Comparative effects of secondary bile acids, deoxycholic and lithocholic acids, on aberrant crypt foci growth in the postinitiation phases of colon carcinogenesis.

The objective of this study was to investigate the effect of deoxycholic (DCA) and lithocholic (LCA) acids on the postinitiation phases of colon cancer. Male Sprague-Dawley rats (n = 170) were injected with azoxymethane (2 injections at 15 mg/kg body wt sc given 1 wk apart) and fed a control (CON) AIN-93 diet. Two weeks after the second azoxymethane injection, 10 animals were killed and aberrant crypt foci (ACF) were enumerated. The remaining animals were randomly assigned to four diet groups: 1) CON, 2) DCA, 3) LCA, and 4) high fat (HF, a positive control group). Bile acid diets consisted of 0.2% by weight DCA or LCA; HF diets consisted of 20% fat (5% soybean oil + 15% beef tallow by weight). Animals were killed at Weeks 3, 12, and 20 (from 1st carcinogen injection), and number and growth features of ACF and adenomatous lesions were enumerated in the colon. At Week 12, ACF number and small, medium, and large (1-3, 4-6, and > or = 7 crypts/focus, respectively) ACF were higher in the HF group than in the DCA, LCA, and CON groups (p < or = 0.05). By Week 20, ACF number and small, medium, and large ACF were similar in the LCA and HF groups, whereas the response was similar in the DCA and CON groups. Average crypt multiplicity was higher in the HF and LCA groups than in the DCA and CON groups (p < or = 0.05). Microadenoma (MA) incidence was higher in the HF group than in the CON and LCA groups (p < or = 0.05). Regional distribution patterns for ACF number were similar to MA and tumor distribution patterns within the CON, DCA, and HF groups. In the LCA group, ACF number and MA showed a proximal predominance in regional distribution, whereas tumors showed a distal predominance. HF diets provided the most stimulatory environment, immediately enhancing the number and growth of ACF and MA incidence. In conclusion, HF and LCA diets exerted distinct effects on postinitiation phases of colon cancer, whereas the DCA diet did not.

Effects of the trichothecene mycotoxin T-2 toxin on neurotransmitters and metabolites in discrete areas of the rat brain.

T-2 toxin has been shown to affect the central nervous system. Only recently have attempts been made to characterize the neurochemical perturbations associated with T-2 intoxication. To examine the effect of T-2 on regional brain biogenic monoamines and selected metabolites, male rats were dosed orally with T-2 toxin in corn oil at 0.1, 1.0 or 2.5 mg/kg body weight. At 2, 6 and 10 hr post-dosing, rats were killed, brains were collected and stored at -80 degrees until analysed. Brain nuclei, including nucleus raphe magnus, paraventricular nucleus, locus coeruleus, substantia nigra, medial forebrain bundle, nucleus accumbens and olfactory tubercle, were analysed. T-2 treatment increased 5-hydroxy-3-indoleacetic acid and serotonin throughout the brain, and produced a transient increase in norepinephrine in the nucleus raphe magnus and a temporary decrease in the substantia nigra. Regional dihydroxyphenylacetic acid concentration was affected, with increased DOPAC observed in the locus coeruleus, medial forebrain bundle and paraventricular nucleus of the hypothalamus, and decreased DOPAC in the olfactory tubercles. No regional changes in epinephrine or dopamine were observed. Few treatment differences were observed, with the 0.1 mg/kg body weight T-2, 2% of the LD50, significantly affecting brain monoamines. It had been suggested that neurological manifestations of T-2 toxin are the result of brain hypoxia; however, the altered brain monoamine profile observed at doses that do not alter heart function, suggests the brain is a primary site of trichothecene action.

Effect of T-2 toxin on blood-brain barrier permeability monoamine oxidase activity and protein synthesis in rats.

Systemic exposure to T-2 toxin disrupts brain biogenic monoamine metabolism. Although the mechanisms underlying these neurochemical perturbations are unclear, we have suggested that they are a reflection of increased blood-brain barrier (BBB) permeability, or altered protein synthesis that affects brain enzyme activities. Accordingly, BBB permeability, in vitro protein synthesis and in vitro monoamine oxidase (MAO) activity were examined in rats after either acute, or 7-day exposure to T-2. Membrane permeability was assessed from the recovery of systemically administered [14C]mannitol and [14C]dextran with [3H]water as the diffusible reference, either 2 hr post-intraperitoneal (i.p.) injections of 0, 0.2 and 1 mg T-2/kg body weight or following a 7-day exposure to diets containing 0 and 10 ppm T-2. Protein synthesis, determined by [14C]leucine incorporation, and MAO activity, determined by H2O2 production, were observed either 2 hr post-ip injection of 0 and 1 mg T-2/kg body weight or following a 7-day exposure to diets containing 0, 2.5 and 10 ppm T-2. Permeability increases were observed in all brain regions examined for mannitol, but not for dextran following T-2 i.p. The effect of dietary T-2 was more modest, affecting mannitol uptake in two brain regions, the cerebellum and pons plus medulla regions. Protein synthesis was significantly decreased by i.p. administration of T-2, while dietary treatment significantly reduced MAO enzyme activity. Collectively, the effect of T-2 toxin on BBB permeability, protein synthesis and MAO enzyme activity may account for the neurochemical imbalance observed in T-2 intoxication.

Modulation of colonic xenobiotic metabolizing enzymes by feeding bile acids: comparative effects of cholic, deoxycholic, lithocholic and ursodeoxycholic acids.

Primary and secondary bile acids such as cholic (CHA), deoxycholic (DCA) and lithocholic (LCA) acids have been shown to increase colon tumorigenesis. It has been suggested that inhibition of xenobiotic metabolizing enzymes such as glutathione S-transferase (GST) and UDP-glucuronyltransferase (UGT) by bile acids may be a factor in the development of colon cancer. While enzyme inhibition has been demonstrated in vitro, it is unclear whether feeding bile acids modulates colonic GST and UGT in vivo. To test this notion, male, Sprague-Dawley rats (n = 100) were assigned to a control (CON) or test diets containing 0.2% CHA, DCA, LCA or ursodeoxycholic acid (UDCA). After 5 weeks, colonic tissue was harvested and used for enzyme and cell proliferation measurements. The response to bile acids varied with the enzyme measured and appeared isoenzyme specific. GST-alpha activity was lower in the bile acid fed groups compared with CON. While GST-mu was lower in the LCA-fed group, GST-pi was lower in the DCA-, CHA- and UDCA-fed groups. Unlike GST, both UGT and NADPH-cytochrome P-450 reductase (CYC) activities were increased by bile acids. The proliferative response of the colonic epithelium varied with the bile acids and was regionally specific. These data demonstrate that feeding bile acids alters the activity of colonic phase I and II enzymes; however, the physiological effect of these enzymatic perturbations is yet to be determined.

Tumor-enhancing effects of cholic acid are exerted on the early stages of colon carcinogenesis via induction of aberrant crypt foci with an enhanced growth phenotype.

The objective of the study was to establish whether cholic acid (CHA) enhanced colonic tumor incidence in the early phase of carcinogenesis. Male, Sprague-Dawley rats (n = 180) were injected twice with azoxymethane (AOM) (15 mg x kg(-1) body weight x week(-1), s.c., given 1 week apart). Following the first AOM injection, animals were randomly assigned to two groups, control AIN-93G diet (CON) or control diet containing 0.2% CHA by weight (CHA). Three weeks after the first injection, 20 animals (10 animals/group) were killed and aberrant crypt foci (ACF) were enumerated. The remaining animals were further subdivided and animals randomly assigned to CON or CHA diets, creating four treatments: CON-CON, CON-CHA, CHA-CHA, and CHA-CON. After 3, 12, and 20 weeks (following the first carcinogen injection), the animals were killed and the number and crypt multiplicity of ACF enumerated. Macroscopic tumors were evaluated at week 20. Total ACF were not different between groups. Average crypt multiplicity and medium (4-6 crypts/focus) and large (> or = 7 crypts/focus) ACF were greater in CHA-CHA and CHA-CON compared with CON-CON and CON-CHA (p < 0.01). Transient exposure to CHA (CHA-CON) was sufficient to induce development of ACF with an accelerated growth phenotype and elicit a tumor-enhancing effect. CHA-CHA had the highest tumor incidence (82.8%, p < 0.05) followed by CHA-CON (56.7%, p < 0.05), and tumor multiplicity and number of tumors per rat in CHA-CON were similar to CHA-CHA (2.29 and 1.3 versus 2.33 and 1.9, respectively). Delayed intervention with CHA (CON-CHA) produced a tumor outcome similar to CON-CON (31 and 30%, respectively), it did not enhance colonic tumor incidence. Taken collectively these results suggest CHA was effective in enhancing colon carcinogenesis during early phases and ineffective in post-initiation phases.

Phenobarbital and 3-methylcholanthrene treatment alters phase I and II enzymes and the sensitivity of the rat colon to the carcinogenic activity of azoxymethane.

It has been hypothesized that cancer risk may be influenced by phase I and II drug-metabolizing enzyme systems. This study attempted to determine the relationship between colon phase I and II enzyme activity and the subsequent induction of aberrant crypt foci (ACF), preneoplastic lesions by azoxymethane (AOM), a colon-specific carcinogen. Phenobarbital (PB) and 3-methylcholanthrene (MC) treatment (prototype hepatic inducers of phase I and II enzymes) provided the framework to study the induction of phase I and II enzymes in the rat colonic mucosa. Following induction for five consecutive days, the animals were given a single injection of AOM. Phase I and II enzymes were determined fluorometrically and spectrophotometrically and ACF were identified microscopically. Phase I and II xenobiotic metabolizing enzymes were induced in the rat colonic mucosa by prototype hepatic inducers. A lower number of ACF and crypt multiplicity was observed in animals induced with MC than in those in the non-induced and PB groups. Altered levels of phase I and II enzymes in the colon during preinitiation stages were associated with modulation in the growth of ACF, putative preneoplastic lesions.

Dietary Iodine and selenium interact to affect thyroid hormone metabolism of rats.

The interaction of dietary selenium and iodine on the activities of the selenoenzymes, selenium-dependent glutathione peroxidase (GSH-Px), and type I deiodinase (DI-I), and the thyroid hormones thyroxine (T4) and triiodothyronine (T3) were studied. Male weanling Sprague-Dawley rats were fed an AIN-93G diet for 6 wk with modified selenium and iodine concentration as follows: three levels each of iodine and selenium (0.03, 0.2 added and 1.0 added mg iodine/kg diet, and 0.05, 0.18 added and 1.0 added mg selenium/kg diet) were used in a 3 x 3 factorial design. Renal, but not hepatic, DI-I activity was lower in rats with low selenium intake than in controls. Circulating T3 concentration was not affected by the dietary levels of iodine or selenium. Unlike in liver, kidney and erythrocytes, thyroidal GSH-Px activity was not lower than in controls in rats with low selenium intake, but was significantly higher when iodine intake was low. Significant interactions of iodine and selenium on serum T4 and thyroidal GSH-Px activity were observed. Serum T4 was maintained at control levels when both dietary iodine and selenium were low, but not when iodine alone, or selenium alone, was low. Activity of thyroidal GSH-Px was lowest in rats fed a diet containing high iodine and low selenium. The results suggest that high iodine intake, when selenium is deficient, may permit thyroid tissue damage as a result of low thyroidal GSH-Px activity during thyroid stimulation. A moderately low selenium intake normalized circulating T4 concentration in the presence of iodine deficiency.

Effect of dietary protein on hepatic and extrahepatic phase I and phase II drug metabolizing enzymes.

Weanling male rats were fed low (LP, 7.5%), standard (SP, 15%) or high protein (HP, 45%) diet for 7 or 14 days ad libitum, and cytochrome c reductase (CYC) and UDP-glucuronosyltransferase (UGT) enzyme activities were determined in intestine, kidney and liver microsomes. HP diet increased CYC activity in intestine and kidney, while LP diet had no effect. Hepatic CYC activity declined with decreasing level of dietary protein. Liver and intestine UGT activities were higher on an LP diet, while kidney enzyme activities were higher on an HP diet. UGT activity toward alpha-naphthol, a UGT1 isoform substrate, was modulated by dietary protein in all tissues, while UGT activity toward 4-hydroxybiphenyl, a substrate for a second UGT1 isoform, was affected only in the intestine. The duration of feeding affected CYC and UGT activities in the intestine. This observation may be explained by the dynamic nature of intestinal tissue. The observation of unique tissue and enzyme responses suggests that generalizations regarding metabolic response to diets based on hepatic studies or single enzymes, may be erroneous.

A method for the determination of type I iodothyronine deiodinase activity in liver and kidney using 125I-labelled reverse triiodothyronine as a substrate.

OBJECTIVES: A detailed method for the determination of iodothyronine deiodinase type 1 (DI-1) activity is described. The objective of the present method development was to consolidate the effective procedures of previous methods and produce an efficient assay that can be easily reproduced. DESIGN AND METHODS: This method uses a 5',-125I labelled rT3 as substrate and ion-exchange chromatography to separate released ionic iodine. Released 125I- collected in the eluate is counted, and the results used to calculate DI-1 activity. RESULTS: Results were found to be linear for tissue homogenates containing 3-11 mg protein.mL-1. Day-to-day coefficient of variation of liver homogenate was determined to be 13%. CONCLUSIONS: This method was found to be reliable, reproducible, and sample sizes as small as 10 microL could be readily assayed. The use of centrifuge filter units to contain the ion-exchange medium decreased handling of the material, and potential sources of error.

Walking program reduces elevated cholesterol in women postmenopause.

OBJECTIVE: To examine the effect of a moderate exercise regimen on total serum cholesterol, triglyceride, high density lipoprotein cholesterol (HDL-C), low density lipoprotein cholesterol (LDL-C), body composition and cardiovascular fitness (VO2 max) in mildly hyperlipidemic women, postmenopause. DESIGN: Randomized assignment to walking (n = 24) or control (n = 16) groups. SETTING: Community based intervention. PARTICIPANTS: Over 300 volunteers were screened to obtain the sample of 40 eligible women. Participants were mildly hyperlipidemic, postmenopause (mean age 62.0 +/- 5.7 years), sedentary, nonsmokers and not on hormone replacement therapy. Results are reported for the 25 subjects (15 walkers, 10 controls) who completed the study. INTERVENTION: Exercisers walked an average of 54.3 +/- 7.7 mins/day, 4.9 +/- 1.7 days/week, at an intensity of 54% maximum heart rate reserve, for six months. Participants were counselled not to change their diets. MAIN RESULTS: Total serum cholesterol, triglyceride, total serum cholesterol: HDL-C ratio, weight and fat mass decreased significantly in the walkers compared with the controls (P < 0.05), as did body mass index (P < 0.01). Walking resulted in a significant increase in VO2 max (P < 0.01). Changes in serum lipids were significantly related to changes in body fat, but not to change in aerobic fitness. There were no changes in dietary intake. CONCLUSIONS: A moderate intensity exercise program induces favourable alterations in total serum cholesterol and other atherogenic indices in hyperlipidemic women postmenopause, and these changes are related more to loss of body fat than to increased fitness level.

Effect of dietary T-2 toxin on biogenic monoamines in discrete areas of the rat brain.

Acute T-2 toxin treatments alter biogenic monoamine concentrations in the brain; however, these perturbations have not been well documented or demonstrated in feeding trials. In this study, the effect of dietary T-2 toxin on regional brain concentrations of biogenic monoamines and their metabolites was investigated in male rats fed a semi-synthetic diet containing 0, 2.5 or 10 ppm T-2 toxin for either 7 or 14 days. Reduction in feed consumption, feed efficiency and weight gain was observed in rats fed either 2.5 or 10 ppm T-2 toxin. This effect was transient in animals fed the 10 ppm T-2 toxin diet, with feed consumption, feed efficiency and weight gain improving significantly during wk 2. T-2 toxin affected brain biogenic monoamine concentrations. In the nucleus raphe magnus, serotonin, 5-hydroxy-3-indoleacetic acid and norepinephrine increased in a dose-dependent manner, and dopamine increased transiently. In the substantia nigra of rats fed 10 ppm T-2, epinephrine increased after 7 days and norepinephrine decreased after 14 days, when compared with controls. Dihydroxyphenylacetic acid concentrations in the paraventricular nucleus and medial forebrain bundle were lower in T-2 toxin-treated rats than in control animals. The observed effects of T-2 toxin on brain monoamines and the resulting neurochemical imbalance may account for the physiological manifestation of trichothecene intoxication.

The response of obese females to low impact exercise and diet counselling.

Obese women enrolled in a 24 week low impact exercise program were assigned to either an exercise or an exercise plus diet counselling treatment. Overweight volunteers were recruited to serve as the control group. No significant changes were observed in maximal oxygen uptake, serum lipids, body composition, self esteem or physical body image during the program. A significant negative correlation was found between changes in serum HDL-C and changes in percent body fat. Maximal oxygen uptake was significantly and positively related to self esteem and physical body image. Participants who received diet counselling were less likely to drop out of the program than other members of the exercise group, and were more likely to attend classes. Changes in dietary intake were similar in all exercising subjects, regardless of counselling. These results suggest that a short term program of low impact exercise may not be sufficient to produce significant adaptations in this population.

Effect of UDP-glucuronyltransferase induction on zearalenone metabolism.

Experiments were conducted to determine the UDP-glucuronyltransferase (UDP-GT) isoenzyme which catalyzes zearalenone (Z) conjugation, and the effect of increased enzyme activity on Z metabolism. In competitive enzyme assays, the activity of rat liver UDP-GT towards Z was inhibited by 1-naphthol (NA), a GT1 substrate, and 4-hydroxybiphenyl (HB), a GT2 substrate. When enzyme activity was induced with either 3-methylcholanthrene (MC), a GT1 inducer, or phenobarbital (PB), a GT2 inducer, increased UDP-GT activity towards Z, NA and HB was observed. UDP-GT induction by PB increased urinary excretion of conjugated alpha-zearalenol. These results indicate that UDP-GT isoenzymes have overlapping substrate specificities, and that Z detoxification may be enhanced by UDP-GT enzyme induction, resulting in increased urinary excretion of conjugated alpha-zearalenol.

Measurement of the relative binding affinity of zearalenone, alpha-zearalenol and beta-zearalenol for uterine and oviduct estrogen receptors in swine, rats and chickens: an indicator of estrogenic potencies.

1. The relative binding affinity of zearalenone, alpha-zearalenol, and beta-zearalenol for estrogen receptors was determined in the pig, rat and chicken. 2. Similar relative binding patterns were observed, with alpha-zearalenol exhibiting greater affinity than zearalenone and beta-zearalenol the least binding affinity in all species. 3. The relative binding affinity of alpha-zearalenol was greater in pig, than in rat and significantly greater than in chicken. 4. Interspecies differences in zearalenone sensitivity may be due to the binding affinity of alpha-zearalenol for estrogen receptors and differences in zearalenone metabolites formed.

Zearalenone metabolism and excretion in the rat: effect of different doses.

Young female rats were orally dosed with either 1 or 100 mg zearalenone kg-1 body weight; zearalenone and metabolites were measured in a 96-h collection of urine and feces by HPLC analysis. Dose had little effect on metabolites formed, or excretion route. In both treatment groups, about 55% of the oral dose was excreted in the feces, while the urine was also a major route of excretion accounting for 15-20% of the administered dose. Zearalenone and metabolites were excreted mainly in the free form, with the production of alpha-zearalenol, the most potent estrogenic metabolite, being greater than 10% of the zearalenone dose.

Effect of the trichothecene deoxynivalenol on brain biogenic monoamines concentrations in rats and chickens.

Male Sprague-Dawley rats (180 g) and 28-day-old Single Comb White Leghorn Cockerels (300 g) were orally dosed with deoxynivalenol (DON) at 2.5 mg kg-1 body weight. In the first experiment, whole brains were collected at 2, 6, 12, 24 and 48 hours after the toxin treatment and analyzed for brain biogenic monoamines by high-performance liquid chromatography with electrochemical detection. Although several interesting trends were observed, DON did not influence whole brain concentrations of monoamine neurotransmitters or their metabolites in either species, at any time. In a second experiment, brains were collected 24 hours postdosing, dissected into 5 brain regions (pons and medulla oblongata, cerebellum, hypothalamus, hippocampus and cerebral cortex), and analyzed. DON treatment resulted in significantly elevated concentrations of serotonin (HT) and 5-hydroxyindole-3-acetic acid (HIAA) in all brain regions of the rat. However, this was not seen in poultry, where DON treatment resulted in a decrease in norepinephrine (NE) in the hypothalamus and hippocampus, and a decrease in dopamine (DA) in the pons and medulla oblongata region. These results suggest that DON influences brain biogenic amine metabolism, and that there may be intraspecies differences in the central effects of this mycotoxin.

Effect of T-2 toxin on brain biogenic monoamines in rats and chickens.

Two experiments were conducted to determine the effect of T-2 toxin on brain biogenic monoamines and their metabolites. Male rats (180 g) and cockerels (28 day, 300 g) were orally dosed with T-2 toxin at 2.5 mg kg-1 body weight. In the first experiment, whole brains were collected 2, 6, 12, 24 and 48 h postdosing and analyzed for monoamines by high performance liquid chromatography with electro-chemical detection. T-2 toxin did not influence whole brain concentrations of monoamines in either species. In the second experiment, brains were collected 24 h postdosing, dissected into five brain regions, and analyzed for monoamines. T-2 toxin treatment resulted in increased serotonin and 5-hydroxy-3-indoleacetic acid in all brain regions of the rat. However, this was not seen in poultry where T-2 toxin treatment resulted in an increase in 5-hydroxy-3-indoleacetic acid, no alteration in serotonin concentration and a decrease in regional norepinephrine and dopamine concentrations. These results suggest that T-2 toxin influences brain biogenic amine metabolism and that there is an intraspecies difference in the central effects of this mycotoxin.

Comparison of the trichothecenes deoxynivalenol and T-2 toxin for their effects on brain biogenic monoamines in the rat.

Male Sprague-Dawley rats (180 g) were orally dosed with deoxynivalenol (DON) and T-2 toxin at 2.5 mg kg-1 body weight. Brains were collected 24 h postdosing, dissected into five brain regions and analyzed for biogenic monoamines by high performance liquid chromatography with electrochemical detection. DON and T-2 toxin treatment resulted in significantly elevated concentrations of the indoleamines, serotonin (HT) and 5-hydroxy-3-indoleacetic acid (HIAA) in all brain regions examined, whereas norepinephrine (NE) and dopamine (DA) regional concentrations were not significantly altered. These results indicate that DON and T-2 toxin influence brain biogenic monamine metabolism, and suggest that the central nervous system (CNS) actions of these trichothecenes are similar.

Rapid, sensitive liquid chromatographic method for determination of zearalenone and alpha- and beta-zearalenol in wheat.

A rapid, sensitive liquid chromatographic (LC) method is described for quantitative determination of zearalenone and alpha- and beta-zearalenol in wheat. The procedure incorporates an internal standard, zearalenone oxime, to facilitate quantitation and automated analysis. A sample, buffered with pH 7.8 phosphate, is extracted with water-ethanol-chloroform (2 + 50 + 75) and cleaned up. The final residue is dissolved in LC mobile phase and injected onto a reverse phase RP-18 column under the following conditions: water-methanol-acetonitrile (5 + 3 + 2) mobile phase; fluorescence (excitation wavelength 236 nm, 418 nm cut-off emission filter) and UV (254 nm, range 0.0025 AU) detectors. The limit of detectability (twice background) is 0.5 ng for zearalenone and alpha-zearalenol standards on the fluorescence detector and 4 ng for beta-zearalenol on the UV detector, which is equivalent to 20 micrograms zearalenone and 20 micrograms alpha-zearalenol/kg, and 160 micrograms beta-zearalenol/kg feed. Standard curves are linear over the range 0-35 ng zearalenone and alpha-zearalenol on the fluorescence detector and 0-50 ng beta-zearalenol on the UV detector. Recoveries of all compounds are 87.5-101% in the range 0.1-3.0 mg/kg (ppm).