals (P < 0.05) (Fig. 1). No difference (P > 0.05) was

observed in the release of NO by peritoneal macrophages inany of the treatment groups (data not shown).Only chicks in the 0.75% So treatment group given for

28 days showed a significantly reduced weight in the bursa ofFabricius (Fig. 2) (P < 0.05). No significant differences wereobserved in the weight of other lymphoid organs (i.e. thymusand spleen), in treated birds compared with the control group.

Humoral immune response

All pre-vaccination sera tested by ELISA were negative forNDV (titers < 399 were considered negative), indicating nolevels of maternal antibodies that could influence the vaccine

challenge. Antibodies to NDV were detected 7 and 14 daysafter the vaccination (Table 1). Birds receiving one dose ofcommercial vaccine 1 week after challenge exhibited positivetiters, with some exceptions in animals from the 0.50% and

0.75% groups; however no statistical differences were detectedamong groups. However, 14 days after vaccination antibodyproduction was enhanced in all groups of animals.

There were no differences in relative weight of thymus inchicks treated for 42 days (Fig. 3). Conversely, spleen weights

0.0

2.5

5.0

7.5

10.0(a) (b)

Control0.25% So0.50% So0.75% So

H2O2 without PMA H2O2 with PMA

*

*

Groups

nM

ols

Fig. 1. Effect of administration of Senna occidentalis seeds on spon-taneous (a) and phorbol myristate-acetate-induced H2O2 release (b) byperitoneal macrophages in broiler chicks given rations containing 0.0(control), 0.25%, 0.50% and 0.75% So seeds for 28 days. Resultsrepresent means ± SEM. *Significantly different from control(P < 0.05).

0.0

0.1

0.2

0.3

0.4

0.5

Control

0.25% So

0.50% So

0.75% So

Thymus Spleen Bursa of fabricius

g10

0g

–1 o

f B

.W.

**

Fig. 2. Effect of administration of Senna occidentalis seeds on relativeweight of lymphoid organs in broiler chicks given rations containing0.0 (control), 0.25%, 0.50% and 0.75% So seeds for 28 days. Resultsrepresent means ± SEM. *Significantly different from control(P < 0.05).

Immunotoxic Effects of So Seeds in Broiler Chicken 181

were reduced in chicks treated with 0.50% and 0.75% So seeds(P < 0.05 and P < 0.01 respectively) when compared withcontrol group. Moreover, only birds treated with 0.75% Soseeds had a lower relative weight of the bursa of Fabricius

compared with controls (P < 0.05).

Cellular immune response

Although no statistical differences observed on these data,visual observations during both evaluation periods, 8

and 24 h after PHA-injection, showed that injection of

PHA into the chicks� feet resulted in smaller tumourdevelopment in the 0.75% So group compared withcontrols (Table 2).

Total feed intake, body weight gain and feed conversion ratio

Feed intake, body weight gain and feed conversion ratio for0–42 days of age are given in Table 3. The food consumptionwas reduced in birds treated with 0.75% So seed compared

with the control group (P < 0.05); similarly, feed intake in thePF group was also reduced (P < 0.05). Body weight gain wasdiminished in the 0.75% So and the PF groups (P < 0.01)compared with birds from control group. Conversely, body

weight gain of chicks treated with 0.25% of So improved(P < 0.05) when compared with controls. However, feedconversion was enhanced in broiler chicks in the PF and

0.75% groups (P < 0.01) when compared with controlbirds; there was no change in feed conversion in birdsgiven 0.25% So.

Pathology

The histological evaluation of birds treated with 0.75% So for42 days showed depleted lymphoid cells on the periateriolarsheath of the spleen (Fig. 4a). Although the epithelial tissue ofthe bursa of Fabricius was unchanged, we noted alterations in

lymphoid nodules characterized by depleted lymphocytes, inboth the cortical and the medullar area (Fig. 4c). Pathologyalso revealed the presence of apoptotic cells and immunoblasts

cells. The cortico-medullar border of the bursa of Fabriciusfollicles was disrupted which indicates marked lymphoid organatrophy. Conversely, the diffuse lymphoid tissues, liver,

kidneys and other organs from all treated birds did not showabnormal histology (data not shown). Furthermore, thecardiac and skeletal muscles did not show lesions caused bySo seeds.

Table 1. Serum antibody response following vaccination (at 28 days of age) with commercial Newcastle vaccine (Hitchner B1) of chicks givenrations containing 0.0 (control), 0.25%, 0.50% and 0.75% Senna occidentalis (So) seeds for 42 days

Age (days)

Groups

Control (n ¼ 16) Pair-feda (n ¼ 16) 0.25% of So (n ¼ 16) 0.50% of So (n ¼ 16) 0.75% of So (n ¼ 16)

28 9.0 ± 3.0 12.3 ± 3.4 14.4 ± 4.5 14.8 ± 5.1 11.2 ± 4.835 412.5 ± 60.5 406.2 ± 66.1 430.7 ± 66.7 396.1 ± 70.6 301.3 ± 4842 5196.9 ± 1099 7104.9 ± 955 6041.4 ± 755 6649.0 ± 1919 7354.2 ± 1349

Titers > 399 are considered positive.Results represent mean ± SEM.aChicks from the pair-fed group received an amount of diet equivalent to that consumed by birds from the group treated with 0.75% of So seeds.

0.0

0.1

0.2

0.3

0.4

Control

Pair-fed

0.25% So

0.50% So

0.75% So

Thymus Spleen Bursa of fabricius

g10

0g

–1 o

f B

.W.

* **

*

Fig. 3. Effect of administration of Senna occidentalis seeds on relativeweight of lymphoid organs in broiler chicks untreated (control andpair-fed group) and given rations containing 0.25%, 0.50% and 0.75%So seeds for 42 days. Results represent means ± SEM. *Significantlydifferent from control (P < 0.05); **Significantly different fromcontrol (P < 0.01).

Table 2. Cellular immune response of chicks given rations containing 0.0 (control), 0.25%, 0.50% and 0.75% Senna occidentalis (So) seeds for42 days

Hours after PMA challenge

Skin thickness measure (mm)

Control (n ¼ 8) Pair-feda (n ¼ 8) 0.25% of So (n ¼ 8) 0.50% of So (n ¼ 8) 0.75% of So (n ¼ 8)

8 1.22 ± 0.2 1.23 ± 0.1 1.20 ± 0.1 1.14 ± 0.2 0.93 ± 0.124 0.79 ± 0.1 0.79 ± 0.1 0.87 ± 0.1 0.74 ± 0.2 0.61 ± 0.1

Results represent mean ± SEM.aChicks from the pair-fed group received an amount of diet equivalent to that consumed by birds from the group treated with 0.75% of So seeds.

182 I. M. Hueza et al.

Discussion

It is well known that damage in the immune system generally

can be produced with lower doses of xenobiotics than thosewhich induce overt toxic effects in other systems (Sjoblad,1988). Previous studies, conducted in this laboratory, revealedthat prolonged administration of So seeds in concentrations of

1% in the diet can cause lesions in cardiac and skeletal musclein broiler chicks (Haraguchi et al., 1998a,b). In contrast tothese other results, at the concentrations employed here

(0.25%, 0.50% and 0.75% So seeds) did not induce lesionscharacteristic of So toxicosis.

Many studies revealed that So exposure, regardless of the

animal species, promotes anorexia and a remarkable loss ofbody weight (Torres et al., 1971; Barth et al., 1994; Haraguchiet al., 1998a,b; Tasaka et al., 2000). Moreover, it is well known

that nutritional deficiencies such as lack of protein, energy,copper, zinc, folic acid, vitamins and other compounds canimpair the immune system, and, increase susceptibility toinfectious diseases (Scrimshaw and SanGiovanni, 1997). Thus,

the PF group was added to this study to determine if abnormalimmune responses are due to the direct effect of the plant or ifthey result from dietary deficiencies. While birds from both the

0.75% So and PF groups had reduced feed intakes, and lower

body weight gain, only chicks treated with So seeds developedalterations in lymphoid organs (i.e. decrease in relative organweight and histological changes). This suggests that there is adirect toxic effect of the plant on these organs and that the

results are not due to nutritional deficiency.Electron microscopy studies of affected muscle fibres

revealed the presence of mitochondrial damage promoted by

So, denoting that this myopathy is secondary to impairment ofmitochondria function (Graziano et al., 1983; Calore et al.,1997, 1999, 2000; Haraguchi et al., 1998a; Tasaka et al., 2000);

however, the exact mechanism of action of So toxin onmitochondrial metabolism is still unknown. On the otherhand, a study conducted by Lewis and Shibamoto (1989) withSenna obtusifolia showed that this toxic plant can cause

impairment of mitochondrial metabolism inhibiting NADHoxidation and electron transport in mitochondria, resulting inuncoupling of oxidative phosphorylation.

The mitochondrial respiratory chain uses electron flow todrive adenosine triphosphate (ATP) synthesis. Briefly, theelectrons are passed to the terminal electron acceptor oxygen

which is then reduced to water. Associated with the movementof electrons along the electron chain transport is the movementof protons (H+) from the mitochondrial matrix into the

Fig. 4. Histopathology of spleen(a) and bursa of Fabricius (c) fromchicks fed a ration with 0.75%Senna occidentalis seeds for42 days and from untreated chicks(b and d). Spleens from treatedbirds showed notable depletion oflymphoid cells on periateriolarsheath. HE. Bar ¼ 100 lm. Bursaof Fabricius from chicks fed aration of So is characterized bydepleted lymphocytes, and disrup-tion of the cortico-medullar bor-der, denoting marked lymphoidorgan atrophy HE. Bar ¼ 30 lm.

Table 3. Total feed intake, bodyweight gain and feed conversionratio of chicks given rations con-taining 0.0 (control), 0.25%, 0.50%and 0.75% Senna occidentalis (So)seeds for 42 days

GroupsTotal feed intake(kg/replicate)

Total body weightgain (kg/replicate)

Total feedconversion

Control (n ¼ 6)a 13.7 ± 0.5 5.6 ± 0.2 2.43 ± 0.08Pair-fedb (n ¼ 6) 11.8 ± 0.4* 3.7 ± 0.1** 3.17 ± 0.07**0.25% of So (n ¼ 6) 14.5 ± 0.3 6.5 ± 0.2* 2.28 ± 0.060.50% of So (n ¼ 6) 12.8 ± 0.5 5.6 ± 0.2 2.29 ± 0.070.75% of So (n ¼ 6) 11.8 ± 0.4* 3.6 ± 0.2** 3.28 ± 0.11**

Results represent mean ± SEM.aResults are means of six replicates (n ¼ 6) obtained by group weighing all four chickens from each cage.bChickens from the pair-fed group received an amount of diet equivalent to that consumed by birds fromthe group treated with 0.75% of So seeds.*P < 0.05; **P < 0.01 versus control group.

Immunotoxic Effects of So Seeds in Broiler Chicken 183

intramembrane spaces. The movement of H+ through theATPase provides energy to support ATP synthesis (Stryer,1995). If a site-specific defect exists in electron transport,

electrons will leak from the respiratory chain and cause aunivalent reduction of oxygen that results in the formation ofsuperoxide (O2.

)) which is converted to H2O2 in the presence

of superoxide dismutase (Chance et al., 1979).The present study clearly shows increased H2O2 production

by peritoneal macrophages of birds treated with 0.75% So

seeds, but no further enhanced effects were observed in othermacrophage activities. This suggests that H2O2 results fromthe mitochondria and not from any possible pro-inflammatoryeffect of the plant. Actually, So seeds are employed in several

popular medicinal preparations used for different therapeuticpurposes including anti-inflammatory drugs (Sadique et al.,1987).

The mitochondria, besides being the organelle in whichoxidative phosphorylation and ATP production occur, is thelocale where several important aspects of apoptosis arise. We

hypothesize that some caspases activators, such as the cyto-chrome c, could be released from the mitochondria to thecytosol, leading to disturbance of the mitochondrial trans-

membrane potential, resulting in subsequent activation of pro-apoptotics genes, eventual disruption in electron transport anduncoupling of oxidative phosphorylation, and ultimately celldeath.

We further hypothesize that the reduced weight of the spleenand bursa of Fabricius and associated histological changeswith rarefaction of splenocytes and reduced follicles with

depleted cells area either in the cortical or in the medullarregion of the bursa of Fabricius result from an apoptoticprocess triggered by the plant toxin. Conversely, the thymus

was not affected by So. This lack of toxicity may be related tothe lack of a cellular immune response after PHA challenge,suggesting that So seed had no toxic effect on T-lymphocytepopulations.

Despite the histological changes observed in the spleen andbursa of Fabricius of birds treated with So seeds, noimpairment in antibody anti-NDV production was found.

The antibody production occurs mainly in the spleen and inlymphoid tissue dispersed throughout the body, and to a lesserextent in the bursa of Fabricius and bone marrow (Fairbrother

et al., 2004). Xenobiotics tend to cause prominent toxic effectsonly in organs to which the compounds have a higher affinity(Rozman and Klaassen, 2001). Thus, we suggest that dian-

throne has an affinity only for the spleen and bursa ofFabricius; consequently there was normal antibody productionfrom the dispersed lymphoid tissue which was unaffected by Soexposure.

The present study clearly showed that So administration tobroiler chicks can promote immunotoxic effect in lymphoidorgans such as the spleen and bursa of Fabricius, corrobor-

ating results from Silva et al. (2003). These results, however,show that despite alterations in acquired immune responses,antibody production and cellular immune responses were not

compromised. Even so, we can not assume that So seeds donot have any toxic effects on the immune system, because it ispossible that immunosuppression occurs at higher doses thanused in this study. Thus, future experiments will be conducted

in our laboratory, administering higher So doses to investigatethis question. In addition, we will develop toxicokinetic

protocols to evaluate the specific affinity of dianthrone foravian lymphoid tissues.

Acknowledgements

The authors gratefully thank Dr Jim Pfister from the Poison-ous Plant Research Laboratory (PPRL) - U.S. Department of

Agriculture – Agriculture Research Service – Utah (USA) forhis assistance and suggestions. This study was supported bygrants from Fundacao de Amparo a Pesquisa do Estado de

Sao Paulo – FAPESP, Brazil (Proc. No. 02/00080-0).

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