Effects of exogenous inositol hexakisphosphate (InsP(6)) on the levels of InsP(6) and of inositol trisphosphate (InsP(3)) in malignant cells,tissues and biological fluids

InsP(6) is abundant in cereals and legumes. InsP(6) and lower inositol phosphates, in particular InsP(3), participate in important intracellular processes. In addition, InsP(6) possess significant health benefits, such as anti-cancer effect, kidney stones prevention, lowering serum cholesterol. Because of the insensitivity of existing methods for determination of non-radiolabeled inositol phosphates, little is known about the natural occurrence, much less on the concentrations of InsP(6) and InsP(3) in biological samples. Using gas chromatography-mass detection analysis of HPLC chromatographic fractions, we report a measurement of unlabeled total InsP(3) and InsP(6) (a) as they occur within cells culture, tissues, and plasma, and (b) their changes depending on the presence of exogenous InsP(6). When rats were fed on a purified diet in which InsP(6) was undetectable (AIN-76A) the levels of InsP(6) in brain were 3.35 +/- 0.57 (SE) micromol.kg(-1) and in plasma 0.023 +/- 0.008 (SE) micromol.l(-1). The presence of InsP(6) in diet dramatically influenced its levels in brain and in plasma. When rats were given an InsP(6)-sufficient diet (AIN-76A + 1% InsP(6)), the levels of InsP(6) were about 100-fold higher in brain tissues (36.8 +/- 1.8 (SE)) than in plasma (0.29 +/- 0.02 (SE)); InsP(6) concentrations were 8.5-fold higher than total InsP(3) concentrations in either plasma (0.033 +/- 0.012 (SE)) and brain (4.21 +/- 0.55 (SE)). When animals were given an InsP(6)-poor diet (AIN-76A only), there was a 90% decrease in InsP(6) content in both brain tissue and plasma (p < 0.001); however, there was no change in the level of total InsP(3). In non-stimulated malignant cells (MDA-MB 231 and K562) the InsP(6) contents were 16.2 +/- 9.1 (SE) micromol.kg(-1) for MDA-MB 231 cells and 15.6 +/- 2.7 (SE) for K 562 cells. These values were around 3-fold higher than those of InsP(3) (4.8 +/- 0.5 micromol.kg(-1) and 6.9 +/- 0.1 (SE) for MDA-MB 231 and K562 cells respectively). Treatment of malignant cells with InsP(6) resulted in a 2-fold increase in the intracellular concentrations of total InsP(3) (9.5 +/- 1.3 (SE) and 10.8 +/- 1.0 (SE) micromol.kg(-1) for MDA-MB 231 and K562 cells respectively, p < 0.05), without changes in InsP(6) levels. These results indicate that exogenous InsP(6) directly affects its physiological levels in plasma and brain of normal rats without changes on the total InsP(3) levels. Although a similar fluctuation of InsP(6) concentration was not seen in human malignant cell lines following InsP(6) treatment, an increased intracellular levels of total InsP(3) was clearly observed.     

Dietary phytate and mineral bioavailability

The relation between the dietary phytate (InsP₆), mineral status and InsP₆ levels in the organism, using three controlled diets (AIN-76A, AIN-76A + 1% phytate, AIN-76A + 6% carob seed germ), are studied. AIN-76A is a purified diet in which InsP₆ is practically absent. No important or significant differences in the mineral status (Zn, Cu, Fe) of blood, kidneys, liver, brain and bone, were observed, except iron in the brain. Thus, the amounts of iron found in the brain of rats fed AIN-76A + 1% InsP₆ were significantly inferior to those found in rats fed AIN-76A diet. The amounts of InsP₆ found in organs of rats fed AIN-76A diet became very low or even undetectable while the ones found in rats fed diets that contained 1% and 0.12% (AIN-76A + 6% carob seed germ) InsP₆, were considerably higher and similar. Moreover the majority of rats fed AIN-76A diet exhibited calcifications at the corticomedullary junctions, whereas no calcifications were detected in rats fed the other two diets. From these results, it can be deduced that there was no important adverse effects on mineral status as a consequence of the presence of InsP₆ in the studied diets. Besides, considering that a 0.12% InsP₆ contained in the AIN-76A purified diet through the addition of a 6% of carob seed germ to this diet, produced the same beneficial effects as the direct addition of a 1% of InsP₆ and no negative effects on mineral status was observed, it can be concluded that the value of the presence of InsP₆ at adequate amounts in the diet is remarkable and must be favourably considered.     

Phytate prevents tissue calcifications in female rats

The AIN-76 A, a purified rodent diet, has a propensity to cause kidney calcifications in female rats which is not observed with non-purified rodent diets, suggesting a nutritional factor that avoids these calcifications. One candidate is phytate, which inhibits crystallisation of calcium salts and is practically absent in purified diets. Therefore, the effects on calcification of kidney tissue of phytate addition to the AIN-76 A diet using female Wistar rats were studied. The rats were assigned to three groups: AIN-76 A, AIN-76 A + 1% phytate and standard nonpurified chow. Urinary phytate of the AIN-76 A fed group was undetectable. Urinary phytate of AIN-76 A + 1% phytate and standard fed groups did not differ and was significantly higher than in the AIN-76 A group. The concentrations of calcium and phosphorus in kidneys were greater in the AIN-76 A group than in AIN-76 A + 1% phytate and standard groups. Only rats of the AIN-76 A group displayed mineral deposits at the corticomedullary junction. These findings demonstrated that the absence of phytate in the AIN-76 A diet is one of the causes of renal calcification in female rats.     

Vitamin K deficiency of germfree mice caused by feeding standard purified diet sterilized by gamma-irradiation.

Germfree mice died when they were fed a purified diet of AIN-76 formula sterilized by gamma-irradiation. Vitamin K deficiency was suspected and this study was performed to confirm the cause of the death. Germfree mice were fed purified diets of AIN-76 or AIN-93M formula, which were pelleted and sterilized by gamma-irradiation at a dose of 50 kGy. One half of the mice fed the AIN-76 diet died within two weeks and the surviving animals were also in poor health, while 91% of mice fed the AIN-93M diet survived. No hemorrhage was observed grossly in any organs of the surviving animals. Histologically, degeneration with inflammatory cell infiltration was observed as well as hemorrhage and fibrosis in the heart muscles of mice fed the AIN-76 diet. No microscopic lesions were observed in the other organs. Prothrombin time (PT) and activated partial thromboplastin time (APTT) were extremely prolonged when mice were fed the AIN-76 diet. The animals totally recovered when they were intragastrically administered 1 microg/day of vitamin K(3) from the third day of feeding of the AIN-76 diet, except for PT and APTT which were still slightly longer than in mice fed the AIN-93M diet. The concentration of vitamin K(3) supplied in the AIN-76 diet decreased to an undetectable level after gamma-irradiation, while the AIN-93M diet contained 240 microg/kg of vitamin K(1). These results indicate that the deaths of the germfree mice fed the gamma-irradiated AIN-76 diet were caused by vitamin K deficiency. Vitamin K deficiency may cause fatal degeneration of cardiac muscle cells.     

Regulation of the type III InsP(3) receptor by InsP(3) and ATP

Many hormones and neurotransmitters raise intracellular calcium (Ca(2+)) by generating InsP(3) and activating the inositol 1,4, 5-trisphosphate receptor (InsP(3)R). Multiple isoforms with distinct InsP(3) binding properties () have been identified (). The type III InsP(3)R lacks Ca(2+)-dependent inhibition, a property that makes it ideal for signal initiation (). Regulation of the type III InsP(3)R by InsP(3) and ATP was explored in detail using planar lipid bilayers. In comparison to the type I InsP(3)R, the type III InsP(3)R required a higher concentration of InsP(3) to reach maximal channel activity (EC(50) of 3.2 microM versus 0.5 microM for the types III and I InsP(3)R, respectively). However, the type III InsP(3)R did reach a 2.5-fold higher level of activity. Although activation by InsP(3) was isoform-specific, regulation by ATP was similar for both isoforms. In the presence of 2 microM InsP(3), low ATP concentrations (<6 mM) increased the open probability and mean open time. High ATP concentrations (>6 mM) decreased channel activity. These results illustrate the complex nature of type III InsP(3)R regulation. Enhanced channel activity in the presence of high InsP(3) may be important during periods of prolonged stimulation, whereas allosteric modulation by ATP may help to modulate intracellular Ca(2+) signaling.     

Modulation of DNA modification (I-compound) levels in rat liver and kidney by dietary carbohydrate, protein, fat, vitamin, and mineral content.

I-compounds are DNA modifications detected by 32P-postlabeling that increase with age in rodents without known carcinogen exposure. Diet type (natural ingredient versus purified) greatly influences patterns and levels of I-compounds. To test the hypothesis that I-compound formation is affected, also, by dietary macro- and micronutrients, effects of carbohydrate, protein, fat, vitamin, and mineral content on rat liver and kidney I-compounds were determined. Female Sprague-Dawley rats were fed basic or modified AIN-76A purified diets for 3-6 months. High protein (HP) diet (50%, w/w) increased I-compound levels in liver but not kidney. High carbohydrate (HC) diet (78%) produced a significant increase in the polar as well as total I-compound levels in both tissues. High fat diets (20%) elicited significantly lower levels of liver I-compounds than HC, HP, and basic diets. There were few significant differences between high polyunsaturated (safflower oil) and saturated fat (lard) diet groups. No qualitative differences in I-compound profiles were observed in either tissue. In rats fed basic diet supplemented with vitamins and/or minerals, increased vitamin content reduced the levels of polar I-compounds in liver. No extra diet-induced adducts were observed; all effects were of a quantitative nature. These data provide direct evidence that nutrients significantly influence I-compound levels and support the hypothesis that normal metabolism of nutrients leads to the production of small amounts of DNA-reactive electrophiles. These observations suggest a novel mechanism where nutrient composition of the diet may play a role in development of neoplasia and other adverse health effects.     

Increased levels of inositol hexakisphosphate (InsP6) protect HEK293 cells from tumor necrosis factor (alpha)- and Fas-induced apoptosis

The overexpression of inositol 1,3,4-trisphosphate 5/6-kinase has recently been shown to protect HEK293 cells from tumor necrosis factor alpha (TNF(alpha))-induced apoptosis. This overexpression leads to an increase in the levels of both inositol 1,3,4,5,6-pentakisphosphate (InsP5) and inositol 1,2,3,4,5,6-hexakisphosphate (InsP6). Cells that overexpress InsP5 2-kinase have increased levels of InsP6 and are also protected from TNFalpha-induced apoptosis; furthermore, cells that express an RNA interference construct to the 2-kinase are deficient in InsP6 and are sensitized to TNFalpha-induced apoptosis. Therefore the protective effect of 5/6-kinase on TNFalpha-mediated apoptosis is due to an increase of InsP6 or to a metabolite derived from InsP6. Furthermore, we find that the InsP6 also protects from Fas-mediated apoptosis. No effect was seen in the endocytic rate of transferrin receptor, caspase 8 activity, or TNF receptor number at the cell surface. Cells that overexpress 2-kinase do show an increase in the amount of receptor-interacting protein (RIP), while cells with reduced InsP6 levels show relatively less RIP, providing a possible mechanism for the effect on apoptosis.     

The mouse bioassay for the detection of estrogenic activity in rodent diets: III. Stimulation of uterine weight by dextrose, sucrose and corn starch

We have shown previously that mice fed the American Institute of Nutrition (AIN-76A) purified diet experience a significant increase in uterine:body weight (U:BW) ratios when compared to the U:BW ratios of mice fed a closed formula natural ingredient diet (Certified Rodent Chow #5002) for 7 days. The AIN-76A purified diet contains 5% corn oil and 65% carbohydrates with 50% of the carbohydrates coming from sucrose or dextrose and 15% from corn starch. The objective of this study was to determine whether the fat and carbohydrate content contributed to the unexpected uterine growth promoting activity observed in mice fed the AIN-76A diet. Estrogen bioassays were performed using CD-1 mice weaned at 15 days of age and assigned randomly to the negative control diet (Certified Rodent Chow #5002) or to the positive control diet (#5002) containing 4 or 6 ppb DES for comparison or to the test diets. The test diets were prepared by adding sucrose, dextrose, corn starch, corn oil or soybean oil to the #5002 negative control diet at 10% w/w concentration. Uterine:BW ratios were determined at 7 days post-feeding. The uterine weights and the U:BW ratios of mice fed the test diets containing dextrose, corn starch, or corn oil, were increased significantly (P less than 0.05) over those of mice fed the negative control diet. The uterine weights and U:BW ratios of mice fed the test diets containing sucrose or soybean oil also were increased over those of mice fed the negative control diet. These increases in uterine weights and U:BW ratios were similar to the increases in uterine weights and U:BW ratios of mice fed the positive control diet containing 4 ppb DES. It was concluded that the fats and carbohydrates caused preferential increases in uterine weights and in U:BW ratios and may account for the estrogen-like uterine growth promoting activity observed in mice fed the AIN-76A purified diet.     

Heat-treatment, phytase and fermented liquid feeding affect the presence of inositol phosphates in ileal digesta and phosphorus digestibility in pigs fed a wheat and barley diet

The aim was to evaluate the effect of heat-treatment, microbial phytase addition and feeding strategy (dry feeding v. fermented liquid feeding) on degradation of phytate (myo-inositol hexakisphosphate, InsP6) and formation and further degradation of lower inositol phosphates (myo-inositol pentakisphosphate-myo-inositol bisphosphate, InsP5-InsP2) at the distal ileum of pigs. Furthermore, the apparent ileal digestibility/degradability (AID) of phosphorus (P), InsP6-P and calcium (Ca) and the apparent total tract digestibility (ATTD) of P and Ca were studied. Pigs were fitted with a T-shaped ileal cannula for total collection of digesta at 2 h intervals during an 8 h sampling period after feeding the morning meal. Each period lasted for 2 weeks: 8 days of adaptation followed by 3 days of total collection of faeces and 3 days of total collection of ileal digesta. The experiment was designed as a 4 × 4 Latin square with four pigs fed four diets. A basal wheat/barley-based diet was fed either as non-heat-treated or heat-treated (steam-pelleted at 90°C). The heat-treatment resulted in an inactivation of plant phytase below detectable level. Diet 1 (non-heat-treated basal diet fed dry); diet 2 (heat-treated basal diet fed dry); diet 3 (as diet 2 but with microbial phytase (750 FTU/kg as fed) fed dry); diet 4 (as diet 3 fed liquid (fermented for 17.5 h nighttime and 6.5 h daytime at 20°C with 50% residue in the tank)). Chromic oxide (Cr2O3) was included as marker and ATTD was determined both by total collection of faeces (ATTDTotal) and Cr2O3 (ATTDCr). InsP6 was completely degraded in diet 4 before feeding resulting in no InsP6-P being present in ileal digesta. InsP6-P concentration in ileal digesta decreased with increasing dietary levels of plant or microbial phytase in pigs fed the dry diets. Consequently, AID and ATTD of P and Ca were greatest for pigs fed diet 4 followed by diets 3, 1 and 2. The ATTD of P depended on the used method as ATTDTotal of P was 72%, 61%, 44% and 34%, whereas ATTDCr of P was 65%, 52%, 38% and 23% for diets 4, 3, 1 and 2, respectively. In all pigs the ileal concentration of InsP5-InsP2-P was extremely small, and thus unimportant for maximisation of ATTD of plant P. In conclusion, fermented liquid feeding with microbial phytase seems to be an efficient approach to improve ATTD of plant P compared with dry feeding. This opens up for further reductions in P excretion.     

Spontaneous channel activity of the inositol 1,4,5-trisphosphate (InsP3) receptor (InsP3R). Application of allosteric modeling to calcium and InsP3 regulation of InsP3R single-channel gating

The InsP3R Ca2+ release channel has a biphasic dependence on cytoplasmic free Ca2+ concentration ([Ca2+]i). InsP3 activates gating primarily by reducing the sensitivity of the channel to inhibition by high [Ca2+]i. To determine if relieving Ca2+ inhibition is sufficient for channel activation, we examined single-channel activities in low [Ca2+]i in the absence of InsP3, by patch clamping isolated Xenopus oocyte nuclei. For both endogenous Xenopus type 1 and recombinant rat type 3 InsP3R channels, spontaneous InsP3-independent channel activities with low open probability Po ( approximately 0.03) were observed in [Ca2+]i < 5 nM with the same frequency as in the presence of InsP3, whereas no activities were observed in 25 nM Ca2+. These results establish the half-maximal inhibitory [Ca2+]i of the channel to be 1.2-4.0 nM in the absence of InsP3, and demonstrate that the channel can be active when all of its ligand-binding sites (including InsP3) are unoccupied. In the simplest allosteric model that fits all observations in nuclear patch-clamp studies of [Ca2+]i and InsP3 regulation of steady-state channel gating behavior of types 1 and 3 InsP3R isoforms, including spontaneous InsP3-independent channel activities, the tetrameric channel can adopt six different conformations, the equilibria among which are controlled by two inhibitory and one activating Ca2+-binding and one InsP3-binding sites in a manner outlined in the Monod-Wyman-Changeux model. InsP3 binding activates gating by affecting the Ca2+ affinities of the high-affinity inhibitory sites in different conformations, transforming it into an activating site. Ca2+ inhibition of InsP3-liganded channels is mediated by an InsP3-independent low-affinity inhibitory site. The model also suggests that besides the ligand-regulated gating mechanism, the channel has a ligand-independent gating mechanism responsible for maximum channel Po being less than unity. The validity of this model was established by its successful quantitative prediction of channel behavior after it had been exposed to ultra-low bath [Ca2+].     

Effect of the inositol polyphosphate InsP(6) on DNA-PK-dependent phosphorylation

Inositol hexakisphosphate (InsP(6)) is a member of the inositol polyphosphate group that participates in numerous intracellular signaling pathways. Cheung and colleagues previously reported that InsP(6) stimulated double-strand break repair by nonhomologous end joining (NHEJ) in cell-free extracts and that InsP(6) binding by the Ku70/80 subunit of the DNA-dependent protein kinase (DNA-PK) was required for stimulation of NHEJ in vitro. This report describes InsP(6)-dependent phosphorylation of two NHEJ factors, XRCC4 and XLF, in partially purified human cell extracts. XRCC4 and XLF are known substrates for DNA-PK, which does not require InsP(6) for protein kinase activity. Consistent with a role for DNA-PK in these reactions, InsP(6)-dependent phosphorylation of XRCC4 and XLF was DNA dependent and not observed in the presence of DNA-PK inhibitors. Depletion of the Ku70/80 DNA-, InsP(6)-binding subunit of DNA-PK resulted in loss of InsP(6)-dependent phosphorylation and showed a requirement for Ku70/80 in these reactions. Complementation of Ku70/80-depleted reactions with recombinant wild-type Ku70/80 restored InsP(6)-dependent phosphorylation of XRCC4 and XLF. In contrast, addition of a Ku70/80 mutant with reduced InsP(6) binding failed to restore InsP(6)-dependent phosphorylation. While additional protein kinases may participate in InsP(6)-dependent phosphorylation of XRCC4 and XLF, data presented here describe a clear requirement for DNA-PK in these phosphorylation events. Furthermore, these data suggest that binding of the inositol polyphosphate InsP(6) by Ku70/80 may modulate the substrate specificity of the phosphoinositide-3-kinase-related protein kinase DNA-PK.     

Laboratory diet profoundly alters gene expression and confounds genomic analysis in mouse liver and lung

Nutritional studies in laboratory animals have long shown that various dietary components can contribute to altered gene expression and metabolism, but diet alone has not been considered in whole animal genomic studies. In this study, global gene expression changes in mice fed either a non-purified chow or a purified diet were investigated and background metal levels in the two diets were measured by ICP-MS. C57BL/6J mice were raised for 5 weeks on either the cereal-based, non-purified LRD-5001 diet or the purified, casein-based AIN-76A diet, as part of a larger study examining the effects of low dose arsenic (As) in the diet or drinking water. Affymetrix Mouse Whole Genome 430 2.0 microarrays were used to assess gene expression changes in the liver and lung. Microarray analysis revealed that animals fed the LRD-5001 diet displayed a significantly higher hepatic expression of Phase I and II metabolism genes as well as other metabolic genes. The LRD-5001 diet masked the As-induced gene expression changes that were clearly seen in the animals fed the AIN-76A diet when each dietary group was exposed to 100 ppb As in drinking water. Trace metal analysis revealed that the LRD-5001 diet contained a mixture of inorganic and organic As at a total concentration of 390 ppb, while the AIN-76A diet contained approximately 20 ppb. These findings indicate that the use of non-purified diets may profoundly alter observable patterns of change induced by arsenic and, likely, by other experimental treatments, particularly, altering gene and protein expression.     

Regulation of the type III InsP(3) receptor by InsP(3) and calcium

It has been proposed that the inositol 1,4,5-trisphosphate receptor (InsP(3)R) type III acts as a trigger for InsP(3)-mediated calcium (Ca(2+)) signaling, because this InsP(3) isoform lacks feedback inhibition by cytosolic Ca(2+). We tested this hypothesis in RIN-m5F cells, which express predominantly the type III receptor. Extracellular ATP increases Ca(2+) in these cells, and we found that this effect is independent of extracellular Ca(2+) but is blocked by the InsP(3)R antagonist heparin. There was a dose-dependent increase in the number of cells responding to ATP and two-photon flash photolysis of caged-Ca(2+) heightened the sensitivity of RIN-m5F cells to this increase. These findings provide evidence that Ca(2+) increases the sensitivity of the InsP(3)R type III in intact cells and supports the idea that this isoform can act as a trigger for hormone-induced Ca(2+) signaling.     

Oat lipids-induced covalent DNA modifications (I-compounds) in female Sprague-Dawley rats, as determined by 32P-postlabeling.

Previous studies have shown that the presence of oats in the diet contributes to formation of I-compounds (age-dependent covalent DNA modifications detected by 32P-postlabeling assay) in female Sprague-Dawley rat liver DNA. The current study explored the possible ingredients in oats responsible for the observed effects on DNA. Feeding AIN-76A diet containing 5% oat lipids (obtained by methanol extraction and dissolved in trioctanoin) in place of corn oil for 2 months successfully induced the formation of 3 oats-specific (spots 2-4) and 4 natural ingredient diet-specific I-compounds (spots 6-9) in liver DNA. Barley, an oatlike cereal, induced 3 of these spots at very low intensities but not the 3 oats-specific I-spots. Oral administration of oat lipids to weanling rats of both sexes for 7 days elicited trace amounts of the oats-specific spots and spot 9 in liver DNA. However, when oat lipids were given at 6 or 9 weeks of age, the oats-specific spots were detected at high levels in female but not in male rats. These oats-related DNA modifications were also present in 6-week-old female rats which had received oat lipids p.o. for 2 or 3 days or i.p. for 4 days. Rats given trioctanoin or extracts from natural ingredient Wayne diet (lacking oats) did not show any of these spots. On the other hand, rats treated with extracts from an oats-containing Teklad diet displayed a trace amount of one of these I-compounds. Oat lipids did not induce any extra spots in rat kidney DNA. Feeding of AIN diet supplemented with oats to female Syrian hamsters did not elicit any renal or hepatic DNA alterations, as detected by 32P-postlabeling. Rats fed oat lipids-supplemented AIN diet or Purina diet showed the highest levels of I-compounds overall in liver among all dietary groups and these two groups also had significantly higher hepatic DNA synthesis rates. Oat lipids enhanced kidney DNA synthesis also. The total hepatic or renal cytochrome P-450 contents were not significantly affected by different diets. These results demonstrate a novel link between a natural dietary ingredient and covalent DNA modifications and shed light on the origins of certain I-compounds.     

Mechanisms by which resistant starches and non-starch polysaccharide sources affect the metabolism and disposition of the food carcinogen, 2-amino-3-methylimidazo[4,5-f]quinoline

Although both non-starch polysaccharides (NSP) and resistant starches (RS) are included in current definitions of dietary fibre, our previous work has suggested fundamental differences in the way in which these two classes of material affect the disposition and absorption of a dietary carcinogen. The present studies explore whether different effects on carcinogen metabolism could play a role in the contrasting patterns seen previously. Groups of female Wistar rats were pre-fed for 4 weeks one of five types of defined diet (AIN-76). The control diet contained 35% maize starch and no dietary fibre. The RS-containing diets had all the maize starch substituted with either Hi-maize or potato starch. In the NSP-containing diets, 10% of the maize starch was substituted with dietary fibre in the form of either lignified plant cell walls (wheat straw) or soluble dietary fibre (apple pectin). Pre-fed rats were gavaged with the food carcinogen, [2-14C] 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), and plasma and urinary metabolites characterized using HPLC at various time intervals after administration. After 4 h gavage, plasma from rats on both RS-containing diets contained significantly higher levels of intact IQ and lower levels of the major metabolites, IQ-5-O-glucuronide and IQ-5-sulfate, as compared with plasma from the negative control group at this time. In contrast, plasma from animals on the NSP-containing wheat straw diet (and to a lesser extent the apple pectin diet) showed significantly lower levels of intact IQ, and significantly higher levels of the two major metabolites, as compared with those from the control rats. These different metabolite profiles were also reflected in different urinary excretion profiles. Urine from rats pre-fed RS-containing diets revealed significantly slower metabolite excretion as compared with urine from rats that had been given the NSP-containing diets. Western blotting methodologies also profiled differences between the effects of these two types of dietary fibre in the expression of xenobiotic metabolizing enzymes. We conclude that changes in activity and expression of xenobiotic metabolising enzymes could play a role in the contrasting effects of these two types of dietary fibre on carcinogen uptake and disposition.     

Do mammals make all their own inositol hexakisphosphate?

A highly specific and sensitive mass assay for inositol hexakisphosphate (InsP6) was characterized. This centres around phosphorylating InsP6 with [32P]ATP using a recombinant InsP6 kinase from Giardia lambia, followed by HPLC of the 32P-labelled products with an internal [3H]InsP7 standard. This assay was used to quantify InsP6 levels in a variety of biological samples.Concentrations of InsP6 in rat tissues varied from 10-20 microM (assuming 64% of wet weight of tissue is cytosol water), whereas using the same assumption axenic Dictyostelium discoideum cells contained 352 +/- 11 microM InsP6. HeLa cells were seeded at low density and grown to confluence, at which point they contained InsP6 levels per mg of protein similar to rat tissues. This amounted to 1.952 +/- 0.117 nmol InsP6 per culture dish, despite the cells being grown in serum shown to contain no detectable(less than 20 pmol per dish) InsP6. These results demonstrate that mammalian cells synthesize all their own InsP6. Human blood was analysed, and although the white cell fraction contained InsP6 at a concentration comparable with other tissues, in serum and platelet-free plasma no InsP6 was detected (<1 nM InsP6). Human urine was also examined, and also contained no detectable (<5 nM) InsP6. These results suggest that dietary studies purporting to measure InsP6 at micromolar concentrations in human plasma or urine may not have been quantifying this inositol phosphate. Therefore claims that administrating InsP6 in the diet or applying it topically can produce health benefits by increasing extracellular InsP6 levels may require reassessment.     

ACh and 5-HT induced changes in the concentration of cytosolic inositol trisphosphate (InsP3) and inositol bisphosphate (InsP2) in the ABRM of Mytilus edulis L.

1. For determination of the phosphoinositides and inositol phosphates present in anterior byssus retractor muscle (ABRM) of Mytilus edulis fiber bundles of this muscle were incubated with [3H]-inositol. Close-to-equilibrium labelling was achieved after 14-17 hr of incubation. 2. The phosphoinositides formed during incubation were identified as phosphatidylinositolphosphates by thin layer chromatography and as glycerophosphoryl esters by anion-exchange chromatography after deacylation. Besides PtdIns, PtdInsP and PtdInsP2 two labelled products are formed, which could not be identified. 3. Inositol phosphates were separated by anion-exchange chromatography. InsP, InsP2 and InsP3 are present, while InsP4 seemed to be absent. 4. Incubation of pre-labelled fibers with ACh induces the accumulation of InsP3 and InsP2 immediately. While 5-Ht accomplishes the accumulation after a lag time of 25 sec. The concentration of cytosolic InsP does not change.     

Inhibition of DNA adduct formation of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine and 2-amino-3-methylimidazo[4,5-f]quinoline by dietary indole-3-carbinol in female rats.

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) are two important heterocyclic amines formed in proteinaceous foods during the cooking process. Both PhIP and IQ are carcinogenic in several strains of rats. PhIP induces mammary tumors in female F344 rats, while IQ induces principally mammary and liver tumors in female Sprague-Dawley rats. Both PhIP and IQ are activated enzymatically, first by N-hydroxylation, catalyzed by CYP1A1 and CYP1A2, and subsequently by esterification (O-acetylation or sulfation), to yield DNA adducts. Such DNA adduct formation, and persistence of adducts, is related to initiation of carcinogenesis, while inhibition of this process leads to prevention of carcinogenesis. Indole-3-carbinol (I3C), a constituent of cruciferous vegetables, has chemopreventive properties in various systems; it probably acts by induction of detoxification enzymes. We have examined the effect of dietary I3C on DNA adduct formation by PhIP in female F344 rats and on that by IQ in female Sprague-Dawley rats. In experiment 1, F344 rats were maintained on AIN-76A diet containing 0.1% (w/w) I3C and then given p.o. doses (10 or 50 mg/kg) of PhIP. These doses are known to induce CYP1A1 and CYP1A2. Groups of animals (4/time point) were euthanized 1, 2, 6, and 16 days later, and their blood (for isolation of white blood cells), mammary glands, liver, stomach, small intestine, cecum, colon, heart, lungs, kidneys, and spleen were removed for DNA isolation and quantitation of PhIP-DNA adducts by 32P-postlabeling. PhIP-DNA adduct formation was inhibited (40-100%) by I3C in virtually all organs, including the mammary gland (the target organ), at both doses of PhIP, and at almost all time points. In a second experiment, Sprague-Dawley rats were fed either control AIN-76A diet or this diet containing 0.02% I3C or 0.1% I3C for a total of 42 days. IQ was added to the diets (0.01%, w/w) from day 15 to day 42, after which all rats received diet free of IQ and I3C. Groups of animals (4/time point) were killed on days 43 and 57. In addition to the organs removed in experiment 1, the pancreas, uterus, and ovaries were also removed, and IQ-DNA adducts were quantitated by 32P-postlabeling. Both dietary concentrations of I3C inhibited IQ-DNA adduct formation in most organs (except in lungs, kidneys, and pancreas) on both days 43 and 57; in liver, stomach, mammary gland, and spleen, inhibition was evident only on day 43. Inhibitions ranged from 22.6 to 86.6% with the 0.02% I3C diet and from 32.2 to 89.6% with the 0.1% I3C diet. I3C diets did not affect rate of adduct removal in either experiment. It is concluded that dietary I3C inhibits PhIP- and IQ-DNA adduct formation in both target and nontarget organs of female rats, even with high doses of PhIP when CYP1A1 and CYP1A2, the enzymes responsible for the initial activation (N-hydroxylation) of PhIP, are expected to be induced.