Implications of apoptosis for toxicity, carcinogenicity, and risk assessment: fumonisin B(1) as an example.

The rates of cell proliferation and cell loss in conjunction with the differentiation status of a tissue are among the many factors contributing to carcinogenesis. Nongenotoxic (non-DNA reactive) chemicals may affect this balance by increasing proliferation through direct mitogenesis or through a regenerative response following loss of cells through cytotoxic (oncotic) or apoptotic necrosis. In a recent NTP study in Fischer rats and B6C3F(1) mice, the mycotoxin fumonisin B(1) caused renal carcinomas in male rats and liver cancer in female mice. In an earlier study in male BD-IX rats, fumonisin B(1) caused hepatic toxicity and hepatocellular carcinomas. An early effect of fumonisin B(1) exposure in these target organs is apoptosis. However, there is also some evidence of oncotic necrosis following fumonisin B(1) administration, especially in the liver. Induction of apoptosis may be a consequence of ceramide synthase inhibition and disruption of sphingolipid metabolism by fumonisin B(1). Fumonisin B(1) is not genotoxic in bacterial mutagenesis screens or in the rat liver unscheduled DNA-synthesis assay. Fumonisin B(1) may be the first example of an apparently nongenotoxic (non-DNA reactive) agent producing tumors through a mode of action involving apoptotic necrosis, atrophy, and consequent regeneration.

Dietary vitamin A modulates the concentrations of RRR-alpha-tocopherol in plasma lipoproteins from calves fed milk replacer.

The practice of supplementing milk replacers fed to neonatal calves with high concentrations of vitamin A has raised concerns regarding the effect of excess vitamin A on the bioavailability of vitamin E. A 4 x 2 factorial experiment evaluated the effects of four dietary amounts of vitamin A [0, 1.78 [National Research Council (NRC)(6) requirement, control], 35.6 and 71.2 micromol daily as retinyl acetate] and two forms of vitamin E (RRR-alpha-tocopherol and RRR-alpha-tocopheryl acetate, 155 micromol daily) on plasma RRR-alpha-tocopherol and RRR-gamma-tocopherol and RRR-alpha-tocopherol associated with plasma lipoproteins (Lp) from milk replacer-fed Holstein calves from birth to 28 d of age. The VLDL, LDL, HDL and very high-density lipoprotein (VHDL) fractions were separated by ultracentrifugal flotation, and the amount of vitamin E associated with each fraction was determined by normal-phase HPLC. The amount and distribution of RRR-alpha-tocopherol in Lp fractions were unaffected by the form of dietary vitamin E. Plasma and Lp RRR-alpha-tocopherol concentrations increased with age (P < 0.0001) and were maximal at 28 d of age. Concentrations of RRR-alpha-tocopherol associated with Lp were 25% (P < 0.01) to 39% (P < 0.0001) lower in calves fed 35.6 and 71.2 micromol of vitamin A daily than in control calves at 28 d of age. The RRR-gamma-tocopherol concentrations were unaffected by dietary vitamin A (P >/= 0.05). In conclusion, dietary vitamin A modulated the amount and distribution of RRR-alpha-tocopherol in the circulation of milk replacer-fed neonatal calves. Because of the essential antioxidant role of vitamin E, the health-related consequences associated with the depression of the LP RRR-alpha-tocopherol concentrations in calves fed vitamin A at 35.6 and 71.2 micromol need to be investigated.

Probiotic spectra of lactic acid bacteria (LAB).

Lactic acid bacteria (LAB) and their probio-active cellular substances exert many beneficial effects in the gastrointestinal tract. LAB prevent adherence, establishment, and replication of several enteric mucosal pathogens through several antimicrobial mechanisms. LAB also release various enzymes into the intestinal lumen and exert potential synergistic effects on digestion and alleviate symptoms of intestinal malabsoption. Consumption of LAB fermented dairy products with LAB may elicit antitumor effects. These effects are attributed to the inhibition of mutagenic activity; decrease in several enzymes implicated in the generation of carcinogens, mutagens, or tumor-promoting agents; suppression of tumors; and the epidemiology correlating dietary regimes and cancer. Specific cellular components in LAB strains seem to induce strong adjuvant effects including modulation of cell-mediated immune responses, activation of reticuloendothelial system, augmentation of cytokine pathways and regulation of interleukins, and tumor necrosis factors. Oral administration of LAB is well tolerated and proven to be safe in 143 human clinical trials and no adverse effects were reported in any of the total 7,526 subjects studied during 1961-1998. In an effort to decrease the reliance on synthetic antimicrobials and control the emerging immunocompromised host population, the time has come to carefully explore the prophylactic and therapeutic applications of probiotic LAB.

beta-carotene: friend or foe?

This symposium focused on the research which documents benefit and toxicity in beta-carotene supplementation. Reflecting on past and current studies, the panel of experts discussed: (1) the potential harm of a high intake of beta-carotene on selected populations, (2) biochemical antioxidant/prooxidant mechanisms of beta-carotene at the cellular level, (3) potential benefits of other carotenoids and antioxidants, and (4) future directions for research in beta-carotene and other antioxidants.

Interrelationships of food, nutrition, diet and health: the National Association of State Universities and Land Grant Colleges White Paper.

Nutrition and food science have each enhanced the development of an abundant, nutritious, safe food supply. A healthy diet should contain all of the required nutrients and sufficient calories to balance energy expenditure and provide for growth and maintenance throughout the life cycle. Importantly, dietary factors are associated with 5 of the 10 leading causes of death, including coronary heart disease, certain types of cancer, stroke, noninsulin dependent diabetes mellitus and atherosclerosis. National health care expenditures for 1990 totaled $666 billion of which 30% are related to inappropriate diet. Identification of external factors that contribute to premature death would aid preventive efforts, improve the quality of life, and reduce health care costs. Even though genetic predisposition increases susceptible people's risk for many of these chronic diseases, these conditions may be diminished or prevented by improvements in the American diet. Each stage of the life cycle has specific nutrient needs. Throughout infancy, childhood and adolescence nutrients are required to meet the growth processes as well as cognitive function. During pregnancy nutrients are required for both mother and developing infant needs. Adult nutrition focuses on tissue maintenance, nutrient and energy needs, and disease prevention. As the population of elderly increase in number and greater age, nutritional needs must be met to minimize certain disease states and assure the quality of life. Nutrition associated health risks have been identified for coronary heart disease, cancer and diabetes mellitus. Recommendations for each includes a decrease in dietary fat, awareness of caloric intake and enhancement of nutrient density including an increase in fruit and vegetables. These recommendations also impact obesity and diminish the compounding of other disease states affected by excessive body weight. Calcium intake at early ages affects development of bone density and manifestation of osteoporosis. Current gaps in knowledge are also identified that could improve health. Numerous nutrients are being examined for their regulation of specific gene expressions and in the processes of transcription and translation. To offer food products with greater nutrient density or improved functional health ingredients, modification of existing foods is needed to assure an improved diet. Policies to improve health require integration of nutrition needs with economic growth and development, agriculture and food production, processing, marketing, health care and education, and includes changing life styles and food choices. Increased research support is required to achieve national health goals with emphasis on nutrition and food sciences. Education methods must be improved to better inform consumers, to encourage food producers and manufactures to produce healthier foods, to assure training of future professionals and to provide legislators with the basis to make informed decisions. Recommendations to CFERR are identified. Improved quality and availability of nutritious foods will result in a healthier, more productive population. A decrease in the occurrence and duration of chronic disease should diminish the cost of health care and allow these resources to further benefit the nation. International concerns about undernutrition include 780 million people who are malnourished, lacking sufficient food to meet their basic nutritional needs for protein and energy, and 2 billion people who subsist on diets lacking essential nutrients needed for growth, development and physiological maintenance. National concerns about undernutrition exist based on incomplete data identified by indices of hunger and characterized by an increased demand for food assistance for women, children and the elderly. Major health problems in the US impacted by diet and nutrition include coronary heart disease, atherosclerosis, some types of cancer, non-insulin dependent diabetes mellitus, hypert

Formation of beta-glucuronides and of beta-galacturonides of various retinoids catalyzed by induced and noninduced microsomal UDP-glucuronosyltransferases of rat liver.

The complex of microsomal UDP-glucuronosyl transferases (UGT; EC 2.4.1.17) of rat liver catalyzes the formation of retinoyl beta-glucuronide (RAG) from retinoic acid (RA) and retinyl beta-glucuronide (ROG) from retinol (ROL) in the presence of UDP-glucuronic acid (UDPGA). The relative rates of formation of beta-glucuronides in noninduced microsomes were: 9-cis RA > 13-cis RA > all-trans 4-oxo RA > TTNPB > all-trans RA > CD-367 > 13-cis ROL > 9-cis ROL > acitretin > all-trans ROL. Michaelis constants (KM) for all-trans RA, 13-cis ROL, TTNPB and UDPGA were 130 microM, 300 microM, 210 microM and 2.6 microM, respectively. Galacturonides of RA, but not detectably of ROL, were formed from UDP-galacturonic acid at 11-30% the rate of the beta-glucuronides, whereas UDP derivatives of nonionized sugars did not serve as substrates. Pretreatment of rats with 3-methylcholanthrene markedly increased RAG formation in the absence of detergent (Triton X-100), but less so in its presence. Clofibrate pretreatment doubled the rate of RAG formation, whereas phenobarbital pretreatment showed little effect. N-Ethylmaleimide (5 microM) minimized cis-trans isomerization without significantly affecting glucuronidation. Rates of glucuronidation of retinoids clearly depend both on their isomeric states and on their chemical structures. Different UGT enzymes might well act on different geometric isomers of RA.

The fear of healthy eating: understanding the paranoia.

Scientific research continues to point to the health benefits of including more fruits and vegetables in the diet. At the same time, some groups claim that pesticides used in their production pose significant health risks. This article reviews some of the position statements, describes the principles of risk and hazard assessment, and identifies regulatory processes to assure the safe use of pesticides.

Dietary magnesium depletion: p-nitroanisole metabolism and glucuronidation in rat hepatocytes and hepatic microsomal membranes.

Male Sprague-Dawley rats were pair fed either a control magnesium diet (480 mg/kg) or a magnesium-deficient diet (30 mg/kg) for 10 days to produce magnesium depletion. Serum magnesium concentration declined 60%, but tissue magnesium deficiency was not apparent. Hepatic parenchymal cells and microsomal membranes were isolated to evaluate in vitro metabolism of p-nitroanisole and conjugation of its metabolite, p-nitrophenol (pNP). O-Demethylation of p-nitroanisole was decreased slightly in both magnesium depletion hepatocytes and microsomal membranes. However, pNP glucuronide formation was specifically decreased 50% in the magnesium depletion cells at all p-nitroanisole concentrations. Similar results were found in the microsomal reaction system measuring pNP conjugation. Thus, early stages of magnesium depletion may result in a specific decrease in the pNP UDP-glucuronyl transferase activity.

The anomaly of pyridine nucleotide synergism in carbon tetrachloride metabolism.

Carbon tetrachloride metabolism was examined in hepatic microsomes isolated from control and phenobarbital-treated Sprague-Dawley rats to determine the mechanism of pyridine nucleotide synergism. An NADPH generator increased metabolism two fold as determined by lipid peroxidation. Addition of NADH to the reaction system did not alter the maximum velocity, but did decrease the Km for NADPH from 61 microM to 7.6 microM in control and from 21 microM to 6.3 mM PB microsomes. Addition of NAD+ produced an increase in metabolism similar to NADH. Substrates and competitive inhibitors of nucleotide pyrophosphatase also enhanced CCl4 metabolism. A high correlation (r = 0.947) was indicated between the percent inhibition of nucleotide pyrophosphatase and the percent synergism of NADPH-catalyzed CCl4 metabolism. Thus, pyridine nucleotide synergism in CCl4 metabolism appears to result from the increased availability of NADPH produced by a decreased degradation of the NADPH by the nucleotide pyrophosphatase.

Microsomal metabolism of carbon tetrachloride: participation of pyridine nucleotide synergism.

The role of pyridine nucleotide synergism in CCl4 metabolism was evaluated for its potential contribution to enhanced lipid peroxidation. Male Sprague-Dawley rats receiving either no treatment (control) or treatment with phenobarbital (PB) were used to prepare hepatic microsomes. Metabolism was evaluated in the presence and absence of an NADPH generator system and in the presence and absence of NADH. The generator system produced a greater extent of metabolism for both control and PB microsomes. NADH-catalyzed CCl4 metabolism occurred to a similar extent in control and PB microsomes, amounting to 9-10% and 5-6% of the NADPH rate in control and PB microsomes, respectively. Synergism by NADH occurred at the lowest concentrations of NADPH, apparently decreasing the Km for NADPH and having little effect on the Vmax. Addition of NAD+ produced synergism, as did the addition of 5' AMP, an inhibitor of nucleotide pyrophosphatase. Thus, the synergistic increase in CCl4 metabolism produced by NADH may occur in part from an increased availability of NADPH, as a result of decreased degradation, rather than by electron donation from NADH.

A rapid method of preparing hepatic parenchymal cells for studying drug metabolism.

Parenchymal cells were prepared from the livers of male Sprague-Dawley rats by collagenase perfusion and purified by a self-generating Percoll gradient. The method consisted of mixing 31% Percoll and 5 x 10(6) cells/ml, followed by centrifugation at 10,000 x g for 10 min. A self-generated gradient provided a rapid and efficient recovery of highly viable parenchymal cells. The parenchymal cells were determined to be very stable during incubation at 37 degrees C for at least 2 h. Cell integrity was evaluated by trypan blue dye exclusion, lactate dehydrogenase leakage, and membrane peroxidation. In addition, drug metabolism and conjugation were evaluated as markers of intracellular integrity. With increasing p-nitroanisole (pNA) concentration, the formation of p-nitrophenol (pNP) increased. The rate of sulfation was maximal at a pNA concentration of 0.25 mM and decreased greatly above 1.0 mM. Glucuronidation increased from 0.25 mM to a maximum rate of 2.0 mM pNA. Above 1.0 mM pNA, nonconjugated pNP increased proportionately to the decrease in sulfation. These results indicate that the cell integrity was maintained, and that these cells can be used as a model for studying drug metabolism.

Nutritional requirements of the aged.

The age of the population continues to increase. At the turn of the century, 4% (about 3.1 million people) were 65 years of age or older. Today the percentage has increased to 12%, or 27.4 million people, and the prediction for the 21st century may increase to 17 to 20%. It should be noted that the number of individuals 85 years of age and older, as well as those over the age of 100, is increasing rapidly. The nutritional problems of the elderly are related to physiological decline, to low economic status and limited food consumption, and to a multitude of disease processes and the therapeutic regimens prescribed to cure or treat those illnesses. Examined as a group, the elderly at first appear to be less uniform in their health status than younger members of the population. However, only 10% of the elderly population contributes to the disproportionate (30%) expenditure of health care services. A majority of the elderly are normal, healthy individuals. Yet, with each decade of life the percentage of the elderly population needing additional care increases. The national nutritional surveys (National Food Consumption Survey and the National Health and Nutrition Examination Surveys, I and II) used entirely different methodologies, but reported very similar findings. The mean intake of this population was actually quite good, revealing low intakes of calcium and iron in the elderly female population. However, the standard deviation of the intake data strongly suggests a major portion of the population is at nutritional risk. For more than 30% of the population, nutrient intakes below two thirds of the recommended dietary allowances (RDA) occurred for calories, calcium, and vitamin A, while for more than 20% of the population, iron and vitamin C were at risk. Although the data evaluating the effects of age on the nutritional requirements of the elderly are limited, careful interpretation of the existing assessment and intervention efforts can provide some basic guidelines. Generalities about calorie intake must be avoided, and emphasis must be placed on calorie needs. Enhanced activity should be encouraged. Protein intake for the majority of elderly exceeds the RDA, but with increased age a greater amount of high quality protein is needed to maintain nitrogen balance. The group at greatest risk is the poor elderly who may not be able to afford enough high quality protein. Increased consumption of complex carbohydrates serves several functions including decreased caloric density and enhanced nutrient intake. Decreasing total dietary fat decreases caloric intake and indirectly decreases dietary cholesterol.(ABSTRACT TRUNCATED AT 400 WORDS)

Effect of aluminum on the hepatic mixed function oxidase and drug metabolism.

The effect of aluminum injection on the hepatic mixed function oxidase was examined in male Wistar rats. A cannula was surgically implanted in both the control and aluminum treated animals to provide a common port for aluminum injection. In addition, the control animals were pair-fed to the aluminum treated animals. The treated animals accumulated aluminum at about 0.1 mg/gm dry weight of liver/day. At 14 days, the cytochrome P-450 was decreased 20%, but the other components, cytochrome b5 and cytochrome reductases, were unchanged. By day 21 both cytochrome P-450 and cytochrome b5 were reduced 25%. Although NADPH cytochrome c reductase was not affected, the other flavoprotein, NADH cytochrome c reductase, was reduced. Drug metabolism, O-demethylation of p-nitroanisole and p-hydroxylation of aniline, was not affected at 14 days. However, at 21 days O-demethylation was not affected, but aniline hydroxylation was decreased, indicating an affect of aluminum on a specific isoenzyme of cytochrome P-450. Uniquely, the nonactivated glucuronyl transferase activity was fourfold greater in the aluminum treated animals. The increase was greater than cation activation and was similar to the detergent activated activity. Thus, aluminum infusion does produce specific alterations in microsomal function, including drug metabolism and conjugation.

Nutritional parameters that alter hepatic drug metabolism, conjugation, and toxicity.

A major concern of contemporary medicine is the adverse effects resulting from the use of prescribed and over-the-counter pharmacologic agents. In many cases more than one drug is taken at the same time, which increases the risk of overloading the detoxification mechanisms. If the individual has poor nutritional status, the system becomes even more inefficient. The liver contains the most important of these detoxification systems: the cytochrome P-450-dependent mixed function oxidase (MFO) and several conjugation enzymes, e.g., sulfotransferase, glucuronyl transferase, and glutathione transferase, which convert lipophilic compounds to more water-soluble products to enhance their excretion. The balance of these reactions determines the rate of metabolism and clearance of xenobiotic agents, and regulates in part the degree of intracellular damage. Nutritional factors, including proteins, carbohydrates, fats, vitamins, and minerals, affect the efficiency of these reactions. Changes in intracellular metabolism can alter not only the enzyme levels but also the availability of their cofactors, e.g., NADPH, UDPGA (uridine diphosphate glucuronic acid), PAPS (3'-phosphoadenosine-5'-phosphosulfate), and GSH. Diets restricted in calories, protein, or essential fatty acids, as well as those having low quality protein or high sugar content, can affect the component enzymes, cytochrome P-450 and the cytochrome P-450 reductase, and the MFO activity toward a variety of drugs. In addition, deficiencies of specific vitamins (riboflavin, ascorbic acid, and vitamins A and E) and minerals (iron, copper, zinc, and magnesium) affect the components and activities of the system in unique ways. Insight into the regulation of the hepatic detoxification mechanism can be gained by using nutrient variables to perturb the system.

The effect of nutritional factors on hepatic drug and toxicant metabolism.

A variety of foreign agents, including environmental chemicals, food additives, natural food constituents, and pharmaceuticals, enter the body every day. Many of these compounds must undergo metabolism by specific enzymes within the body to facilitate their clearance. The most important of the enzyme systems, which is found in the liver, includes the cytochrome P-450 dependent mixed function oxidase, as well as several conjugation enzymes, including sulfotransferase, glucuronyl transferase, and glutathione-S-transferase. The balance of the reactions determines the rate of metabolism and clearance of the agents, as well as the degree of intracellular damage or toxicity. Specific dietary factors, such as proteins, carbohydrates, vitamins, and minerals, affect the efficiency of these reactions.

Dietary concerns associated with the use of medications.

Medications can affect, and be affected by, nutrition. The interactions between drugs and various components in the diet are in part responsible for the erratic drug responses observed in patients. Because of the diversity of food consumption patterns among individuals and the widespread use of drugs, an array of varied and complicated side effects can occur. When vitamins, minerals, or other food components alter drug utilization or when drugs induce nutritional deficiencies, the effect poses a risk to the patient. Drugs can also affect many nutrition-related factors, such as appetite, taste acuity, and gastrointestinal function. Dietary habits or metabolic changes should be examined carefully and evaluated in any patient who unexpectedly gains or loses weight while taking drugs. Medicated patients need to be aware of good nutrition practices and to understand the importance of dietary modifications associated with certain diseases. A nutritious diet not only makes an important contribution to the health of those individuals but also reduces the risk of nutrition disorders or altered drug efficacy. Nutrition counseling should be considered for medicated patients to help them make rational decisions about their food choices.

Multiple drug metabolism: p-nitroanisole reversal of acetone enhanced aniline hydroxylation.

The unique effect of acetone on the p-hydroxylation of aniline was evaluated in microsomes prepared from control, phenobarbital- and 3-methylcholanthrene-pretreated animals. The existence of multiple forms of the hydroxylase was evaluated as an explanation of the acetone enhancement. Simultaneous metabolism of aniline in the presence of either p-nitroanisole (pNA) or ethylmorphine (EM) was evaluated to probe the participation of different mixed function oxidase systems. Aniline inhibited both N- and O-demethylation, while pNA and EM both inhibited p-hydroxylation of aniline. Acetone decreased the individual demethylation reactions, but enhanced aniline hydroxylation. In multiple drug reactions, acetone decreased N-demethylation and proportionately increased aniline p-hydroxylation. On the other hand, p-nitroanisole blocked the acetone enhancement of aniline metabolism. Kinetic evaluation of the acetone and p-nitroanisole effects on aniline metabolism indicated that each agent increased the apparent Km' by 4- to 5-fold for aniline in the hydroxylation reaction, but only acetone increased the Vmax'. From the Eadie-Scatchard analysis of the rates of aniline hydroxylation, acetone appeared to produce a biphasic increase in the hydroxylation above 0.75 mM aniline, even in the presence of pNA. Thus, multiple forms of the aniline p-hydroxylase are indicated by their altered activities in the presence of other drugs, and acetone seemed to specifically alter a species having a higher Km' for aniline.