Shift from a dairy product-rich to a dairy product-free diet: influence on cytotoxicity and genotoxicity of fecal water--potential risk factors for colon cancer.

Several epidemiologic studies have suggested that dairy product intake is associated with a decreased incidence of colon cancer. To determine whether the cytotoxicity and genotoxicity of the aqueous portion of human stool (two potential risk markers for the disease) were affected by a change in dairy product intake, 18 healthy male and female volunteers were randomly divided into two groups. In a crossover design, the volunteers shifted from their normal dairy product-rich diet to a dairy product-free diet. Nutritional analysis of the food consumed during the study period showed a significant decrease in energy intake from 9000 to 7866 kJ/d because of a decreased intake of protein and fat. Carbohydrate and fiber intakes remained unchanged during the intervention. Calcium intake decreased significantly from 1488 to 372 mg/d, with similar significant decreases in phosphate and vitamin D intakes. Cytotoxicity of fecal water, analyzed by the HT-29 cytotoxicity assay, indicated a significant decrease in cell survival from 34% to 20% when dairy products were excluded from the participants' diets. Single-cell gel electrophoresis (COMET assay), used to analyze genotoxicity of fecal waters, indicated no differences brought about by the dietary intervention. In conclusion, our findings indicate that a shift from a dairy product-rich to a dairy product-free diet resulted in a significant effect on an accepted risk marker for colon cancer and may suggest that the mechanism by which dairy products are protective is at the level of tumor promotion rather than initiation.

Shift from a mixed to a lactovegetarian diet: influence on acidic lipids in fecal water--a potential risk factor for colon cancer.

Although there have recently been reports in the literature indicating that vegetarian-type diets are protective against the development of human colon cancer, this is still far from clear. It was also recently indicated that the concentration of acidic lipids in the aqueous phase of stool constitutes a risk factor for the development of colon cancer. Thus, we examined the effect of a change from a mixed to a lactovegetarian diet on this fecal variable. The dietary change caused a decrease in the total concentration of soluble fecal fatty acids (4310 +/- 3020 to 1080 +/- 1040 mumol/L, p less than 0.05) and deoxycholic acid (125 +/- 42 to 73 +/- 35 mumol/L, p less than 0.05). However, there was no change in either the total bile acid concentration in (164 +/- 54 to 107 +/- 41 mumol/L) or the cellular toxicity of (0.94 +/- 0.55 to 1.60 +/- 0.63 mumol/L, relative survival) the aqueous phase of stool. Thus, the consumption of a lactovegetarian diet may reduce certain risk factors of potential significance in colon carcinogenesis.

Cellular toxicity of fecal water depends on diet.

To determine whether concentrations of potentially toxic lipids in the aqueous phase of human stool are responsive to changes in dietary fat, calcium, and fiber, 20 male volunteers were placed on a high-fat, low-calcium, low-fiber or a low-fat, high-calcium, high-fiber diet for 4 days. To assess toxicity of the fecal fractions, we examined the ability of fecal supernatants to lyse human erythrocytes. Bile acid concentrations in fecal water from the low-fat group were reduced significantly from 180 +/- 60 microM to 100 +/- 70 microM; in the high-fat group, increased from 190 +/- 60 microM to 250 +/- 100 microM. Erythrocyte lysis was 76% for the high-fat group, 37% for the low-fat group. There was a significant weak correlation between aqueous bile acid concentration and cell lysis. Results suggest that diet can influence concentrations of detergents in the aqueous phase of human stool and the potential toxicity of this fraction to cell membranes.

Studies of the vivo uptake of Ga-67 by an experimental abscess: concise communication.

The blocking of Ga -67 plasma protein-binding sites-by administration of scandium citrate, ferric citrate, and a colloidal hydrous ferric oxide preparation-reduced the uptake of Ga-67 in normal soft tissues and also that in the viable portion of an experimental abscess. On the other hand, enhancement of Ga-67 plasma protein binding by administration of rabbit apotransferrin increased Ga-67 uptake in both abscess and normal soft tissues. These results indicate that the pathways of Ga-67 from blood into inflammatory processes and normal soft tissues may be similar. However, when Ga-67 plasma protein binding was increased by inducing anemia, a markedly decreased Ga-67 uptake in the abscess resulted, whereas uptake in normal soft tissue was still elevated. It is possible that the discrepancy between the effects of apotransferrin and anemia on abscess-tissue uptake of Ga-67 resulted from a secondary effect produced by anemia, i.e., a decrease in the macrophage population in the abscess. Taken as a whole, the results obtained suggest that Ga-67 leaves the blood and enters inflammatory lesions by pathways that are probably quite different from those in a soft-tissue tumor, and that the routes for abscesses may be similar to those occurring in normal soft tissues.

Studies of the in vivo entry of Ga-67 into normal and malignant tissue.

Previous studies of the effect of scandium on the tissue distribution of Ga-67 suggest that Ga-67 makes its initial in vivo entry into normal and malignant tissues by different routes. (Scandium blocking of plasma protein Ga-67 binding increased Ga-67 excretion, decreased its uptake in normal tissues, but had little effect on rodent tumors.) In further studies we have used other methods to alter the plasma binding of Ga-67. Iron saturation of plasma produced effects on Ga-67 tissue distribution similar to those observed with scandium. On the other hand, increasing Ga-67 plasma binding through induction of anemia and administration of apotransferrin produced the reverse of the effects observed with scandium and iron. We conclude that the initial in vivo entry of Ga-67 into tumor tissue involves mainly an unbound or loosely bound form of Ga-67, whereas its uptake by normal soft tissues is strongly promoted by its binding to transferrin.

The effect of scandium on the tissue distribution of Ga-67 in normal and tumor-bearing rodents.

In rats and mice the intravenous administration of scandium before or with Ga-67 produces an increase in Ga-67 excretion and bone deposition, coupled with pronounced decreases in the uptake of Ga-67 in soft tissues. These effects result from the blocking by scandium of Ga-67 plasma-protein binding sites, which forces Ga-67 into an unbound or loosely bound state. This increases Ga-67 excretion and bone deposition, which in turn acts to produce greatly reduced Ga-67 uptake in soft tissues. When tumor-bearing rats and mice are administered scandium, similar effects occur, but the uptake of Ga-67 by tumor tissue remains unchanged. This suggests that Ga-67 enters tumor and normal soft tissues by different routes. With tumor, an unbound or loosely bound form of gallium is primarily involved, whereas with normal soft tissues this route is apparently of minor importance.

Effects on Cell Proliferation, Activator Protein-1 and Genotoxicity by Fecal Water from Patients with Colorectal Adenomas.

BACKGROUND: The free water phase of feces (fecal water) may mediate the effects of diet on colon carcinogenesis. We examined the effects of fecal water from adenoma patients and controls on three parameters in colonocytes believed to be relevant to tumorigenesis, i.e. genotoxicity in intact cells and on isolated DNA, proliferative activity and activator protein-1 (AP-1) activity. METHODS: Genotoxicity in intact colonic cells was assayed using the single-cell gel electrophoresis assay (`comet' assay) and on isolated DNA using double-stranded DNA from the X-174 RF plasmid. Cell proliferation was assessed using the commercially available `alamar blue' proliferation kit and AP-1 activity using cells transiently transfected with an AP-1-luciferase reporter construct. RESULTS: The data showed that lipid extracts of fecal water samples from the adenoma patients had a significantly higher capacity to induce cell proliferation than those from controls, and that this effect could be explained to a large extent by the concentrations of deoxycholic and chenodeoxycholic acids in the fecal water using regression models. No difference between patients and controls was observed for induction of AP-1 activity or induction of DNA strand breaks in intact cells. However, induction of DNA strand breaks in isolated DNA was significantly higher for the fecal waters from patients than for those from controls, which could be explained in part in a regression model by concentrations of lithocholic acid in fecal water and fecapentaene-12 in feces. CONCLUSIONS: Our results support the hypothesis that the biochemistry of fecal waters from adenoma patients and controls differs.

N-acetyl derivative as the major active metabolite of 2-amino-6-methyldipyrido[1,2-a:3',2'-d]imidazole in rat bile.

2-Amino-6-methyldipyrido[1,2-a:3',2'-d]imidazole (Glu-P-1) is a mutagen and carcinogen isolated from a glutamic acid pyrolysate. When this 14C-labeled compound was administered to male F344 rats at a dose of 0.3 mCi (20.8 mg)/kg b.w., 70% of the radioactivity was excreted into the bile in 24 h. On HPLC analysis of this bile, several metabolites of Glu-P-1 were found with unmetabolized Glu-P-1. One of the mutagenic metabolites was identified as N-acetyl-Glu-P-1. This metabolite had a specific mutagenic activity of about one quarter of that of Glu-P-1 and its amount in the bile corresponded to a few percent of the dose of Glu-P-1 administered.

Steroid metabolism by rat nasal mucosa: studies on progesterone and testosterone.

The metabolism of [4-14C]progesterone and [4-14C]testosterone by slices of the nasal mucosa from rats was studied. As shown by gas chromatography-mass spectrometry there was a preferential formation of reduced progesterone-metabolites (5 alpha-pregnane-3,20-dione, 3 alpha- and 3 beta-hydroxy-5 alpha-pregnane-20-one, 20 alpha- and 20 beta-hydroxypregn-4-en-3-one, 2 alpha,3 alpha-dihydroxy-5 alpha-pregnane-20-one, 3 alpha,16 alpha-dihydroxy-5 alpha-pregnane-20-one) and reduced testosterone-metabolites (4-androstene-3,17-dione, 5 alpha-dihydrotestosterone, 3 alpha-hydroxy-5 alpha-androstane-17-one, and 5 alpha-androstane-3 alpha, 17 beta-diol, 2 alpha-hydroxy-5 alpha-dihydrotestosterone, 5 alpha-androstane-2 alpha,3 alpha, 17 beta-triol) indicating the presence of 5 alpha-reductase, 3 alpha-, 3 beta-, 17 beta-, 20 alpha- and 20 beta-hydroxysteroid oxidoreductase activities in this tissue. Progesterone-metabolites hydroxylated at positions 2 alpha, 6 alpha, 6 beta, 15 alpha and 16 alpha and testosterone-metabolites hydroxylated at positions 1 beta, 2 alpha, 6 beta, 15 beta and 16 alpha were also identified, indicating the presence of several steroid hydroxylases in the nasal mucosa. Autoradiography of the nasal region of rats injected with [4-14C]progesterone or [4-14C]testosterone showed a selective localization of radioactivity in the mucosa covering the olfactory region of the nasal cavity.

Involvement of the intestinal microflora in the formation of sulphur-containing metabolites of caffeine.

1. The urine of conventional and germ-free rats was examined, after an oral dose of caffeine, with regard to the presence of sulphur-containing conjugates of this compound. 2. Only the methyl sulphoxide analogue was detected. 3. The ratio of the quantities of this metabolite in conventional and germ-free rat urine was 42:1. 4. The proposed mechanism for the formation of the methyl sulphoxide analogue of caffeine involves the biliary excretion of a mercapturic acid pathway metabolite of caffeine with subsequent metabolism by the intestinal microflora.

Biliary metabolites of the anti-inflammatory drug 2-acetamido-4-(chloromethyl)thiazole.

The mechanism for the formation of a class of sulfur-containing conjugates of xenobiotics was further investigated in this report. The major biliary metabolites of 2-acetamido-4-(chloromethyl)thiazole in the rat were found to be the mercapturic acid conjugate of 2-acetamido-4-methylthiazole and the glucuronic acid conjugate of 2-acetamido-4-(mercaptomethyl)thiazole. When these two compounds were introduced directly into the cecum of the rat, 2-acetamido-4-[(methylsulfinyl)methyl]thiazole and 2-acetamido-4-[(methylsulfonyl)methyl]thiazole were found as urinary metabolites. These results give strong support to a proposed mechanism in which intestinal microfloral metabolism of biliary metabolites, together with enterohepatic circulation, is necessary for the formation and urinary excretion of the 4-(methylthiomethyl), 4-(methylsulfinyl-methyl), and 4-(methylsulfonylmethyl) analogs of 2-acetamido-4-(chloromethyl)thiazole in the rat.

Studies on the substrate specificity and inducibility of cytochrome P-450meg.

The cytochrome P-450-dependent steroid 15 beta-hydroxylase system in Bacillus megaterium A.T.C.C. 13368 was investigated with regard to its appearance in the cell with respect to the growth curve of the organism, with regard to its inducibility by a number of agents (among them some of the classical inducers of the mammalian liver microsomal cytochrome P-450 system) and with regard to its capacity to convert non-steroidal substances into oxygenated compounds. The enzyme was found to reach a maximum concentration in the cell during the stationary phase of the growth curve. Of all the agents tested as inducers, none showed any capacity to induce cytochrome P-450meg. Finally, of the substances tested as substrates only aniline (p-hydroxylation) was metabolized by the microbial enzyme system. This conversion might be related to the general oxygenase activity of haemoproteins. It is concluded that the substrate specificity of the B. megaterium hydroxylase system is narrow.

Metabolism of the dietary carcinogen TRP-P-1 in rats.

The heterocyclic amine 3-amino-1,4-dimethyl-5H-pyrido(4,3-b)indole (Trp-P-1) was administered as a single oral dose to conventional, bile-fistulated and germ-free rats. There was rapid excretion of Trp-P-1 and its metabolites via the bile, urine and feces. The pattern of metabolites did not differ markedly between the three excretory routes. While there was considerable excretion of unmetabolized Trp-P-1, at the dose level used, there was also extensive metabolism to primary ring-hydroxylated and N-acetylated metabolites which were polar enough to be excreted without undergoing conjugation reactions. Four of the metabolites exhibited mutagenic activity towards Salmonella typhimurium TA98 in the presence of S9 mix. However, all showed a lower mutagenicity than the parent compound. Studies with the bile-fistulated animals indicated that enterohepatic circulation was occurring with Trp-P-1. The intestinal microflora did not appear to have a major role to play in the metabolism of this heterocyclic amine but they did lead to the formation of 'bound fecal residues' in the gut of the conventional animals.

Increased excretion of a brain depressor amine in infantile coeliac disease and in healthy infants on a high protein milk diet.

Urinary excretion of piperidine, a heterocyclic pressor amine of gut bacterial origin and nicotine-like activity in the brain, has been estimated by a gas chromatography method in healthy men and women, in normal breast-fed and formula-fed infants and in infants with untreated coeliac disease. The excretion of piperidine cannot usually be detected during the first week of life. The amount present in urine increases upon weaning with higher excretion in formula-fed than in breast-fed infants at four to six months of age. When premature infants fed on human milk are weaned, the urinary content of piperidine rises from undetectable amounts to normal for age. The high content present in untreated coeliac disease may be responsible for the initial mental depression commonly seen in this disease and suggests that piperidine is one of the "auto-intoxicating" substances arising from the bacterial decomposition of protein postulated by Metchnikoff in 1903 but hitherto unidentified.