Perinatal manipulation of α-linolenic acid intake induces epigenetic changes in maternal and offspring livers.

Previous studies indicated that the intake of α-linolenic acid (ALA) can alter the concentration of both ω-6 and ω-3 fatty acids in both mother and offspring, with consequences on postnatal brain development. This study describes the association between maternal ALA availability during gestation and lactation, and alterations in the Fads2 DNA methylation in both maternal and offspring livers, at the end of lactation period. Both Fads2 promoter and intron 1 DNA methylation were increased in the groups receiving postnatal flaxseed oil containing 50% ALA (mothers or pups), while bivariate analysis indicated a significant association of the Fads2 epigenetic status in the liver between each mother and its offspring. In addition, Fads2 expression was negatively correlated with promoter methylation at the individual level in maternal livers (P<0.05). This study also indicated that the interplay between ALA availability during gestation and lactation can differentially alter the expression of desaturases and elongases involved in ω-6 and ω-3 metabolic pathways. In summary, when considering the perinatal dietary ALA requirements in mice, both gestation and lactation periods should be considered as having distinct roles in modulating the metabolism of ω-6 and ω-3 fatty acids in maternal mouse livers.

Maternal α-linolenic acid availability during gestation and lactation alters the postnatal hippocampal development in the mouse offspring.

The availability of ω-3 polyunsaturated fatty acids is essential for perinatal brain development. While the roles of docosahexaenoic acid (the most abundant ω-3 species) were extensively described, less is known about the role of α-linolenic acid (ALA), which is the initial molecular species undergoing elongation and desaturation within the ω-3 pathways. This study describes the association between maternal ALA availability during gestation and lactation, and alterations in hippocampal development (dentate gyrus) in the mouse male offspring, at the end of lactation (postnatal day 19, P19). Postnatal ALA supplementation increased cell proliferation (36% more proliferating cells compared to a control group) and early neuronal differentiation, while postnatal ALA deficiency increased cellular apoptosis within the dentate gyrus of suckling pups (61% more apoptotic cells compared to a control group). However, maternal ALA deficiency during gestation prevented the increased neurogenesis induced by postnatal supplementation. Fatty acid analysis revealed that ALA supplementation increased the concentration of the ω-3 species in the maternal liver and serum, but not in the brain of the offspring, excepting for ALA itself. Interestingly, ALA supplementation also increased the concentration of dihomo γ-linolenic acid (a ω-6 species) in the P19 brains, but not in maternal livers or serum. In conclusion, postnatal ALA supplementation enhances neurogenesis in the dentate gyrus of the offspring at postnatal day 19, but its beneficial effects are offset by maternal ALA deficiency during gestation. These results suggest that ALA is required in both fetal and postnatal stages of brain development.

Maternal dietary choline deficiency alters angiogenesis in fetal mouse hippocampus.

We examined whether maternal dietary choline modulates angiogenesis in fetal brain. Pregnant C57BL/6 mice were fed either a choline-deficient (CD), control (CT), or choline-supplemented diet (CS) from days 12 to 17 (E12-17) of pregnancy and then fetal brains were studied. In CD fetal hippocampus, proliferation of endothelial cells (EC) was decreased by 32% (p < 0.01 vs. CT or CS) while differentiated EC clusters (expressing factor VIII related antigen (RA)) increased by 25% (p < 0.01 vs. CT or CS). These changes were associated with > 25% decrease in the number of blood vessels in CD fetal hippocampus (p < 0.01 vs. CT and CS), with no change in total cross-sectional area of these blood vessels. Expression of genes for the angiogenic signals derived from both endothelial and neuronal progenitor cells (NPC) was increased in CD fetal hippocampus VEGF C (Vegfc), 2.0-fold, p < 0.01 vs. CT and angiopoietin 2 (Angpt2), 2.1-fold, (p < 0.01 vs. CT)). Similar increased expression was observed in NPC isolated from E14 fetal mouse brains and exposed to low (5 microM), CT (70 microM), or high choline (280 microM) media for 72 h (low choline caused a 9.7-fold increase in relative gene expression of Vegfc (p < 0.001 vs. CT and high) and a 3.4-fold increase in expression of Angpt2, (p < 0.05 vs. CT and high). ANGPT2 protein was increased 42.2% (p < 0.01). Cytosine-phosphate-guanine dinucleotide islands in the proximity of the promoter areas of Vegfc and Angpt2 were hypomethylated in low choline NPC compared to CT NPC (p < 0.01). We conclude that maternal dietary choline intake alters angiogenesis in the developing fetal hippocampus.

Dietary choline reverses some, but not all, effects of folate deficiency on neurogenesis and apoptosis in fetal mouse brain.

In mice, maternal dietary folate, a cofactor in 1-carbon metabolism, modulates neurogenesis and apoptosis in the fetal brain. Similarly, maternal dietary choline, an important methyl-donor, also influences these processes. Choline and folate are metabolically interrelated, and we determined whether choline supplementation could reverse the effects of folate deficiency on brain development. Timed-pregnant mice were fed control (CT), folate-deficient (FD), or folate-deficient, choline-supplemented (FDCS) AIN-76 diets from d 11 to 17 (E11-17) of pregnancy, and on E17, fetal brains were collected for analysis. Compared with the CT group, the FD group had fewer neural progenitor cells undergoing mitosis in the ventricular zones of the developing mouse brain septum (47%; P < 0.01), hippocampus (29%; P < 0.01), striatum (34%; P < 0.01), and anterior and mid-posterior neocortex (33% in both areas; P < 0.01). In addition, compared with CT, the FD diet almost doubled the rate of apoptosis in the fetal septum and hippocampus (P < 0.01). In the FDCS group, the mitosis rates generally were intermediate between those of the CT and FD groups; mitosis rates in the septum and striatum were significantly greater compared with the FD group and were significantly lower than in the CT group only in the septum and neocortex. In the FDCS group, the hippocampal apoptosis rate was significantly lower than in the FD group (P < 0.01) and was the same as in the CT group. In the septum, the apotosis rate in the FDCS group was intermediate between the CT and FD groups' rates. These results suggest that neural progenitor cells in fetal forebrain are sensitive to maternal dietary folate during late gestation and that choline supplementation can modify some, but not all, of these effects.

Deletion of murine choline dehydrogenase results in diminished sperm motility.

Choline dehydrogenase (CHDH) catalyzes the conversion of choline to betaine, an important methyl donor and organic osmolyte. We have previously identified single nucleotide polymorphisms (SNPs) in the human CHDH gene that, when present, seem to alter the activity of the CHDH enzyme. These SNPs occur frequently in humans. We created a Chdh(-/-) mouse to determine the functional effects of mutations that result in decreased CHDH activity. Chdh deletion did not affect fetal viability or alter growth or survival of these mice. Only one of eleven Chdh(-/-) males was able to reproduce. Loss of CHDH activity resulted in decreased testicular betaine and increased choline and PCho concentrations. Chdh(+/+) and Chdh(-/-) mice produced comparable amounts of sperm; the impaired fertility was due to diminished sperm motility in the Chdh(-/-) males. Transmission electron microscopy revealed abnormal mitochondrial morphology in Chdh(-/-) sperm. ATP content, total mitochondrial dehydrogenase activity and inner mitochondrial membrane polarization were all significantly reduced in sperm from Chdh(-/-) animals. Mitochondrial changes were also detected in liver, kidney, heart, and testis tissues. We suggest that men who have SNPs in CHDH that decrease the activity of the CHDH enzyme could have decreased sperm motility and fertility.

Choline deficiency alters global histone methylation and epigenetic marking at the Re1 site of the calbindin 1 gene.

Maternal choline availability is essential for fetal neurogenesis. Choline deprivation (CD) causes hypomethylation of specific CpG islands in genes controlling cell cycling in fetal hippocampus. We now report that, in C57BL/6 mice, CD during gestational days 12-17 also altered methylation of the histone H3 in E17 fetal hippocampi. In the ventricular and subventricular zones, monomethyl-lysine 9 of H3 (H3K9me1) was decreased by 25% (P<0.01), and in the pyramidal layer, dimethyl-lysine 9 of H3 (H3K9me2) was decreased by 37% (P<0.05). These changes were region specific and were not observed in whole-brain preparations. Also, the same effects of CD on H3 methylation were observed in E14 neural progenitor cells (NPCs) in culture. Changes in G9a histone methyltransferase might mediate altered H3K9me2,1. Gene expression of G9a was decreased by 80% in CD NPCs (P<0.001). In CD, H3 was hypomethylated upstream of the RE1 binding site in the calbindin 1 promoter, and 1 CpG site within the calbindin1 promoter was hypermethylated. REST binding to RE1 (recruits G9a) was decreased by 45% (P<0.01) in CD. These changes resulted in increased expression of calbindin 1 in CD (260%; P<0.05). Thus, CD modulates histone methylation in NPCs, and this could underlie the observed changes in neurogenesis.

Dietary docosahexaenoic acid supplementation modulates hippocampal development in the Pemt-/- mouse.

The development of fetal brain is influenced by nutrients such as docosahexaenoic acid (DHA, 22:6) and choline. Phosphatidylethanolamine-N-methyltransferase (PEMT) catalyzes the biosynthesis of phosphatidylcholine from phosphatidylethanolamine enriched in DHA and many humans have functional genetic polymorphisms in the PEMT gene. Previously, it was reported that Pemt(-/-) mice have altered hippocampal development. The present study explores whether abnormal phosphatidylcholine biosynthesis causes altered incorporation of DHA into membranes, thereby influencing brain development, and determines whether supplemental dietary DHA can reverse some of these changes. Pregnant C57BL/6 wild type (WT) and Pemt(-/-) mice were fed a control diet, or a diet supplemented with 3 g/kg of DHA, from gestational day 11 to 17. Brains from embryonic day 17 fetuses derived from Pemt(-/-) dams fed the control diet had 25-50% less phospholipid-DHA as compared with WT (p < 0.05). Also, they had 60% more neural progenitor cell proliferation (p < 0.05), 60% more neuronal apoptosis (p < 0.01), and 30% less calretinin expression (p < 0.05; a marker of neuronal differentiation) in the hippocampus compared with WT. The DHA-supplemented diet increased fetal brain Pemt(-/-) phospholipid-DHA to WT levels, and abrogated the neural progenitor cell proliferation and apoptosis differences. Although this diet did not change proliferation in the WT group, it halved the rate of apoptosis (p < 0.05). In both genotypes, the DHA-supplemented diet increased calretinin expression 2-fold (p < 0.05). These results suggest that the changes in hippocampal development in the Pemt(-/-) mouse could be mediated by altered DHA incorporation into membrane phospholipids, and that maternal dietary DHA can influence fetal brain development.

Dose response effects of dermally applied diethanolamine on neurogenesis in fetal mouse hippocampus and potential exposure of humans.

Diethanolamine (DEA) is a common ingredient of personal care products. Dermal administration of DEA diminishes hepatic stores of the essential nutrient choline and alters brain development. We previously reported that 80 mg/kg/day of DEA during pregnancy in mice reduced neurogenesis and increased apoptosis in the fetal hippocampus. This study was designed to establish the dose-response relationships for this effect of DEA. Timed-pregnant C57BL/6 mouse dams were dosed dermally from gestation day 7-17 with DEA at 0 (controls), 5, 40, 60, and 80 mg/kg body/day. Fetuses (embryonic day 17 [E17]) from dams treated dermally with 80 mg/kg body/day DEA had decreased neural progenitor cell mitosis at the ventricular surface of the ventricular zone (hippocampus, 54.1 +/- 5.5%; cortex, 58.9 +/- 6.8%; compared to controls; p < 0.01). Also, this dose of DEA to dams increased rates of apoptosis in E17 fetal hippocampus (to 177.2 +/- 21.5% of control; measured using activated caspase-3; p < 0.01). This dose of DEA resulted in accumulation of DEA and its metabolites in liver and in plasma. At doses of DEA less than 80 mg/kg body/day to dams, there were no differences between treated and control groups. In a small group of human subjects, dermal treatment for 1 month with a commercially available skin lotion containing 1.8 mg DEA per gram resulted in detectable plasma concentrations of DEA and dimethyldiethanolamine, but these were far below those concentrations associated with perturbed brain development in the mouse.

Diethanolamine alters neurogenesis and induces apoptosis in fetal mouse hippocampus.

Diethanolamine (DEA) is present in many consumer products such as shampoo. Dermal administration of DEA diminishes hepatic stores of the essential nutrient choline, and we previously reported that dietary choline deficiency during pregnancy reduces neurogenesis and increases apoptosis in the hippocampus of fetal rats and mice. Therefore, DEA could also alter brain development. Timed-pregnant C57BL/6 mice were dosed dermally from gestation day 7 through 17 with DEA at 0, 20, 80, 160, 320, and 640 mg/kg body/day. At doses of DEA > 80 mg/kg body/day, we observed decreased litter size. In fetuses (embryonic day 17) collected from dams treated dermally with 80 mg/kg body/day DEA, we observed decreased neural progenitor cell mitosis at the ventricular surface of the ventricular zone of the hippocampus [to 56+/-14% (se) histone 3 (H3) phosphorylation as compared to controls; P < 0.01]. We also observed increased apoptosis in fetal hippocampus (to 170+/-10% of control measured using TUNEL and to 178+/-7% of control measured using activated caspase 3; P < 0.01). Thus, maternal exposure to DEA reduces the number of neural progenitor cells in hippocampus by two mechanisms, and this could permanently alter memory function in offspring of mothers exposed to this common ingredient of shampoos and soaps.

Choline deficiency increases lymphocyte apoptosis and DNA damage in humans.

BACKGROUND: Whereas deficiency of the essential nutrient choline is associated with DNA damage and apoptosis in cell and rodent models, it has not been shown in humans. OBJECTIVE: The objective was to ascertain whether lymphocytes from choline-deficient humans had greater DNA damage and apoptosis than did those from choline-sufficient humans. DESIGN: Fifty-one men and women aged 18-70 y were fed a diet containing the recommended adequate intake of choline (control) for 10 d. They then were fed a choline-deficient diet for up to 42 d before repletion with 138-550 mg choline/d. Blood was collected at the end of each phase, and peripheral lymphocytes were isolated. DNA damage and apoptosis were then assessed by activation of caspase-3, terminal deoxynucleotide transferase-mediated dUTP nick end-labeling, and single-cell gel electrophoresis (COMET) assays. RESULTS: All subjects fed the choline-deficient diet had lymphocyte DNA damage, as assessed by COMET assay, twice that found when they were fed the control diet. The subjects who developed organ dysfunction (liver or muscle) when fed the choline-deficient diet had significantly more apoptotic lymphocytes, as assessed by the activated caspase-3 assay, than when fed the control diet. CONCLUSIONS: A choline-deficient diet increased DNA damage in humans. Subjects in whom these diets induced liver or muscle dysfunction also had higher rates of apoptosis in their peripheral lymphocytes than did subjects who did not develop organ dysfunction. Assessment of DNA damage and apoptosis in lymphocytes appears to be a clinically useful measure in humans (such as those receiving parenteral nutrition) in whom choline deficiency is suspected.

Dietary choline deficiency alters global and gene-specific DNA methylation in the developing hippocampus of mouse fetal brains.

The availability of choline during critical periods of fetal development alters hippocampal development and affects memory function throughout life. Choline deficiency during fetal development reduces proliferation and migration of neuronal precursor cells in the mouse fetal hippocampus and these changes are associated with modifications in the protein levels of some cell cycle regulators and early differentiation markers. We fed C57 BL/6 mouse dams diets deficient or normal in choline content from days 12 to 17 of pregnancy, and then collected fetal brains on embryonic day 17. Using laser-capture micro-dissection we harvested cells from the ventricular and subventricular zones of Ammon's horn and from the prime germinal zone of the dentate gyrus (hippocampus). In the ventricular and subventricular zones from the choline-deficient group, we observed increased protein levels for kinase-associated phosphatase (Kap) and for p15(INK4b) (two cell cycle inhibitors). In the dentate gyrus, we observed increased levels of calretinin (an early marker of neuronal differentiation). In fetal brain from mothers fed a choline-deficient diet, DNA global methylation was decreased in the ventricular and subventricular zones of Ammon's horn. We also observed decreased gene-specific DNA methylation of the gene (Cdkn3) that encodes for Kap, correlating with increased expression of this protein. This was not the case for p15(INK4b) or calretinin (Cdkn2b and Calb2, respectively). These data suggest that choline deficiency-induced changes in gene methylation could mediate the expression of a cell cycle regulator and thereby alter brain development.

Maternal dietary choline availability alters the balance of netrin-1 and DCC neuronal migration proteins in fetal mouse brain hippocampus.

Alterations in maternal dietary choline availability during days 12-17 of pregnancy led to an increase in the level of immunoreactive netrin-1 and a decrease in the level of DCC protein in the developing fetal mouse brain hippocampus compared with controls. Changes in the expression of cell migration cues during development could account for some of the lifelong consequences of maternal dietary choline availability for cognitive and memory processes.

Gene expression profiling of choline-deprived neural precursor cells isolated from mouse brain.

Choline is an essential nutrient and an important methyl donor. Choline deficiency alters fetal development of the hippocampus in rodents and these changes are associated with decreased memory function lasting throughout life. Also, choline deficiency alters global and gene-specific DNA methylation in several models. This gene expression profiling study describes changes in cortical neural precursor cells from embryonic day 14 mice, after 48 h of exposure to a choline-deficient medium. Using Significance Analysis of Microarrays, we found the expression of 1003 genes to be significantly changed (from a total of 16,000 total genes spotted on the array), with a false discovery rate below 5%. A total of 846 genes were overexpressed while 157 were underexpressed. Classification by gene ontology revealed that 331 of these genes modulate cell proliferation, apoptosis, neuronal and glial differentiation, methyl metabolism, and calcium-binding protein classes. Twenty-seven genes that had changed expression have previously been reported to be regulated by promoter or intron methylation. These findings support our previous work suggesting that choline deficiency decreases the proliferation of neural precursors and possibly increases premature neuronal differentiation and apoptosis.

Regulation of choline deficiency apoptosis by epidermal growth factor in CWSV-1 rat hepatocytes.

Previous studies show that acute choline deficiency (CD) triggers apoptosis in cultured rat hepatocytes (CWSV-1 cells). We demonstrate that prolonged EGF stimulation (10 ng/mL x 48 hrs) restores cell proliferation, as assessed by BrdU labeling, and protects cells from CD-induced apoptosis, as assessed by TUNEL labeling and cleavage of poly(ADP-ribose) polymerase. However, EGF rescue was not accompanied by restoration of depleted intracellular concentrations of choline, glycerphosphocholine, phosphocholine, or phosphatidylcholine. In contrast, we show that EGF stimulation blocks apoptosis by restoring mitochondrial membrane potential (Delta Psi(m)), as determined using the potential-sensitive dye chloromethyl-X-rosamine, and by preventing the release and nuclear localization of cytochrome c. We investigated whether EGF rescue involves EGF receptor phosphorylation and activation of the down-stream cell survival factor Akt. Compared to cells in control medium (CT, 70 micromol choline x 48 hrs), cells in CD medium (5 micromol choline) were less sensitive to EGF-induced (0-300 ng/mL x 5 min) receptor tyrosine phosphorylation. Compared to cells in CT medium, cells in CD medium treated with EGF (10 ng/mL x 5 min) exhibited higher levels of phosphatidylinositol 3-kinase (PI3K)-dependent phosphorylation of AktSer473. Inactivation of PI3K was sufficient to block EGF-stimulated activation of Akt, restoration of mitochondrial Delta Psi(m), and prevention of cytochrome c release. These studies indicate that stimulation with EGF activates a cell survival response against CD-apoptosis by restoring mitochondrial membrane potential and preventing cytochrome c release and nuclear translocation which are mediated by activation of Akt in hepatocytes.

Folic acid deficiency during late gestation decreases progenitor cell proliferation and increases apoptosis in fetal mouse brain.

In mice and rats, maternal dietary choline intake during late pregnancy modulates mitosis and apoptosis in progenitor cells of the fetal hippocampus and septum. Because choline and folate are interrelated metabolically, we investigated the effects of maternal dietary folate availability on progenitor cells in fetal mouse telencephalon. Timed-pregnant mice were fed a folate-supplemented (FS), control (FCT) or folate-deficient (FD) AIN-76 diet from d 11-17 of pregnancy. FD decreased the number of progenitor cells undergoing cell replication in the ventricular zones of the developing mouse brain septum (46.6% of FCT), caudate putamen (43.5%), and neocortex (54.4%) as assessed using phosphorylated histone H3 (a specific marker of mitotic phase) and confirmed by bromodeoxyuridine (BrdU) labeling of the S phase. In addition, 106.2% more apoptotic cells were found in FD than in FCT fetal septum. We observed 46.8% more calretinin-positive cells in the medial septal-diagonal band region of FD compared with pups from control dams. FS mice did not differ significantly from FCT mice in any of these measures. These results suggest that progenitor cells in fetal forebrain are sensitive to maternal dietary folate during late gestation.

Choline availability during embryonic development alters progenitor cell mitosis in developing mouse hippocampus.

Previously, we reported that dietary choline influences development of the hippocampus in fetal rat brain. It is important to know whether similar effects of choline occur in developing fetal mouse brain because interesting new experimental approaches are now available using several transgenic mouse models. Timed-pregnant mice were fed choline-supplemented (CS), control (CT) or choline-deficient (CD) AIN-76 diet from embryonic day 12 to 17 (E12-17). Fetuses from CD dams had diminished concentrations of phosphocholine and phosphatidylcholine in their brains compared with CT or CS fetuses (P < 0.05). When we analyzed fetal hippocampus on day E17 for cells with mitotic phase-specific expression of phosphorylated histone H3, we detected fewer labeled cells at the ventricular surface of the ventricular zone in the CD group (14.8 +/- 1.9) compared with the CT (30.7 +/- 1.9) or CS (36.6 +/- 2.6) group (P < 0.05). At the same time, we detected more apoptotic cells in E17 hippocampus using morphology in the CD group (11.8 +/- 1.4) than in CT (5.6 +/- 0.6) or CS (4.2 +/- 0.7) group (P < 0.05). This was confirmed using terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-digoxigenin anti-digoxigenin fluorescein conjugate antibody nick end-labeling (TUNEL) and activated caspase-3 immunoreactivity. We conclude that the dietary availability of choline to the mouse dam influences progenitor cell proliferation and apoptosis in the fetal brain.

Activation of a caspase-dependent oxidative damage response mediates TGFbeta1 apoptosis in rat hepatocytes.

Activation of transforming growth factor-beta type 1- (TGFbeta1) mediated signaling occurs in response to cell injury affecting stem-type cells and hepatocytes in liver. In this work we used WB stemlike liver epithelial cells and p53-defective CWSV-1 nontumorigenic rat hepatocytes to investigate the possible roles of caspases and oxidative stress in TGFbeta1 signaling. TGFbeta1 significantly increased the level of 4-hydroxy-2-nonenal (4-HNE), a stable product of lipid peroxidation. In addition, TGFbeta1-treated cells exhibited activation of caspases that accompanied by enhanced cleavage of the caspase substrate poly(ADP)-ribose polymerase (PARP) and induction of apoptosis. WB cells were twice as sensitive as sensitive as CWSV-1 cells to induction of TGFbeta1 apoptosis. TGFbeta1-apoptosis was significantly reduced when cells were treated with TGFbeta1 in the presence of inhibitors of caspase-1, -3, -8, and -9. Importantly, in addition to suppression of apoptosis, treatment of cells with the caspase-3 inhibitor Z-DEVD-FMK in the presence of TGFbeta1 suppressed the formation 4-HNE and restored mitotic activity. Together, these data suggest TGFbeta1 induces activation of a caspase signaling cascade that includes an oxidative damage response, PARP cleavage, and apoptosis that do not require intact p53 in rat hepatocytes.

Choline availability during embryonic development alters the localization of calretinin in developing and aging mouse hippocampus.

Choline availability in the diet during pregnancy alters fetal brain biochemistry with resulting behavioral changes that persist throughout the lifetime of the offspring. In the present study, the effects of dietary choline on the onset of GABAergic neuronal differentiation in developing fetal brain, as demarcated by the expression of calcium binding protein calretinin, are described. In these studies, timed-pregnant mice were fed choline supplemented, control or choline deficient AIN-76 diet from day 12-17 of pregnancy and the brains of their fetuses were studied on day 17 of gestation. In the primordial dentate gyrus, we found that pups from choline deficient-dams had more calretinin protein (330% increase), and pups from choline supplemented-dams had less calretinin protein (70% decrease), than did pups from control-dams. Importantly, decreased calretinin protein was still detectable in hippocampus in aged, 24-month-old mice, born of choline supplemented-dams and maintained since birth on a control diet. Thus, alterations in the level of calretinin protein in fetal brain hippocampus could underlie the known, life long effects of maternal dietary choline availability on brain development and behavior.

Mitochondrial and microsomal derived reactive oxygen species mediate apoptosis induced by transforming growth factor-beta1 in immortalized rat hepatocytes.

Transforming growth factor-beta1 (TGFbeta1) is a multifunctional cytokine that is over expressed during liver hepatocytes injury and regeneration. SV40-transformed CWSV-1 rat hepatocytes that are p53-defective undergo apoptosis in response to choline deficiency (CD) or TGFbeta1, which mediates CD-apoptosis. Reactive oxygen species (ROS) are essential mediators of apoptosis. We have shown that apoptosis induced by TGFbeta1 is accompanied by ROS generation and the ROS-trapping agent N-acetylcysteine (NAC) inhibits TGFbeta1-induced apoptosis. While persistent induction of ROS contributes to this form of apoptosis, the source of ROS generated downstream of TGFbeta1 is not clear. The mitochondria and the endoplasmic reticulum both harbor potent electron transfer chains that might be the source of ROS essential for completion of TGFbeta1-apoptosis. Here we show that CWSV-1 cells treated with cyclosporine A, which prevents opening of mitochondrial membrane pores required for ROS generation, inhibits TGFbeta1-induced apoptosis. A similar effect was obtained by treating these cells with rotenone, an inhibitor of complex 1 of the mitochondrial electron transfer chain. However, we demonstrate that TGFbeta1 induces cytochrome P450 1A1 and that metyrapone, a potent inhibitor of cytochrome P450 1A1, inhibits TGFbeta1-induced apoptosis. Therefore, our studies indicate that concurrent with promoting generation of ROS from mitochondria, TGFbeta1 also promotes generation of ROS from the cytochrome P450 electron transfer chain. Since inhibition of either of these two sources of ROS interferes with apoptosis, it is reasonable to conclude that the combined involvement of both pathways is essential for completion of TGFbeta1-induced apoptosis.

Lack of significant genotoxicity of purified soy isoflavones (genistein, daidzein, and glycitein) in 20 patients with prostate cancer.

BACKGROUND: Genistein may be useful in the prevention or treatment of prostate cancer; however, it causes genetic damage in cultured human cells. OBJECTIVE: The objective was to assess the potential genotoxicity of a purified soy unconjugated isoflavone mixture in men with prostate cancer. DESIGN: Twenty patients with prostate cancer were treated with 300 mg genistein/d for 28 d and then with 600 mg/d for another 56 d. In peripheral lymphocytes, DNA strand breaks were assessed as nuclear tail moment, chromosomal damage was assessed as micronucleus frequency (MF), and translocations of the MLL gene (11q23) were assessed by using fluorescence in situ hybridization. Values are also reported for 6 healthy men. The studies were performed under Investigational New Drug application no. 54 137 at a tertiary referral academic medical center. RESULTS: No changes in group average or individual nuclear tail moment and MF were observed. We observed a single elevated MF value in one subject that exceeded a clinical threshold set before we initiated the study. A significant decrease in average COMET tail moment was observed on day 28 relative to day 0. We detected no genistein-induced rearrangements of the MLL gene in the 3 subjects we studied with this technique. MF increased significantly in lymphocytes exposed in vitro to unconjugated genistein at concentrations > or = 100 micromol/L. Total genistein never exceeded a peak concentration of 27.1 micro mol/L, and unconjugated genistein never exceeded a peak concentration of 0.32 micromol/L. CONCLUSION: Although isoflavones are capable of inducing genetic damage in vitro, a similar effect was not observed in subjects treated with a purified soy unconjugated isoflavone mixture.