Avaliou-se a eventual atividade quimiopreventiva por parte da vitamina A (VA) quando administrada a ratas Sprague-Dawley durante a etapa de pós-iniciação da carcinogênese mamária induzida pelo 7-12 dimetilbenz(a)antraceno (DMBA). Com exceção de 10 animais que constituíram um grupo controle à parte de ratas consideradas normais [grupo normal (N)], e que não foram submetidas a qualquer procedimento experimental durante todo o estudo, 40 ratas com 50 dias de idade receberam o agente carcinogênico DMBA, por meio de entubação gástrica na dose de 60 mg/kg/peso corpóreo. Após 2 semanas, as ratas iniciadas receberam durante 12 semanas consecutivas, por entubação gástrica, 0,25 mL/100 g de peso corpóreo de óleo de milho (grupo OM; controle, n=20) e 2,5 mg/100 g de peso corpóreo de vitamina A (grupo VA, n=20), sendo, então, eutanasiadas. Não houve diferenças (p>0,05) entre os grupos OM e VA quanto à latência de aparecimento da primeira neoplasia mamária, incidência, multiplicidade e peso médio das neoplasias mamárias, todas classificadas como malignas. Em comparação ao grupo OM, o grupo VA apresentou maior concentração (p<0,05) de retinol no tecido mamário neoplásico e hepático, além de maiores concentrações hepáticas (p<0,05) de palmitato de retinila. Não se detectou palmitato de retinila em neoplasias mamárias de ambos os grupos. Não houve diferença (p>0,05) entre neoplasias mamárias do grupo OM e o tecido mamário do grupo N quanto à metilação global do DNA. Em comparação a neoplasias mamárias do grupo OM, neoplasias do grupo VA apresentaram menor (p<0,05) metilação global do DNA. Conclui-se que a VA não apresentou atividades quimiopreventivas e seu metabolismo encontra-se alterado em neoplasias mamárias. / The potential chemopreventive activity of vitamin A (VA) was evaluated when administered to Sprague-Dawley rats during the stage of post-initiation of mammary carcinogenesis induced by 7-12 dimethylbenz(a)anthracene (DMBA). Except for 10 animals that constituted a separate control group of normal rats [normal group (N)], that were not subjected to any experimental procedure throughout the study, 40 rats with 50 days of age received the carcinogen DMBA by gavage at the dose of 60 mg/kg/body weight. After 2 weeks, the rats received for 12 consecutive weeks, by gavage, 0.25 mL/100 g body weight of corn oil (OM group, control, n = 20) or 2.5 mg/100 g body weight of vitamin A (VA group, n = 20), being then euthanized. There were no differences (p> 0.05) between the OM and VA groups regarding the latency of onset of first breast neoplasm, incidence, multiplicity and average weight of breast tumors, all classified as malignant. Compared to the OM group, the VA group had a higher concentration (p <0.05) of retinol in neoplastic breast tissue and liver as well as higher concentrations (p <0.05) of retinyl palmitate in the liver. Retinyl palmitate was not detected in breast tumors of both groups. There was no difference (p> 0.05) between breast tumours of OM group and mammary tissue of N group regarding global DNA methylation. Compared to breast tumors of OM group, breast tumors of VA group had lower (p <0.05) global DNA methylation. VA did not show chemopreventive activity when administered to rats during the stage of post-initiation of mammary carcinogenesis. Furthermore, the metabolism of VA is altered in breast tumors.

No presente estudo avaliou-se a atividade quimiopreventiva do farnesol (FR) e geraniol (GR), isoprenóides presentes em frutas e ervas, quando administrados a ratos Wistar durante as etapas de iniciação e/ou seleção/promoção do modelo de hepatocarcinogênese do \"hepatócito resistente\" (RH). No Protocolo Experimental 1, os animais receberam durante 8 semanas consecutivas, continuamente durante as etapas de iniciação e seleção/promoção, por entubação gástrica e dissolvido em óleo de milho (OM): FR (25 mg/100 g de peso corpóreo [p.c.]; grupo FR) ou GR (25 mg/100 g de p.c.; grupo GR). Além disso, 1 grupo recebeu durante o mesmo período, por entubação gástrica, apenas OM (0,25 mL/100 g de p.c.; grupo OM; controle). Duas semanas após o início dos tratamentos, todos os grupos foram submetidos ao modelo do RH. Esse consistiu na aplicação intraperitoneal de uma dose do agente iniciante dietilnitrosamina (DEN, 20 mg/100 g de p.c.), seguida, 2 semanas após, da aplicação de 4 doses consecutivas de 2-acetilaminofluoreno (2-AAF; 2,5 mg/100 g de p.c.) e de uma hepatectomia parcial (HP) a 70%, acrescida de 2 doses de 2-AAF (2 mg/100 g de p.c.) 2 e 4 dias após a cirurgia. Decorridas 6 semanas após a iniciação com DEN, os animais foram sacrificados administrando-se, entretanto, 2 h. antes desse procedimento 5-bromo-2-desoxiuridina (BrdU) (10 mg/100 g de p.c.). De acordo com a análise macroscópica dos fígados, e em comparação ao grupo OM, verificou-se que o FR inibiu a incidência (p<0,05) e número médio (p<0,05) de lesões pré-neoplásicas (LPN) hepáticas visíveis à macroscopia. No caso do grupo GR, observou-se apenas sugestão de redução da incidência e número médio dessas LPN visíveis à macroscopia. Em relação à análise morfométrica das LPN hepáticas positivas para a enzima glutationa S-transferase forma placentária (GST-P) totais (persistentes + em remodelação), observou-se que em comparação ao grupo OM, o FR reduziu o tamanho (p<0,05) e área do corte ocupada (p<0,05) por essas lesões. O GR, por sua vez, reduziu o tamanho (p<0,05) das LPN GST-P positivas totais, observando-se, também, sugestão de redução pelo isoprenóide da área do corte ocupada pelas mesmas. Em comparação ao grupo OM, o FR e o GR foram capazes de inibir (p<0,05) a proliferação celular nas LPN, enquanto apenas o GR induziu (p<0,05) a apoptose nas mesmas. Além disso, danos no DNA hepático foram menores (p<0,05) nos animais tratados com FR ou GR, em comparação aos tratados com OM (controles). O tratamento com FR, mas não com GR, resultou em inibição (p<0,05) das concentrações plasmáticas de colesterol total. A análise, por \"western blot\", da expressão hepática do receptor nuclear ativado pelo farnesóide X (FXR) não revelou diferenças (p>0,05) entre os diferentes grupos. No Protocolo Experimental 2, os ratos receberam apenas durante 2 semanas consecutivas na fase de iniciação, e por entubação gástrica, FR (25 mg/100 g p.c.; grupo FRi), GR (25 mg/100 g p.c.; grupo GRi) ou OM (0,25 mL/00 g p.c.; grupo OMi, controle), sendo então submetidos ao modelo do RH, conforme descrito para o Protocolo Experimental 1. O sacrifício dos animais ocorreu 6 semanas após iniciação com DEN. De acordo com a análise macroscópica dos fígados, não foram constatadas diferenças entre os diferentes grupos (p>0,05) quanto à incidência de LPN hepáticas visíveis à macroscopia. Em comparação ao grupo OMi (controle), observou-se nos grupos FRi e GRi sugestão de maior número de LPN hepáticas visíveis à macroscopia. Também em comparação ao grupo OMi (controle), observou-se no grupo GRi menor (p<0,05) número de LPN hepáticas GST-P positivas totais (persistentes + em remodelação), e no grupo FRi sugestão de menor número dessas LPN GST-P positivas totais. Além disso, foram observadas nos grupos FRi e GRi, em comparação ao grupo OMi, LPN hepáticas GST-P positivas totais (persistentes + em remodelação) maiores (p<0,05), bem como sugestão de maior área do corte ocupada por essas LPN GST-P positivas. Não foram observadas diferenças (p>0,05) entre os diferentes grupos quanto à concentração hepática de DNA. No Protocolo Experimental 3, os ratos receberam inicialmente uma dose de DEN (20 mg/100 g de p.c.). Duas semanas após, os animais passaram a receber por entubação gástrica, durante 6 semanas consecutivas em período compreendendo a etapa de seleção/promoção: FR (25 mg/100 g p.c.; grupo FRs/p), GR (25 mg/100 g p.c.; grupo GRs/p) ou OM (0,25 mL/100 g p.c.; grupo Oms/p; controle). Nesse experimento, as administrações de 2-AAF e a realização da HP ocorreram 4 semanas após a iniciação com DEN. O sacrifício dos animais ocorreu após 8 semanas da iniciação com DEN. Em comparação ao grupo OMs/p (controle), observou-se nos grupos FRs/p e GRs/p sugestão de menor número médio de LPN hepáticas visíveis à macroscopia. Não foram constatadas diferenças (p>O,05) entre os diferentes grupos quanto à incidência de LPN hepáticas visíveis à macroscopia; quanto ao número, tamanho e área do corte ocupada por LPN hepáticas GST-P positivas totais (persistentes + em remodelação); e quanto à concentração hepática de DNA. De acordo com os resultados do estudo, considerou-se pronunciada a atividade quimiopreventiva do FR quando administrado a ratos Wistar continuamente durante as etapas de iniciação e seleção/promoção do modelo de hepatocarcinogênese do RH (Protocolo Experimenta! 1). Nessas mesmas condições, considerou-se moderada a atividade quimiopreventiva do GR. Inibições da proliferação celular e de danos no DNA parecem estar envolvidas com as ações anticarcinogênicas do FR e GR, enquanto que a indução da apoptose parece ser mecanismo de ação específico do GR. Além disso, as ações protetoras do FR e GR não parecem envolver alterações na expressão do receptor nuclear FXR. Finalmente, quando administrados especificamente durante a etapa de iniciação (Protocolo Experimental 2) ou de seleção/promoção (Protocolo Experimental 3), ambos os isoprenóides não foram capazes de apresentar atividades quimiopreventivas efetivas. Dessa forma, em ratos Wistar submetidos ao modelo do RH, é necessária a administração contínua de FR ou GR durante as etapas de iniciação e seleção/promoção para a ocorrência de atividades quimiopreventivas. / In the present study, the chemopreventive activity of farnesol (FR) and geraniol (GR), isoprenoids present in fruits and herbs, was evaluated when administered to Wistar rats during the initiation and/or selection/promotion phases of the \"resistant hepatocyte\" (RH) model of hepatocarcinogenesis. In Experimental Protocol 1, animals received during 8 consecutive weeks, continuously during the initiation and selection/promotion phases, by gavage and dissolved in corn oil (CO): FR (25 mg/100g9 body weight [b.w.]; FR group) or GR (25 mg/100 g de b.w.; GR group). Moreover, 1 group received during the same period, by gavage, only CO (0,25 mL/100 g de b.w.; CO group; controls). Two weeks after the beginning of the treatments, all groups were submitted to the RH model. Initiation was obtained by administration of a single intraperitoneal dose of diethylnitrosamine (DEN; 20 mg/100 g b.w.) followed, 2 weeks after, by the administration of 4 consecutive doses of 2-acetylaminofluorene (2-AAF; .2.5 mg/100 b.w.) and by a partial (70%) hepatectomy (PH). Finally, 2 and 4 days after PH, 2 additional 2-AAF doses (2 mg/100 g b.w.) were administered. Six weeks after initiation with DEN, the animals were anesthetized and sacrificed by exsanguination. Two hours before sacrifice, the rats received 5-bromo-2\'-deoxyuridine (10 mg/100 g b.w.). According to the macroscopic examination of the livers, and compared to CO group, FR inhibited the incidence (P<0.05) and mean number (P<0.05) of visible hepatic preneoplastic lesions (PNL). Regarding GR group, only a suggestion of inhibition of visible PNL incidence and mean number was observed. Morphometrical analysis of total (persitent and remodeling) glutathione S-transferase (GST-P) positive PNL showed that compared to CO group, FR group presented with smaller total GST-P positive PNL (p<0.05) that occupied a smaller area of the liver section (p<0.05). Also compared to CO group, GR group presented with smaller total GST-P positive PNL (p<0.05) and a suggestion of reduction of the liver section area occupied by these LPN was observed. Compared to CO group, FR and GR inhibited (p<0.05) PNL cell proliferation, whereas only GR induced (p<0.05) apoptosis in these PNL. Furthermore, hepatic DNA damage was lower (p<0.05) in FR or GR treated animals, compared to CO treated ones (controls). Animal treatment with FR, but not with GR, inhibited (p<0,05) total plasma cholesterol levels. Farnesoid X activated receptor (FXR) expression analysis by western blot did not reveal differences (p>0,05) between the different groups. In the Experimental Protocol 2, rats received only for 2 consecutive weeks during the initiation phase, and by gavage: FR (25 mg/100 g body weight b.w.; FRi group), GR (25 mg/100 g de b.w.; GRi group) or CO (0,25 mL/100 g de b.w.; COi group; controls) being submitted to the RH model as described for Experimental Protocol 1. Six weeks after initiation with DEN, the animals were sacrificed. According to the macroscopic examination of the livers, no differences (p>0.05) were observed among the different groups regarding the incidence of visible PNL. In FRi and GRi groups a suggestion of higher number of visible PNL was observed, when compared to COi group (controls). Also compared to COi group, GRi group presented with smaller (p<0.05) number of total (persistente + remodeling) GST-P positive PNL, whereas in FRi group a suggestion of smaller number of these visible PNL was observed. Moreover, compared to COi group, FRi and GRi groups presented with total (persistent + remodelling) GST-P positive PNL with greater (p<0,05) size, and a suggestion of greater area of the liver section occupied by these GST -P positive PNL was observed. No differences (p>0.05) among the different groups were observed regarding hepatic DNA concentration. In Experimental Protocol 3, rats were first initiated with DEN (20 mg/100 g de b.w.). After 2 weeks, animals received by gavage for 6 consecutive weeks during the selection/promotion phase: FR (25 mg/100 g body weight b.w.; FRs/p group), GR (25 mg/100 g de b.w.; GRs/p group) or CO (0,25 Ml/100 g de b.w.; COs/p group; controls). In this experiment animals received 2-AAF doses and were submitted to PH 4 weeks after initiation with DEN. Six weeks after initiation with DEN, the animals were sacrificed. Compared to COs/p group (controls), a suggestion of smaller visible PNL mean number was observed in FRs/p e GRs/p groups. No differences (p>0.05) among the different groups were observed regarding visible PNL incidence; regarding number, size and liver section occupied by total (persistent + remodeling) GST-P positive PNL; and regarding hepatic DNA concentration. According to the results of the study, FR chemopreventive activity was considered pronounced when administered to Wistar rats continuously during the initiation and selection/promotion phases of the RH model of hepatocarcinogenesis (Experimental Protocol 1). In these same conditions, GR chemopreventive activity was considered moderate. Cell proliferation and DNA damage inhibition seem to be involved with FR and GR anticarcinogenic actions, whereas apoptosis induction seems to represent a GR specific mechanism. Furthermore, FR and GR protective actions do not seem to involve alterations in FXR expression. Finally, when administered specifically during the initiation (Experimental Protocol 2) or selection/promotion (Experimental Protocol 3) phase, both isoprenoids did not present effective chemopreventive activity. Thus, in Wistar rats submitted to the RH model, FR or GR should be administered continuously during the initiation and selection/promotion phases in order to obtain chemopreventive activities.

O carcinoma hepatocelular (HCC) é a 3&#170; causa de morte por neoplasias no mundo, sendo também o 5º tipo de neoplasia mais frequente. Seu tratamento limitado juntamente com o mau prognóstico tornam importante a adoção de medidas preventivas. A prevenção das etapas iniciais da hepatocarcinogênese pela administração de compostos bioativos de alimentos (CBAs) atualmente vem sendo observada em diversos estudos. Sugere-se que o processo de carcinogênese envolva alterações genéticas e epigenéticas, como a hipometilação do DNA e a desacetilação de histonas. Nesse sentido, o tratamento com ácido fólico (AF), precursor indireto de grupamento metil, e tributirina (TB), inibidora de enzimas desacetilases de histonas, quando em associação, poderia agir em adição potencializando os efeitos quimiopreventivos dos mesmos comparados às suas ações isoladas. A eventual atividade quimiopreventiva da TB (100mg/100g de peso corpóreo), do AF (0,08mg/100g de peso corpóreo), ambos com a metade da dose utilizada na literatura, e da associação entre os mesmos (AS) foi analisada durante a etapa de promoção da hepatocarcinogênese em ratos Wistar submetidos ao modelo do hepatócito resistente (HR). Para avaliação de GST-P, da proliferação celular, da apoptose, bem como a localização da histona H3K9 foi utilizada a técnica de imunohistoquímica; para a quantificação de H3K9, western blot. Em relação à análise macroscópica, os grupos tratados apresentaram menor número de nódulos em relação a MD (sem significância estatística), além de menor porcentagem de LPN menores do que 1mm nos grupos TB e AS. Na análise microscópica foi observado que os grupos TB, AF e AS apresentaram menor número de lesões pré neoplásicas persistentes (pLPN) em relação ao grupo controle isocalórico (MD, maltodextrina), assim como menor porcentagem de área ocupada por pLPN e por lesões em remodelação (rLPN). Quanto a proliferação celular, os grupos AF e AS apresentaram menor número de núcleos em fase S/mm2 quando comparados aos grupos MD e TB. Em relação à apoptose, os grupos TB e AS apresentaram maior número de hepatócitos em apoptose e corpúsculos apoptóticos/mm2 quando comparados aos grupos MD e AF. No resultado da metilação global do DNA não houve diferença entre os grupos. Em adição, os grupos AF e AS apresentaram maiores concentrações séricas de folato; o mesmo não ocorrendo para B12. No que diz respeito à acetilação de H3K9, os grupos TB e AS apresentaram maior porcentagem de acetilação quando comparados aos grupos N, MD e AF. Em conclusão, os grupos tratados com TB, AF e a associação entre eles apresentaram quimioprevenção pronunciada, sendo que o grupo AS apresentou mecanismos aditivos dos tratamentos isolados. / The hepatocellular carcinoma (HCC) is the third leading cause of death by cancer in the world, and the fifth most frequent type of neoplasm. Its limited treatment along with poor prognosis make it important to adopt preventive measures. The prevention of the initial stages of hepatocarcinogenesis by the administration of bioactive food compounds (BFCs) is currently being observed in several studies. It is suggested that the process of carcinogenesis involves genetic and epigenetic modifications, such as DNA hypomethylation and deacetylation of histones. Accordingly, treatment with folic acid (FA), indirect precursor of methyl grouping, and tributyrin (TB), a histone deacetylases inhibiting enzyme, when associated could act in addition to enhance the chemopreventive effects compared to their isolated actions. The possible chemopreventive activity of TB (100mg/100g body weight), FA (0.08 mg/100 g body weight) and the association between them (AS) were studied during the promotion stage of hepatocarcinogenesis in Wistar rats submitted to resistant hepatocyte model (RH).For evaluation of GST-P, cell proliferation, apoptosis, and the location of histone H3K9 was used immunohistochemistry, to quantify H3K9, western blot. Regarding the macroscopic analysis, the treated groups showed a lower number of nodules compared to MD (not statistically significant), and a lower percentage smaller than 1mm in groups TB and AS. Under microscopic examination the groups TB, FA and AS showed a lower number of persistent pre-neoplastic lesions (pLPN) compared to the isocaloric control group (MD, maltodextrin), as well as a lower percentage of the area occupied by pLPN and remodeling lesions (rLPN). As cell proliferation, FA and AS groups had fewer S phase nuclei/mm2 compared to MD and TB groups. Regarding apoptosis, TB and AS groups showed higher numbers of hepatocytes in apoptosis and apoptotic corpuscles/mm2 compared to MD and FA groups. Global DNA methylation did not differ between groups. In addition, AF and AS groups had higher serum concentrations of folate, which did not occur for B12.Concerning the H3K9 acetylation, the groups TB and AS showed higher percentage of acetylation when compared with the groups N, MD and FA. In conclusion, the groups treated with TB, FA and the association of both showed pronounced chemoprevention, and the AS group showed additive mechanisms compared with isolated treatments.

O câncer é um dos principais problemas de saúde pública no mundo. Dentre as neoplasias primárias que acometem o fígado, o carcinoma hepatocelular (HCC) é a mais frequente. Diversos fatores de risco predispõem ao HCC, entre eles a doença hepática gordurosa não alcóolica (NAFLD). Segundo estudos prévios do grupo, a &#946-ionona (BI), apresenta potencial quimiopreventivo na hepatocarcinogênese, promovendo redução de lesões preneoplásicas (LPN). Assim pretendeu-se investigar se a NAFLD potencializaria o desenvolvimento de LPN em ratos Wistar submetidos ao modelo do hepatócito resistente (RH) na etapa de iniciação/promoção inicial da hepatocarcinogênese e se a BI apresenta efeito quimiopreventivo nesse contexto. Para tanto, os animais foram distribuídos em 4 grupos experimentais: grupo não-tratado (NT), grupo submetido ao RH (RH), grupo submetido ao RH e ao modelo de NAFLD, que consiste na administração de emulsão hiperlipídica, (AS) e grupo AS tratado com BI (AS + BI). Em uma primeira instância, 5 animais pertencentes aos grupos NT, AS,AS + BI foram eutanasiados após 6 semanas de administração de emulsão hiperlipídica, antes da aplicação do modelo RH, para se confirmar o desenvolvimento da NAFLD. Foi possível observar que a administração de emulsão hiperlipídica durante 6 semanas foi suficiente para o desenvolvimento de esteatose hepática. Após as 6 semanas foi introduzido o modelo do RH concomitante à administração da emulsão hipercalipídica até o fim do experimento na 13a semana . Após 13 semanas o grupo AS apresentou maiores (p<0,05) valores de focos de inflamação, hepatócitos balonizados e grau de esteatose hepática em relação ao grupo AS+BI, assim como maiores (p<0,05) níveis séricos de triacilgliceróis, colesterol, HDL, LDL, VLDL, e maior (p<0,05) valor de MDA em relação ao grupo RH embora sem diferença estatística. O grupo AS também apresentou maior (p<0,05) incidência, número total e multiplicidade de nódulos, além de maior (p<0,05) número e tamanho de LPN persistentes (pLPN) e índice de proliferação quando comparado aos grupos RH e AS+BI. O grupo AS + BI, por sua vez, demonstrou menores (p<0,05) valores de escore de células ovais e menores valores de comprimentos de cometa e danos no DNA quando comparado ao grupo AS, embora sem diferença estatista para este último parâmetro. Em relação à expressão gênica, o grupo AS apresentou menores (p<0,05) valores de expressão do gene Hmgcr em relação ao grupo RH e maiores (p<0,05) valores dos genes Insig 1 e Thy 1 quando comparados ao grupo AS+BI. Portanto, no contexto de esteatose hepática associada ao modelo do RH, a administração de BI durante a etapa de iniciação/promoção em ratos Wistar resultou em atividade quimiopreventiva que se deu pela diminuição de pLNP, redução da proliferação celular e do número de células ovais, consideradas potenciais alvos para o desenvolvimento da hepatocarcinogênese, entretanto os genes analisados parecem não serem modulados pela BI. / Cancer is a major public health problem in the world. Among the primary neoplasms affecting the liver, hepatocellular carcinoma (HCC) is the most frequent. Several risk factors predispose to HCC, including the nonalcoholic fatty liver disease (NAFLD). According to previous studies of the group, the &#946-ionone (BI), has potential chemopreventive in hepatocarcinogenesis, promoting reduction of preneoplastic lesions (LPN). Thus we investigated whether NAFLD would increase the development of LPN in Wistar rats resistant hepatocyte model (RH) at the stage of initiation / promotion of hepatocarcinogenesis and if BI has chemopreventive effect in this context. Therefore, the animals were divided into 4 groups: non-treated group (NT), the group submitted to HR (HR), the group submitted to HR and NAFLD model, consisting of the fatty emulsion administration (AS) and AS group treated with BI (AS + BI). In a first point, 5 animals belonging to the groups NT, AS, AS + BI were euthanized after 6 weeks of administration of fat emulsion prior to application of the HR model, to confirm the development of NAFLD. It was observed that the administration of fatty emulsion for 6 weeks was sufficient to the development of hepatic steatosis. After 6 weeks it was introduced into the model HR the concomitant administration of fatty emulsion until the end of the experiment at 13 weeks. In the endpoint, the AS group had higher (p <0.05) of serum triacylglycerols, cholesterol, HDL, LDL, VLDL although no statistical difference inrelation to RH group, and increased (p <0.05) amount of MDA in relation to the group RH. The AS group also had higher (p <0.05) incidence, multiplicity and total number of nodes and greater (p <0.05) number and size of persistent LPN (pLPN) and proliferation index when compared to HR groups and AS + BI. AS + BI group. It was observed in AS+BI group lower (p <0.05) cell oval score values compared to AS group. In addition the AS+BI group showed lower values of the comet length and DNA damage compared to the AS group, although no statistical differences. In relation to gene expression, the AS group showed lower(p <0.05) HMGCR gene expression values in relation to HR group and higher (p <0.05) expression of Insig genes 1 and Thy 1 compared to group AS + BI.Therefore, in the context of hepatic steatosis associated with HR model BI for administration to the stage of initiation / promotion in rats resulted in chemopreventive activity was due to decrease in area of pLNP, reducing cell proliferation, and the number of oval cells, as potential targets for the development of hepatocarcinogenesis, however the genes do not seem to be modulated analyzed by BI.

Magister Scientiae (Medical Bioscience) - MSc(MBS) / Laboratory studies, epidemiological investigations and human clinical trials indicate that flavonoids have important effects on cancer chemoprevention and therapy. Flavonoids may interfere in several steps that lead to cancer development but may also lead to toxicity as the inhibition of carcinogen-activating enzymes may also cause potential toxic flavonoid-drug interactions. However, the potential toxicity of these dietary components has not been well studied. The use of polyphenol enriched supplements prepared from South African herbal teas, rooibos(Aspalathus linearis)and honeybush (Cyclopia spp.) are being marketed to alleviate symptoms that are known to be “cured” by the herbal teas. However, there is a lack of information regarding the possible health promoting effects of these polyphenol-enriched extracts on xenobiotic metabolism. In the present study, the modulating effects of aspalathinenriched rooibos and mangiferin-enriched C. genistoides and C. subternata extracts on the gene expression of xenobiotic metabolising enzymes (XMEs) were investigated in vivo in the rat liver and kidneys. An in vitro study, utilising a primary rat hepatocyte cell model, was included to further evaluate changes in the expression of selected XMEs by the herbal tea extracts, including their major polyphenolic constituents, aspalathin and mangiferin. The use of the in vitro primary hepatocytes assay as a predictive cell model for the modulation of the expression of XMEs genes by the herbal tea extracts in vivo was critically evaluated.In the liver and kidneys, the C. subternata polyphenol-enriched herbal tea extract effected the majority of changes regarding the expression of XMEs genes when compared to the rooibos and C. genistoides. Variations in the modulation of gene expression of the XMEs by the herbal tea extracts were related to differences in their polyphenol constituents, although non-polyphenolic constituent could also be involved.Overall the herbal teas regulated alcohol,energy, drug and steroid metabolism in the liver, whereas in the kidneys the gene expression of phase I, phase II, steroid metabolising enzymes, as well as drug transporters were modulated. It would appear that the herbal teas are likely to exhibit both beneficial and adverse effects in vivo,depending on the specific organ, the xenobiotic and/or drug that are involved. The primary rat hepatocytes model display varying effects with respect to modulating gene expression of specific XMEs by the polyphenol-enriched herbal tea extracts. Apart from the reduction in 17 -hydroxysteroid dehydrogenase gene expression care should be taken to directly extrapolate the in vitro findings to changes that prevail in vivo.However, interesting results regarding the masking effect of complex mixture on the modulation of XME gene expression of individual polyphenols were encountered. In addition differences in the dose and duration of exposure between the in vitro and in vivo studies were not comparable and should be further explored to validate the in vitro primary hepatocytes model to predict changes in vivo. Future studies should investigate the effects of the herbal tea extracts, its polyphenols and metabolites on XME induction at a protein level as well as varying herb-drug-enzyme interactions.

Philosophiae Doctor - PhD / The aim of this study was to determine whether dietary fats constituting specific ω-6/ω-3 fatty acids (FA) ratio has chemopreventive modulating effects on the development of colon cancer. Western diets intake of saturated FA (SATS) and ω-6 polyunsaturated FA (PUFA) are very high relative to low ω-3 PUFA consumption. This high ω-6 and low ω-3 FA intake, resulting in a high ω-6/ω-3 FA ratio, appears to have a promoting effect on disease outcome, whilst increased ω-3 FA intake exhibiting anti-cancer effects. An animal cancer model was employed to evaluate the effects of dietary fat ratios on chemically induced carcinogenesis during cancer promotion. This was to determine whether the FA diets have a promoting or inhibitory effect on early neoplastic lesions by quantifying aberrant crypt foci (ACF) development and monitoring the crypt cells proliferative and apoptotic indices. The expressions of genes associated with changes in cells redox balance were also assessed. Common dietary fats were combined to produce the dietary fat ratios: sunflower oil (S), borage oil (B) and fish oil (F). Combinations of these oils generated the different ω-6/ω-3 FA ratios: SB (ω-6/ω-3: 38:1), SF (ω-6/ω-3: 13:1) and SBF (10:1). To represent the Western diet's high ω-6/ω-3 FA ratio profile, S (ω-6/ω-3: 501: 1) was used as a control, and canola oil and olive oil as additional reference. The dietary fats had no toxic effects on the liver and kidney based on serum clinical biochemical measurements. Diets containing borage oil (SB and SBF diets), canola and olive oil decreased (p<0.05) the crypt multiplicity of large (≥7 crypts/focus) ACF, exhibiting anti-cancer effects by decreasing (p<0.05) the proliferative activity of the rat colon crypts. Borage oil's protective effect resulted from the enhanced supply of C18:3ω-6 that has anti-inflammatory and anti-proliferative properties. The observed decrease (p<0.05) in apoptosis in the ACF was also facilitated by the up- and down-regulation of DNA repair and DNA replication associated genes, Xpa and Ercc2 by borage oil, respectively. Canola oil and olive had the largest inhibitory effect on suppressing crypt multiplicity by reducing (p<0.05) proliferation in the colon. Both oils effected the up-regulation (p<0.05) of the expression of several oxidative stress and anti-oxidant defence genes mediating the regulation of cell proliferation. The increased supply of C18:1ω-9 (canola and olive) and total polyphenolic content (olive) protected cells against oxidative stress induced apoptosis, which provided interesting interactive effects between FA and polyphenolic oil constituents that should be further elucidated. In contrast, the fish oil containing (SF diet) and the control sunflower (S diet) increased (p<0.05) the total ACF and colon crypt multiplicity (≥7 crypts/focus) when compared to the SB, SBF, olive oil and canola oil diets. An increased resistance to oxidative stress induced apoptosis appears to facilitate fish oil’s enhancing effect on crypt multiplicity despite the increased supply of LC ω-3 FA, which are prone to oxidation and leads to increased oxidative stress. This protective effect on crypt multiplicity and ACF development was mainly due to enhanced cellular antioxidant and DNA repair responses through the up-regulation (p<0.05) of Gpx4 and Nudt1, which favoured the increase (p<0.05) of crypt cells proliferation.The in vitro study demonstrated that oil ratio emulsions (S: ω-6/ω-3 = 249:1; SB: ω-6/ω-3 = 28:1; SF: ω-6/ω-3 = 12:1 and SBF: ω-6/ω-3 = 12:1) had differential effects on the survival indices of HT-29 and Caco-2 colon cancer cells. Contrary to the in vivo model, fish oil (SF and SBF emulsions) significantly (p<0.05) reduced the viability and proliferation of both cell lines, with the HT-29 cells showing greater sensitivity to the oil’s anti-proliferative effect. The HT-29 cells exposure to increased levels of C20:5ω-3 and C22:6ω-3 predisposes it to lipid peroxidation that increases the potential for cell removal via apoptosis. However, apoptotic effects were absent due to the HT-29 cells removal via necrosis as the cells energy status (ATP production) was significantly (p<0.05) depleted. Similar to the animal cancer model, borage oil (SB and SBF emulsions) had a reducing (p<0.05) effect on cell proliferation in both cell lines. However, as ATP was decreased (p<0.05), the S, SF and SBF emulsions resulted in an increased (p<0.05) apoptotic response in the Caco-2 cells in a dose dependent manner. This response resulted from the altered FA and lipid composition effected by the oil emulsions. Increased (p<0.05) incorporation of C20:5ω-3 and C22:6ω-3 in membrane phospholipid, phosphatidylethanolamine (PE), resulted in a significant decrease (p<0.05) in total SATS and MUFA content. A decrease (p<0.05) in membranes ω-6/ω-3 FA ratio was noted as well. This effect seems to selectively favour the induction of apoptosis by borage oil (SB and SBF). Similarly, an increase (p<0.05) in the PC/PE ratio by all oil emulsions, and a decrease (S and SB) and increase (SF and SBF) (p<0.05) in the chol/PL ratio appears to facilitate apoptosis too. A different threshold of the FA and lipid composition parameters elicits the inhibition of cell proliferation utilising lower oil emulsion concentrations. Therefore, the dietary supply of fats characterised by a defined low ω-6/ω-3 FA ratio can selectively modulate the growth indices of colon cancer. Specific oil ratio combinations by incorporating borage oil and fish oil hereby provide a selective strategy for chemoprevention in the colon, although underlying interactions and threshold effects of specific FA seems to prevail that should be further unravelled. / Medical Research Council (MRC) and Cancer Association of South Africa (CANSA)

<p>Colorectal cancer is one of the leading causes of cancer deaths in the United States and multiple modifiable factors contribute to colorectal carcinogenesis. Gut microbiota are believed to play key roles in colon cancer development. Dietary factors may modulate gut microbiota composition, which may potentially have impact on carcinogenesis. Thus, it is reasonable to develop dietary interventions to effectively prevent colorectal cancer development through alteration of gut microbiota. In this thesis, the first objective is to evaluate the effect of vitamin E forms and metabolites, i.e., δ-tocotrienol (δTE), γ-tocotrienol (γTE) and δTE-13’-COOH (δTE-13’), respectively, on gut microbiota in mice. Healthy male balb/c mice were supplemented with a δTE/γTE mixture or δTE-13’ by gavage for two weeks, while control mice received soybean oil. We isolated DNAs from fecal samples and used 16S rRNA gene sequencing to evaluate the impact of these compounds on gut microbiota compositions. Further, we also examined the effect on short chain fatty acids (SCFAs). We observed that supplementation of δTE-13’ increased microbial richness using the Faith index. On the other hand, supplementation did not separate the microbial communities from the control group. But, these compounds managed to alter the relative abundances of several taxa that might present chemopreventive activities against colon cancer. Specifically, <i>Desulfovibrio</i>, a sulfur-reducing bacterium, was decreased after δTE/γTE supplementation. <i>Eubacterium coprostanoligenes</i> group, a group of microbes that can reduce circulating cholesterol, was increased after δTE/γTE supplementation. In addition, several members from the <i>Lachnospiraceae</i> family were elevated under δTE/γTE and δTE-13’ supplementation, and these microbes are known to produce SCFAs and maintain colonic health. However, the measurement of SCFAs showed that supplementation of δTE/γTE and δTE-13’ did not change SCFAs compared with controls. In the second project, I investigated anti-proliferative effects of combining δTE or δTE-13’ with sodium butyrate (NaBu) on human colorectal carcinoma HCT116 cells. Our data showed promising additive effects against cell growth. Collectively, these results indicate that δTE/γTE and δTE-13’ can modulate gut microbiota under healthy conditions, which provides insights into potential chemopreventive activities of these vitamin E forms. Our cell-based studies also showed additive anticancer effects of combining δTE or δTE-13’ with NaBu, which provides rationale to further develop combination of butyrate producers with vitamin E forms for cancer prevention.</p>

Nutritional factors play an important role in the prevention and promotion of colorectal cancer. Vitamin E is a generic term that describes a group of lipid-soluble chain-breaking antioxidants that includes tocopherols and tocotrienols. Vitamin E occurs in nature as eight structurally related forms that include four tocopherols and four tocotrienols. Vitamin E is a potent membrane-soluble antioxidant. Antioxidants like vitamin E (tocopherols) may prevent colon cancer through several different cellular and molecular mechanisms. Vitamin E in the American diet is primarily available in plant-oil rich foods such as vegetable oils, seeds and nuts and these foods vary widely in their content of α-tocopherol and γ-tocopherol [1]. Vitamin E may help prevent colon cancer by decreasing the formation of mutagens arising from the oxidation of fecal lipids, by decreasing oxidative stress in the epithelial cells of the colon and by molecular mechanisms that influence cell death, cell cycle and transcriptional events. Most epidemiological, experimental and clinical studies have evaluated the α-isoform and not the γ-isoform of vitamin E. Recent epidemiological, experimental and mechanistic evidence suggests that γ-tocopherol may be a more potent cancer chemopreventive agent than α-tocopherol. The differences in chemical reactivity, metabolism and biological activity may contribute to these differences in the effects of γ-tocopherol when compared with α-tocopherol. The rationale supporting the development of γ-tocopherol as a colorectal cancer preventive agent is reviewed here.

Phytochemicals are recognized as playing an important role in cancer prevention by fruits and vegetables. The avocado is a widely grown and consumed fruit that is high in nutrients and low in calories, sodium, and fats. Studies have shown that phytochemicals extracted from the avocado fruit selectively induce cell cycle arrest, inhibit growth, and induce apoptosis in precancerous and cancer cell lines. Our recent studies indicate that phytochemicals extracted with chloroform from avocado fruits target multiple signaling pathways and increase intracellular reactive oxygen leading to apoptosis. This review summarizes the reported phytochemicals in avocado fruit and discusses their molecular mechanisms and targets. These studies suggest that individual and combinations of phytochemicals from the avocado fruit may offer an advantageous dietary strategy in cancer prevention.

Colorectal carcinoma is an important, feasible and attractive target for chemoprevention because a) it is a major cause of mortality in the United States and in other developed countries worldwide, b) there is a high mortality associated with advanced disease, c) there is a well described molecular carcinogenesis pathway and d) recent advances in molecular genetics will improve the ability to identify high-risk subjects. Epidemiological data, colonoscopic screening and advances in molecular genetics has made possible the identification and selection of subjects at increased risk of developing colorectal cancer. Due to this new information it may be possible to impede malignant cellular transformation with drugs. Such intervention with relatively simple maneuvers, such as a low daily dose of aspirin, can potentially reduce mortality from colorectal cancer. Prospective trials need to confirm experimental and epidemiological data supporting the efficacy of aspirin and other NSAID as chemopreventive agents before they can be used in the general population at risk. To use cancer chemopreventives effectively and safely in an asymptomatic population, the risks should be minimized and the benefits maximized by determination of optimal dose, schedule and chemopreventive mechanism of the NSAID. By linking the putative mechanism of drug action to effect endpoints, we expect to know whether the chemopreventive intervention is likely to be effective in a given individual.