Fecal mutagens and Bacteroides fragilis levels in the feces of dimethylhydrazine-treated rats: influence of diet

In a study designed to investigate the effects of dietary synergisms on 1,2-dimethylhydrazine (DMH)-induced rat colon carcinogenesis, fecal pellets were examined for the presence of direct-acting fecal mutagens and levels of Bacteroides fragilis group organisms. Intraperitoneal injections of DMH at 10 mg/kg were given for 16 weeks (weeks 3-18) to 160 male F344 rats consuming 4 supplemental dietary factors in all possible combinations. The dietary factors examined were wheat bran (15%), cholesterol (1%), beef tallow (18%) and indole-3-carbinol (IC) (0.1%). Feces were collected 3, 10, 17, 24 and 31 weeks after commencing the dietary treatments and dichloromethane extracts were assayed using the Salmonella typhimurium TA100 without metabolic activation. The numbers of B. fragilis group organisms were enumerated in feces collected at the same time. Most feces samples were negative for mutagens but extracts from weeks 17-31 showed a significant mutagenic response from the IC factor in the diet. The fecal levels of B. fragilis were significantly increased by the inclusion of cholesterol in the diets. The B. fragilis counts and fecal mutagen production were not correlated (r = 0.09), although species of the B. fragilis group have been implicated in the production on human fecal mutagens.     

Urolithiasis in rats consuming a dl bitartrate form of choline in a purified diet

Urolithiasis appeared in rats maintained to study the effects of nutrients and methylmercury on development and aging. After a year, the mortality rate was approximately 10%, and by 2 years, it had increased to nearly 30%. Clinical signs and urinary tract pathology were examined as a function of diet, duration on diet, gender, methylmercury exposure, genetics, and other potential risk factors by using survival analyses and qualitative comparisons. Urolithiasis in female rats appeared 15 weeks after beginning a purified diet and after 5 weeks for male rats. After 97 weeks, the mortality rate of female rats was 22% and for male rats was 64%. Lifetime urolithiasis-associated mortality was about 2% in a group of rats that consumed the contaminated diet for < 30 weeks. No urolithiasis occurred in siblings or cohorts of the rats described here that were maintained on a standard rodent chow containing choline chloride. Urolithiasis was traced to racemic, rather than levo-, bitartaric acid in some purified diets shipped in 2001 and 2002. It is unknown when the impurity first appeared in the diet, so estimates of exposure duration are upper limits. Chronic methylmercury exposure increased vulnerability. Some families (dam + offspring) had multiple cases of urolithiasis, but probability models constructed to evaluate familial clustering revealed no evidence for a genetic predisposition to urolithiasis apart from gender. Removing racemic tartaric acid did not decrease mortality once rats had been on the diet for 20 to 30 weeks, but it helped when exposure duration was shorter.     

The fluidity of the serum phospholipids of Fulani pastoralists consuming a high-fat diet

The semi-nomadic cattle Fulani of northern Nigeria consume a diet rich in saturated fatty acids. Since the quality of an individual's dietary fat can influence the fatty acid composition of their membrane phospholipids (PL), we investigated the effect consumption of relatively large amounts of saturated fat might have on the fatty acid composition and fluidity of the serum PL of the Fulani. We obtained blood serum from 112 Fulani pastoralists (38 males, 74 females) 15-77 years of age and determined the serum fatty acid composition of the total PL fraction of each specimen. Our results indicate that the PL of the Fulani were enriched for saturated fatty acids. The unsaturated/saturated fatty acid ratio was 1.02 for the Fulani PL compared to 1.22-2.08 for seven other reference groups drawn from published reports. In addition, the mean melting point (MMP) of the fatty acyl chains of the serum PL of the Fulani was considerably higher than that of the reference populations (MMP, 30.6 degrees C versus 21.3-26.1 degrees C, respectively). The double bond index (DBI) of the serum PL of the Fulani was much lower than that of the PL of the groups against which comparisons were made (DBI, 0.98 versus 1.24-1.43, respectively). Since serum PL and tissue PL are in dynamic equilibrium, these findings suggest that the tissue PL of the Fulani we studied has considerably less fluid character than those of other populations. Since a variety of membrane functions depend on the fluid property of the acyl chains of their constituent PL, it is conceivable that certain critical membrane-dependent systems, including receptor-ligand interactions, solute transport, enzyme activity and lateral movement of macromolecules, are affected in the Fulani.     

Exercise effects on chromium excretion of trained and untrained men consuming a constant diet

Chromium excretion of eight trained and five sedentary men was determined on rest days and after exercise to exhaustion at 90% of maximum O2 consumption (VO2max) to determine if degree of physical fitness affects urinary Cr losses. Subjects were fed a constant daily diet containing approximately 9 micrograms Cr/1,000 kcal. VO2max of the trained runners was in the good or above range based on their age and that of the sedentary subjects was average or below. While consuming the control diet, basal urinary Cr excretion of subjects who exercise regularly was significantly lower than that of the sedentary control subjects, 0.09 +/- 0.01 and 0.21 +/- 0.03 microgram/day (mean +/- SE), respectively. When subjects consumed self-chosen diets, basal urinary Cr excretion of the trained subjects was also significantly lower than that of the untrained subjects. Daily urinary Cr excretion of trained subjects was significantly higher on the day of a single exercise bout at 90% VO2max compared with nonexercise days, 0.12 +/- 0.02 and 0.09 +/- 0.01 microgram/day, respectively. Urinary Cr excretion of sedentary subjects was not altered after controlled exercise. These data demonstrate that basal urinary Cr excretion and excretion in response to exercise are related to VO2max and therefore degree of physical fitness.     

Synthesis of hepatic secretory proteins in normal adults consuming a diet marginally adequate in protein

The plasma concentration and hepatic synthesis rates of albumin, transthyretin, very low-density lipoprotein apolipoprotein B-100 (VLDL-apoB-100), high-density lipoprotein apolipoprotein A-1, fibrinogen, alpha1-antitrypsin, and haptoglobin were measured in six normal adults before and after consuming a protein intake of 0.6 g. kg body wt(-1). day(-1) for 7 days. The synthesis of hepatic proteins was measured from the incorporation of [(2)H(5)]- phenylalanine, following prime/continuous infusion, using plasma VLDL-apoB-100 isotopic enrichment to represent the precursor pool. Synthesis of albumin declined by 50% (P < 0.001) following the lower-protein diet, VLDL-apoB-100 declined by 20% (P < 0.001), and apoA-1 declined by 16% (P < 0.05). By contrast, synthesis increased for fibrinogen (50%, P < 0.05) and haptoglobin (90%, P < 0.001). This pattern of change, with decreased synthesis of nutrient transport proteins and increased formation of acute-phase proteins, suggestive of a low-grade inflammatory response, was accompanied by increased plasma concentration of the inflammatory cytokine interleukin 6 (30%, P < 0.05). The pattern of change in the synthesis of hepatic secretory proteins following 7 days on the low-protein diet may be of functional relevance for lipid transport and the capacity to cope with stress.     

The effect of diet on ouabain binding to erythrocytes from obese subjects

Ouabain binding to erythrocyte membranes is increased in obese subjects. Three study groups are compared: 14 reference subjects, 102 +/- 16% of ideal weight; 9 obese on unrestricted diets, 207 +/- 16% of ideal weight; 11 obese on restricted diets, 202 +/- 35% of ideal weight. A reproducible (CV = 11.3%) ouabain-binding assay is used to measure Na+-K+ ATPase sites in erythrocyte membranes. The number of binding sites per red blood cell for obese subjects on unrestricted diets, 431 +/- 30, is greater than for the reference group, 346 +/- 66 (p less than 0.01), or for obese subjects on restricted diets, 371 +/- 68 (p less than 0.05). These data suggest that caloric intake influences the number of Na+-K+ ATPase sites. Scatchard plots indicate only one type of binding site for ouabain with an affinity constant of about 3 X 10(8) M-1.     

Sources of error in the isotopic cholesterol balance method in African green monkeys consuming a cholesterol-free diet

Six African green monkeys were labeled intravenously with [1,2-(3)H]cholesterol while consuming a cholesterol-free liquid formula diet. The plasma cholesterol specific activity was compared with the specific activity of the biliary cholesterol and bile acids and with the fecal neutral steroids in order to determine whether the traditional isotopic balance method was valid for the calculation of endogenous cholesterol excretion. The specific activity of biliary cholesterol and bile acids averaged 10-15% lower than plasma cholesterol specific activity. Fecal cholesterol and coprostanone specific activities were similar to that of the biliary cholesterol, but the specific activity of fecal coprostanol was approximately 25% lower. This suggests that biliary cholesterol and bile acids were derived from a pool of hepatic cholesterol that did not completely equilibrate with the whole body exchangeable cholesterol pool. In addition, there was further reduction in the specific activity of coprostanol, the major fecal neutral steroid, presumably by cholesterol synthesized in the lower intestine and preferentially converted to coprostanol. As a result, the traditional isotopic balance procedure underestimated endogenous neutral steroid excretion by 46% and bile acid excretion by 31% in African green monkeys fed the cholesterol-free diet. Within 7 days after the addition of 1 mg cholesterol/kcal to the diet, the specific activities of plasma and biliary cholesterol and biliary bile acids were identical and there was no difference in the specific activities of the individual fecal neutral steroids. Thus, the traditional isotopic balance procedure (DPM fecal neutral steroids + bile acids/specific activity [DPM/mg] plasma cholesterol) can be used for calculation of endogenous cholesterol excretion in cholesterol-fed animals during the nonsteady state when plasma cholesterol concentrations are rapidly increasing, as well as after a new steady state has been achieved.-Henderson, G. R., and R. W. St. Clair. Sources of error in the isotopic cholesterol balance method in African green monkeys consuming a cholesterol-free diet.     

Endogenous mutagens derived from amino acids

L-Cysteine, glutathione and the therapeutically used L-cysteine precursor, N-acetyl-L-cysteine, induced strong mutagenic effects in Salmonella typhimurium (reversion of the his- strains TA97, TA92 and TA104), when tested in the presence of subcellular kidney preparations. The tyrosine metabolites, levodopa (an ortho-hydroquinone) and homogentisic acid (a para-hydroquinone) reverted various his- strains as well. This mutagenicity did not require the presence of mammalian enzymes, and was relatively weak. The induction of gene mutations was also studied in mammalian cells (V79 Chinese hamster cells), using acquisition of resistance toward 6-thioguanine as the marker. L-Cysteine and N-acetyl-L-cysteine were found to be inactive, levodopa was weakly mutagenic, and homogentisic acid was strongly mutagenic (enhancing the mutation frequency 135-fold above background at an exposure concentration of 50 microM). This finding is striking as the urinary concentration of homogentisic acid is about 1000 times higher in patients with a genetic defect in homogentisic acid 1,2-dioxygenase (alkaptonuria). Genotoxic and carcinogenic effects of other amino acids and metabolites, reported in the literature, are discussed as well.     

Release of mutagens from finished leather

Extracts of a leather widely used in the furniture and dress-making industries were tested for their mutagenic activity in the Salmonella/microsome assay. Extracts obtained after vigorous treatment of leather samples in a Soxhlet apparatus with toluene or ethanol were mutagenic in strain TA98 of S. typhimurium in the absence of S9 mix. The analysis of extracts of leather at various intermediate stages of processing showed that the mutagenic activity appeared after the coloration process. The responsible compound was identified to be an azo dye (Color Index: Acid Brown 83) whose mutagenic potency was about 4 revertants/micrograms.     

Analysis of the fecal microflora of human subjects consuming a probiotic product containing Lactobacillus rhamnosus DR20

The composition of the fecal microflora of 10 healthy subjects was monitored before (6-month control period), during (6-month test period), and after (3-month posttest period) the administration of a milk product containing Lactobacillus rhamnosus DR20 (daily dose, 1.6 x 10(9) lactobacilli). Monthly fecal samples were examined by a variety of methods, including bacteriological culture analysis, fluorescent in situ hybridization with group-specific DNA probes, denaturing gradient gel electrophoresis of the V2-V3 region of 16S rRNA genes amplified by PCR, gas-liquid chromatography, and bacterial enzyme activity analysis. The composition of the Lactobacillus population of each subject was analyzed by pulsed-field gel electrophoresis of bacterial DNA digests in order to differentiate between DR20 and other strains present in the samples. Representative isolates of lactobacilli were identified to the species level by sequencing the V2-V3 region of their 16S rRNA genes and comparing the sequences obtained (BLAST search) to sequences in the GenBank database. DR20 was detected in the feces of all of the subjects during the test period, but at different frequencies. The presence of DR20 among the numerically predominant strains was related to the presence or absence of a stable indigenous population of lactobacilli during the control period. Strain DR20 did not persist at levels of >10(2) cells per g in the feces of most of the subjects after consumption of the product ceased; the only exception was one subject in which this strain was detected for 2 months during the posttest period. We concluded that consumption of the DR20-containing milk product transiently altered the Lactobacillus and enterococcal contents of the feces of the majority of consumers without markedly affecting biochemical or other bacteriological factors.     

Skeletal muscle respiratory capacity is enhanced in rats consuming an obesogenic Western diet

Obesity-induced lipid oversupply promotes skeletal muscle mitochondrial biogenesis. Previous investigations have utilized extreme high-fat diets (HFD) to induce such mitochondrial perturbations despite their disparity from human obesogenic diets. Here, we evaluate the effects of Western diet (WD)-induced obesity on skeletal muscle mitochondrial function. Long-Evans rats were given ad libitum access to either a WD [40% energy (E) from fat, 17% protein, and 43% carbohydrate (30% sucrose); n = 12] or a control diet (CON; 16% of E from fat, 21% protein, and 63% carbohydrate; n = 12) for 12 wk. Rats fed the WD consumed 23% more E than CON (P = 0.0001), which was associated with greater increases in body mass (23%, P = 0.0002) and adiposity (17%, P = 0.03). There were no differences in fasting blood glucose concentration or glucose tolerance between diets, although fasting insulin was increased by 40% (P = 0.007). Fasting serum triglycerides were also elevated in WD (86%, P = 0.001). The maximal capacity of the electron transfer system was greater following WD (37%, P = 0.02), as were the maximal activities of several mitochondrial enzymes (citrate synthase, β-hydroxyacyl-CoA dehydrogenase, carnitine palmitoyltransferase). Protein expression of citrate synthase, UCP3, and individual respiratory complexes was greater after WD (P < 0.05) despite no differences in the expression of peroxisome proliferator-activated receptor (PPAR)α, PPARδ, or PPARγ coactivator-1 mRNA or protein abundance. We conclude that the respiratory capacity of skeletal muscle is enhanced in response to the excess energy supplied by a WD. This is likely due to an increase in mitochondrial density, which at least in the short term, and in the absence of increased energy demand, may protect the tissue from lipid-induced impairments in glycemic control.     

Adaptation of the doubly labeled water method for subjects consuming isotopically enriched water

Gretebeck, Randall J., Dale A. Schoeller, Rick A. Socki,Janis Davis-Street, Everett K. Gibson, Leslie O. Schulz, and Helen W. Lane. Adaptation of the doubly labeled water methodfor subjects consuming isotopically enriched water. J. Appl. Physiol. 82(2): 563-570, 1997.The use ofdoubly labeled water (DLW) to measure energy expenditure is subject toerror if the background abundance of the oxygen and hydrogen isotopetracers changes during the test period. This study evaluated theaccuracy and precision of different methods by which such backgroundisotope changes can be corrected, including a modified method thatallows prediction of the baseline that would be achieved if subjectswere to consume water from a given source indefinitely. Subjects inthis study were eight women (4 test subjects and 4 control subjects)who consumed for 28 days water enriched to resemble drinking water aboard the United States space shuttle. Test subjects and control subjects were given a DLW dose on days1 and 15,respectively. The change to an enriched water source produced a bias inexpenditure calculations that exceeded 2.9 MJ/day (35%), relative tocalculations from intake-balance. The proposed correction based on thepredicted final abundance of ¹⁸Oand deuterium after equilibration to the new water source eliminated this bias, as did the traditional use of a control group. This newmodified correction method is advantageous under field conditions whensubject numbers are limited.

     

Loss of molar occlusion and mandibular morphology in adults in an ancient human population consuming a coarse diet

The purpose of the study is to investigate the link between number of molar teeth retained in occlusion and mandibular morphology in adults in an ancient, high dental wear human population. The study material comprises skeletons from Mediaeval Wharram Percy, England (N = 50 female, 69 male adults). It was hypothesized that adults retaining fewer occluding molars would show reduction in mandibular dimensions, particularly in the ascending ramus and gonial regions where the main muscles of mastication have their insertions. Molar occlusal status is assessed using the concept of functional units. Mandibular morphology is assessed using a suite of ten linear measurements plus the mandibular angle. Results show no evidence for any association between number of molars retained in occlusion and mandibular angle. There was an association between mandibular size and number of molars retained in occlusion, with smaller mandibular dimensions in those retaining fewer occluding molars. Some measurements were affected more than others so that there was also some shape alteration. Alteration of mandibular dimensions was more clearly demonstrable in females than in males. Only in females could significant reduction in the ascending ramus and gonial regions be demonstrated. Reasons for the apparent difference in response to loss of molar occlusion between male and female mandibles are unclear, but sex differences in bony metabolism mediated by hormonal factors may be implicated. Results suggest that care should be exercised when including mandibles from individuals showing loss of molar occlusion in morphological studies. Am J Phys Anthropol 152:383–392, 2013. © 2013 Wiley Periodicals, Inc.     

Effect of coenzyme Q10 supplementation on cardiac hypertrophy of male rats consuming a high-fructose,low-copper diet

Administration of coenzyme Q₁₀ to humans and animals has a beneficial effect on a number of cardiac diseases. The purpose of the present study was to determine if coenzyme Q₁₀ treatment could ameliorate cardiac abnormalities associated with the carbohydrate × copper interaction in rats. Weanling male rats were provided with a copper-deficient diet (0.6 μg Cu/g) containing either 62.7% starch (S−Cu) or fructose (F−Cu) for 5 wk. Half of the rats provided with the F−Cu diet were given daily oral supplements of 300 mg coenzyme Q₁₀/kg body weight (F−Cu+Q). Heart hypertrophy, liver enlargement, or pancreatic atrophy were not affected by, nor was body growth or anemia improved by, supplementation with coenzyme Q₁₀ when compared to rats fed only the F−Cu diet. Hearts from rats fed the F−Cu diet had severe inflammation, degeneration, fibrosis, and giant mitochondria with abnormal cristae. Hearts from F−Cu+Q rats had similar mitochondrial changes as the F−Cu rat hearts but without any apparent degenerative changes. None of the F−Cu+Q rats, but 30% of the F−Cu rats, died during the study as a result of heart rupture. These observations show that whereas coenzyme Q₁₀ treatment did not prevent the cardiac hypertrophy of the carbohydrate × copper interaction, it did play a role in maintaining the integrity of the heart. This work was presented in part at the 2nd International Symposium on Metal Ions in Biology and Medicine, Loutraki, Greece, May 15–22, 1992 (Metal Ions (1992), J. Anastassopoulou, P. Collery, J.C. Etienne, T. Theophanides, eds., John Libbey Eurotext, Montrouge, France, pp. 402–407).     

Metabolic epidemiology of colon cancer: fecal mutagens in healthy subjects from rural Kuopio and urban Helsinki, Finland

Fecal mutagenic activity and dietary pattern of rural and urban Finnish population groups with distinct risk for the development of colon cancer were studied in a low-risk population in rural Kuopio and an intermediate-risk population in urban Helsinki. The average daily intake of protein and fat was the same in the two groups but the frequency of consumption of whole-grain cereals and whole-grain bread, as well as the amount of fiber from the bread were higher in Kuopio as compared to Helsinki. Fecal samples collected for 2 days were incubated under anaerobic conditions at 37 degrees C for 96 h, extracted with hexane: peroxide-free diethyl ether, partially purified on a silica Sep-Pak cartridge, and assayed for mutagenic activity using the Salmonella typhimurium/mammalian microsome system. The fecal mutagenic activity was observed with the tester strains TA98 and TA100 with and without microsomal activation in both the population groups. The percentage of samples showing fecal mutagenic ratio greater than 3 with TA98 and TA100 with microsomal activation, was higher in volunteers from Helsinki than in Kuopio.     

Iron status biomarkers in iron deficient women consuming oily fish versus red meat diet

Specific recommendations for anemic individuals consist in increasing red meat intake, but the population at large is advised to reduce consumption of red meat and increase that of fish, in order to prevent the risk of developing cardiovascular disease. This study aimed to determine the effects of consuming an oily fish compared to a red meat diet on iron status in women with low iron stores. The study was designed attending the Consolidated Standards of Reporting Trials (CONSORT) statement guidelines. It was a randomised crossover dietary intervention study of two 8-week periods. Twenty-five young women with low iron stores completed the study. Two diets containing a total of 8 portions of fish, meat and poultry per week were designed differing only in their oily fish or red meat content (5 portions per week). At the beginning and the end of each period blood samples were taken and hemoglobin, hematocrit, serum ferritin, serum iron, serum transferrin, serum transferrin receptor-2 and the Zn-protoporphyrin/free-protoporphyrin ratio were determined. Food intake and body weight were monitored. During the oily fish diet, PUFA intake was significantly higher (p=0.010) and iron intake lower (mean±SD, 11.5±3.4 mg/dayvs. 13.9±0.1 mg/day, p=0.008), both diets providing lower mean daily iron intake than recommended for menstruating women. Although there were no significant differences after 16 weeks, serum ferritin moderately decreased and soluble transferrin receptor increased with the oily fish, while changes with the red meat diet were the opposite. In conclusion, an oily fish diet compared to a red meat diet does not decrease iron status after 8 weeks in iron deficient women.     

16S ribosomal DNA analysis of the faecal lactobacilli composition of human subjects consuming a probiotic strain Lactobacillus acidophilus NCFM

AIMS: The aims of this study were to evaluate the ability of exogenous Lactobacillus acidophilus strain NCFM to survive through the human gastro-intestinal (GI) tract, and to evaluate the selectivity of Rogosa SL medium for faecal lactobacilli. METHODS AND RESULTS: The composition of the faecal lactobacilli of 10 healthy subjects was monitored for two weeks prior to, two weeks during and two weeks after the administration of the Lact. acidophilus strain NCFM consumed with skim milk (daily dose 10(10) viable cells). Fresh faecal samples were collected, processed and cultured on Rogosa SL selective medium for lactobacilli enumeration. Colonies demonstrating various morphologies were identified and purified for 16S ribosomal DNA sequence analysis for speciation of colonial genotype. The species composition of cultivable faecal lactobacilli changed considerably during consumption of the strain NCFM. CONCLUSIONS: The probiotic Lact. acidophilus strain NCFM can survive through the human GI tract, but cannot colonize itself during the two-week consumption. Rogosa SL medium is selective for faecal lactobacilli. However, genetic analysis is required for colony speciation. SIGNIFICANCE AND IMPACT OF THE STUDY: It is demonstrated that continuous consumption is necessary to maintain a high population of the probiotic strain, and that the Rogosa SL medium is reliable.     

Possible mutagens derived from lipids and lipid precursors

Free radicals can initiate the oxidative decomposition of cellular membranes by lipid peroxidation. In this process a great variety of reactive aldehydes are produced intracellularly. Some of them, such as 4-hydroxynonenal or malonaldehyde, are biologically very active and might be involved in free radical-mediated DNA damage. A short review of the effects of aldehydic lipid peroxidation products on isolated DNA, their genotoxic effect in prokaryotes and eukaryotes and their in vivo carcinogenicity is given. Additionally own experiments on cytotoxic and genotoxic effects of 4-hydroxynonenal, 2-nonenal and nonanal in primary cultures of rat hepatocytes are reported. 4-Hydroxynonenal was highly cytotoxic at 100 microM, at subcytotoxic concentrations of 0.1-10 microM 4-hydroxynonenal increased the frequency of micronuclei, chromosomal aberrations and sister-chromatid exchange. 2-Nonenal and nonanal were not cytotoxic at 100 microM, the maximum dose tested. At 100 microM 2-nonenal led to a slight increase in micronuclei; chromosomal aberrations were not significantly altered. Nonanal had no detectable genotoxic effects. The level of endogenous 4-hydroxynonenal in tissues is in the range of 0.1-3.0 microM and can increase to 10 microM in conditions of oxidative stress; such levels appear to be sufficiently high to produce DNA damages, whether such damages are transient or irreversible is not known.