Concentration and ratio of essential fatty acids influences the inflammatory response in lipopolysaccharide challenged mice.

The goal of this study was to evaluate the role of both the % of dietary, 18-carbon PUFA (2.5%, 5% and 10%) and the n-6:n-3 ratio (1:1, 10:1 and 20:1) on the acute inflammatory response. Mice were fed diets for 8 weeks and injected intraperitoneally with LPS to induce acute inflammation. After 24h mice were sacrificed and plasma cytokines measured. Diets significantly affected the erythrocyte PUFA composition and the effect of PUFA ratio was more prominent than of PUFA concentration. The % dietary PUFA affected feed efficiency (p<0.05) and there was a PUFA×ratio interaction with body fat (p<0.01). In mice fed high %kcal from PUFA, those given a low n-6:n-3 ratio had more body fat than those fed a high ratio. Of the twelve cytokines measured, eleven were significantly affected by the % PUFA (p<0.05), whereas five were affected by the ratio (p<0.05). For seven cytokines, there was a significant PUFA×ratio interaction according to a two way ANOVA (p<0.05). These data indicate that dietary polyunsaturated fatty acids can affect LPS induced-inflammation.

Membrane-rich milk fat diet provides protection against gastrointestinal leakiness in mice treated with lipopolysaccharide.

Milk fat globule membrane is a protein-lipid complex that may strengthen the gut barrier. The main objective of this study was to assess the ability of a membrane-rich milk fat diet to promote the integrity of the gut barrier and to decrease systemic inflammation in lipopolysaccharide (LPS)-challenged mice. Animals were randomly assigned to one of 2 American Institute of Nutrition (AIN)-76A formulations differing only in fat source: control diet (corn oil) and milk fat diet (anhydrous milk fat with 10% milk fat globule membrane). Each diet contained 12% calories from fat. Mice were fed diets for 5 wk, then injected with vehicle or LPS (10mg/kg of BW) and gavaged with dextran-fluorescein to assess gut barrier integrity. Serum was assayed for fluorescence 24h after gavage, and 16 serum cytokines were measured to assess the inflammatory response. Gut permeability was 1.8-fold higher in LPS-challenged mice fed the control diet compared with the milk fat diet. Furthermore, mice fed the milk fat diet and injected with LPS had lower serum levels of IL-6, IL-10, IL-17, monocyte chemotactic protein (MCP)-1, interferon (IFN)-γ, tumor necrosis factor (TNF)-α, and IL-3 compared with LPS-injected mice fed the control diet. The results indicate that the membrane-rich milk fat diet decreases the inflammatory response to a systemic LPS challenge compared with corn oil, and the effect coincides with decreased gut permeability.

Iron at the center of ferritin, metal/oxygen homeostasis and novel dietary strategies.

Bioiron - central to respiration, photosynthesis and DNA synthesis and complicated by radical chemistry with oxygen - depends on ferritin, the super family of protein nanocages (maxi-ferritins in humans, animals, plant, and bacteria, and mini-ferritins, also called DPS proteins, in bacteria) for iron and oxygen control. Regulation of ferritin synthesis, best studied in animals, uses DNA transcription and mRNA translation check points. Ferritin is a member of both the "oxidant stress response" gene family that includes thioredoxin reductase and quinine reductase, and a member of the iron responsive gene family that includes ferroportin and mt-aconitase ferritin DNA regulation responds preferentially to oxidant response inducers and ferritin mRNA to iron inducers: heme confers regulator synergy. Ferritin proteins manage iron and oxygen, with ferroxidase sites and iron + oxygen substrates to form mineral of both Fe and O atoms; maxi-ferritins contribute more to cellular iron metabolism and mini-ferritins to stress responses. Iron recovery from ferritin is controlled by gated protein pores, possibly contributing to iron absorption from ferritin, a significant dietary iron source. Ferritin gene regulation is a model for integrating DNA/mRNA controls, while ferritin protein function is central to molecular nutrition cellular metabolism at the crossroads of iron and oxygen in biology.

Cellular regulation and molecular interactions of the ferritins.

Controlling iron/oxygen chemistry in biology depends on multiple genes, regulatory messenger RNA (mRNA) structures, signaling pathways and protein catalysts. Ferritin, a protein nanocage around an iron/oxy mineral, centralizes the control. Complementary DNA (antioxidant responsive element/Maf recognition element) and mRNA (iron responsive element) responses regulate ferritin synthesis rates. Multiple iron-protein interactions control iron and oxygen substrate movement through the protein cage, from dynamic gated pores to catalytic sites related to di-iron oxygenase cofactor sites. Maxi-ferritins concentrate iron for the bio-synthesis of iron/heme proteins, trapping oxygen; bacterial mini-ferritins, DNA protection during starvation proteins, reverse the substrate roles, destroying oxidants, trapping iron and protecting DNA. Ferritin is nature's unique and conserved approach to controlled, safe use of iron and oxygen, with protein synthesis in animals adjusted by dual, genetic DNA and mRNA sequences that selectively respond to iron or oxidant signals and link ferritin to proteins of iron, oxygen and antioxidant metabolism.

Iron at the center of ferritin, metal/oxygen homeostasis and novel dietary strategies

Bioiron _ central to respiration, photosynthesis and DNA synthesis and complicated by radical chemistry with oxygen _ depends on ferritin, the super family of protein nanocages (maxi-ferritins in humans, animals, plants and bacteria, and mini-ferritins, also called DPS proteins, in bacteria) for iron and oxygen control. Regulation of ferritin synthesis, best studied in animals, uses DNA transcription and mRNA translation check points. Ferritin is a member of both the "oxidant stress response" gene family that includes thioredoxin reductase and quinine reductase, and a member of the iron responsive gene family that includes ferroportin and mt-aconitase ferritin DNA regulation responds preferentially to oxidant response inducers and ferritin mRNA to iron inducers; heme confers regulator synergy. Ferritin proteins manage iron and oxygen, with ferroxidase sites and iron + oxygen substrates to form mineral of both Fe and O atoms; maxi-ferritins contribute more to cellular iron metabolism and mini-ferritins to stress responses. Iron recovery from ferritin is controlled by gated protein pores, possibly contributing to iron absorption from ferritin, a significant dietary iron source. Ferritin gene regulation is a model for integrating DNA/mRNA controls, while ferritin protein function is central to molecular nutrition cellular metabolism at the crossroads of iron and oxygen in biology.

Cancer-protective properties of high-selenium broccoli.

Selenium (Se) from high-Se garlic reduces the incidence of chemically induced mammary tumors, and Se from high-Se broccoli reduces colon cancer. However, the ability of Se from high-Se broccoli to protect against mammary cancer has not been tested. Also, the sprout form of broccoli contains many secondary plant compounds that are known to reduce cancer risk, but the anticarcinogenic activity of broccoli sprouts has not been investigated. The present studies examined the ability of high-Se broccoli or high-Se broccoli sprouts to protect against chemically induced mammary or colon cancer. In one experiment, Sprague--Dawley rats that consumed diets containing 3.0 microg of Se/g supplied as high-Se broccoli had significantly fewer mammary tumors than rats fed 0.1 microg of Se as selenite with or without the addition of regular broccoli. In the second experiment, Fisher F-344 rats fed 2.0 microg of Se/g of diet supplied as either high-Se broccoli florets or high-Se broccoli sprouts had significantly fewer aberrant colon crypts than rats fed 0.1 or 2 microg of Se/g of diet supplied as selenite with or without the addition of low-Se broccoli. These data demonstrate that the cancer-protective effect of Se in high-Se broccoli extends to mammary cancer and the protective forms of broccoli against colon cancer include high-Se broccoli sprouts.

Areas with high concentrations of selenium in the soil and forage produce beef with enhanced concentrations of selenium.

Beef provides a significant portion of human dietary selenium (Se), and it is possible that modest portions of beef produced in areas with high-Se soil and forage could provide the entire Recommended Dietary Allowance (RDA) for Se. The present study has addressed the environmental conditions that resulted in the production of high-Se beef. One hundred and thirty-eight cull cows were obtained from 21 ranches in five distinct geographic regions that, on the basis of soil parent material, reports of Se deficiency, and previous soil and forage Se surveys, were likely to have high or low Se concentrations in the soil. Grass and soil samples were taken from ranch sites, and hair, whole blood, skeletal muscle, diaphragm muscle, and liver samples were obtained from the animals. Hair and whole blood samples were taken 1 day prior to shipping. Selenium concentrations of all samples were determined by hydride generation atomic absorption spectroscopy. Geographic origin affected Se content of all samples (p < 0.05). Selenium concentrations in soil (r = 0.53; p < 0.01) and grass (r = 0.63; p < 0.01) were correlated to Se content of skeletal muscle. Selenium concentrations in whole blood, diaphragm, hair, and liver also were significantly correlated to Se content of skeletal muscle (p < 0.01). Cows that received Se in mineral supplements did not have significantly higher concentrations of Se in sampled tissues (p > 0.05). Results of this study suggest that the greatest source of variation in Se content of bovine skeletal muscle was the geographic region from which the beef originated and not production or management practices. Results also demonstrated that a 100 g serving of high-Se beef could provide 100% of the RDA for Se.

A comparison of low volume, Draize and in vitro eye irritation test data. III. Surfactant-based formulations.

The third phase in a series of investigations of the relationship between low volume eye test (LVET) data, Draize eye irritation test data, and comparable data from in vitro assay protocols is presented. These investigations utilize Draize eye test and in vitro endpoint data generated previously as part of the CTFA Evaluation of Alternatives Program. LVET data were generated de novo using the same 25 representative surfactant-based personal-care formulations. In general, these formulations were minimally to moderately irritating. The linear correlation between maximum average score as determined by the Draize test (MAS) and the LVET (LVET-MAS) was 0.87; LVET-MAS values were typically about 30% lower then corresponding MAS values. Comparison of in vitro assay performance with that of the LVET was determined by statistical analysis of the relationship between LVET-MAS and in vitro endpoint. Regression modelling was the primary means of enabling such a comparison, the objective being to predict LVET-MAS for a given test material (and to place upper and lower prediction bounds on the range in which the LVET-MAS is anticipated to fall with high probability) based on observation of an in vitro score for that material. The degree of 95% confidence in prediction is quantified in terms of the relative widths of prediction intervals constructed about the fitted regression curves. Twenty in vitro endpoints were shown to have the greatest agreement with the LVET (these endpoints included those with low discordance rates relative to the Draize test) and were therefore selected for regression modelling. Although prediction interval widths tended to be narrower when predicting LVET-MAS compared with predicting MAS, the confidence with which the selected in vitro endpoints predicted both LVET-MAS and MAS for surfactant-based formulations was greatest when values were close to the lower or upper limits of the observed irritation range (i.e. 95% prediction interval widths were most narrow in these areas). Overall precision of LVET-MAS prediction for surfactant-based formulations was similar to that previously reported for hydroalcoholic formulations and considerably better than was reported for oil/water emulsions.

A comparison of low volume, draize and in vitro eye irritation test data. II. Oil/water emulsions.

The second phase in a series of investigations of the relationship between low volume eye test (LVET) data, Draize eye irritation test data, and comparable data from in vitro eye irritation test protocols is presented. These investigations utilize Draize eye test and in vitro endpoint data generated previously as part of the CTFA Evaluation of Alternatives Program. LVET data were generated de novo using the same 18 representative oil/water based personal-care formulations. In general, these formulations were minimally to mildly irritating; only three were classified as moderate eye irritants. The linear correlation between maximum average score as determined by the Draize test (MAS) and the LVET (LVET-MAS) was 0.85; LVET-MAS values were typically about half the corresponding MAS values. Comparison of in vitro assay performance with that of the LVET was determined by statistical analysis of the relationship between LVET-MAS and each in vitro endpoint. Regression modelling was the primary means of enabling such a comparison, the objective being to predict LVET-MAS for a given test material (and to place upper and lower 95% prediction bounds on the range in which the LVET-MAS is anticipated to fall with high probability) based on observation of an in vitro score for that material. The degree of confidence in prediction is quantified in terms of the relative widths of prediction intervals constructed about the fitted regression curves. Sixteen endpoints were shown to have the greatest agreement with the LVET (all but two were selected for modelling when compared with the Draize procedure). While the lower maximum average scores values (compared with the Draize test) in the LVET led to lower variability in LVET-MAS compared to MAS, the upper and lower bounds on predicted LVET-MAS values conditional on observed in vitro scores were still wide. Because there was overlap in the range of scores determined by the prediction bounds for many formulations, each of the selected endpoints was frequently unable to distinguish between test formulations in terms of statistically different predicted LVET-MAS values. In summary, none of the in vitro endpoints evaluated were able to reliably predict values of LVET-MAS among the set of oil/water emulsions considered here.

Comparison of low-volume, Draize and in vitro eye irritation test data. I. Hydroalcoholic formulations.

The first phase in a series of investigations of the relationship between low-volume eye test (LVET) data, Draize eye irritation test data, and comparable data from 25 in vitro assay protocols is presented. These investigations utilize Draize eye test and in vitro assay data generated previously as part of the Cosmetic, Toiletry and Fragrance Association (CTFA) Evaluation of Alternatives Program. LVET data were generated de novo using the same 10 representative hydroalcoholic personal-care formulations. The linear correlation between maximum average score (MAS) as determined by the Draize test and the LVET (LVET-MAS) was 0.93. Comparison of in vitro assay performance with that of the LVET was determined by statistical analysis of the relationship between LVET-MAS and in vitro endpoint. As in the CTFA program, regression modelling is the primary means of enabling such a comparison. The objective is to predict LVET-MAS for a given test material (and to place upper and lower prediction interval bounds in the range in which the LVET-MAS is anticipated to fall with high probability) conditional on observing an in vitro assay score for that material. The degree of confidence in prediction is quantified in terms of the relative widths of prediction intervals constructed about the fitted regression curves. Four assays [EYTEX MPA (membrane partition assay), HET-CAM (hen's egg test-chorioallantoic membrane HET-CAM) I, neutral red release and HET-CAM II] were shown to have the greatest agreement with the LVET. These assays were also among those with low discordance rates relative to the Draize test. Prediction of LVET-MAS values from experimentally determined in vitro scores was more accurate for hydroalcoholic formulations with lower rather than higher irritancy potential.

The CTFA Evaluation of Alternatives Program: an evaluation of in vitro alternatives to the Draize primary eye irritation test. (Phase III) surfactant-based formulations.

The CTFA Evaluation of Alternatives Program is an evaluation of the relationship between data from the Draize primary eye irritation test and comparable data from a selection of promising in vitro eye irritation tests. In Phase III, data from the Draize test and 41 in vitro endpoints on 25 representative surfactant-based personal care formulations were compared. As in Phase I and Phase II, regression modelling of the relationship between maximum average Draize score (MAS) and in vitro endpoint was the primary approach adopted for evaluating in vitro assay performance. The degree of confidence in prediction of MAS for a given in vitro endpoint is quantified in terms of the relative widths of prediction intervals constructed about the fitted regression curve. Prediction intervals reflect not only the error attributed to the model but also the material-specific components of variation in both the Draize and the in vitro assays. Among the in vitro assays selected for regression modeling in Phase III, the relationship between MAS and in vitro score was relatively well defined. The prediction bounds on MAS were most narrow for materials at the lower or upper end of the effective irritation range (MAS = 0-45), where variability in MAS was smallest. This, the confidence with which the MAS of surfactant-based formulations is predicted is greatest when MAS approaches zero or when MAS approaches 45 (no comment is made on prediction of MAS > 45 since extrapolation beyond the range of observed data is not possible). No single in vitro endpoint was found to exhibit relative superiority with regard to prediction of MAS. Variability associated with Draize test outcome (e.g. in MAS values) must be considered in any future comparisons of in vivo and in vitro test results if the purpose is to predict in vivo response using in vitro data.

Protein binding of several drugs in serum and plasma of healthy subjects.

The protein binding of phenytoin, propranolol, salicylic acid, warfarin, and bilirubin was determined in heparinized plasma and serum obtained from the same healthy adult subjects. There were no significant differences in the free fraction values of these compounds in plasma and serum. Addition of heparin to serum had no significant effect on protein binding. Contact with Vacutainer stoppers increased the free fraction of the weak base propranolol in serum and plasma but no significant effect on the protein binding of the other, weakly acidic compounds tested. The lack of differences in the protein binding of phenytoin, salicylic acid, warfarin, and bilirubin in human plasma and serum is in contrast with the pronounced differences observed previously in rat serum and plasma.

Role of the pituitary in cyproheptadine-induced pancreatic beta-cell toxicity.

Hypophysectomized rats given cyproheptadine (40 mg/kg) for 10 days exhibited a loss of pancreatic immunoreactive insulin and ultrastructural changes in the cytoplasm of beta-cells. Sham-operated animals given cyproheptadine showed identical changes in pancreatic beta-cells except that cytoplasmic involvement progressed to the formation of large vacuoles. The pituitary is not directly involved with the cyproheptadine-induced depletion of pancreatic insulin but plays a role in the formation of large cytoplasmic vacuoles.

Isomeric specificity of diphenylmethylpiperidine in the production of rat pancreatic islet cell toxicity.

The relationship between chemical structure and the pancreotoxic potential of positional isomers of diphenylmethylpiperidine was investigated in rats. The chemical synthesis of these compounds and their N-methylated analogs is reported. Oral administration of 4-diphenylmethylpiperidine and its N-methylated derivative (130 and 260 micronmoles/kg) to rats for 14 days resulted in hyperglycemia, reduced pancreatic insulin content and the formation of large vacuoles in the cytoplasm of pancreatic islet cells. No effect on beta cell morphology or insulin content was observed after administration of 2- and 3-diphenylmethylpiperidine and their N-methylated analogs.

Studies on the biological disposition of diethylstilbestrol in rats and humans.

Studies were conducted to elucidate the processes involved in the biological disposition of diethylstilbestrol (DES). The apparent biological half-life of [14C] DES decreased with increasing age in immature rats. The half-life of DES in adult rats (14 min) was reached between the 15th and 25th day after birth. Comparison of the intestinal absorption of [14C] DES and [14C]DES monoglucuronide showed the unconjugated drug to be absorbed much more rapidly. Hydrolysis of the conjugate took place in the lower intestine, and this permitted the drug to undergo enterohepatic cycling.

Disposition of cyproheptadine in rats, mice, and humans and identification of a stable epoxide metabolite.

Radioactivity was excreted in the urine and feces of rats, mice, and humans after a dose of 14C-cyproheptadine. The major metabolite in rat urine was unconjugated, but the majority of radioactive materials in mouse and human urine were conjugated with glucuronic acid. Identification of the rat urinary metabolite of cyproheptadine as an epoxide was accomplished with mass spectrometry and other methods. The rat metabolite was 10.11 -epoxydesmethylcyproheptadine and accounted for about 25% of a 45-mg dose of cyproheptadine per kg. Only a small amount of this epoxide was found in mouse urine, and none was apparent in the urine of two humans who received 5 mg of the drug. Dihydrodiols, which could arise by epoxide hydrase hydrolysis of possible 10.11-epoxy metabolites, were not found in the urine of any of the species studied. The spoxide found in rat urine appears to be unusually stable to in vivo hydrolysis. Possible implications of these results in the species-selective pancreotoxicity of cyproheptadine in the rat are presented.