Ewa Skopiñska-Ró¿ewska1,3, Barbara J. Ba³an1, Ewa Sommer1, Joanna Chorostowska-Wynimko1, Janusz Bany2,
Aleksander Wasiutyñski3, Andrzej K. Siwicki3
1Department of Laboratory Diagnostics and Immunology, National Institute of Tuberculosis and Lung Diseases, Warsaw, Poland; 2Department of Pharmacology and Toxicology , Military Institute of Hygiene and Epidemiology, Warsaw, Poland; 3Department of Pathology, Medical University of Warsaw, Warsaw, Poland; 4Department of Microbiology and Clinical Immunology, Warmia and Mazury University in Olsztyn, Olsztyn, Poland Key words: mice, pregnancy, chocolate feeding, progeny, immunity Chocolate which contains flavonoids, a group of compounds which may exhibit biological activity, is made of Theobroma cacao beans. It is strongly suggested that certain dietary flavonoids may have the potential to reduce the risk of many diseases. Flavonoids have strong antioxidantpotential, so they may help to maintain the cardiovascular health and can influence immune response. Polyphenols from cacao liquor can inhibitmitogen-induced proliferation of T cells and polyclonal Ig production by B cells in vitro. They also influence IL-2 secretion by T cells. Chocolateis used by people as a form of self-medication for dietary deficiencies (e.g. magnesium) or to balance low levels of neurotransmitters involved inthe regulation of mood, food intake and compulsive behaviours. However, our previous research performed in mice showed that chocolate andsubstances contained in it have many unfavourable effects on organism during its development, e.g. they lower embryos mass and their tissuesangiogenic activity. Moreover, such treatment was found to cause the shortening of the limbs in offspring of mice fed chocolate during pregnan-cy, and disturbed the process of bone mineralization. The aim of the present paper is to examine the possible influence of chocolate feeding ofpregnant and lactating mice on the cellular and humoral immunological response of their six-week old progeny.
We have found, that a daily dose of 400 mg of chocolate given to pregnant and lactating mice resulted in the enhancement of humoral immu- nity and suppression of cell-mediated immunity of their 6 week-old progeny.
all chocolate contains numerous alkaloids (theobromine,caffeine, theophylline), and flavonoids. The subclass of Chocolate enjoys a very long history. It is thought, that flavonoids are flavanols to which belong catechin, epicate- the ancient civilizations, Olmeks, Mayas and Aztecs, living chin, quercetin and epigallocatechin, the so-called in Central America, first began to grow cacao trees, and monomeric flavan-3-ols, and corresponding to them produce chocolate drink from their beans. The drink made oligomers procyanidins. Besides the chocolate and the by them was considered to be magic, because it improved cocoa, flavanols appear also in green tea, red wine and many the mood, raised energy, protected against diseases or pre- fruits. However, chocolate contains most of all polyphenols vented them. Owing to its magic power it was drunk by that occur in all consumed food-products [Scalbert, 2004].
rulers, and it lent religious solemnities. The Greek name of Catechines make one tenth of polyphenols found in the the cacao tree – Theobroma cacao, directly means the chocolate. The remaining nine tenth are tannins and proan- draught of Gods (Theos – God, broma – the drink). Cocoa thocyanins – polymers of high molecular weight, which are beans first appeared in Europe in 1502. Christopher weakly absorbed from the alimentary tract. Procyanidins Columbus is attributed to be the discoverer of cocoa beans.
extracted from Theobroma cacao have antiviral (against Hernando Cortez, a Spanish conquistador contributed HSV and HIV), and antibacterial properties. Flavanols to the spread of cacao trees plantations by bringing cocoa have strong antioxidant potential. Their influence on the beans to Spain in 1519. Towards the end of the XVIth cen- production of prostaglandins which are part of inflammato- tury chocolate conquered France and Italy and from there ry reactions, modify the synthesis of the nitric oxide, reached Holland and the other parts of Europe. The first decreases the aggregation of thrombocytes, and it is sug- hard bar of chocolate appeared in the XIXth century gested that chocolate polyphenols may lower the risk of var- ious diseases of the circulatory system, inflammatory dis- Chocolate contains many different substances and chem- eases and cancer [Ba³an, 2002; Natsume et al., 2000; Nelson ical compounds essential to the human organism. Among & Sharpless, 2003; Osakabe et al., 2000; Rein et al., 2000; them are proteins, carbohydrates, microelements such as Richelle et al., 2001; Sanbongi et al., 1998; Wang et al., 2000].
calcium, magnesium, iron, and others as niacin. But first of Still, some authors described anti-angiogenic effects of Author’s address for correspondence: Prof. Ewa Skopiñska-Ró¿ewska, Department of Laboratory Diagnostics and Immunology, National Insti-tute of Tuberculosis and Lung Diseases, P³ocka 26, 01-138 Warsaw, Poland; tel.: 0600479495; e-mail: [email protected] or [email protected] green tea catechins, some of these catechins are present local graft – versus host response was highly significantly also in cocoa [Cao & Cao, 1999; Kim, 2003; Lamy et al., 2002; Ling et al., 1997]. In our previous studies we observed No differences in relative spleen weight (6.6±0.33 for that chocolate feeding of pregnant mice resulted in epigal- control spleens and 6.7±0.35 for spleens of progeny of locatechin-related embryonic angiogenesis suppression, and chocolate-fed mice) and in microscopic picture of spleen influenced development of offspring limbs and their bones tissues were seen between experimental and control groups.
mineralization [Skopiñski et al., 2003, 2004]. TABLE 1. The effect of chocolate feeding of pregnant Balb/c mice on The aim of the present paper is to examine the possible the anti-SRBC antibodies production of their 6-weeks old offspring.
influence of chocolate feeding of pregnant and lactatingmice on the cellular and humoral immunological response MATERIALS AND METHODS
Our experiments were performed on 2–3-month-old female Balb/c mice, fed during pregnancy and lactation with TABLE 2. The effect of chocolate feeding of pregnant Balb/c mice on 400 mg of dark chocolate (Wedel) administered with wheat the GvH activity of their 6 weeks-old offspring splenic lymphocytes in crisp in addition to standard laboratory chow. Control mothers were fed wheat crisp and standard chow. The prog-eny was sacrificed at the 6th week after birth. In this group, the spleens were collected, weighed, half of each spleen wasfixed in 4% buffered formaldehyde and processed for microscopic analysis. From the second part of spleen, the lymphocytes were isolated and their activity was evaluated in the local graft-versus-host (GvH) reaction. Some of 6-week-old mice were immunized i.p. with 0.2 mL of 10% sheep red blood cells (SRBC). Seven days after, mice were anaesthetized with chloral hydrate and theperipheral blood from retro-orbital plexus has been collect-ed. Serum was isolated and frozen at -20°C. Animals were DISCUSSION
handled according to the Polish law on the protection ofanimals and NIH standards. All experiments were accepted Chocolate contains flavonoids, a group of compounds, which may exhibit biological activity. It is strongly suggested In the haemagglutination assay the anti-SRBC titre was that certain dietary flavonoids may have the potential to evaluated. Sera were inactivated at 56°C for 30 min, serially reduce the risk of various diseases. Flavonoids have strong diluted, and supplemented with 0.5% SRBC. After 60-min antioxidant potential, so they may help to maintain the car- incubation at room temperature, the samples were cen- diovascular health and can influence immune response trifuged for 10 min at 150 ´ g, and vigorously mixed.
[Osakabe et al., 2000, 2002]. Polyphenols from cacao liquor Haemagglutination was evaluated in the optical micro- can inhibit mitogen-induced proliferation of T cells and poly- scope – as the last dilution with at least 3 cells conglomer- clonal Ig production by B cells in vitro. This effect is dose- ates present in at least 3 consecutive fields. Results were -dependent. They also can inhibit IL-2 secretion by T cells expressed as the mean antibodies titre in the treated group [Sanbongi et al., 1997]. Chocolate appears to support heart health also by positively affecting the way some blood com- The local graft-versus-host reaction (GvH) test (lym- ponents function, especially platelets [Rein et al., 2000].
phocyte-induced angiogenesis, LIA test) was performed Chocolate is used by people as a form of self-medication for according to Sidky and Auerbach [1975] with some modifi- dietary deficiencies (e.g. magnesium) or to balance low levels cations [Skopiñska-Ró¿ewska et al., 2002 ]. Briefly, spleno- of neurotransmitters involved in the regulation of mood, food cytes at a concentration of 1´106 cells/injection were graft- intake and compulsive behaviours (e.g. serotonin and ed intradermally into anaesthetized F1 (Balb/c ´ C3H) dopamine) [Bruinsma & Taren, 1999]. However, it is known crossbreeds mice. After 72 h, the mice were sacrified and that dopamine at non-toxic levels, strongly and selectively the newly-formed blood vessels were identified and counted inhibits the vascular permeabilizing and angiogenic activities in the dissection microscope at 6 ´ magnification, in central of VEGF, the important cytokine of embryonic angiogenesis.
Dopamine acts through D2 dopamine receptors to induce The statistical significance of difference from the con- endocytosis of VEGF receptor 2. This receptor is critical for trol was studied by Student t and Mann-Whitney tests.
promoting angiogenesis. The action of dopamine is specificfor VEGF and do not affect other mediators of microvascu- lar permeability or endothelial cell proliferation and migra-tion [Basu et al., 2001]. Also such flavonoids as catechin and Chocolate feeding of mice during pregnancy and lacta- epicatechin contained in chocolate may exert antiangiogenic tion period resulted in profound changes in the immuno- activity through inhibition of IL-8 production, or/and logical response of their progeny. Antibody production was because of the inhibition of growth, migration and tube for- highly significantly enhanced, and lymphocyte activity in mation of endothelial cells [Tang & Meydani, 2001].
Chocolate contains high levels of monomeric, oligomeric, w: Wp³yw substancji naturalnych na uk³ad odpornoœcio- and polymeric catechins. Plasma concentrations of epicatechin wy (ed. E. Skopiñska-Ró¿ewska). Fundacja Pomocy and also theobromine are increased markedly after chocolate Zdrowiu-Medycyna Naturalna, Warszawa, pp. 111–129 consumption. Plasma level of this substances reaches the max- imum 2–3 h after consumption of chocolate and is highly cor- 2. Basu S., Nagy J.A., Pal S., Vasile E., Eckelhoefer I.A., related with the dose of chocolate [Richelle et al., 1999].
Bliss V.S., Manseau E.J., Dasgupta P.S., Dvorak H.F., Pregnant mouse is a very good model to investigate the Mukhopadhyay D., The neurotransmitter dopamine influence of various drugs, xenobiotics and products of nat- inhibits angiogenesis induced by vascular permeability ural origin on the immunological response and the immune factor vascular endothelial growth factor. Nat. Med., system of her progeny. This practical model was used by us many times previously [Skopiñska-Ró¿ewska et al., 1996, 3. Bruinsma K, Taren D.L., Chocolate: food or drug? J.
2003; Kamiñski et al., 1998, 2000]. Our previous studies Am. Diet. Assoc., 1999, 99, 1249–1256.
have demonstrated the inhibitory action of chocolate and 4. Cao Y., Cao R., Angiogenesis inhibited by drinking tea.
theobromine on tumour and embryonic angiogenesis. We have observed significant inhibition of embryos growth and 5. Chorostowska-Wynimko J., Skopiñska-Ró¿ewska E., also the effect of shortening of limbs and bones in 4-week- Sommer E., Ba³an B.J., Skopiñski P., Demkow U., old offspring of female mice fed chocolate during pregnan- ¯ukowska M., Gil M., Some immunotropic effects of cy and lactation. In other study we have observed that theophylline and theobromine. Eur. Resp. J., 1995, 8, chocolate given to mice during pregnancy may disturb the processes of bone mineralization in offspring. 400 mg of 6. Chorostowska-Wynimko J., Skopiñska-Ró¿ewska E., chocolate given to pregnant mice has significantly sup- Sommer E., Rogala E., Skopiñski P., Wojtasik E., The pressed angiogenic activity of 18-days embryos tissues and multiple effects of theobromine on the fetus develop- this effect was correlated with epigallocatechin concentra- ment and postnatal status of the immune system. Int. J.
tion [Skopiñski et al., 2003, 2004].
Tissue Reactions, 2004, 26, in press.
Except for flavonoids, chocolate contains some methy- 7. Kamiñski P., Skopiñska-Ró¿ewska E., W¹sik M., Bany J., loxantines (theobromine, caffeine, etc.). There is proved Barcz E., Sommer E., Marianowski L., Thymus and with no doubt the strong immunomodulatory and the anti- lymph node cell CD4 and CD8 marker expression and inflammatory activity of methyloxantines. Their activity is their angiogenic activity of offspring of salbutamol- exerted through the activation and function of various -treated pregnant C3H mice. Pharmacol. Res., 2000, 41, From our previous research it results also that both the 8. Kamiñski P., Skopiñska-Ró¿ewska E., Bany J., W¹sik M., theophylline and the theobromine significantly inhibit the Sokolnicka I., Barcz E., Rogala E., Sommer E., Braj- ability of mononuclear blood cells of healthy donors, czewska-Fisher W., Filewska M., Ba³an B.J., Skopiñski patients suffering from diabetic retinopathy or rheumatoid P., Marianowski L., The effect of salbutamol treatment arthritis, as well as of the cells isolated from various human on the cellular immunity of the offspring of pregnant cancers, to the induction of neovascularisation in mice mice: spleen cell activity. Drugs Exptl. Clin. Res., 1998, cutaneous test. We also observed some effects of theo- bromine on the fetus development and the postnatal status 9. Kim M.H., Flavonoids inhibit VEGF/bFGF-induced of the immune system [Chorostowska et al., 1995, 2004; angiogenesis in vitro by inhibiting the matrix-degrading Skopiñski et al., 1998; Skopiñska-Ró¿ewska & Skopiñski, proteases. J. Cell. Biochem., 2003, 89, 529–538.
2001]. We suppose that immunological abnormalities 10. Lamy S., Gingras D., Beliveau R, Green tea catechins observed by us in the present work are partly connected inhibit vascular endothelial growth factor receptor pho- with the influence of theobromine and partly with catechins sphorylation. Cancer Res., 2002, 15, 62, 381–385.
11. Ling Y.C., Lin-siau S.Y., Chen C.F., Lin J.K., Suppres- sion of extracellular signals and cell proliferation through CONCLUSION
EGF receptor binding by (-)-epigallocatechin gallate inhuman A431 epidermoid carcinoma cells. J. Cell. Bio- As far as eating of chocolate, for its wholesome value is advisable for men and not pregnant women, insomuch preg- 12. Natsume M., Osakabe N., Yamagishi M., Takizawa T., nant women and children should themselves beware for the Nakamura T., Miyatake H., Hatano T., Yoshida T., Ana- possibility of appearing unwanted side effects of eating lyses of polyphenols in cocoa, and chocolate normal- phase and reversed-phase HPLC. Biosci. Biotechnol.
Biochem., 200,64, 2581–2587.
13. Nelson B.C., Sharpless K.E., Quantification of the pre- dominant monomeric catechins in black chocolate stan- The study was supported by grant 3PO5EO372 of the dard reference material by LC/APCI-MS. J. Agric. Food State Committee for Scientific Research.
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