Tesi sul tema "Rat colon"

The enteric nervous system controls the gut through the release of specific neurotransmitter and neuromodulators at specific sites such as mucosal secretory cell or smooth muscle cell. In the present study, we have examined the response to one of these neurohumoral agents, Neurotensin, in the rat distal colon. Neurotensin is a paracrine and endocrine modulator of the digestive tract. Even though these effects have been seen in colonic preparations, there are very few functional studies of the effects of Neurotensin in the rat colon, especially the distal colon. In the current study we propose the following hypothesis that Neurotensin will lead to contractile effect on basal tone and phasic contraction in the distal rat colon and will mediate this process primarily through the NT1 receptor. This hypothesis is based on evidence from the mixed action of Neurotensin in other regions of the gut and the more widespread distribution of the NT1 receptor. We have identified two specific aims to investigate this hypothesis. Aim 1 is to investigate the role of Neurotensin in tonic contraction and phasic contraction of the distal rat colon. In this aim, we will expose distal rat colon strips to varying doses of Neurotensin and record changes in basal tone and phasic activity. For our second aim, we will investigate the receptors mediating these responses to Neurotensin. In this aim, we will introduce NT1, NT2, and nonspecific inhibitors to distal rat colon and observe modulation in Neurotensin effects. We will also determine the existence of the receptors via Western Blot. The rat distal colon did respond in a dose-response fashion to varying doses of Neurotensin, but elicited different effects dependent on the strip preparation. When the mucosa was intact, circular muscle responded with an inhibitory effect to phasic activity, but there was little to no change in tonic activity. When the mucosa was removed, the circular muscle responded to Neurotensin by eliciting an increase in tonic activity, but had no effect on phasic activity. The use of SR48692, a specific NT1 receptor inhibitor, showed that the effects that were observed due to Neurotensin were not mediated through the NT1 receptor. With the use of SR142948, a non-selective NT1/NT2 inhibitor, the effects of Neurotensin was completely abolished. This led us to believe that the observed effects were mediated through a Neurotensin receptor and that receptor is likely the NT2 receptor. This was confirmed by the use of the specific NT2 receptor antagonist, levocabastine. The existence of the receptor in rat colon had to be confirmed in order to ensure that the effects observed were mediated through the NT2 receptor and not from an outside mediator. Western Blot analysis confirmed the existence of the NT2 receptor within the mucosa, within the muscle, and within the intact preparation of the distal rat colon. Although these results conflict with our hypothesis, it provides for an interesting template and avenue of exploration.

A l'aide de la technique d'electromyographie chronique, l'auteur a etudie la motricite du colon chez le rat et son controle medullaire. Il a pu definir un profil moteur colique et determiner le role respectif du systeme sympathique et parasympathique de la substance p et des opioides dans le controle medullaire de la motricite colique

Preliminary studies were undertaken to investigate whether or not added iron (0, 35, 880 ppm of iron as ferrous fumarate) and fat type (corn oil, beef tallow, or menhaden oil ) influenced the oxidation of the rat diet during storage . Iron level affected thiobarbituric acid (TBA) values only in the menhaden oil diets. Storage for 4 d did not affect TBA values of the diets . Neither food intake nor body weight of the rats was affected by the different diets, suggesting all diets were equally acceptable to rats. The effects of iron supplementation and fat type on in vivo lipid peroxidation in rat colon were studied. Semi-synthetic diets were formulated to contain 15% (wt/wt) total fat, an amount comparable with human diets, as either 15% corn oil (C), 1% corn oil + 14% beef tallow (B), or 1% corn oil + 14% menhaden oil (M). Diets of each fat type were formuIated with ferrous fumarate to contain 35 ppm iron, a level sufficient to meet the requirement of the rats, or 880 ppm iron, a level similar to that found in iron-fortified breakfast cereals. During a 6-wk study, each of 6 groups of 10 male weanling Sprague-Dawley rats was fed one of the 6 diets (C35, C880, B35, B880, M35, M880). Lipid peroxidation products in the colon mucosa and in the feces were measured as thiobarbituric acid reactive substances (TBARS), and other possible physiological changes were monitored by measuring body weight, and iron levels of the feces and colon mucosa, and by observing the histology of the colon. At the beginning of the trial, each group of rats had similar body weights and TBARS in the feces. After the feeding trial, groups of rats remained similar in body weight, and no histological changes were observed in the colon. However, rats fed the different dietary fats had different (p < 0.05) TBARS in the feces and colon mucosa (BThus, the type of dietary fat was a significant determinant of in vivo lipid peroxidation, independent of dietary iron level. Rats fed the high iron diets had higher TBARS in both the feces and colon mucosa. When compared by dietary fat type, rats fed high iron diets had higher TBARS in the mucosa only if they were also fed the menhaden oil diet. Thus, dietarv iron was a significant determinant of in vivo lipid peroxidation only in combination with menhaden oil. The long-term intake of iron-fortified foods with high menhaden oil may lead to significant increased in vivo lipid peroxidation.

Electrophysiological recordings were made from myenteric neurones of the rat colon and were combined with immunohistochemical and intracellular staining techniques with a view to gaining an insight into the nature of regional and species differences in neuronal properties and the determination of enteric neuronal function. Immunohistochemical studies indicated that immunoreactivity (IR) to the biologically active mediators VIP and NPY were contained within three distinct neuronal populations, namely those containing either VIP- or NPY-IR and those containing both VIP- and NPY-IR. Regional differences in numbers of immunoreactive neurones and the extent of co-localization of the two neuropeptides indicated the possibility of regional differences in function. Intracellular recordings of >500 myenteric neurones revealed three electrophysiological classes of neurones (S/Type 1, AH and Type 3 neurones; impalement ratio 5:4:1) based on their active and passive properties and their synaptic inputs. Presynaptic tetanic stimulation evoked either slow membrane depolarizations or hyperpolarizations, but these were not a prominent feature of myenteric neurones being detected in only 8% of neurones. Muscarinic presynaptic autoinhibition of acetylcholine output, detectable as changes in fast ESP amplitude, was not commonly observed. Two presynaptic 5-HT receptors were, however, identified, a 5-HT_1A receptor and 5-HT₄ receptor that respectively inhibited and facilitated acetycholine output.

Tumour immunity is a balance between immune mediators that promote tumor progression versus mediators that promote tumor rejection. Infiltrating lymphocytes in human colorectal cancer tissues are independent prognostic factors for a better survival and a high number of cytotoxic CD8+ T-cells have been associated with a better prognosis in terms of a longer and disease free survival for the patient. In our syngeneic rat model we induce colon carcinoma subperitoneally by injecting a colon cancer cell line BN7005, a cell line expressing the epitope (Lewis Y) for the BR96 antibody. Tumours are dissected out and treated with different fixatives and then either frozen, snap-frozen or embedded in paraffin followed by sectioning. Immunohistochemistry using monoclonal antibodies against the tumour infiltrating leukocytes was performed on the tissue. The results were seen as an infiltration of different leukocytes in the tumours.

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior
O objetivo desse estudo foi verificar o efeito da suplementaÃÃo intragÃstrica com fibra solÃvel e prebiÃtico (frutooligossacarÃdeos - FOS), do ponto de vista inflamatÃrio e metabÃlico, no tecido cÃlico normal ou na vigÃncia da colite induzida por Ãcido trinitrobenzeno sulfÃnico (TNBS), em ratos. Foram usados 96 ratos da linhagem Wistar, machos, com peso mÃdio de 300g. Foram distribuÃdos aleatoriamente em dois grupos, com 48 animais. Um Grupo Estudo Sem Colite (GESC) submetido à aplicaÃÃo transanal de Ãgua ou outro Grupo Estudo Com Colite (GEC) induzida por TNBS, 20 mg mais etanol à 50%. Cada grupo foi redistribuÃdo em 3 subgrupos, de acordo com a substÃncia utilizada por infusÃo intragÃstrica: 1(Ãgua), 2(Fibra solÃvel) e 3(FOS). Todos os animais receberam essas soluÃÃes durante 14 dias antes da aplicaÃÃo transanal de Ãgua ou da induÃÃo da colite, atà o dia determinado para eutanÃsia (7 e 14 dias). Foram realizadas avaliaÃÃes do peso dos animais, relaÃÃo peso/tamanho do colo, escores macroscÃpicos e microscÃpicos, e dosagens de metabÃlitos no tecido cÃlico in vivo (concentraÃÃes de corpos cetÃnicos [3-hidroxibutirato, acetoacetato], piruvato, lactato, ATP e relaÃÃo [acetoacetato]/[3-hidroxibutirato] e [piruvato]/[lactato]. Foram avaliados o GESC, GEC, e os subgrupos Ãgua, do grupo sem colite (ESC1) e com colite (EC1). NÃo houve alteraÃÃes significantes nos diversos parÃmetros inflamatÃrios avaliados no GESC. No GEC, houve aumento na relaÃÃo peso do colo/comprimento do colo no subgrupo FOS, no 14 dia (ρ<0,05). Com relaÃÃo ao peso dos animais, os do subgrupo GEC1, apresentaram reduÃÃo significante no 7 dia. Houve aumento na relaÃÃo peso/tamanho do colo, no escores macro e microscÃpico no subgrupo GEC1 no 7 e 14 dias (ρ<0,05). Quanto à avaliaÃÃo metabÃlica, no GESC foi evidenciado aumento nas concentraÃÃes de acetoacetato no subgrupo fibra solÃvel no 14 dia (ρ<0,05). Houve reduÃÃo nas concentraÃÃes de piruvato no subgrupo fibra solÃvel e FOS no 14 dia (ρ<0,05). As concentraÃÃes de ATP foram reduzidas nos subgrupos fibra solÃvel e FOS, no 7 e 14 dias (ρ<0,05). No GEC, as concentraÃÃes do piruvato apresentaram-se elevadas no subgrupo fibra solÃvel, no 14 dia (ρ<0,05). As concentraÃÃes de ATP diminuÃram nos subgrupos fibra solÃvel e FOS (ρ<0,05). Comparando os subgrupos GESC1 com GEC1, houve elevaÃÃo nas concentraÃÃes dos corpos cetÃnicos no GEC1 no 7 dia (ρ<0,05). Houve reduÃÃo nos nÃveis de piruvato no GEC1 no 7 e 14 dias (ρ<0,05). Observou-se reduÃÃo nas relaÃÃes [acetoacetato]/[3-hidroxibutirato] e [piruvato]/[lactato] no GEC1, no 7 dia (ρ<0,05). Conclui-se, portanto, que a suplementaÃÃo intragÃstrica com fibra solÃvel ou com FOS, nÃo alteram os parÃmetros inflamatÃrios avaliados no 7 e 14 dias, nos colos normais ou na vigÃncia de colite, porÃm eleva a oferta de precursores para produÃÃo de energia, alterando as concentraÃÃes in vivo de ATP, tanto no tecido cÃlico normal, como na vigÃncia da colite.
The aim of this study was research the metabolic and inflammatory findings in normal colon and trinitrobenzene sulphonic acid-induced (TNBS) colitic rats which received soluble fiber and fructooligosaccharides-FOS. Ninety six Wistar male rats were used, average weight 300g. They were distributed in two groups, of 48 animals each. One group with animals without colitis (GESC) submitted to transanal water enema and another group with colitis (GEC) produced by TNBS, 20mg. Each group was distributed into three subgroups, according to the used substance: 1(water), 2(soluble fiber), 3(FOS). All the animals received 3 ml such substances twice daily into the stomach through an intragastric catether during 14 days before the transanal water enema and the colitis production until the euthanasia day (7th and 14th. Days). The animals were evaluated concerning to the weight, colon weight/size relationship, macroscopic and microscopic scores and the metabolites colon in vivo concentrations (ketone bodies [3-hydroxybutirate, acetoacetate], piruvate, lactate and ATP). The cell redox estate was also evaluated by the [acetoacetate]/[3-hydroxybutirate] and [piruvate]/[lactate] relationship. GESC and GEC were evaluated, equally the subgroups water from the group without colitis (ESC1) comparing to the colitis group (EC1). No significant differences were found in the various inflammatory evaluated parameters in GESC group. An increased colon weight/size relationship was found in GEC group in the subgroup FOS, on the 14th day (p<0,05). Concerning to the animals weight from the subgroup GEC1, a significant decreasing was found on the 7th day. An increased colon weight/size relationship was found in macroscopic and microscopic scores in the subgroup GEC1 on the 7th and 14th days (p<0,05). Concerning to the metabolic evaluation, an increased acetoacetate concentration was found in GESC, in the fiber subgroup on the 14th day (p<0,05). Decreased piruvate concentrations in the fiber and FOS subgroups were demonstrated on the 14th day (p<0,05). The ATP concentrations were reduced in the fiber and FOS subgroups on the 7th and 14th days (p<0,05). The piruvate concentrations were increased in GEC group, in the fiber subgroup, on the 14th day (p<0,05). The ATP concentrations decreased in the fiber and FOS subgroups (p<0,05). Comparing the subgroups GESC1 with GEC1, an increased cetonic corps concentration was found in GEC1 on the 7th and 14th days (p<0,05). There was reduction of piruvate levels in GEC1 on the 7th and 14th days (p<0,05). There was also reduction in acetoacetate/3-hidroxibutirate and piruvate/lactate relationship in GEC1, on the 7th day (p<0,05). Itâs concluded that the soluble fiber or FOS intragastric intake doesnât change the evaluated inflammatory parameters on the 7th and 14th days in normal colons or during colitis process, but it increases the precursors offering for energy production, modifying the ATP in vivo concentrations in normal colonic tissue and during colitis process.

Els objectius d'aquesta tesi han estat l'estudi de la funció de les cèl·lules intersticials de Cajal (ICCs) en l'activitat marcapassos i en la transmissió neuromuscular, dins el tracte gastrointestinal. L'estudi s'ha realitzat en el còlon perquè en aquesta part del tracte gastrointestinal els mecanismes marcapassos i de neurotransmissió són poc coneguts. El model de rata s'ha utilitzat perquè estudis previs, realitzats al nostre laboratori, van mostrar-hi un marcapassos d'origen miogènic, també descrit en el còlon humà. A més a més, les rates Ws/Ws tenen una mutació en el gen c-kit i presenten una afectació de certes línies cel·lulars, incloses les ICCs. Per tant, hem utilitzat tres grups d'animals: rates Sprague-Dawley, mutants Ws/Ws i les seves germanes controls +/+. Per investigar l'activitat marcapassos i la neurotransmissió vam realitzar: (i) tincions immunohistoquímiques amb anticossos c-kit i nNOS per marcar ICCs i neurones nitrèrgiques, respectivament; (ii) experiments d'activitat mecànica en bany d'òrgans; (iii) enregistraments elèctrics amb la tècnica de microelèctrodes intracel·lulars.
Els nostres estudis mostren dues xarxes d'ICCs en el còlon de rata, a nivell dels plexes d'Auerbach (ICC-AP, 100 cèl·lules/mm2) i submuscular (ICC-SMP,
50-60 cèl·lules/mm2). Les ICCs intramurals (ICC-IM) van trobar-se distribuïdes de forma paral·lela entre les cèl·lules musculars llises. Resultats similars van ser obtinguts en rates Sprague-Dawley i +/+. En canvi, les rates mutants Ws/Ws van presentar una marcada disminució d'immunoreactivitat al c-kit. Les rates Sprague-Dawley van presentar una activitat elèctrica espontània formada per ones lentes i despolaritzacions cícliques, seguint un patró regular. Aquesta activitat elèctrica dóna lloc a contraccions d'alta i baixa freqüència, respectivament. Les contraccions d'alta i baixa freqüència varien en relació a l'orientació de la tira de múscul (orientació circular o longitudinal) i també en relació al segment estudiat (còlon proximal, mig i distal). L'activitat elèctrica i mecànica de les rates +/+ va ser similar a la presentada per les rates Sprague-Dawley. Les rates Ws/Ws, en canvi, van mostrar una alteració dels patrons mecànic i elèctric, presentant potencials d'acció irregulars que donaven lloc a contraccions incoordinades.
La neurotransmissió va ser estudiada mitjançant: i) la distribució i quantificació de les neurones nNOS; ii) el potencial post unió inhibitori (IJP) i la relaxació mecànica induïda per l'estimulació elèctrica de camp; iii) la presència d'un to neural. Les neurones nNOS positives es van trobar a la regió del plexe d'Auerbach formant una xarxa de ganglis i branques, acompanyades per fibres primes que van trobar-se paral·leles a les cèl·lules llises musculars ( en major nombre en la capa muscular circular). El còlon mig va ser la zona més inervada (130 cèl·lules/mm2). En les rates Ws/Ws, es va observar una lleugera reducció en el nombre de neurones nitrèrgiques. Tant en rates Sprague-Dawley, com Ws/Ws i +/+, l'estimulació elèctrica de camp va causar un IJP i relaxació mecànica, degut a l'alliberació d'ATP i NO. A més a més, es va evidenciar un to neural funcional (inhibitori), provocat per l'alliberació tònica d'ATP i NO.
Per tant, podem concloure que: (i) la presència de dues xarxes d'ICCs és un element indispensable per la correcta funció marcapassos en el còlon; (ii) les ICC-SMP serien responsables de les ones lentes de les quals s'originen les contraccions d'alta freqüència; (iii) les ICC-AP serien l'origen de les despolaritzacions cícliques que provoquen les contraccions de baixa freqüència; (iv) les propietats mecàniques de cada tira serien les responsables dels patrons motors que asseguren el trànsit, propulsió i barreja.; i finalment (v) les ICCs c-kit positives són elements no essencials per la neurotransmissió a no ser que aquesta funció la pogués acomplir les cèl·lules intersticials c-kit negatives o bé que es produís un canvi del fenotip en les rates mutants Ws/Ws.
The objective of this research project was to investigate the role of interstitial cells of Cajal (ICCs) as pacemaker cells and as mediators of neuromuscular transmission in the gastrointestinal tract. We have studied the rat colon because in this part of the gastrointestinal tract the pacemaker and neurotransmission mechanisms are poorly understood. The rat is used as the animal model because previous work from our laboratory showed that a myogenic pacemaker is present, similarly to what it has been described in the human colon. Moreover, rats with a mutation in the c-kit gene show impairment of the development of certain classes of cell lineages, including ICCs. Accordingly, we have used three groups of animals: (i) Sprague-Dawley; (ii) Ws/Ws mutant rats and (iii) +/+ rats (the siblings of Ws/Ws mutant rats). To study the pacemaker activity and neurotransmission we have performed: (i) immunohistochemical stainings with c-kit and neuronal nitric oxide synthase (nNOS) antibodies to label ICCs and nitrergic neurons respectively; (ii) mechanical experiments with muscle bath technique and (iii) intracellular electrical recordings with microelectrode technique.
Our results demonstrate that ICCs are present in the rat colon forming two networks at the level of the Auerbach's (ICC-AP, around 100 cells/mm2) and submuscular plexuses (ICC-SMP, about 50-60 cells/mm2). Intramural ICCs (ICC-IM) were found running parallel and between smooth muscle cells. Similar results were obtained both in Sprague-Dawley and +/+ rats. In contrast, a strong reduction in c-kit immunoreactivity was found in Ws/Ws rats. The spontaneous electrical activity in Sprague-Dawley rats consisted of slow waves superimposed with cyclic depolarizations forming a regular pattern. These two electrical events were responsible for high frequency (HF) and low frequency (LF) contractions, respectively. HF and LF contractions varied in relation to the orientation of the muscle strip (circular or longitudinal orientation) and the segment studied (proximal, mid and distal colon). The electrical and mechanical activities found in +/+ rats were very similar to those described in Sprague-Dawley rats. In contrast, Ws/Ws rats showed an impairment of the electrical and mechanical patterns, characterized by irregular action potentials, which cause uncoordinated contractions.
Neurotransmission was studied with by: (i) the distribution and quantification of nNOS positive neurons; (ii) the inhibitory junction potential (IJP) and mechanical relaxation induced by electrical field stimulation and (iii) the presence of a neural tone. nNOS positive neurons were found at the Auerbach's plexus (AP) region forming a network of nerve strands and ganglia. Moreover, fine fibers were found running parallel to smooth muscle cells, especially in the circular muscle layer. The mid colon was the most innervated segment (about 130 cells/mm2). A slight reduction in nNOS positive neurons was observed in Ws/Ws rats compared to control rats.
Electrical field stimulation caused an IJP and mechanical relaxation due to the release of ATP and NO. Moreover, a functional neural tone due to the ongoing release of ATP and NO was present. These results were observed in the three groups of animals: Sprague-Dawley, +/+ and Ws/Ws rats.
We conclude that: (i) the presence of two ICC networks is a crucial element responsible for the pacemaker activity in the colon; (ii) the ICC-SMP network is probably responsible for the slow wave activity that originates HF contractions; (iii) the ICC-AP network might be responsible for the cyclic depolarizations that trigger LF contractions; (iv) the mechanical properties of each strip are responsible for the motor events that ensure transit, propulsion and mixing and (v) c-kit positive ICC cells are not essential elements for neurotransmission, unless this function could be accomplished by c-kit negative interstitial cells or alternatively, a change in the phenotype of Ws/Ws animals occurs.

Cette these decrit le clonage de deux sous-unites du canal sodium epithelial sensible au diuretique amiloride dans le colon de rat. La premiere sous-unite de 699 acides amines a ete isolee par clonage par expression dans l'oocyte de xenope. Elle est capable de generer dans l'oocyte de xenope une faible activite canal sodium possedant la meme pharmacologie et la meme selectivite que le canal natif. Une proteine similaire a ete clonee dans le poumon humain et son expression est fortement augmentee dans le poumon de rat au moment de la naissance. La seconde sous-unite de 650 acides amines a ete isolee par homologie avec la premiere sous-unite. Elle ne possede pas d'activite canal sodium par elle-meme dans l'oocyte de xenope mais elle potentialise l'activite obtenue avec la premiere sous-unite. Le canal sodium sensible a l'amiloride est en fait constitue par au moins trois sous-unites homologues. Seule l'expression des trois sous-unites dans l'oocyte de xenope est capable de generer un important courant sodium. Ces sous-unites ne sont homologues a aucun canal ionique connu et possedent une structure nouvelle pour les canaux ioniques. Elles sont homologues a une famille de proteines du nematode c. Elegans, appelees degenerines, qui sont impliquees dans la transduction mecanique et certaines formes de neurodegenerescence. La regulation a long terme par l'aldosterone de l'activite du canal sodium sensible a l'amiloride dans le colon de rat est due a la neo-synthese des deux dernieres sous-unites de ce canal

L'oliva i l'oli d'oliva verge són un dels ingredients fonamentals que caracteritzen la Dieta Mediterrània, definida en la dècada dels anys 60 a partir dels costums alimentaris dels habitants de l'illa de Creta ("The Seven Countries Study"). Per altra banda, tot i que la poma no era una fruita habitual a l'illa de Creta, no deixa de ser una fruita que com a tal forma part de la Dieta Mediterrània. A més a més, és consumida a nivell mundial, està disponible durant tot l'any i és apta per a totes les butxaques. En les últimes dècades, els genetistes de plantes han treballat i segueixen treballant en la millora de noves varietats de poma de polpa vermella ("red-fleshed apples") ja que són més atractives pel seu consum degut a la seva coloració rosa-vermella, com a conseqüpencia de la presència d'antocians en la polpa. La present Tesi Doctoral s'ha centrat en tres objectius: Objectiu 1. Avaluar l'impacte de la suplementació de la dieta amb els principals compostos fenòlics de l'oliva/oli d'oliva enfront la concentració d'antioxidants endògens (α-tocoferol) en múscul i fetge. Objectiu 2. Estudiar durant dues campanyes consecutives el perfil fitoquímic (compostos fenòlics, antocians, triterpens, àcids orgànics i àcid ascòrbic) de noves varietats de poma de polpa vermella obtingudes mitjançant millora varietal, utilitzant com a referència diferents varietats de poma de polpa blanca ('Golden Smoothee', 'Granny Smith'...). Objectiu 3. Determinar l'impacte de la suplementació de la dieta amb poma de polpa vermella, utilitzant com a referència poma de polpa blanca, en el desenvolupament i progressió del càncer de còlon (CC) induït químicament en un model murí. En relació a l'Objectiu 1, s'ha realitzat una suplementació de la dieta en model de rata Wistar amb compostos fenòlics d'oliva (oleuropeïna, hidroxitirosol i secoiridoids), de farigola, i amb una combinació d'aquests (5 mg /kg pes /dia), durant un període de 21 dies. Els resultats han mostrat que la dieta suplementada amb alguns compostos fenòlics d'oliva i timó han augmentat de forma significativa la concentració d'α-tocoferol en fetge de rates femelles. A més a més , s'ha observat un efecte sinèrgic entre els fenols d'oliva i de la farigola. Aquest increment observat de la concentració hepàtica de α-tocoferol, suggereix el possible potencial de la suplementació dietètica amb compostos fenòlics com a estratègia per al control de la malaltia hepàtica més comuna en l'actualitat: l'hepatopatia grassa no alcohòlica. En relació a l'Objectiu 2, els resultats han mostrat que les varietats de poma "red-fleshed" estudiades contenen una quantitat de flavanols més baixa, una concentració d'àcids orgànics similar a la varietat 'Granny Smith', i una major concentració d'antocians (cianidina-3-O-galactòsid) i dihidrochalcones (floretin xilosil-glucòsid) en comparació a les varietats de polpa blanca. A més a més, s'ha detectat un important efecte campanya en la concentració dels diferents compostos fenòlics en totes les varietats de poma estudiades, tot i que el comportament observat difereix entre la pell i la polpa. A diferència d'altres metabòlits secundaris, la variació inter-campanya d'antocians en les pomes de polpa vermella no ha mostrat una relació amb les condicions climàtiques de l'any (pluviometria i temperatura mitjana). Pel que fa al model experimental de CC (Objectiu 3), dut a terme en rates mascle Wistar, els resultats han mostrat que la suplementació de la dieta amb 'Golden Smoothee' (polpa blanca) aconsegueix una inhibició del 41.3% de focus amb criptes aberrants (lesió preneoplàsica CC). D'altra banda, els nivells de mucina colònica s'han mantingut normals per efecte de la suplementació amb 'Golden Smoothee' i amb la cianidina-3-O-galactòsid (principal antocià de la poma). Aquests resultats reforcen el refrany Gal·lès "An apple a day keeps the doctor away" en la prevenció del desenvolupament de càncer de còlon.
La aceituna y el aceite de oliva virgen son uno de los ingredientes fundamentales que definen a la Dieta Mediterránea, definida en la década de los años 60 a partir del patrón alimentario de los habitantes de la isla de Creta (“The Seven Countries Study”). Por otra parte, aunque la manzana no era una fruta habitual en la isla de Creta, no deja de ser una fruta que como tal forma parte de la Dieta Mediterránea. Además, es consumida a nivel mundial, está disponible durante todo el año y es asequible económicamente. En las últimas décadas, los genetistas de plantas han trabajado y trabajan en la mejora de nuevas variedades de manzana de pulpa roja (“red-fleshed apples”) para hacerlas más atractivas para su consumo, en base a la presencia de antocianos en la pulpa. La presente Tesis Doctoral se ha centrado en tres objetivos: Objetivo 1. Evaluar el impacto de la suplementación de dieta con los principales compuestos fenólicos de la aceituna/aceite de oliva sobre la concentración de antioxidantes endógenos (α-tocoferol) en músculo e hígado. Objetivo 2. Estudiar durante dos campañas consecutivas el perfil fitoquímico (compuestos fenólicos, antocianos, triterpenos, ácidos orgánicos y ácido ascórbico) de nuevas variedades de manzana de pulpa roja obtenidas mediante mejora varietal, utilizando como referencia diferentes variedades de manzana de pulpa blanca ('Golden Smoothee', 'Granny Smith'...). Objetivo 3. Determinar el impacto de la suplementación de la dieta con manzana de pulpa roja, utilizando como referencia manzana de pulpa blanca, sobre el desarrollo y progresión del cáncer de colon (CC), inducido químicamente en un modelo murino. En relación al Objetivo 1, se ha realizado suplementación de la dieta en modelo de rata Wistar con compuestos fenólicos de oliva (oleuropeína, hidroxitirosol y secoiridoides), de tomillo, y con una combinación de estos (5 mg/kg de peso/día), durante un periodo de 21 días. Los principales resultados han mostrado que la dieta suplementada con algunos compuestos fenólicos de oliva y tomillo aumentó de forma significativa la concentración de α-tocoferol en hígado de ratas hembras. Además, se ha observado un efecto sinérgico en la combinación de fenoles de oliva y de tomillo. Este incremento de la concentración hepática de α-tocoferol observado sugiere el potencial de la suplementación dietética con compuestos fenólicos, como estrategia para el control de la enfermedad hepática más común en la actualidad, la hepatopatía grasa no alcohólica. En relación al Objetivo 2, los resultados han mostrado que las variedades de manzana “redfleshed” estudiadas contienen una cantidad de flavanoles más baja, una concentración de ácidos orgánicos similar a la variedad ‘Granny Smith’, y una mayor concentración de antocianos (cianidina-3-O-galactósido) e dihidrocalconas (floretin xilosil-glucósido), en comparación a las variedades de pulpa blanca. Además, se ha detectado un importante efecto campaña en la concentración de los diferentes compuestos fenólicos en todas las variedades de manzana estudiadas. Aunque el efecto campaña difiere entre la piel y la pulpa. A diferencia de otros metabólitos secundarios, la variación inter-campaña de antocianos en las manzanas de pulpa roja no mostró una relación clara con las condiciones climáticas del año (pluviometría y temperatura media). En cuanto al modelo experimental de CC (Objetivo 3), llevado a cabo en ratas macho Wistar, los resultados han mostrado que la suplementación de dieta con ‘Golden Smoothee’ (pulpa blanca) consigue una inhibición del 41.3% de focos con criptas aberrantes (lesión preneoplásica CC). Por otra parte, los niveles de mucina colónica se han mantenido normales por efecto de la suplementación con ‘Golden Smoothee’ y con cianidina-3-O-galactósido (principal antociano de la manzana). Estos resultados refuerzan el refrán Galés "An apple a day keeps the doctor away" en la prevención del desarrollo de cáncer de colon.
The olive and virgin olive oil are one of the fundamental ingredients that characterize the Mediterranean Diet, defined in the 1960s from the eating habits of the inhabitants of the island of Crete (“The Seven Countries Study”). On the other hand, even though the apple was not an usual fruit on the island of Crete, it is still a fruit that as such is part of the Mediterranean Diet. Also, it is consumed worldwide, it is available all year round and it is economically affordable. In recent decades, plant geneticists have worked and are still working on the improvement of new varieties of red-fleshed apples which are more attractive for consumption, due to their red-pink flesh colour as consequence of the presence of anthocyanins in the flesh. This present Doctoral Thesis has focused on three objectives: Objective 1. Evaluate the impact of dietary supplementation with the main phenolic compounds of olive/olive oil on the concentration of endogenous antioxidants (α-tocopherol) in the muscle and in the liver. Objective 2. Study for two consecutive harvest seasons the phytochemical profile (phenolic compounds, anthocyanins, triterpenes, organic acids and ascorbic acid) of new varieties of redfleshed apples obtained through varietal improvement, using as reference different varieties of white-fleshed apples ('Golden Smoothee', 'Granny Smith'...). Objective 3. Tackle the impact of dietary supplementation with red-fleshed apples, using as a reference white-fleshed apples, on the development and progression of colon cancer (CC), chemically induced in a murine model. As regards as Objective 1, dietary supplementation has been carried out in the Wistar rat model with olive phenolic compounds (oleuropein, hydroxytyrosol and secoiridoides), thyme, and with a combination of these (5 mg /kg body weight /day), for a period of 21 days. The main results have shown that the supplemented diet with some phenolic compounds of olive and thyme have significantly increased the concentration of α-tocopherol in the liver of female rats. In addition, a synergistic effect was observed in the combination of olive and thyme phenols. The observed increase in the hepatic concentration of α-tocopherol suggests the potential of the dietary supplementation with phenolic compounds, as a strategy for the control of the currently most common liver disease: the non-alcoholic fatty liver disease. In relation to Objective 2, the results have shown that the “red-fleshed” apple varieties studied contain a lower amount of flavanols, a concentration of organic acids similar to the 'Granny Smith' variety, and a higher concentration of anthocyanins (cyanidin-3-O-galactoside) and dihydrocalcones (phloretin xylosyl glycoside), compared to white-fleshed cultivars. Besides, an important season effect has been detected in the concentration of the different phenolic compounds in all the apple varieties studied. Even though, the harvest season effect differed between the skin and the flesh part. Unlike other secondary metabolites, the inter-season variation of anthocyanins in red-fleshed apples has not shown a clear relationship with the climatic conditions of the year (rainfall and average temperature). Regarding the experimental model of CC (Objective 3), carried out in male Wistar rats, the results have shown that the dietary supplementation with 'Golden Smoothee' (white-fleshed) achieves a 41.3% inhibition of foci with aberrant crypts (preneoplastic lesion CC). On the other hand, the levels of colonic mucin have remained normal as a result of the supplementation with ‘Golden Smoothee’ and with cyanidin-3-O-galactoside (main anthocyanin of apples). These results reinforce the Welsh saying "An apple a day keeps the doctor away" in preventing the development of colon cancer.

Dans ce travail, les modifications du metabolisme energetique des cellules epitheliales intestinales sont etudiees: lors du developpement intestinal chez le porc nouveau-ne. Dans ce modele, le jejunum, contrairement a l'ileon, subit une maturation importante entre la naissance et 2 jours d'allaitement en l'absence de stimulation de l'epithelium colique chez le rat depourvu de flore intestinale (rat axenique). Nous avons mis en evidence in vivo (par la technique d'utilisation du 2-deoxyglucose) et in vitro (sur cellules epitheliales isolees) une augmentation entre la naissance et deux jours de vie de l'utilisation de glucose specifique a la partie proximale de l'intestin grele. De plus, nous avons montre le role de l'hexokinase et du transport baso-lateral de glucose dans ce developpement. Chez le rat axenique, nous avons mis en evidence une augmentation de l'utilisation de la glutamine dans les colonocytes isoles a partir de ce modele. La capacite a utiliser le butyrate est, elle, conservee. Toutefois, la capacite a produire des corps cetoniques a partir de ce substrat est plus faible. Cela est imputable a une diminution de l'hmg coa synthase, cette enzyme etant regulee au niveau de l'expression de son gene

La toxine thermostable d'escherichia coli (sta) perturbe les mecanismes actifs sur lesquels repose la fonction absorptive du colon. In vitro, l'activite de ces mecanismes peut etre electrophysiologiquement mesuree par la valeur du courant de court-circuit (isc) que genere l'epithelium. Sta entraine une augmentation du isc. Cette augmentation est environ trois fois plus grande dans le colon proximal que dans le colon distal. L'action secretagogue de sta est mimee par le 8-br-gmpc. La mesure des flux unidirectionnels revele que sta diminue l'absorption de sodium et de chlorures dans les segments distaux, alors qu'elle baisse l'absorption de sodium et active la secretion de chlorure dans les segments proximaux. Au niveau du colon distal uniquement, l'indometacine inhibe l'action de sta et du 8-br-gmpc. Le mecanisme d'action de sta est different dans les deux segments. Au niveau du segment distal elle consiste en l'inhibition d'une phosphodiesterase de l'ampc par le gmpc. L'individualite des parties distale et proximale est confirmee par l'heterogeneite de leurs reponses aux voies secretagogues ampc et calcium dependantes ainsi que par une etude morphologique

Purification des recepteurs mineralocortiroide (ou recepteur de l'aldosterone de type i) par chromatographie d'affinite. Ces recepteurs ont ete caracterises dans le rein du rat surrenalectomise, le colon humain normal et cancereux et le rein de lapin surrenalectomise. Parmi les agonistes et antagonistes de l'aldosterone etudies, seul un divise en 3 de la dioxycorticosteione presentait les qualites d'affinite et de specificite pour en faire un bon liganol d'affinite. Le gel synthetise avec ce derive s'est avere hautement specifique et selectif et a permis de purifier les recepteurs mineralocorticoides d'unifacteur 1000

L’administration par voie orale des médicaments reste encore de nos jours la voie royale de la prise des médicaments car moins onéreuse et plus adaptée au confort du patient. Mais cette voie reste toujours inaccessible pour certains médicaments comme les médicaments biologiques et les bio similaires voir certains anticancéreux et antirétroviraux.Le but de ce travail est d’améliorer la biodisponibilité par voie orale des médicaments à faible biodisponibilité par la mise au point d’un promoteur d’absorption. Pour y arriver nous avons adopté comme stratégie de développer un promoteur qui agit à la fois sur le passage passif et sur le passage actif des médicaments. Les études in vitro ont été réalisées en chambre de perméation d’Ussing adaptées par la société Biomécatronics SAS (BéthuneFrance). Dans la première partie de ce travail (Brevet), nous avons montré que l’utilisation d’une composition pharmaceutique et/ou diététique comprenant un extrait de plante(Hibiscus sabdariffa) pouvait augmenter la biodisponibilité in vitro des médicaments et des xénobiotiques qui passent par la voie paracellulaire comme le cisplatine (21 fois),l’oxaliplatine (11fois), la fluorescéine isothiocyanate-Dextran 4000 (3 fois), mais également les médicaments connus pour leur transport actif par la voie transcellulaire comme l’Efavirenz (7 fois) et l’Atazanavir (4 fois). Dans la seconde partie de ce travail, nous avons cherché à vérifier si notre promoteur d’absorption des médicaments a un effet sur la couche de mucus intestinale.Cette couche peut être un facteur limitant de passage des médicaments au travers de la barrière intestinale.Dans un premier temps (article 1), nous avons induit l’augmentation de la couche mucus au niveau du colon de rat après un prétraitement pendant une semaine avec le métronidazole. Puis nous avions confirmé (article 2) que l’administration par voie orale de deux antibiotiques le Cotrimoxazole (CTX) et le métronidazole (MTZ) pendant une semaine augmente la couche de mucus au niveau du côlon ; aussi nous avons montré qu’il existe une relation entre l’augmentation de la couche de mucus et la diminution de la conductance qui est l’index de transport passif des ions, des électrolytes et de certaines molécules à faibles poids moléculaires.De plus l’augmentation de la couche de mucus au niveau de l’intestin est responsable de la diminution du passage transépithélial des deux antirétroviraux dont l’utilisation est recommandée en première ligne par l’OMS (le.Ritonavir et l’Atazanavir) surles sujets porteurs du VIH (virus de l’immunodéficience humain). Après les traitements auMTZ et au CTX la sécrétion de l’Atazanavir augmente respectivement dans le côlon proximal de 2 et 4 fois et dans le côlon distal de 3 et 5 fois. On obtient également une sécrétion du Ritonavir de 5 et 10 fois dans le proximal et de 2 et 5 fois plus dans le distal.Le travail se poursuit par l’étude de l’effet de notre promoteur d’absorption des médicaments sur la couche de mucus intestinal.En conclusion, ce travail montre que l’on peut augmenter la biodisponibilité in vitroen utilisant les promoteurs de l’absorption des xénobiotiques qui agissent à la fois au niveau du transport passif et actif
Oral dosing is still seen as the silver bullet of drug administration, as it is cheaper andbetter adapted to patient comfort. However, oral route is still inaccessible to many drugssuch as biologics and biosimilars respectively certain anticancer drugs and antiretrovirals(ARV).The aim of this present study was to find new drugs enhancers that improve the oralbioavailability of drugs and xenobiotics. All the studies were realized in vitro using Ussingchambers technic. To achieve the set objective we used the strategy to develop drugenhancer which can modulate at the same time transcellular and paracellular pathways.In the first part of this study (patent) we have shown that the use of a pharmaceutical and /or a dietetic formulation containing a plant extract (Hibiscus sabdariffa) could increase thebioavailability in vitro in rats not only of cisplatin (21 fold), oxaliplatin (11 fold) andFluorescein Isothiocyanate-Dextran 4000 (FD4, 3 fold). All that drugs were transportedthrough intestinal barrier using paracellular pathway. In addition the study showed thatthis formulated enhancer can increased the bioavailability of Efavirenz (7 fold) andAtazanavir (4 fold) which are active transported.In order to assess the effect of new drugs enhancer on mucus thickness that limits thetransport of xenobiotic through intestinal barrier, we decide to evaluate his effect on passiveand active transport of drugs.In the second part of this study we have shown that after a week of pre-treatment of ratswith Metronidazole (MTZ, publication 1) and Cotrimoxazole (CTX, publication 2), the twomost commonly used antibiotics in the prophylaxis against opportunistic infections in HIV /AIDS, both increase colonic mucus thickness that affect directly passive intestinalpermeability by reducing conductance an index of passive transport through intestinalepithelium. In addition those antibiotics also entail a change in the transepithelialconductance and ARV fluxes. After MTZ and CTX treatment the secretion of Atazanavir(ATZ) increases respectively in the proximal colon by 2 to 4 fold and in the distal colon by 3to 5 fold respectively. Ritonavir (RTV) is poorly absorbed in control, after a week of pretreatmentwith MTZ and CTX one rather notices a secretion of RTV 5 to 10 fold higher in theproximal and 2 to 5 fold higher in the distal colon. The next study will be conducted toevaluate the effect of new drugs enhancer on mucus thickness layer.In conclusion, oral bioavailability of drugs and xenobiotics can be enhanced bypharmaceutical composition that contains herbal extract which increase passive and activetransport of drugs through intestinal barrier

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Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)
Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)
Yacon (Smallanthus sonchifolius) é uma raiz originária da região dos Andes que tem se destacado pelos seus compostos bioativos principalmente frutanos como futooligossacarídeos e inulina. O presente projeto teve como objetivo determinar a atividade quimioprotetora da ingestão de Yacon sobre o desenvolvimento de lesões pré-neoplásicas (focos de criptas aberrantes-FCA) induzidas pela dimetilhidrazina (DMH) em ratos Wistar machos. Os animais foram divididos em seis grupos com 5 a 12 animais cada. Os animais dos Grupos 1 a 4 e Grupos 5 e 6 receberam respectivamente, quatro injeções subcutâneas de DMH (40 mg/Kg) e solução de EDTA (veículo da DMH) nas duas semanas iniciais do experimento respectivamente. Os animais receberam ração basal até a sexta semana do experimento e a partir desta os animais dos grupos 2, 3, 4, 5 receberam ração acrescida de extrato de Yacon a 0,5%, 1%, 1% e 1%, respectivamente. Os animais do grupo 4 receberam Lactobacilus casei (2,5 x 1010 de UFC por Kg de ração). O sacrifício ocorreu na vigésima semana de experimento para análise de focos de criptas aberrantes (FCA) e tumores. Nossos resultados mostraram uma redução no número, multiplicidade de FCA e no número de adenocarcinomas invasivos nos grupos tratados com 1% yacon (G3) e na combinação simbiótica (G4), (0,05 < p < 0, 001). A multiplicidade de tumores (invasivos e não invasivos) foi significativamente menor no grupo tratado com a combinação simbiótica (p < 0,02). Observou-se também uma redução significativa nas taxas de proliferação celular tanto em criptas colônicas como em tumores nos grupos tratados com 1% yacon (G3) e na combinação simbiótica (G4), p < 0.001. Os resultados sugerem que a ingestão de extrato de yacon exerce atividade quimiopreventiva contra carcinogênese de cólon
Yacon (Smallanthus sonchifolius) is a tuberous root native to the Andean region of South America which contains high amounts of inulin-type fructans. The present study investigated the beneficial potential of yacon root intake on development of colon carcinogenesis induced by 1,2-dimethylhydrazine (DMH) in male Wistar rats. Six groups were studied: Groups 1–4 were given four s.c. injections of DMH (40 mg/kg b.w.) twice a week, during two weeks, whereas Groups 4 and 5 received similar injections of EDTA solution (DMH vehicle). After 6 weeks of DMH-initiation, groups were fed with basal diet (G1 and G6) or basal diet containing dried extract of yacon root at 0.5% (G2), 1.0% (G3 and G5) or a synbiotic formulation (G4) (1.0% yacon root plus Lactobacillus casei at 2.5 x 1010 CFU per g diet) for 13 weeks. At 20 week, all animals were killed and the colons were analyzed for development of aberrant crypt foci (ACF) and tumor. A significant reduction in number and multiplicity of ACF and in number of invasive adenocarcinomas was observed in the groups orally treated with 1.0% yacon root (G3) or their synbiotic formulation (G4) (0.05 < p < 0.001). Tumor multiplicity (noninvasive plus invasive) was significantly lower solely in group fed with symbiotic formulation (p < 0.02). Also, a reduction in cell proliferation indexes in colonic crypt and tumor were observed in groups orally treated with 1.0% yacon root (G3) or their synbiotic formulation (G4) (p < 0.001). The findings this study suggest that yacon root intake may have potential as chemopreventive agent against colon carcinogenesis

A majority of the arid grasslands in the western U.S. have been dramatically altered by anthropogenic influences resulting in degradation and desertification. Within the arid grasslands of North America a guild of burrowing herbivorous rodents that includes kangaroo rats (Dipodomys spp.) and prairie dogs (Cynomys spp.) is often considered integral to arid grassland maintenance. As part of the larger guild of burrowing herbivorous rodents, kangaroo rats are considered to be an important keystone guild whose role as ecosystem engineers and habitat modifiers complements that of prairie dogs. Together these species organize and structure arid grassland ecosystems and the biodiversity therein, by providing a mosaic of microhabitat patches, thus increasing overall heterogeneity. In an area where black-tailed prairie dogs (C. ludovicianus) were reintroduced, I used Giving-up Density (GUD) to assess the indirect effects black-tailed prairie dogs might have on the foraging patterns of resident kangaroo rats (D. spectabilis and D. merriamii). My objective was to compare and contrast kangaroo rat foraging GUD within and along the boundary of a on a recently established black-tailed prairie dog colony with that in the surrounding unmodified native habitat. This enabled assessment of whether black-tailed prairie dogs had an influence on the perceived quality of the habitat by kangaroo rats. Kangaroo rats visited off-colony feeding trays more frequently, and collected a greater mean mass of seed per tray as well. This indicates that the kangaroo rats perceived the area off the prairie dog colony as having a lower foraging cost than on the colony or along the colony edge. I conclude that from the perspective of the seed-eating kangaroo rat, the colony is not viewed as high quality habitat. What impact the reintroduction and management of one keystone species might have on another keystone species deserves additional consideration as we attempt to restore arid grassland ecosystems.

Stress-related functional bowel disease is a common, often incapacitating, problem in humans; the symptomatology of stress-related intestinal dysfunction is: (1) impaired small intestinal transit and motility, and (2) increased large intestinal transit and, commonly, diarrhea. The etiology of stress-induced intestinal dysfunction is completely unresolved, and the lack of an appropriate animal model has hindered studies of causality. We compared a number of stressors and their resultant effects on intestinal transit, a measure of the propulsive motor activity of the gut, in the rat. We found that the response of the intestine to stress, and the neural systems activated by stress, were dependent on the type and duration of stress, as well as the animal strain, and gender. We developed a model, acute wrapping restraint stress, to fully characterize the effects of stress on intestinal transit. Wrap restraint stress is a nonulcerogenic model in which rats are subjected to acute restraint by wrapping them in a harness of paper tape to restrict, but not prevent movement of the upper body and forelimbs. Transit was evaluated by the geometric center method, in which a radiomarker (⁵¹Cr) is instilled directly into the proximal duodenum and proximal colon via a surgically placed intestinal cannula, in fasted, adult female Sprague Dawley rats (150-200g). Subjecting animals to 35 min. of wrap restraint stress resulted in (1) inhibition of small intestinal transit, and (2) increased large intestinal transit and increased fecal output. The effects of stress on intestinal transit in rats resembled symptoms associated with stress in humans, suggesting that wrap restraint stress may be suitable as a model of stress-induced intestinal dysfunction. We found a close correlation between stress-induced intestinal dysfunction and stress-activation of endocrine systems. Stress-induced changes in intestinal function was strongly influenced by circadian variations in endocrine levels, suggesting that stress-induced intestinal dysfunction may be hormonally mediated. However, neither pituitary nor adrenal factors mediated the effects of stress on the gut. To evaluate the role of corticotropin-releasing factor (CRF), the major hypothalamic factor released in response to stress, in stress-induced intestinal dysfunction, we studied the effects of exogenous CRF on intestinal transit. CRF resulted in (1) a potent, dose-dependent inhibition of small intestinal transit, (2) a dose-dependent increase in large intestinal transit, and (3) increased fecal excretion. The effects of exogenously administered CRF closely paralleled the effects of stress on intestinal transit and on ACTH secretion in the rat. Blockade of CRF receptors by means of an antagonist, α helical CRF (9-41), prevented the effects of stress on colonic transit and fecal excretion. These data strongly suggest that endogenous CRF may mediate the effects of wrap restraint stress on intestinal motor activity and coordination in the rat.

Colon cancer is the third most common cancer in the United States. However, evidence indicates that a proper diet abundant in fruits and vegetables may be protective against colon cancer development. Bioactive compounds in fruits and vegetables, such as flavonoids and limonoids, have been shown to possess anti-proliferative and antitumorigenic effects in various in vitro and in vivo models of cancer. Since there are few animal studies involving flavonoids and limonoids and colon cancer, this experiment investigated the potentially protective effects of four citrus flavonoids and one limonoid mixture against the promotion stage of chemically-induced colon cancer in rats. Male SD rats (n =60; 10 rats/group) were assigned to receive diets containing 0.1% apigenin, 0.02% naringenin, 0.1% hesperidin, 0.01% nobiletin, 0.035% limonin glucoside/obacunone glucoside mixture, or a control diet (0% flavonoid/limonoid). Rats received the diets for 10 wk and were injected with azoxymethane (15 mg/kg) at wk 3 and 4. The excised colons were evaluated for aberrant crypt foci (ACF) formation, cell proliferation (PCNA assay), apoptosis (TUNEL assay), and iNOS and COX-2 expression. When compared to the control diet, apigenin lowered the number of high multiplicity ACF (> 4 AC/focus) by 57% (P<0.05) and tended to lower the proliferative index (28%; P=0.07), while naringenin lowered both the number of high multiplicity ACF by 51% (P<0.05) and the proliferative index by 32% (P<0.05). Both apigenin and naringenin increased apoptosis of surface colon cells (78% and 97%, respectively; P<0.05) when compared to control diet. Hesperidin, nobiletin, and the limoninglucoside/obacunone glucoside mixture did not have any effects on the above variables measured in this model of colon carcinogenesis. The colonic mucosal protein levels of iNOS or COX-2 were not different among the six diet groups. Evidence suggests that high multiplicity ACF are indicative of future tumor development in both humans and rats. Furthermore, dysregulated proliferation and apoptosis may also lead to tumorigenesis. Therefore, the ability of dietary apigenin and naringenin to reduce high multiplicity ACF, lower proliferation, and increase apoptosis may contribute toward colon cancer prevention. However, their protection is not due to their influence on iNOS and COX-2 protein levels.