Exposure of newborn rats to pharmacologically active compounds may permanently alter carcinogen metabolism.

Administration of phenobarbital to mother rats during early lactation causes long-term, perhaps permanent, alteration of hepatic microsomal mixed-function oxidase activity and aflatoxin B1 adduct formation in the adult male offspring. These findings suggest that perinatal exposure to pharmacologically active compounds may be a determinant of cancer risk.

Gossypol detoxication by fungi.

An unidentified Diplodia isolate representative of several fungi tested and grown for 13 days on gossypol-containing cottonseed reduced the amount of free gossypol by 90 percent and detoxified the cottonseed for weanling rats and chickens. Residual free gossypol was also less toxic than equivalent amounts of normal free gossypol.

Nutritional Renaissance and Public Health Policy.

The science of nutrition has long been entrapped in reductionist interpretation of details, a source of great confusion. However, if nutrition is defined as the integration of countless nutrient factors, metabolic reactions and outcomes, biologically orchestrated as in symphony, its relevance for personal and public health would be less confusing and more productive. This more wholistic interpretation may be observed at the cellular and physiological levels and may be described, in part, by the concept of pleiotropy (multiple cell-based effects from one nutrient source), together with its more expansive cousin, epitropy (multiple cell-based effects from multiple nutrients). There are many consequences. First, wholistic interpretation helps to explain the profound but little-known health benefits of whole plant-based foods (not vegan or vegetarian) when compared with whole animal-based foods and/or with the nutritionally variable convenience foods (generally high in fat, salt, refined carbohydrates and low in complex carbohydrates). Second, wholistic interpretation explains why the U.S. Dietary Guidelines and related public policies, which are primarily conceived from reductionist reasoning, serve political agendas so effectively. If diet and health advisories were to acknowledge the biological complexity of nutrition, then make greater use of deductive (top down) instead of inductive (bottom up) reasoning, there would be less confusion. Third, wholistic nutrition, if acknowledged, could greatly help to resolve the highly-polarized, virtually intractable political debate on health care. And fourth, this definition tells why nutrition is rarely if ever offered in medical school training, is not one of the 130 or so medical specialties, and does not have a dedicated research institute at U.S. National Institutes of Health. Nutrition is a wholistic science whereas medical practice is reductionist, a serious mismatch that causes biased judgement of nutrition. But this dichotomy would not exist if the medical practice profession were to understand and adopt wholistic interpretation. Reductionist research, however, is crucially important because its findings provide the granular structure for wholistic interpretation-these two philosophies are inescapably interdependent. Evidence obtained in this manner lends strong support to the suggestion that nutrition is more efficacious and far more affordable in maintaining and restoring (treating) health than all the pills and procedures combined. Admittedly, this is a challenging paradigm for the domain of medical science itself.

Relative contribution of dietary protein level and aflatoxin B1 dose in generation of presumptive preneoplastic foci in rat liver.

Male weanling F344 rats were orally gavaged with aflatoxin B1 (AFB1) in daily doses of 200, 235, 270, 300, and 350 micrograms/kg/day for a total of 10 doses over a 12-day period, and then 1 week after the last dose they were fed diets of varying protein (casein) content to compare the contribution of AFB1 dose and dietary protein level on the development of presumptive preneoplastic gamma-glutamyltransferase-positive (GGT+) foci in rat liver. All animals were fed the same 20% dietary casein level during the dosing period. One week after the end of the dosing period, one-half of the animals in each dose group were then continued on the 20% casein diet for the entire 12-week foci-development period; the remaining half in each dose group were fed lower levels of dietary casein during the foci-development period for the increasing AFB1 dose groups (20, 16, 12, 8, and 4% casein for the 235-, 250-, 270-, 300-, and 350-micrograms/kg/day groups, respectively). The AFB1 dose groups used were determined in a preliminary experiment. In this previous experiment, a clearly discernible threshold dose at about 100-150 micrograms AFB1/kg/day (below which no GGT+ foci were observed) and a steep slope between 150 and 400 micrograms/kg/day were produced. In the second experiment, while the expected positive slope of (AFB1) dose versus (GGT+ foci) response relationship was found for animals fed the 20% casein diet, the dose response for the animals fed the lower levels of casein was eliminated, providing evidence that nutrient intake during the postdosing foci development is more rate limiting toward the development of these preneoplastic lesions than is the carcinogen dose.

Dietary protein intervention during the postdosing phase of aflatoxin B1-induced hepatic preneoplastic lesion development.

The effects of high and low dietary protein intervention on the growth and development of hepatic preneoplastic lesions were examined. Inbred male F344 rats fed a semipurified (AIN-76) 20% casein diet were given doses orally of aflatoxin B1 (10 doses, each 250 micrograms/kg). One week after the last dose, animals were fed diets containing either 5 or 20% casein. Animals fed a 5% casein diet throughout the 12-week postdosing period had a marked reduction in development of gamma-glutamyltransferase-positive foci. Animals fed a 20% casein diet throughout the same period had the highest response. Groups fed the 5% casein diet for half of the postdosing period and 20% for the other half had intermediate responses. The data show that the high response observed in animals fed a high-protein diet can be inhibited by the postinitiation intervention of a low-protein diet. Likewise, the low response observed in the animals fed a low-protein diet was increased by the introduction of a high-protein diet late in the experiment.

Effect of dietary protein quality on development of aflatoxin B1-induced hepatic preneoplastic lesions.

The effect of the quality of dietary protein on the post-initiation development of aflatoxin B1-initiated putatively preneoplastic foci in Fischer 344 rat liver was compared with the effect of the quantity of dietary protein. Feeding wheat gluten, a low-quality protein, during the postinitiation period (between the end of aflatoxin B1 dosing and the death of the rats) inhibited the development of gamma-glutamyltransferase-positive foci when compared with that in animals fed high-quality protein (casein) diets during the same period. Lysine supplementation of wheat gluten during the postinitiation period enhanced the gamma-glutamyltransferase-positive response to a level comparable with that of the high-quality protein. These results suggest that one can inhibit the development of foci either by decreasing the quantity of protein intake and holding the quality of the protein constant or by decreasing the quality and holding the quantity constant.

Dietary intervention during the postdosing phase of L-azaserine-induced preneoplastic lesions.

The effects of intervention by diets with high or low levels of dietary fat on the development of preneoplastic pancreatic lesions were examined. Wistar rats were treated ip at 14 days of age with a 30-mg/kg dose of L-azaserine [CAS: 115-02-6; diazoacetate serine (ester)] and weaned onto the test diets. Animals fed 5% corn oil had fewer preneoplastic lesions compared to animals fed 20% corn oil throughout the 4-month posttreatment period. The strong response observed in rats fed 20% corn oil could be markedly reduced by intervention with a 5% corn oil diet halfway through the posttreatment period. Similarly, the low response in animals fed 5% corn oil could be markedly elevated by intervention with a high-fat diet. These results provide evidence for the hypothesis that tumor development may be modified by dietary means.

Effect of dietary intake of fish oil and fish protein on the development of L-azaserine-induced preneoplastic lesions in the rat pancreas.

The effect of dietary intake of fish (menhaden) oil and fish (cod) protein on the development of pancreatic preneoplastic lesions was examined in male Wistar rats. Fourteen-day-old animals were given a single ip injection of 30 mg L-azaserine/kg body weight [CAS: 115-02-6; diazoacetate serine (ester)]. At 21 days of age they were weaned and maintained on dietary treatment for 4 months. Fish protein did not appear to produce a significantly different preneoplastic response when compared to casein as a protein source. However, a 20% menhaden oil diet, rich in omega 3 fatty acids, produced a significant decrease in the development of both the size and number of preneoplastic lesions when compared to a 20% corn oil diet rich in omega 6 fatty acids. This study provides evidence that fish oils, rich in omega 3 fatty acids, may have potential as inhibitory agents in cancer development.

Effect of dietary omega-3 and omega-6 fatty acids on development of azaserine-induced preneoplastic lesions in rat pancreas.

We examined the effect of varying the ratio of dietary omega-3 (omega 3) to omega-6 (omega 6) on the development of pancreatic preneoplastic lesions in male Wistar rats given azaserine at 14 days of age. As the ratio of dietary omega 3 to omega 6 fatty acids increased in a diet totaling 20% by weight of fat, the development of preneoplastic atypical acinar cell nodules (AACNs) at 4 months after dosing with azaserine decreased significantly. In addition, serum levels of prostaglandin thromboxane B2, prostaglandin E2, and 6-keto-prostaglandin F1 alpha decreased significantly. The fatty acid composition of the rbc membrane was also significantly influenced by the ratio of dietary omega 3 to omega 6 fatty acids. In a second experiment, we examined the effect of dietary intervention with a different type of fat (corn oil or menhaden oil) 2 months into the 4-month postdosing period on AACN development at the end of the post-dosing period. Intervention of the omega 6 fatty acid-rich diet with the omega 3 fatty acid-rich diet significantly decreased focal development. The opposite was true when intervention involved substituting the omega 3 fatty acid-rich diet with the omega 6 fatty acid-rich diet.

Effect of high and low dietary protein on the dosing and postdosing periods of aflatoxin B1-induced hepatic preneoplastic lesion development in the rat.

Aflatoxin B1-induced liver lesion development is readily modified by dietary protein intake. Earlier work had shown that low-protein diets enhanced the acutely toxic lesion but depressed the carcinogenic lesion. This study examined the emergence of these lesions as a function of dietary protein intake, particularly with respect to whether the protein modification occurred during or after the aflatoxin B12 dosing period. High (20%) and low (5%) casein diets were fed to growing Fischer 344 rats during the dosing and postdosing periods of aflatoxin B2-induced hepatic preneoplastic lesion development. Focal areas of hepatocellular alteration were identified and quantitated by staining sections of liver for gamma-glutamyl transferase (GGT). Animals fed low casein diets during the dosing period displayed a characteristic spectrum of lesions including hepatomegaly, severe bile duct proliferation, cholangiofibrosis, and a tendency for developing large remodeling GGT-positive foci. These lesions were regarded as symptomatic of acute hepatoxicity. Animals fed high-protein diets during the dosing period had small, densely stained, GGT-positive foci, with only mild bile duct proliferation and no cholangiofibrosis, hepatomegaly, or large, remodeling GGT-positive foci. During the postdosing period, protein modulation markedly influenced the total number of foci. Animals fed high casein diets during this period exhibited an approximate 6-fold increase in the number of foci, regardless of the level of protein fed during the earlier dosing period. The marked increase in foci number (as well as area of liver occupied) in high casein diet animals during the postdosing period is regarded as an increased tendency for tumor development.

Effects of sex difference and dietary protein level on the binding of aflatoxin B1 to rat liver chromatin proteins in vivo.

The risk of aflatoxin-induced hepatocarcinoma is greater in males than in females and is similarly higher in animals fed 20% casein diets than in those fed 5%. In this study, groups of male and female F344 rats were fed either a 5 or 20% casein diet for 6 weeks. Two hr after a 1-mg/kg i.p. dose of [3H]aflatoxin B1, animals were killed and four protein fractions were sequentially extracted from the liver chromatin of each. Within each treatment group, aflatoxin binding to nonhistones was greater than to histones, both before and after dialysis. Comparing treatments, the higher-risk factors were associated with a greater liver content of aflatoxin as well as with increased binding of both nondialyzable and dialyzable aflatoxin to the various fractions. The high degree of correlation between total liver content and adduct formation implicates the former as a major determinant of the latter. Also associated with the higher-risk factors was a shift in the distribution of dialyzable aflatoxin toward greater adduct formation with one of the nonhistone fractions, suggesting the possibility of a role for noncovalent aflatoxin:protein adducts in hepatocarcinogenesis.

Linear dose-response curve for the hepatic macromolecular binding of aflatoxin B1 in rats at very low exposures.

The possibility of a low dose threshold for rat liver macromolecular binding of aflatoxin B1 was investigated. Doses in ng/kg of radiolabeled aflatoxin B1 produced measurable covalent binding of aflatoxin to DNA, RNA, and protein, and the extent of this binding increased linearly over a dose range of 10 to 1000 ng/kg. Macromolecular adduct formation was observed at the lowest dose used (10 ng/kg) which is within the human exposure range. Although diethyl maleate caused a reduction in hepatic glutathione from 5 to 2.3 mumol/g of liver and a slight increase in macromolecular adduct levels, the dose-response curve for macromolecular adduct formation remained linear in both diethyl maleate-pretreated and control groups. These results indicate that macromolecular binding of aflatoxin B1 is essentially a linear function of dose at low exposures and that hepatic glutathione offers little if any protection against this binding.

Dietary lipotropes, hepatic microsomal mixed-function oxidase activities, and in vivo covalent binding of aflatoxin B1 in rats.

Weanling male Sprague-Dawley rats were fed either a nutritionally complete synthetic diet (Diet 1) or a diet marginally deficient in choline and methionine, and lacking folacin (lipotrope deficient, Diet 2) to determine the role of hepatic mixed-function oxidase metabolism of aflatoxin B1 (AFB1) in the Diet 2-induced enhancement of AFB1 hepatocarcinogenesis previously reported. Hepatic microsomal mixed-function oxidase activities, as assayed by ethylmorphine N-demethylation, ethoxycoumarin O-dealkylation, cytochrome c reduction, AFB1 metabolism, and cytochrome P-450 content, were all depressed by Diet 2. Furthermore, the proportion of an i.p. dose of AFB (1 mg/kg) that became covalently bonded to DNA and RNA was similarly reduced when measured 6 hr after administration. The formation of AFB1-protein adducts was not influenced by dietary treatment. The depression of DNA and RNA adduct formation in the Diet 2 animals was probably related to the lower mixed-function oxidase activities and not to an alteration of glutathione levels, which remained unchanged by dietary treatment. These results suggest that the marginally lipotrope-deficient diet does not enhance tumor formation through an increased microsomal activation of AFB1. Alternative hypotheses without data are suggested.

Nonassociation of aflatoxin with primary liver cancer in a cross-sectional ecological survey in the People's Republic of China.

A comprehensive cross-sectional survey was undertaken in The People's Republic of China of possible risk factors for primary liver cancer (PLC) to include 48 survey sites, an approximately 600-fold aflatoxin exposure range, a 39-fold range of PLC mortality rates, a 28-fold range of hepatitis B virus surface antigen (HBsAg+) carrier prevalence, and estimation of exposures for a large number of other nutritional, dietary, and life-style features. PLC mortality was unrelated to aflatoxin intake (r = -0.17) but was positively correlated with HBsAg+ prevalence (P less than 0.001), plasma cholesterol (P less than 0.01), frequency of liquor consumption (P less than 0.01), and mean daily intake of cadmium from foods of plant origin (P less than 0.01). Multiple regression analyses for various combinations of risk factors showed that aflatoxin exposure consistently remained unassociated with PLC mortality regardless of variable adjustment. In contrast, associations of PLC mortality with HBsAg+, plasma cholesterol, and cadmium intake remained, regardless of model specification, while the association with liquor consumption was markedly attenuated (was nonsignificant) with adjustment for plasma cholesterol. The sharp contrast between the findings of no aflatoxin effect upon PLC prevalence in this survey and the positive correlation reported for previous but more restricted surveys is discussed. Based on the results of this survey and the data of laboratory animal and in vitro studies, an explanatory model for the etiology of PLC is proposed, taking into consideration the role of nutrition in the etiology of this disease.

Repression of hepatitis B virus (HBV) transgene and HBV-induced liver injury by low protein diet.

Persistent infection with hepatitis B virus (HBV) is one of the primary risk factors for human hepatocellular carcinoma (HCC). In a human ecological study, we have shown that, in addition to HBV, animal food consumption also significantly contributes to the variance of HCC. To test the interacting effect of HBV and animal food consumption on the development of HCC, we investigated HBV expression in HBV transgenic mice fed three levels of casein diet. HBV expression in transgenic animals was substantially inhibited when dietary casein was reduced from the traditional level of 22% to the level of 6%. Northern analysis revealed that suppression of HBV was derived from both the upstream albumin promoter and the internal HBV promoter. Immunochemical staining of liver sections indicated that only a few hepatocytes around the central vein expressed viral surface antigen (HBsAg) in the 6% casein animals, whereas virtually all hepatocytes stained positively for HBsAg in the 22% dietary casein animals. Serum HBsAg concentrations at 4 months were increased by 1.6-, 2.1-, and 5.1- fold over baseline for animals fed the 6%, 14%, and 22% casein diets, respectively. Correspondingly, liver injury was much less severe in animals fed 6% casein diet than in those fed 14% and 22% casein diets. These results demonstrate that a low casein diet is a potent suppresser of HBV transgene and HBV-induced liver injury, suggesting that diet management may be a practical means to aid in the control HBV infection.

Diet and chronic degenerative diseases: perspectives from China.

A comprehensive ecologic survey of dietary, life-style, and mortality characteristics of 65 counties in rural China showed that diets are substantially richer in foods of plant origin when compared with diets consumed in the more industrialized, Western societies. Mean intakes of animal protein (about one-tenth of the mean intake in the United States as energy percent), total fat (14.5% of energy), and dietary fiber (33.3 g/d) reflected a substantial preference for foods of plant origin. Mean plasma cholesterol concentration, at approximately 3.23-3.49 mmol/L, corresponds to this dietary life-style. The principal hypothesis under investigation in this paper is that chronic degenerative diseases are prevented by an aggregate effect of nutrients and nutrient-intake amounts that are commonly supplied by foods of plant origin. The breadth and consistency of evidence for this hypothesis was investigated with multiple intake-biomarker-disease associations, which were appropriately adjusted. There appears to be no threshold of plant-food enrichment or minimization of fat intake beyond which further disease prevention does not occur. These findings suggest that even small intakes of foods of animal origin are associated with significant increases in plasma cholesterol concentrations, which are associated, in turn, with significant increases in chronic degenerative disease mortality rates.

Association of dietary factors and selected plasma variables with sex hormone-binding globulin in rural Chinese women.

Sex hormone-binding globulin (SHBG) is an important regulator of plasma sex steroids as well as a sensitive indicator of insulin resistance. SHBG may be an important diagnostic measure of risk for pathologies associated with insulin resistance syndrome (IRS) such as non-insulin-dependent diabetes mellitus (NIDDM), obesity, hypertension, dyslipidemia, and atherosclerotic cardiovascular disease. In women, SHBG is also implicated in diverse pathologies such as cancers of steroid-sensitive tissues and hirsutism. Data from an ongoing ecological study linking diet and health in rural China were analyzed to determine the relation of selected plasma variables and diet to plasma concentrations of SHBG. All data represent county mean values, pooled by age and sex, to assess the relation between biochemical and lifestyle characteristics and disease-specific mortality rates at the county level. The study sample consisted of 3250 Chinese women between the ages of 35 and 64 y living in 65 widely dispersed rural counties. Consumption patterns for 21 different food groups were derived from a food-frequency questionnaire and a 3-d dietary survey and subsequently compared. Correlation analyses of county mean values demonstrated a significant association between SHBG and insulin, testosterone, triacylglycerols, body mass index, age at menarche, and several foods. In regression analyses, after adjustments, the strongest predictors of SHBG concentrations were the dietary intake of rice (beta = 0.42, P < 0.01), fish (beta = 0.34, P < 0.05), millet (beta = -0.27, P < 0.01), and wheat (beta = -0.34, P < 0.01). When insulin, testosterone, and triacylglycerols were added to the model only triacylglycerols (beta = -0.26, P < 0.05) remained a significant independent predictor of SHBG. Additional analyses suggested that the consumption of green vegetables was modestly positively correlated with SHBG and negatively with insulin values. Consumption of rice and fish in particular appeared to favorably influence the principle plasma variables associated with a reduction in the risk for IRS pathologies.

Dietary intakes and urinary excretion of calcium and acids: a cross-sectional study of women in China.

The relationship between dietary intakes and urinary calcium was examined in a cross-sectional survey of 764 middle-aged and elderly women with markedly different dietary patterns and lifestyles. Urinary calcium was correlated positively with urinary acids, including titratable acid (r = 0.46, P < 0.0001), ammonia (r = 0.42, P < 0.0001), and sulfate (r = 0.52, P < 0.0001). Urinary excretions of calcium and acids were correlated positively with intakes of animal and nondairy animal protein but were correlated negatively with plant-protein intake, possibly because of the alkaline nature of plant foods. Further analyses showed that urinary calcium and acids were associated positively with acid-forming foods and were associated negatively with plant foods. These results indicate that under free-living conditions urinary calcium excretion is likely determined by the acid-base status of the total diet, including among other factors the contribution of sulfur amino acids to urinary acid production.

Dietary calcium and bone density among middle-aged and elderly women in China.

Associations between dietary calcium and bone status were investigated in 843 Chinese women aged 35-75 y who were selected from five rural counties where dietary calcium varied considerably. Bone mineral content (BMC) and bone mineral density (BMD) at the radius were significantly higher in one of the pastoral counties with higher calcium intake than in the nonpastoral counties. Analysis by individual for all counties combined showed that BMC and BMD were correlated positively with total calcium (r = 0.27-0.38, P < 0.0001), dairy calcium (r = 0.34-0.40, P < 0.0001), and to a lesser extent with nondairy calcium (r = 0.06-0.12, P = 0.001-0.100), even after age and/or body weight were adjusted for. The results strongly indicated that dietary calcium, especially from dairy sources, increased bone mass in middle-aged and elderly women by facilitating optimal peak bone mass earlier in life.

Questioning riboflavin recommendations on the basis of a survey in China.

Riboflavin status was surveyed in adults aged 35-64 y in 65 counties (two communes per county) in the People's Republic of China by erythrocyte glutathione reductase activity coefficients (EGRACs), by urinary excretion of riboflavin 4 h after the oral administration of 5 mg riboflavin (one commune per county), and by direct measurement of food intake over a 3-d period (one commune per county). EGRAC data were highly correlated between sexes (p less than 0.001) and between neighboring communes within the same county (p less than 0.001) and with riboflavin intake (p less than 0.001). The urinary-riboflavin-load test data were not correlated either with EGRAC or with riboflavin intake. Approximately 90% of the survey subjects were found to be deficient by Western and Chinese reference standards and allowances. Upon reconsideration of the Western experimental data originally used to establish these recommendations, it is suggested that riboflavin allowances are set too high both in China and in Western countries.