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INTRODUCTION: The common practice of supplementing folic acid during pregnancy and the absence of such guidelines for vitamin B12 lead to an imbalance of these vitamins, especially in developing countries like India, where many women are vitamin B12 deficient. METHODS: The present study was designed to explore the effect of low vitamin B12 in combination with different levels of folic acid in the parental diet on fetal growth parameters and maternal reproductive performance in a transgenerational manner. The reversibility of these effects was studied by shifting the mice to a regular diet in the F1 generation in the case of transient groups and continued on the same diet in the sustained groups after the dietary exposure in the F0 generation. RESULTS: Vitamin B12 deficiency and different levels of folic acid resulted in the decreased placental and fetal weight of the F1 generation. Surprisingly, a decreased placental weight, low fetal weight, and reduced crown-rump length and head circumference were observed in F2 fetuses of vitamin B12 deficient with folate over-supplemented (BDFO) transient group, i.e. when F1 mice were shifted to normal diet conditions. Reduced follicles in ovaries and alteration in placental pathology in all the F0 groups and BDFO of the F1 transient group were also seen. DISCUSSION: Overall, the study revealed that dietary imbalance of vitamin B12 and folic acid, particularly B12 deficiency with over-supplemented folic acid, negatively affects placental and fetal development and maternal reproductive performance. Such effects are passed on to the next generation too.
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Ácido Fólico , Deficiência de Vitamina B 12 , Feminino , Gravidez , Camundongos , Animais , Placentação , Peso Fetal , Placenta , Vitamina B 12 , Dieta , Desenvolvimento Fetal , HomocisteínaRESUMO
Folate ingestion below and above the physiologic dose has been shown to play a tumorigenic role in certain cancers. Also, excessive folate supplementation after establishment of pre-established lesions led to an advancement in the growth of a few tumors. However, such information has not yet been achieved in the case of HCC. In our study, HepG2 cells were administered with three different concentrations of folic acid i.e. folic acid normal (FN) (2.27 µM), folic acid deficient (FD) (no folic acid), folic acid oversupplementation (FO) (100 µM) for 10 days. Intracellular folate levels were assayed by Elecsys Folate III kit based method. The migratory and invasive abilities were estimated by transwell migration and matrigel invasion methods respectively. FACS was done to evaluate cell viability and apoptosis. Agarose-coated plates were used to access cancer stem cells (CSCs) number. Quantitative RT-PCR and western blotting approaches were used for gene and protein expression of certain tumor suppressor genes (TSGs), respectively. FD cells depicted increased migration, invasion, apoptosis, necrosis and decreased cell viability, CSCs. On the other hand, FO cells showed increased migration, invasion, cell viability and number of CSCs and decreased apoptosis and necrosis. TSGs revealed diminished expression with both FA modulations with respect to FN cells. Thus, FA deficiency as well as abundance enhanced the HCC progression by adapting different mechanisms.
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Carcinoma Hepatocelular , Neoplasias Hepáticas , Carcinogênese , Carcinoma Hepatocelular/genética , Ácido Fólico/metabolismo , Ácido Fólico/farmacologia , Células Hep G2 , Humanos , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/genética , Necrose , SefaroseRESUMO
Background: One of the unique features of placentation is its similarity to tumorigenesis yet being very well regulated. It allows rapid proliferation, migration, and invasion of mononuclear trophoblast cells into the maternal uterus and remodeling the maternal vasculature. This pseudomalignant nature of trophoblastic cells is strictly regulated and its importance becomes evident in abnormal pregnancies that are characterized by aberrant trophoblast proliferation/invasion like preeclampsia. In addition to this, the importance of folic acid supplementation during pregnancy is well documented. We aimed to analyze the molecular and epigenetic regulation of the pseudomalignant nature of placentation via folic acid levels. Methods: Placental tissue samples were collected from different pregnancies in three different gestational stages. We estimated the impact of folic acid levels on global methylation, LINE1 methylation, and expression of DNMTs in all three gestational stages in pregnant women and preeclampsia pregnancies. We also analyzed the effect of folic acid supplementation on trophoblastic invasion using placental derived cells viz, JEG-3 and HTR-8/SVneo cell line and verified the molecular and epigenetic mechanisms involved in this regulation. Results: Development of preeclampsia was observed to be associated with lower folate levels in placental tissue, higher global methylation level, and higher expression of DNMT1and DNMT3A. Folic acid supplementation was found to increase the invasive potential of placental trophoblasts by almost two folds which were associated with the decreased expression of tumor suppressor genes and tissue inhibitors of matrix metalloproteinases; and increased expression of oncogenes, telomerase gene, and matrix metalloproteinases. These folic acid-mediated changes were observed to be regulated by CpG methylation in the case of many genes. Folic acid supplementation was also observed to significantly decrease global methylation in placental trophoblasts related to decreasing expression of DNMT1 and DNMT3A. Conclusion: Lower folic acid levels are associated with preeclampsia development and folic acid supplementation regulates the invasive potential of placental trophoblasts as mediated by various epigenetic changes in the placenta suggesting the protective effect of folic acid against preeclampsia.
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Maternal folic acid and vitamin B12 (B12) status during pregnancy influence fetal growth. This study elucidated the effect of altered dietary ratio of folic acid and B12 on the regulation of H19/IGF2 locus in C57BL/6 mice. Female mice were fed diets with nine combinations of folic acid and B12 for 4 weeks. They were mated and the offspring born (F1) were continued on the same diet for 6 weeks post-weaning and were allowed to mate. The placenta and fetal (F2) tissues were collected at day 20 of gestation. H19 overexpression observed under dietary deficiency of folate combined with normal B12 (B12 normal folic acid-deficient, BNFD) was associated with an increased expression of microRNA-675 (miR-675) in maternal and fetal tissues. Insulin-like growth factor 2 (IGF2) expression was decreased under folic acid-deficient conditions combined with normal, deficient or over-supplemented state of B12 (BNFD, BDFD and BOFD) in fetal tissues along with B12 deficiency combined with normal folic acid (BDFN) in the placenta. The altered expression of imprinted genes under folic acid-deficient conditions was related to decreased serum levels of folate and body weight (F1). Hypermethylation observed at the H19 differentially methylated region (DMR) (in BNFD) might be responsible for the decreased expression of IGF2 in female fetal tissues. IGF2 DMR2 was found to be hypomethylated and associated with low serum B12 levels with B12 deficiency in fetal tissues. Results suggest that the altered dietary ratio of folic acid and B12 affects the in utero development of the fetus in association with altered epigenetic regulation of H19/IGF2 locus.
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Ácido Fólico , RNA Longo não Codificante , Gravidez , Feminino , Animais , Camundongos , Ácido Fólico/metabolismo , Vitamina B 12 , Epigênese Genética , Impressão Genômica , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Camundongos Endogâmicos C57BL , Metilação de DNA , Dieta , Vitaminas , Fator de Crescimento Insulin-Like II/genética , Fator de Crescimento Insulin-Like II/metabolismoRESUMO
Genomic imprinting is important for mammalian development and its dysregulation can cause various developmental defects and diseases. The study evaluated the effects of different dietary combinations of folic acid and B12 on epigenetic regulation of IGF2R and KCNQ1OT1 ncRNA in C57BL/6 mice model. Female mice were fed diets with nine combinations of folic acid and B12 for 4 weeks. They were mated and off-springs born (F1) were continued on the same diet for 6 weeks postweaning and were allowed to mate. The placenta and fetal (F2) tissues were collected at day 20 of gestation. Dietary deficiency of folate (BNFD and BOFD) and B12 (BDFN) with either state of other vitamin or combined deficiency of both vitamins (BDFD) in comparison to BNFN, were overall responsible for reduced expression of IGF2R in the placenta (F1) and the fetal liver (F2) whereas a combination of folate deficiency with different levels of B12 revealed sex-specific differences in kidney and brain. The alterations in the expression of IGF2R caused by folate-deficient conditions (BNFD and BOFD) and both deficient condition (BDFD) was found to be associated with an increase in suppressive histone modifications. Over-supplementation of either folate or B12 or both vitamins in comparison to BNFN, led to increase in expression of IGF2R and KCNQ1OT1 in the placenta and fetal tissues. The increase in the expression of IGF2R caused by folate over-supplementation (BNFO) was associated with decreased DNA methylation in fetal tissues. KCNQ1OT1 noncoding RNA (ncRNA), however, showed upregulation under deficient conditions of folate and B12 only in female fetal tissues which correlated well with hypomethylation observed under these conditions. An epigenetic reprograming of IGF2R and KCNQ1OT1 ncRNA in the offspring was evident upon different dietary combinations of folic acid and B12 in the mice.
Assuntos
Dieta , Epigênese Genética/efeitos dos fármacos , Feto/efeitos dos fármacos , Ácido Fólico/farmacologia , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Placenta/efeitos dos fármacos , RNA Longo não Codificante/genética , Receptor IGF Tipo 2/genética , Vitamina B 12/farmacologia , Animais , Peso Corporal/efeitos dos fármacos , Encéfalo/embriologia , Encéfalo/metabolismo , Relação Dose-Resposta a Droga , Interações Medicamentosas , Feminino , Feto/metabolismo , Ácido Fólico/administração & dosagem , Ácido Fólico/sangue , Deficiência de Ácido Fólico/genética , Deficiência de Ácido Fólico/metabolismo , Impressão Genômica , Homocisteína/sangue , Rim/embriologia , Rim/metabolismo , Fígado/embriologia , Fígado/metabolismo , Masculino , Camundongos , Placenta/metabolismo , Gravidez , Complicações na Gravidez/genética , Complicações na Gravidez/metabolismo , RNA Longo não Codificante/metabolismo , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Receptor IGF Tipo 2/metabolismo , Vitamina B 12/administração & dosagem , Vitamina B 12/sangue , Deficiência de Vitamina B 12/genética , Deficiência de Vitamina B 12/metabolismoRESUMO
Maternal folate and vitamin B12 status during pregnancy may influence development of central nervous system (CNS) in the offspring. Very little attention has been paid to understand the combined effects of both the vitamins during pregnancy. The present study was designed to evaluate the biochemical and behavioral outcomes following alterations in folate and vitamin B12 levels in C57BL/6 mice. The female mice were fed with different combinations of folate and vitamin B12 whereas; males were fed with normal diet for 4 weeks. The mice were mated and the pregnant mice received the same diets as before pregnancy. The F1 male mice were further continued on maternal diet for 6 weeks following neurobehavioral and biochemical assessment. The body weight of the F1 male mice was significantly decreased in the mice that received folate and vitamin B12 deficient diet. Altered cognitive functions were observed in the folate and B12 deficient F1 male mice as assessed by Morris water maze and novel object recognition tests. Spontaneous locomotor activity was decreased in F1 male mice fed with folate and B12 deficient diets. Elevated homocysteine levels and decreased hydrogen sulfide levels were also observed in the brain of F1 male mice on folate and B12 deficient diets. However, GSH and GSSG levels were increased in the brain of the animals supplemented with folate deficient diet with different combinations of B12. The study suggests that exposure of female mice to folate and vitamin B12 deficiency during pregnancy effects in-utero development of fetus, which further leads to behavioral anomalies in adult life and is sufficient to cause impaired cognitive behavior in the subsequent generation. Thus, elucidating the role and importance of maternal dietary folate and B12 ratio during pregnancy.
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Cognição/efeitos dos fármacos , Transtornos da Nutrição Fetal/epidemiologia , Ácido Fólico/metabolismo , Deficiência de Vitamina B 12/metabolismo , Animais , Dieta , Suplementos Nutricionais , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Gravidez , Efeitos Tardios da Exposição Pré-Natal/metabolismo , Vitamina B 12/metabolismo , VitaminasRESUMO
BACKGROUND: Vitamin B12 and folic acid (FA) supplementation (B12-FAS) reduces hematologic toxicity with pemetrexed-based chemotherapy (PEM). However, the basis for recommending 1 week of B12-FAS before PEM initiation has never been proven in a randomized trial. METHODS: An open-label, randomized trial (PEMVITASTART; clinicaltrials.gov identifier NCT02679443) was conducted to compare hematologic toxicity between patients with locally advanced/metastatic nonsquamous non-small cell lung cancer who initiated PEM after 5 to 7 days of B12-FAS (delayed arm [DA]) versus those who received B12-FAS simultaneously (≤24 hours) with PEM initiation (immediate arm [IA]). Every 3 weeks, all enrolled patients received pemetrexed (500 mg/m2 ) AND either cisplatin (65 mg/m2 ) OR carboplatin (area under the curve = 5.0 mg/mL per minute) on day 1 for a maximum of 6 cycles. Supplementation consisted of oral FA 1000 µg daily and intramuscular vitamin B12 1000 µg every 3 weeks. The primary outcome was any grade of hematologic toxicity and secondary outcomes included grade 3/4 hematologic toxicity, the relative dose intensity delivered, and changes in serum levels of B12/FA/homocysteine. RESULTS: Of 161 patients (IA, n = 81; DA, n = 80) recruited, 150 (IA, n = 77; DA, n = 73) received ≥1 cycle and were included in a modified intention-to-treat analysis. Baseline anemia prevalence was 34.7% (IA, 32.5%; DA, 37%; P = .56). The incidence of any grade anemia, leukopenia, neutropenia, and thrombocytopenia was 87% versus 87.7% (P = .90), 37.7% versus 28.8% (P = .25), 20.8% versus 15.1% (P = .36), and 31.2% versus 16.4% (P = .04), respectively, in the IA and DA, respectively. Grade 3/4 cytopenias and median relative dose intensities delivered (pemetrexed, 93.5%; platinum, 91%) were similar in both arms. After cycle 3 (compared with baseline), serum homocysteine levels were lower, whereas FA and B12 levels were higher. In the DA, serum FA and B12 levels on day 1 of cycle 1 (after 5-7 days of B12-FAS) were significantly higher than at baseline, but homocysteine levels were similar. CONCLUSIONS: Simultaneous B12-FAS initiation with a pemetrexed-platinum doublet chemotherapy regimen is feasible and does not lead to enhanced hematologic toxicity. Serum homocysteine levels are unaffected by 5 to 7 days of B12-FAS.
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Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Suplementos Nutricionais , Ácido Fólico/administração & dosagem , Neoplasias Pulmonares/tratamento farmacológico , Neutropenia/prevenção & controle , Vitamina B 12/administração & dosagem , Carboplatina/administração & dosagem , Carcinoma Pulmonar de Células não Pequenas/patologia , Cisplatino/administração & dosagem , Feminino , Seguimentos , Humanos , Neoplasias Pulmonares/patologia , Masculino , Pessoa de Meia-Idade , Neutropenia/induzido quimicamente , Pemetrexede/administração & dosagem , Prognóstico , Taxa de Sobrevida , Tempo para o Tratamento , Vitaminas/administração & dosagemRESUMO
The present study has been designed to determine the effect of folate modulation (deficiency/supplementation) with aging on the promoter methylation of tumor suppressor and proto-oncogenes to understand the underlying mechanism of epigenetic alterations. Folate deficiency was induced for 3 and 5 months in weanling, young and adult groups, and after 3 months of folate deficiency, they were repleted with physiological folate (2 mg/kg diet) and folate oversupplementation (8 mg/kg diet) for another 2 months. The methylation facet in the present study revealed that the combined effect of folate deficiency and aging decreased the methylation index. Folate deficiency with age resulted in the up-regulation of proto-oncogenes (C-MYC and C-JUN) and cell cycle regulator gene Cyclin E as a result of promoter hypomethylation. However, in case of tumor suppressor genes (p53, p15ink4b and p16ink4a), the expression levels were found to be decreased at transcriptional level due to promoter hypermethylation. Upon repletion with physiological folate and folate oversupplementation, we found down-regulation of proto-oncogenes and up-regulation of tumor suppressor genes as a result of promoter hypermethylation and hypomethylation, respectively. Deregulation of these important genes due to folate deficiency may contribute toward the pathogenesis at cellular level.
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Envelhecimento/efeitos dos fármacos , Metilação de DNA/efeitos dos fármacos , Epigênese Genética/efeitos dos fármacos , Ácido Fólico/farmacologia , Fígado/efeitos dos fármacos , Envelhecimento/fisiologia , Animais , Ciclinas/genética , DNA (Citosina-5-)-Metiltransferase 1/genética , DNA (Citosina-5-)-Metiltransferases/genética , DNA Metiltransferase 3A , Regulação da Expressão Gênica/efeitos dos fármacos , Genes Supressores de Tumor/efeitos dos fármacos , Genes myc , Proteínas Quinases JNK Ativadas por Mitógeno/genética , Fígado/fisiologia , Masculino , Ratos Wistar , S-Adenosilmetionina/metabolismo , Tetra-Hidrofolatos/farmacocinética , DNA Metiltransferase 3BRESUMO
Invasive placentation and cancer development shares many similar molecular and epigenetic pathways. Paternally expressed, growth promoting genes (SNRPN, PEG10 and MEST) which are known to play crucial role in tumorogenesis, are not well studied during placentation. This study reports for the first time of the impact of gestational-age, pathological conditions and folic acid supplementation on dynamic nature of DNA and histone methylation present at their differentially methylated regions (DMRs). Here, we reported the association between low DNA methylation/H3K27me3 and higher expression of SNRPN, PEG10 and MEST in highly proliferating normal early gestational placenta. Molar and preeclamptic placental villi, exhibited aberrant changes in methylation levels at DMRs of these genes, leading to higher and lower expression of these genes, respectively, in reference to their respective control groups. Moreover, folate supplementation could induce gene specific changes in mRNA expression in placental cell lines. Further, MEST and SNRPN DMRs were observed to show the potential to act as novel fetal DNA markers in maternal plasma. Thus, variation in methylation levels at these DMRs regulate normal placentation and placental disorders. Additionally, the methylation at these DMRs might also be susceptible to folic acid supplementation and has the potential to be utilized in clinical diagnosis.
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Metilação de DNA , Suplementos Nutricionais , Epigênese Genética , Ácido Fólico/metabolismo , Variação Genética , Placenta/metabolismo , Vilosidades Coriônicas/metabolismo , Feminino , Regulação da Expressão Gênica , Impressão Genômica , Histonas/metabolismo , Humanos , Metilação , Gravidez , Regiões Promotoras Genéticas , RNA Mensageiro/genéticaRESUMO
Pemetrexed is the preferred chemotherapeutic drug for nonsquamous, non-small-cell lung cancer patients whenever the predictive molecular biomarkers for targeted therapy have either not been assessed or are absent. As per manufacturers' instructions, supplementation with folic acid (FA; folate) at a dose of 350 to 1000 µg daily should be started seven days before the first dose of pemetrexed-based chemotherapy and continued during therapy and for 21 days after therapy cessation. Vitamin B12 injections (1000 µg intramuscularly) should also be started one week before the first dose of chemotherapy. However, the evidence for delaying chemotherapy by one week for the purpose of providing vitamin B12 and FA supplementation is not robust. Observational and prospective single-arm studies have not shown any increased toxicity if pemetrexed was started earlier than the recommended duration of supplementation. In a resource-constrained setting, the standard (conventional) approach would lead to one additional visit and a 1-week chemotherapy delay, both of which could be inconvenient for patients. Hence, an open-label, randomized trial (PEMVITASTART [Vitamin Supplementation in NSCLC Patients on Pemetrexed Based Chemotherapy]; ClinicalTrials.gov identifier, NCT02679443) is being undertaken to evaluate whether any differences exist in pemetrexed-related hematologic toxicity among patients who receive delayed initiation of chemotherapy (after 5-7 days of vitamin B12 and FA supplementation [delayed arm]) compared with those for whom vitamin B12 and FA supplementation is started simultaneously (within 24 hours) of chemotherapy initiation (immediate arm). The present report describes the rationale and detailed design of the PEMVITASART trial.
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Antineoplásicos/uso terapêutico , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Ácido Fólico/uso terapêutico , Neoplasias Pulmonares/tratamento farmacológico , Pemetrexede/uso terapêutico , Vitamina B 12/uso terapêutico , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Suplementos Nutricionais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Seleção de Pacientes , Estudos Prospectivos , Projetos de Pesquisa , Resultado do Tratamento , Adulto JovemRESUMO
BACKGROUND: Pemetrexed is the preferred treatment of nonsquamous non-small cell lung cancer (ns-NSCLC). Folic acid supplementation (FAS) (350 to 1000 µg daily PO) is recommended to minimize hematological toxicity (HTox). Elevated total plasma homocysteine (tpHcy) predicts increased risk of HTox with pemetrexed in absence of FAS. The current study aimed to assess prevalence of elevated tpHcy levels at baseline and after pemetrexed treatment. Association of graded tpHcy levels/FAS with toxicity was also assessed. MATERIALS AND METHODS: Retrospective analysis of all ns-NSCLC patients undergoing first-line treatment with pemetrexed-containing platinum doublet over 3½ years was carried out. All eligible patients received pemetrexed (500 mg/m) and cisplatin (65 mg/m) each on D1 of a 3-week cycle. FAS was 400 µg for tpHcy< upper limit of normal (ULN), 700 µg for tpHcy 1 to 2 ULN, and 1000 µg for tpHcy>2 ULN. All patients also received oral ferrous sulphate and injectable vitamin B12. Exact 95% confidence intervals (CI) were calculated for comparison with previously published studies. RESULTS: 75.7% of 111 patients had stage IV disease. Prevalence of tpHcy levels
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Antineoplásicos/toxicidade , Carcinoma Pulmonar de Células não Pequenas/sangue , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Ácido Fólico/uso terapêutico , Hematínicos/uso terapêutico , Homocisteína/sangue , Neoplasias Pulmonares/sangue , Neoplasias Pulmonares/tratamento farmacológico , Pemetrexede/toxicidade , Interações Medicamentosas , Quimioterapia Combinada , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estudos RetrospectivosRESUMO
SCOPE: The present study was designed to identify the molecular mechanism of folate modulation and aging on aberrant liver folate transporter system. METHODS AND RESULTS: An in vivo rat model was used, in which weanling, young and adult rats were given folate deficient diet for 3 and 5 months and after 3 months of folate deficiency, one group received physiological folate repletion (2 mg/kg diet) and another group received over supplemented folate diet (8 mg/kg diet) for another 2 months. In adult group, 3 and 5 months of folate deficiency decreased serum and tissue folate levels with decreased uptake of folate, further associated with decreased expression levels of reduced folate carrier (RFC) and increased expression levels of folate exporter (ABCG2) at both mRNA and protein levels, which in turn regulated by promoter hypermethylation of RFC and promoter hypomethylation of ABCG2 gene. CONCLUSION: Promoter hypermethylation of RFC and promoter hypomethylation of ABCG2 may be attributed to the down regulation of RFC and up regulation of ABCG2 at mRNA and protein levels in conditions of 3 and 5 months of folate deficiency in the adult group.
Assuntos
Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/metabolismo , Envelhecimento/genética , Epigênese Genética , Fígado/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Antígenos de Histocompatibilidade Menor/metabolismo , Proteína Carregadora de Folato Reduzido/metabolismo , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/genética , Animais , Metilação de DNA , Dieta , Suplementos Nutricionais , Ácido Fólico/administração & dosagem , Ácido Fólico/sangue , Deficiência de Ácido Fólico/sangue , Deficiência de Ácido Fólico/tratamento farmacológico , Proteínas de Membrana Transportadoras/genética , Antígenos de Histocompatibilidade Menor/genética , Regiões Promotoras Genéticas , Ratos , Proteína Carregadora de Folato Reduzido/genéticaRESUMO
Folic acid is the key one-carbon donor required for de novo nucleotide and methionine synthesis. Its deficiency is associated with megaloblastic anemia, cancer and various complications of pregnancy. However, its supplementation results in reduction of neural tube defects and prevention of several types of cancer. The intake of folic acid from fortified food together with the use of nutritional supplements creates a state of folate oversupplementation. Fortification of foods is occurring worldwide with little knowledge of the potential safety and physiologic consequences of intake of such high doses of folic acid. So, we planned to examine the effects of acute and chronic folate oversupplementation on the physiology of renal folate transport in rats. Male Wistar rats were procured and divided into two groups. Rats in group I were given semisynthetic diets containing 2 mg folic acid/kg diet (control) and those in group II were given folate-oversupplemented rat diet, i.e., 20 mg folic acid/kg diet (oversupplemented). Six animals from group I and group II received the treatment for 10 days (acute treatment) and remaining six for 60 days (chronic treatment). In acute folate-oversupplemented rats, 5-[(14)C]-methyltetrahydrofolate uptake was found to be significantly reduced, as compared to chronic folate-oversupplemented and control rats. This reduction in uptake was associated with a significant decrease in the mRNA and protein levels of the folate transporters. Results of the present investigation showed that acute oversupplementation led to a specific and significant down-regulation of renal folate uptake process mediated via transcriptional and translational regulatory mechanism(s).
RESUMO
Folic acid is essential for numerous biological functions, ranging from nucleotide biosynthesis to the remethylation of homocysteine. Folic acid is unable to cross the biological membranes by simple diffusion, so there exists a well-developed epithelial folate transport system for the regulation of normal folate homeostasis in the intestine. Any perturbances in the folate uptake system might lead to a state of folate deficiency, which in turn is strongly associated with the risk of various cancers, birth defects and CVD. Countries with obligatory folate fortification of food (USA and Canada) have documented a significant decrease in neural tube defects in newborns. However, the effect of folate oversupplementation on the intestinal absorption of folic acid has not been studied. We studied the process of folate transport and the expression of folate transporters in the rat intestine after folate oversupplementation. Rats were oversupplemented with tenfold the normal requirement of folic acid for periods of 10 and 60 d. Folate uptake in intestinal brush-border membrane vesicles followed saturable kinetics with pH optimum at 5·5. Acute, but not chronic, folate oversupplementation led to a significant down-regulation in intestinal folate uptake at acidic pH optima and was associated with a decrease in Vmax without any significant change in the Km of the folate uptake process. The decrease in folate uptake was also associated with the down-regulation in the protein levels of major folate transporters, proton-coupled folate transporter (PCFT) and reduced folate carrier (RFC), without altering their mRNA levels. Hence, it was concluded that acute folate oversupplementation results in a significant decrease in intestinal folate uptake by down-regulating the expressions of RFC and PCFT, via some post-transcriptional or translational mechanisms.
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Suplementos Nutricionais/efeitos adversos , Deficiência de Ácido Fólico/etiologia , Ácido Fólico/farmacocinética , Mucosa Intestinal/metabolismo , Transportador de Folato Acoplado a Próton/metabolismo , Proteína Carregadora de Folato Reduzido/metabolismo , Complexo Vitamínico B/farmacocinética , Animais , Transporte Biológico , Regulação para Baixo , Ácido Fólico/administração & dosagem , Ácido Fólico/efeitos adversos , Concentração de Íons de Hidrogênio , Absorção Intestinal , Masculino , Modelos Animais , Ratos , Ratos Wistar , Complexo Vitamínico B/administração & dosagem , Complexo Vitamínico B/efeitos adversosRESUMO
Oil feeding is known to increase the secretion of intestinal alkaline phosphatase (IAP) into serum and this phenomenon is shown to be mediated by surfactant-like particles. These lipoprotein particles are secreted by enterocytes and are rich in phosphatidyl choline and IAP. The exact mechanisms underlying this phenomenon are not known. We studied the effect of feeding different oils varying in fatty acid composition, i.e., coconut oil, corn oil, and cod liver oil, on the secretion of IAP into serum. Also, the effect of actinomycin D treatment on this phenomenon was evaluated. Male albino rats were fed 2 ml of various oils. Alkaline phosphatase activity was measured in serum, luminal washings, and other intestinal fractions. Cod liver oil was found to maximally enhance the soluble and membrane-bound IAP as well as secretion of IAP into lumen and serum. Administration of actinomycin D significantly reduced the enzyme activity in serum and various intestinal fractions in both control and cod liver oil-fed rats. These results were further substantiated by 5-bromo-4-chloroindolyl phosphate staining of IAP in acrylamide gels and by western blotting. The effect of cod liver oil feeding was specific for IAP, as there was no change in the activity of another brush border enzyme, sucrase, under these conditions. These findings suggest that fatty acid composition of the oil determines the amount of IAP secretion and there is coordination between IAP synthesis and its secretion for transport into serum in response to oil feeding.
Assuntos
Fosfatase Alcalina/metabolismo , Gorduras na Dieta/farmacologia , Intestinos/enzimologia , Animais , Western Blotting , Óleo de Coco , Óleo de Fígado de Bacalhau/farmacologia , Óleo de Milho/farmacologia , Dactinomicina/farmacologia , Masculino , Óleos de Plantas/farmacologia , Inibidores da Síntese de Proteínas/farmacologia , Ratos , Ratos Wistar , Sacarase/metabolismoRESUMO
BACKGROUND/METHOD: The effect of feeding ethanol for 5 weeks on the lipid composition of the intestinal microvillus membrane (MVM) was studied in rats fed a commercial rat pellet (RP) diet or purified diets containing 10% coconut oil (CCO), corn oil (CO) or fish oil (FO). RESULTS: A low cholesterol/phospholipid ratio and increased saturated fatty acid level were observed in MVM from the CCO or FO groups. Chronic administration of ethanol to RP- or CO-fed animals increased phospholipids, total and free cholesterol, and the triglyceride and ganglioside content of MVM. The free cholesterol and phospholipid content was reduced while the triglyceride level remained unaffected by ethanol treatment in the CCO or FO groups. Ethanol ingestion decreased 10:2 and 20:4 (n-6 fatty acids) but increased the saturated fatty acid content of MVM in all the dietary groups except in CCO-fed animals where the 18:2 level was not affected. An elevated 18:1, but decreased 22:6 percentage was observed in the ethanol-fed FO group. The fatty acid composition of MVM from the CCO-fed group was least affected by ethanol treatment. CONCLUSION: These observations suggest that the type of dietary fat modifies ethanol-mediated alterations in MVM lipid composition.
Assuntos
Colesterol/análise , Gorduras na Dieta/administração & dosagem , Etanol/farmacologia , Microvilosidades/química , Fosfolipídeos/análise , Triglicerídeos/análise , Análise de Variância , Animais , Colesterol/metabolismo , Óleo de Coco , Óleo de Milho/administração & dosagem , Óleos de Peixe/administração & dosagem , Masculino , Fosfolipídeos/metabolismo , Óleos de Plantas/administração & dosagem , Distribuição Aleatória , Ratos , Triglicerídeos/metabolismoRESUMO
The effect of harmaline, a plant alkaloid has been studied on rat intestinal brush border sucrase activity. Stimulation of sucrase activity by Na+ was found to be pH-dependent. At neutral pH, 20 mM Na+ stimulated sucrase activity by reducing K(m) by 30%, while at acidic pH (5.2), the activity increased 4-fold compared to Na+-free enzyme. At 1.0 mM, harmaline markedly inhibited (67%) the enzyme activity at pH 5.2 in the absence of Na+. However, inhibition was reduced in presence of 20 mM sodium, whereas 4.0 mM harmaline was required to inhibit the enzyme activity by 65%. In the absence of Na+ ions, harmaline inhibition of sucrase activity was of competitive type, but it changed to non-competitive type in presence of 20 mM Na+ at pH 5.2. Sucrase-harmaline interactions as a function of pH, both in presence and absence of Na+ revealed a shift in pH optima of the enzyme towards a higher pH in presence of 4 mM and 1 mM harmaline respectively. The observed inhibition was reversible in nature and was only partially overcome by sodium, lithium, potassium, cesium, rubidium and ammonium ions. These findings suggest that harmaline also inhibits rat brush border sucrase and that the presence of Na+ site is not a pre-requisite for the inhibition.