In vitro binding of the food mutagen 2-amino-3-methylimidazo[4,5-f]quinoline to dietary fibers.

The ability of three kinds of fiber to bind to 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), a potent bacterial mutagen formed during the cooking of meat, was investigated to determine interactions among important food components. At pH 6.5 corn bran, wheat bran, and alfalfa meal each bound approximately 50% of the available IQ after incubation for 1 hour. Binding was pH-dependent, occurred optimally between pH 4 and 6, and was probably due to a cation-exchange mechanism. The pH at which IQ is efficiently bound to fiber overlaps the range of pH in the human gastrointestinal tract.

Clastogenic activity of bile acids and organic acid fractions of human feces.

Chloroform extracts of fecal material from 4 subjects on normal mixed western diets were fractionated to obtain an acid fraction and a hexane extract containing neutrals and bases. The acid fraction from at least 2 of the donors induced an elevated frequency of chromosomal aberrations and exchanges in Chinese hamster ovary (CHO) cells. Since acid steroids are expected to be present in the acid fraction, 5 bile acids were assayed for clastogenic activity in CHO cells. Ursodeoxycholic acid induced chromosomal aberrations and exchanges, and this effect was enhanced by the addition of a microsomal S9 mix. However, the enhancement is probably due to physical factors rather than to enzymatic activity.

Formation of mutagens in cooked foods. VI. Modulation of mutagen formation by iron and ethylenediaminetetracetic acid (EDTA) in fried beef.

Mutagen formation during frying of beef is inhibited by the heavy metal chelator ethylenediaminetetracetic acid (EDTA). The addition of 1% EDTA prior to cooking reduces the mutagenicity of the basic extracts to about 60% of control values. The addition of iron as ferrous chloride or ferric chloride at 10 ppm (approximately 50% of endogenous concentrations) doubles the mutagenic activity of beef extracts. Iron, which can be released by denaturation of heme protein, therefore, can modulate the formation of mutagens in beef during cooking.

Testing the environment for dispersed mutagens: use of plant bioconcentrators coupled with microbial mutagen assays.

Mutagens dispersed in ecosystems are usually in low concentration and episodic in occurrence. The possibility of detecting such dispersed mutagens by utilizing indigenous bioconcentrator organisms coupled with a microbial mutagen assay offer a useful screening protocol. There are numerous examples of plant and animal species which concentrate toxic substances from the environment. Body extracts of these bioconcentrators can be suitably fractioned and tested for mutagens with various microbial mutagen assays. The fractions may be tested with a broad range of microbial assays covering numerous genetic end points as well as both with and without mammalian microsomal activation. This kind of environmental screening has an advantage over physicochemical techniques, in that sampling techniques are simpler and a wider chemical spectrum can be screened. There are problems inherent with testing a complex biological extract, however. If a reversion assay is used, the metabolite necessary for growth may be present. Toxins may be introduced, either concentrated from the environment in the same way as the mutagen, or produced by the concentrator itself. Finally, the concentrator may also produce an endogenous mutagen which will give spuriously active extracts. Methods for minimizing some of these difficulties are discussed.

Genotoxicity, carcinogenicity, and mode of action of the fried food mutagen 2-amino-3-methylimidazo[4,5-f]quinoline (IQ).

Because mutagens typified by 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) observed in cooked foods are widely consumed, detailed studies of their biochemical and biological properties including carcinogenicity are most important. IQ induces unscheduled DNA synthesis in liver cells, which when taken together with its powerful mutagenicity in the Salmonella typhimurium test system, predicts carcinogenicity. In female Sprague-Dawley rats, IQ did exhibit potent carcinogenicity for the mammary gland, the ear duct, and to a lesser extent, pancreas and bladder. Data from Japanese laboratories indicate carcinogenicity also to the intestinal tract. Thus, one of the mutagens formed during cooking is a versatile carcinogen that because of extensive human intake requires urgent exploration for specific human cancer risk.

Bile acids, but not neutral sterols, are tumor promoters in the colon in man and in rodents.

Analysis of the etiologic factors and relevant mechanisms involved in carcinogenesis leads to a classification of agents involved in the carcinogenic process as genotoxic or epigenetic. Their mode of action is distinct, especially with regard to dose-response effects and reversibility. The genotoxic carcinogens for colon cancer are unknown, but mutagenic components found in fried beef and fish are under study. Epigenetic agents as promoting factors play a major role in the development of cancer of the colon. Specific nutritional elements associated with colon cancer risk are high fat diets, high cholesterol intake, and low fiber intake. The role of micronutrients as modulators and inhibitors needs to be explored. Through metabolic studies in diverse populations and in reliable animal models, it is now clear that dietary fat and cholesterol control the total flow of bile acids in lumen and a high-fat, high-cholesterol diet increases the total of bile acids in the gut. Bile acids but not neutral sterols have promoting effects and are related to colon cancer risk although bile acids by themselves do not act as complete carcinogens. The effect of dietary fiber such as cereal bran is to increase stool bulk which dilutes the concentration of bile acids. Reducing the concentration of bile acids either by lower dietary fat and cholesterol or by increasing dietary fiber may effectively lower the risk for colon cancer.

Biochemical mechanisms on species differences in gastric carcinogenesis.

The biochemical denitrosation of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) in tissues from four strains of rat, inbred Buffalo, Lewis, B-N, and the random-bred Sprague-Dawley, with different sensitivities to MNNG-induced gastric carcinomas was investigated as a possible explanation for the species/strain differences in MNNG-induced carcinogenesis. An analytical HPLC method was developed to assay denitrosation of MNNG to N-methyl-N'-nitroguanidine (MNG) by cytosolic, microsomal, mitochondrial, and nuclear cell fractions. All the activity was contained in the microsomal and cytosolic fractions, with the major portion occurring in the cytosol. The activity in both fractions was NADPH-dependent, but denitrosation was not reduced by inhibitors of the cytochrome P-450 system. Denitrosation of MNNG post-mitochondrial supernatant (S9) fractions from liver, glandular stomach mucosa, and duodenal mucosa of the four rat strains was determined. In all strains, denitrosation activities were highest in liver. Comparisons between the three strains most sensitive to MNNG-induced gastric carcinogenesis indicated no large differences for any tissue. However, Buffalo, the most resistant strain, did have a higher level of denitrosating activity in all three tissues, which is consistent with the hypothesis that higher levels of detoxifying enzymes may lead to a decreased incidence of tumors. On the other hand, denitrosation accounts for less than 3% of the MNNG that disappears during the incubation period so that the relevance of denitrosation as a mechanism in strain-specific sensitivity to MNNG-induced gastric carcinoma requires additional studies.

Interobserver variation in the diagnosis and grading of dyskaryosis in cervical smears: specialist cytopathologists compared with non-specialists.

AIMS: To compare the assessment of dyskaryosis in cervical smears made by specialist consultant cytopathologists and consultant general histopathologists. METHODS: One hundred and ten cervical smears were circulated to 10 observers from five district general hospital histopathology departments and five major departments of cytopathology. Their responses were analysed by five consultant general histopathologists and five consultant specialist cytopathologists. In 54 of the 110 cases, the histology of a corresponding cervical biopsy specimen was compared with the smear assessments. RESULTS: Specialist cytopathologists were more consistent than non-specialists when diagnosing and grading dyskaryosis. They chose the higher grades of dyskaryosis more frequently than the non-specialists. The cytopathologists recommended referral for colposcopy more frequently, but if they asked for a repeat smear, they wanted it done within three months more frequently than the histopathologists. The specialists were more frequently in agreement with the biopsy grade of intra-epithelial neoplasia than the non-specialists, whose smear diagnoses tended to underestimate the severity of the histopathological abnormality. CONCLUSIONS: This study has shown major differences between specialist and non-specialist cytopathologists in the diagnosis and grading of cervical smears and in the recommended management of patients with abnormal smears. These differences may result in uneven clinical management of women with smear abnormalities. It is therefore important to explore possible strategies for standardising the reporting of cervical smears, such as centralisation of screening services, accreditation in cytopathology for non-specialist consultants, and the value of participation in external quality assessment schemes.

Effect of reactive oxygen species on K+ contractures in the rat diaphragm.

Reactive oxygen species (ROS) are postulated to alter low-frequency contractility of the unfatigued and fatigued diaphragm. It has been proposed that ROS affect contractility through changes in membrane excitability and excitation-contraction coupling. If this hypothesis is true, then ROS should alter depolarization-dependent K+ contractures. Xanthine oxidase (0.01 U/ml) + hypoxanthine (1 mM) were used as a source of superoxide anion eliciting oxidative stress on diaphragm fiber bundles in vitro. Diaphragm fiber bundles from 4-mo-old Fischer 344 rats were extracted and immediately placed in Krebs solution bubbled with 95% O2-5% CO2. After 10 min of equilibration, a K+ contracture (Pre; 135 mM KCl) was induced. Fiber bundles were assigned to the following treatment groups: normal Krebs-Ringer (KR; Con) and the xanthine oxidase system (XO) in KR solution. After 15 min of treatment exposure, a second (Post) K+ contracture was elicited. Mean time-to-peak tension for contractures was significantly decreased in Post vs. Pre (16.0 +/- 0.7 vs. 19.8 +/- 1.0 s) with XO; no change was noted with Con. Furthermore, peak contracture tension was significantly higher (31.5%) in the XO group Post compared with Pre; again, no significant change was found with KR. The relaxation phase was also altered with XO but not with KR. Additional experiments were conducted with application of 1 mM hypoxanthine, with results similar to the Con group. We conclude that the application of ROS altered the dynamics of K+ contractures in the rat diaphragm, indicating changes in voltage-dependent excitation-contraction coupling.

Effect of oral creatine supplementation on power output and fatigue during bicycle ergometry.

Our purpose was to determine the effect of oral creatine supplementation on exercise performance during high-intensity short-duration bicycle sprinting. Power output was recorded for 12 healthy untrained males (age 24.08 +/- 0.53 yr, weight 81.22 +/- 1.32 kg) before and after 5 days of creatine (n = 6) or placebo (n = 6) supplementation. A double-blind research design was employed. Subjects performed maximal sprints against a constant load (111.8 N) for 15 s. Each one-half pedal revolution was magnetically counted, and subsequent measurements of peak power, time to peak power, total work, and the fatigue index were digitized and stored on disk. Mean values for peak power, time to peak power, total work, and fatigue index were 958.01 +/- 40.66 W, 4.09 +/- 0.82 s, 11.28 +/- 0.46 kJ, and 32.1 +/- 1.58% decline from peak power, respectively. No significant differences were observed within or between groups before or after supplementation for any of the mechanical parameters measured (P > 0.05). These findings suggest that oral creatine supplementation does not positively affect power output or fatigue during continuous high-intensity bicycle exercise in untrained men.

Interaction between clenbuterol and run training: effects on exercise performance and MLC isoform content.

The purpose of this study was to determine the separate and combined effects of clenbuterol (CB) administration and interval training on running performance and myosin light-chain (MLC) isoform expression in mouse skeletal muscle. Mice were randomly assigned to one of four treatment groups: 1) control (Con), 2) exercise (Ex), 3) drug (CB), or 4) exercise + drug (Ex + CB). CB and Ex + CB mice were given CB (1.6 mg/kg) orally 4 days/wk. Ex and Ex + CB mice were trained 4 days/wk on a motorized treadmill (3 sets of 3 min, 36-40 m/min, 10-17% grade, 30-s recovery). After 8 wk of treatment, exercise conditioning increased total work performed 58% in the Ex group during a run-to-exhaustion treadmill test, whereas CB decreased total work by 25% in the CB group; in combination with exercise training, CB treatment eliminated the Ex-induced increase in work. Polyacrylamide gel electrophoresis indicated that run training, CB treatment, or a combination did not (P > 0.01) promote changes in fast and slow MLC isoforms in the soleus, gastrocnemius, or tibialis anterior muscles. Although not different from each other after 8 wk, CB and Ex + CB treatments produced significantly greater values than Con and Ex for the following variables: muscle mass (17-46%), total protein (22-50%), and myofibrillar protein (19-53%). It was concluded that CB decreases exercise performance and that the combination of Ex and CB have antagonistic effects on running performance; the two treatments do not interact to diminish the anabolic effects of CB on skeletal muscle and do not alter MLC isoform profiles.

A constant-load ergometer for measuring peak power output and fatigue.

A constant-load cycle ergometer was constructed that allows maximal power output to be measured for each one-half pedal revolution during brief, high-intensity exercise. To determine frictional force, an electronic load cell was attached to the resistance strap and the ergometer frame. Dead weights were attached to the strap's free end. Flywheel velocity was recorded by means of a magnetic switch and two magnets placed on the pedal sprocket. Pedaling resulted in magnetically activated switch closures, which produced two electronic pulses per pedal revolution. Pulses and load cell output were recorded (512 Hz), digitized, and stored on disk via microcomputer. Power output was later computed for each pair of adjacent pulses, representing average power per one-half pedal revolution. Power curves generated for each subject were analyzed for peak power output (the highest one-half pedal revolution average), time to peak power, power fatigue rate and index, average power, and total work. Thirty-eight males performed two 15-s tests separated by 15 min (n = 16) or 48 h (n = 22). Peak power output ranged from 846.0 to 1,289.1 W. Intraclass correlation analysis revealed high test-retest reliability for all parameters recorded on the same or different days (R = 0.91-0.97). No significant differences (P greater than 0.05) were noted between parameter means of the first and second tests. These results indicate that the ergometer described provides a means for conveniently and reliably assessing short-term power output and fatigue.

2,[3]tert-butyl-4-hydroxyanisole does not reduce SCE induction by benzo(a) pyrene in bone marrow cells of C57BL/6 mice.

Recently, a number of publications have suggested that bone marrow cytogenetics may be used to detect anticarcinogenic or antimutagenic activity. In this work, 0.75% 2,[3]-tert-butyl-4-hydroxyanisole (BHA), fed in the diet for 2 weeks, was tested for its ability to reduce the frequency of benzo(a)pyrene (BP)-induced SCE in mouse bone marrow. C57BL/6 male mice, were injected i.p. with BP at 0, 33, 67, and 100 mg/kg body weight. The mean SCE/chromosome +/- s.e.m. for animals on control diet was 0 mg/kg, 0.108 +/- 0.005; 33 mg/kg, 0.225 +/- 0.011; 67 mg/kg, 0.289 +/- 0.012; 100 mg/kg, 0.311 +/- 0.013. The mean SCE/chromosome +/- s.e.m. for animals on the 0.75% BHA diet was 0 mg/kg, 0.105 +/- 0.006; 33 mg/kg, 0.224 +/- 0.009; 67 mg/kg, 0.262 +/- 0.013; 100 mg/kg, 0.326 +/- 0.012. There are no significant differences between animals on the control and BHA diets. Excretion of BP in urine over a 72 hr time period was significantly increased in animals on the BHA diet, at both low and high doses. Water-soluble metabolites accounted for all of this increase. It appears that bone marrow is not a good model for the gastrointestinal tract, and that short-term assays for anticarcinogens or antimutagens are more likely to be predictive if they are done in the target organs.

Effects of Ca(2+)-channel drugs on K(+)-induced respiration in skeletal muscles.

When skeletal muscles are exposed to elevations in extracellular K+, they experience a significant and long-lasting increase in O2 uptake. The basis for this response is unknown but may be related to an influx in extracellular Ca2+ ions during sarcolemmal depolarization. The purpose of this study was to determine if altering Ca2+ entry, either by removal of Ca2+ from the bathing fluid or by exposing muscles to selective Ca(2+)-channel agonists or antagonists, would affect K(+)-induced respiration. Isolated frog sartorii muscles were incubated in normal Ringer's solution (R) or a modified Ringer's containing 10 or 18 mM KCl. O2 uptake increased 83.7% in R+10 mM KCl and 502.2% in R+18 mM KCl. Incubation in Ca(2+)-free R+18 mM KCl containing Ni2+ in place of Ca2+ depressed the metabolic response to elevated K+. O2 uptake increased 234.5% in R+18 mM KCl containing Ni2+ and 80.6% in R+18 mM KCl containing Mg2+. Similarly, addition of the Ca(2+)-channel antagonists (gallopamil (D600) and diltiazem (DILT)) to R+18 mM KCl also depressed the respiratory response to elevated K+. O2 uptake increased 224.2% and 133.1% in R+18 mM KCL containing D600 and DILT, respectively. Conversely, addition of the Ca(2+)-channel agonists (Bay K 8644 (BAY) or palmitoyl carnitine (PC)) to R+10 mM KCl enhanced the metabolic response to elevated K+. O2 uptake increased 278% and 438.9% in R+10 mM KCl containing BAY and PC, respectively. These results indicate that the stimulatory effects of elevated extracellular K+ on skeletal muscle respiration are at least partially dependent on the availability of extracellular Ca2+ and its subsequent entry during membrane depolarization.

Effects of eccentric exercise on trunk extensor torque and lumbar paraspinal EMG.

PURPOSE: Little is known about the effects of eccentric contractions on the function of the lumbar paraspinal muscles. The purpose of this study was to determine the effects of a single bout of eccentric contractions using the trunk extensor muscles on torque and lumbar paraspinal electromyographic (EMG) parameters. METHODS: Twenty healthy men between the ages of 18 and 49 yr participated in the study. Subjects performed a single bout of 50 maximal voluntary concentric (N = 10) or eccentric (N = 10) trunk extension movements while surface EMG signals were recorded from the multifidus and iliocostalis lumborum muscles. A series of isometric contractions were performed both before the exercise protocol and at five additional time points over the following 7 d. RESULTS: During the exercise protocol, peak torque decreased 30% and 24% in the eccentric and concentric groups, respectively, whereas no change occurred in EMG root-mean-square (RMS). There were no group differences in peak torque generation at any of the postexercise protocol time points. Compared with the preexercise protocol values, multifidus EMG was elevated 27% immediately post and 15 min post in the eccentric group. Similarly, compared with the concentric group, multifidus EMG in the eccentric group was increased 34%, 40%, and 25% immediately post, 15 min post, and 1 d after the exercise protocol, respectively. CONCLUSION: Eccentric contractions using the trunk extensor muscles result in higher levels of multifidus EMG activity to produce a given level of torque. This reduction in neuromuscular efficiency persisted for one day with recovery to baseline levels by the third day. Contrary to studies using other muscle groups, no sustained alteration in muscle function was observed.

Fate of the food mutagen 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) in Sprague-Dawley rats. I. Mutagens in the urine.

2-Amino-3-methylimidazo[4,5-f]quinoline (IQ) is a potent bacterial mutagen formed during cooking of beef. IQ was administered intravenously to Sprague-Dawley rats at concentrations ranging from 7.5-50 mg/kg body weight. Urine was collected and analyzed for mutagenicity. Urinary mutagens were found which required activation by S9 mix, and reverted Ames test strains TA98 and TA100, but not TA1535 or TA1537. The amount of urinary mutagen(s) were related to IQ dose administered and were excreted within 48 h. Additional mutagenic activity was not released after incubation with beta-glucuronidase or aryl sulfatase. Analysis of urinary mutagens by HPLC indicates that the majority of mutagenic activity is due to unchanged IQ, but a small peak of mutagenic activity may correspond to N-acetyl or 3-N-demethylated metabolite. Since only 1% of the administered mutagenic activity is recovered in the urine, IQ may be readily detoxified in vivo.

Mutagenicity of fluorene derivatives: a proposed mechanism.

Several derivatives of fluorene, a tricyclic, organic molecule, have been found to induce both frameshift mutations and base-pair substitutions in Salmonella typhimurium strains developed by Ames. Comparisons of the mutagenic potency of these derivatives for several strains of Salmonella suggest the importance of a carbonyl group substituted at the carbon-9 position of mutagenic derivatives, with respect to mutagenic potency. In this study, we present a feasible mechanism for the interaction of mutagenic fluorene derivatives with deoxyribonucleic acid. This mechanism requires the interaction of the mutagenic molecule with carbon-8 of guanine and a second concurrent interaction with the C-4 amino group of an adjacent cytosine residue.

The relationship of Motor-unit activation to isokinetic muscular contraction at different contractile velocities.

This investigation was designed to examine 1) the relationship between motor-unit activation (as recorded by integrated EMG) and speed of contraction and 2) the relationship between mechanical work, power output, peak torque, average torque, and both velocity of movement and integrated electromyographic recordings in the elbow flexor muscles. A series of isokinetic contractions of the elbow flexor muscles was performed by six normal subjects over a range of contractile velocities. Integrated electromyographic discharge and mechanical torque were recorded simultaneously. The results of an analysis of variance, corrected for repeated measures, indicated that both torque and motor-unit electrical activity decreased as contractile velocity increased. The relationship between torque and integrated electromyographic activity was linear and highly significant (r = .95 and r = .93). Implications of a neural interpretation of the in vivo torqu-velocity relationship in muscle are discussed.

Temporal pattern of agonist-antagonist EMG activity during rapid limb movements in man.

Analysis of elbow-extension movements, executed at maximal velocity, show positive correlations of timing of agonist-antagonist EMG activity with both movement velocity and displacement. Results indirectly support the notion that the antagonist musculature provides a braking force to arrest rapid limb movements.