Modulation of toxicity by diet and dietary macronutrient restriction

MUTATION RESEARCH DNAging ELSEVIER

Mutation Research 295 (1993) 151-164

Modulation of toxicity by diet and dietary macronutrient restriction Angelo Turturro *, Peter H. Duffy, Ronald Wilson Hart

/ Division of Biometry and Risk Assessment, HFT-020, National Center for ToxicologicalResearch, Food and Drug Administration, 3900 NCTR Road, Jefferson, AR 72079, USA (Accepted 1 October 1993)

Abstract

Restriction of diet and macronutrients has been reported to modulate the toxicity of numerous chemical agents. Of the various forms of restriction studied, using nutritionally adequate diets, food restriction (FR) appears to be most effective, but protein restriction (PR), fat restriction (FtR), carbohydrate restriction (CbR), and excess of dietary fiber (FE) also affect toxicity and the spontaneous diseases that def'me the background incidence in toxicity tests. The heterogeneity of the dietary macronutrients complicates simple analysis of their effects. Additionally, the interrelationships between these various components in the complex dietary mixture often make experiments difficult to interpret. Despite these complexities, a simple model is presented, which considers the effects of dietary manipulations on the individual steps in the interaction of organism and agent, and puts the varied effects that can occur within an organism into context. Ultimately, many of the effects of dietary modulation on these steps in toxicogenesis can be considered as changing agent exposure and the biologically available dose. The effects of macronutrient restriction are discussed in terms of effects on agent at the interface of organism and toxicant, agent disposition, agent metabolism, and repair of toxicant-induced damage at the level of the genome. After illustrating the influence of these nutritional effects on the chronic bioassay, using mouse liver tumors as an example, the significance of these effects for chronic and short-term testing is discussed. Additionally, methods to address the impact of nutritional factors on toxicity testing are suggested.

Key words: Food restriction; Protein restriction; Fat restriction; Induced toxicity; Fibre; Risk assessment

1. Introduction

Food restriction (FR) is known to significantly inhibit both the incidence rate of background

* Corresponding author. Elsevier Science Publishers B.V.

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disease (Turturro et al., 1993a,b) and the induced toxicity of agents such as aflatoxin B I (AFB1), b e n z o [ a ] p y r e n e (BP), 7,12-dimethylbenz[a]anthracene (DMBA), 3-methylcholanthrene, etc. (Hart et al., 1992). Macronutrient restriction has also been shown to alter induced chtemical toxicity. For example, protein r e s t r i c t e d diets (PR)

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(less than 75% of normal intake) reduced rat liver foci induction by AFB1 (Horio et al., 1991). However, a 50% PR resulted in increased renal adenocarcinomas (Clinton et al., 1992), colon pathology (Clinton et al., 1992) and tumors (Tatsuta et al., 1992) following exposure to azoxymethane. The extent and type of PR is at least partially dependent upon the source of the protein. For instance, the toxicity of methotrexate is high with casein, low with soybean concentrate, and intermediate with corn meal gluten as different protein sources (Funk and Baker, 1991). In addition, restriction of specific amino acids, such as tryptophan (Segall and Timeras, 1976) and methionine (Orentreich et al., 1993), has been reported to extend lifespan (slowing the onset of background disease incidence such as common tumors). Fat restriction (FtR) is also effective in altering both spontaneous tumor incidence and tumors induced by agents such as DMBA (Kritchevsky et al., 1984). Similar to proteins, different fat sources appear to have different effects on the induction of damage by toxicant. For example, beef tallow > menhaden oil > corn oil in stimulating the mutagenic activity of 2-aminofluorene (Tsai and Pence, 1992). Corn oil as a fat is especially interesting because gavage with corn oil is known to reduce the incidence of leukemia in long-term tests while increasing the incidence of pancreatic acinar tumors in rats (Rao and Haseman, 1993). Thus a single fat can appear to have both positive and negative effects depending on what endpoint is being addressed. There is some evidence that significant (> 60%) carbohydrate restriction (CbR) during pregnancy can be detrimental to the fetus (Koski et al., 1993), but interpretation is extremely complicated since higher fat levels were used to keep the diets isoenergetic. Again, similar to the other macronutrients, the source of carbohydrate may be important. For instance, fructose appears to be more supportive of implanted tumor growth than glucose (Yam et al., 1991) and more effective in the induction of nephrocalcinogenesis (Bergstra et al., 1993). For dietary fiber, the situation is reversed as there is evidence that it is the high or 'unrestricted' fiber levels (fiber excess or FE) which have a generally positive impact on spontaneous

disease and toxicity. There is epidemiological evidence that FE is protective against mammary (Van't Veer et al., 1990), stomach (Risch et al., 1985), and colorectal (Giovannucci et al., 1992) neoplasms. FE also inhibited the promotion of N-nitrosomethylurea (NMU)-induced tumors in the gut (Cohen et al., 1991). Often defined as what is 'indigestible', fiber is derived from plant cell wall complexes and is extremely heterogeneous. This impacts on evaluation because, similar to fat and protein, fiber source is important to an evaluation of its effect. For instance, when NMU is given parenterally, pectin exerted a protective effect on the induction of tumors but alfalfa and bran did not (Watanabe et al., 1979). With intrarectal instillation, alfalfa enhanced carcinogenesis, with no effect observed with either pectin or bran (Watanabe et al., 1979). Also, spent barley grain fiber was more effective than cellulose and wheat bran in inhibiting the induction of intestinal tumors by 1,2-dimethylhydrazine (DMH) (McIntosh et al., 1993). The interrelationships between the various macronutrients complicate traditional analysis of the effects of restriction of any one of them. For instance: (1) altering dietary fat levels for FtR significantly affects caloric consumption (Ip, 1991); (2) altering fiber levels affects calorie consumption since fiber fermentation in the cecum leads to a caloric contribution from fiber that may be as much as 80% of the potential energy in the fiber (Eastwood, 1988); and (3) experimenters often use FR, which results in a concomitant caloric, fat and protein restriction (Turturro and Hart, 1992), etc. To help understand the total effect of nutritional modulation, given these interrelationships and the heterogeneity of the dietary components themselves, it is useful to have a model which focuses on the effects of these complex macronutrient alterations on the different stages in the induction of toxicity. Integrating the impact of these manipulations on the individual steps of induction will provide some estimation of the composite effect of nutritional modulations on induced toxicity, allowing better assessment of the risk associated with the compound, especially under the practical conditions of human exposure.

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2. Model

A model suggesting how caloric consumption can alter induced cancer occurrence has recently been presented (Turturro et al., 1993b). In this model, the level of caloric intake is proposed to affect cancer initiation, promotion and progression. Although nutritional modulation, especially FR, is usually thought of as affecting promotion and progression in carcinogenesis, there also appears to be a significant effect of FR on initiation (Chou et al., 1993), and these effects are the focus of this discussion. A modified detail from this model is shown in Fig. 1. The organism, represented by the large box, is exposed to an agent. The folded structure between organism and agent represents the interface between the organism and the environment. This interface contains a number of factors such as (for exposure through the gut) gastric acid in the stomach, intestinal coatings in the large intestine, etc. The solid arrows in the 'organism' suggest agent disposition and metabolism with special emphasis on

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caltn~"v :i~] /.,dl~~ver~/~~ L~,.I"Lo =-~ i,' ~k ....DNA&Repa[ ... r Fig. 1. Model of action of diet on toxicity. The large box represents the organism. The zig-zag represents the agentorganism interface (e.g., gut microfiora, gut contents, skin secretions, etc.). The interior arrows represent disposition and metabolism. Diet (dashed arrows) can be seen to act at many points, with the dashed arrows emphasizing the effect of DNA repair. Also, the effect on the target and its environment are separated to emphasize that these components may be affected differently. Modified from Turturro et al., 1993.

the liver, the target organ (when it is not the liver), and the other organs in the body capable of agent metabolism. The target organ is composed of target cells within a larger matrix of cells in the target organ 'environment' (this approach is similar to one used in Hart and Turturro, 1992). The target organ and its environment are influenced differently by agent and agent metabolites generated in the liver and the other organs. The dotted arrows represent the influence of a major parameter in modulation of genetic damage, DNA repair, while the dashed arrows point out the more general effects of diet modulation. The model utilized here (Fig. 1) is most useful when evaluating the modulation of toxicity by nutritionally adequate diets, i.e., diets that maintain animals in good health for their lifespan. For instance, a 40% FR (i.e., 40% less food than ad libitum) with vitamin supplementation, as conducted in our laboratory, results in no changes in clinical chemistry or pathology that indicate any nutritional deficiencies in rat or mouse (Turturro and Hart, 1992; Witt et al., 1991), and animals with this treatment are, in fact, healthier than the norm. As a counterexample, many of the experiments studying PR involve diets with protein levels so low (e.g., 2 or 3% of diet) that they are inadequate for maintenance of the animals being evaluated and result in early disease or death. Diets deficient in essential nutrients result in deficiency diseases which impact an organism's health, metabolism, homeostasis, etc. In addition, there appears to be a need for essential amino acids and essential fatty acids for tumor induction (Ip, 1991), suggesting that the ability to measure induced toxicity can be compromised by poor nutrition. Therefore, although there is an extensive literature available on the effects of diets which are deficient in one or another essential nutrient, disease induction and the problems in interpreting the induction of toxicity make extrapolation of these observations to animals fed normal balanced diets problematic. As a consequence of this, the term restriction will here be reserved for studies which involve good nutrition. One aspect is consistency with present animal welfare nutritional guidelines to avoid overt deficiency syndromes, with appropri-

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ate nutritional status monitoring. On a practical level, some points are useful to consider. Rodents tolerate high total food (or calories) and high fat well, so vastly different 'control' levels can be used (some FR studies used as their controls animals which had the same body weights as the 50% restricted animals in other studies) (Turturro and Hart, 1993), resulting in very different nutritional states for animals restricted to the same levels in different experiments. For FR, the requirements are different at each age, but we have found a practical limit of 8 c a l / d a y (female) and 10 c a l / d a y (male) for mice and 32 c a l / d a y (female) and 40 c a l / d a y (male) for F-344 rats. Similarly, with normal rodent diets ranging from 5% (cereal diet) to 20% fat, a limit of 3% fat in the diet seems to be the lower limit for FtR, if the fat is not deficient in essential fatty acids. Normal levels of protein in rodent diets vary between 20 and 30%. So, 8 - 1 0 % of the diet as protein seems to be a practical lower limit for PR. It is questionable whether this level (or lower) of PR will support growth in rats. FE much above 10% usually results in gastric problems in rodents, and this is probably a practical upper limit. Studies in our laboratories and others have shown that different species have different requirements, which are important to consider in evaluating diet adequacy. Besides monitoring nutritional state, an indicator that is useful in signaling nutritional problems is any dramatic change in food efficiency (i.e., difference in body weight per gram food ingested) induced by the diet. Thus, if animals are eating more food than controls, yet weigh less (as occurs with some low protein diets) this is indicative that nutritional problems are arising. Vitamin and mineral balance are also important to consider, especially since changing macronutrients can alter mineral metabolism (see below).

3. Effects of diet

3.1. Interface First quantitated in detail in mate F-344 rats (Table 1) (Duffy et al., 1989) and extended to

Table 1 Behavioral effects of FR in male F-344 rats Parameter Water consumption (per g body weight) Food episodes (per day) Average food consumed/episode (g) Water/food consumption (ml)

AL 0.037 11.2_+0.6

Rest. 0.056 3.2+0.5

1.5 -+0.09

3.9 -+ 0.27

0.99

1.34

AL is ad libitum, Rest. is a 40% food restriction. Adapted from Duffy et al., 1989.

female F-344 rats and to B6C3F1 mice of both sexes, there is an effect of FR on food consumption patterns. Animals on unrestricted diets consume small portions of food approximately 11 times a night, while FR animals eat three larger portions. This change in consumption pattern appears to entrain corresponding changes in systems as diverse as certain cytochrome P450s (Manjgaladze et al., 1993) and oncogene expression (Nakamura et al., 1990). Thus, when a chemical agent is given in food, it may exhibit a different exposure pattern under conditions of F R than when food is provided ad libitum. Changes in consumption patterns, and consequent exposure patterns also occur when agent exposure results in aversion to diet (Funk and Baker, 1991). Accompanying the changes seen in feeding pattern is an approximate doubling in water consumption (on a per gram body weight basis) when animals undergo FR (Duffy et al., 1989). Such an increase in water consumption will alter the consumption of water-borne toxicants, as well as nutrient uptake in the gut. FR also changes the barrier function of the skin (Lehman and Franz, 1993). Fig. 2 shows that the permeability of skin to hydrocortisone increases dramatically with FR at 44 weeks of age, a difference which decreases as the animal ages. This effect suggests that FR can result in a higher exposure to lipophilic toxicants in contact with the skin. These data also suggest that a lipophilic drug, such as hydrocortisone, applied to the skin, should penetrate into the body more effectively with FR.

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The effects of PR and FtR on these parameters have not been well characterized. There is some information that high carbohydrate meals are associated with a sedative action in rats and people (Young, 1991), as well as satiety in humans (Klein and Wolfe, 1992), suggesting that CbR would stimulate physical activity similar to what is seen with FR. PR (Weindruch and Walford, 1988), and FtR diets may significantly lower food consumption and, thus, result in the same effects as seen with FR. In addition, there is an effect of protein composition on interface parameters. For instance, use of soy protein results in a 25% quicker transit time through the rat gut when compared to casein (Hara et al., 1992), decreasing both the amount of time the gut is exposed to agents in the food as well as the opportunity for gut microfloral metabolism. An interesting interface interaction with fat appears to be the interaction of corn oil gavage and the gut. Corn oil is often used as a gavage vehicle for lipophilic compounds in toxicity studies. Rao and Haseman (1993) have reported that control (no agent) male F-344 rats exposed to corn oil gavage over the course of a 2-year chronic bioassay, when compared to similar control animals used in feeding studies, have a leukemia

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Age (Mons.) Fig. 3. Leukemia and diet in F-344 males. Leukemia incidence of male F-344 rats serially killed at various ages (12 per time point except at advanced ages) fed an NIH-31 (NAM) 5% fat diet and a diet containing 10% corn oil (CAM) ad libitum (with approximately equal total daily caloric consumption). Note that the two diets have very similar effects when fed ad libitum. CRM is the 10% corn oil diet fed at 40% FR, showing an inhibition of the male-specific tumor incidence to female levels (not shown). Techniques of pathological analysis identical to those reported in Witt et al., 1991.

incidence by the end of the bioassay lowered to that seen in female rats. Evidence that this reduction in male-specific leukemia incidence is not simply a result of a dietary exposure to corn oil itself is shown in Fig. 3. Purina Meal 5770-M-1 (CAM diet) (Turturro et al., 1993) contains corn oil at levels approximately equivalent to the amount of dietary fat and corn oil given to animals using corn oil gavage (i.e., 10% of calories as corn oil) (Rao et al., 1993). NIH-31 diet (NAM) has a fat level similar to that used in the NTP feed studies (5-6% of total diet). In these studies, total caloric consumption is about equal. It can be seen that the dietary corn oil has little effect on the onset of leukemia. It appears that some interaction of corn oil gavage and gut at the interface is involved. It is interesting in this regard that the effects of a 40% FR on the 10% corn oil diet (CRM), which results in a demasculinization of steroid metabolism in males (Manjgaladze et al., 1993), reduces the male incidence of leukemia to approximately the level seen in females. Sex steroid metabolism is very important in the expression of the sexual phenotype. A component of sex steroid disposition involves enterohepatic circulation. The estrogens

156 are conjugated in the liver to glucuronides or sulfates and excreted in the bile. Between 30 and 70% (depending on which estrogen is involved) of the plasma estrogens make their way through the intestinal tract, where they are deconjugated by intestinal bacteria and re-absorbed (Adlercreutz et al., 1979). This circulation may be altered by corn oil gavage on gut microflora, affecting either transport or uptake into the gut. The effect of fiber on the intestinal interface has, by comparison with the other macronutrients, been well characterized. Fiber has been suggested to have an effect as a result of the rheological and colligative properties of the water-soluble components, the surface properties of the water-insoluble components, and the network properties of its swollen hydrated component, including its viscosity, cation exchange, organic acid adsorption and gel filtration (Eastwood, 1992). These characteristics are expressed biologically as effects on: (1) modulation of absorption in the foregut, in general prolonging gastric emptying time and retarding nutrient absorption; (2) modification of sterol metabolism, through either altered lipid absorption, reduced bile acid absorption in the small intestine, altered bile absorption in the cecum, or through fatty acids generated by fermentation; (3) modification of cecal fermentation, which may contribute 1-2 kCal/g (approximately half the calories in protein or carbohydrate), and which is very dependent on the composition of the complex mix in the gut; (4) modification of stool weight, and composition, which can significantly alter the pharmacokinetics of materials excreted in the feces. An example of the effect of dietary fiber on uptake is the response of 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) (a potent carcinogen in meat and fish which is either fried or broiled) to different fibers. Sorghum and wheat bran fiber induce changes in the excretion pattern of MeIQx that suggest that the fibers induce a quicker gastrointestinal transit time and lower intake, than with diets either without fiber or with a soluble fiber, such as pectin (Sj6din et al., 1992).

An example of the effect on dietary fiber composition on sterol metabolism was shown by Reddy et al. (1992). Wheat bran decreased the activity of bile acids, such as fecal deoxycholic acid, the neutral sterols and a number of bacterial enzymes; oat bran reduced only a few bacterial enzyme activities, such as azoreductase; and corn bran increased some bacterial activities, such as that of 7a-dehydroxylase, and decreased others, such as nitroreductase. Also, overt changes in gut flora populations can occur with some fiber manipulations (e.g., DeBethizy et al., 1983). These specific actions of fiber at the gut interface will affect compounds activated and de-activated by factors such as bacterial enzymatic activities. The effects of these changes in enzyme activities with fiber are not limited to changing the metabolism of administered agents. Fiber type alters the lipase-catalyzed hydrolysis of tributyrin, a model fatty acid. Fiber from wheat bran, oat bran and sugarbeet inhibited this enzyme activity, while others, such as pectin and carrageenan, did not (Hendrick et al., 1992). Lipase action is an important step in fat uptake. These data suggest that fiber type can thus directly inhibit fat absorption, limiting the calories available to the animal from fat. Through adsorptive properties, fibers can also interact directly with chemical agents. For example, dietary nitrites can be a source of carcinogenic nitrosamine in the gut (Archer, 1984), and wheat bran may act as a scavenger for the nitrites, reducing their effect (Moiler et al., 1988). However, the total effect is the product of the complex mixture of materials in food. For example, insoluble fibers can adsorb hydrophobic agents, such as 1,8-dinitropyrene (DNP), rendering them unavailable for uptake, while soluble fibers in the diet (with differing capability) can maintain DNP in solution, thereby antagonizing the effect of the insoluble fibers (Harris et al., 1993). The ultimate effect of dietary alterations on the interface is to change the agent exposure by the animal through changes in the exposure pattern, changes in the physiology of the animal, modification of the gut interface and removal of the agent through direct interaction with dietary

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components. Therefore, in toxicity tests, to accurately quantitate the agent exposure, the exposure level has to be modified to account for the changes at the interface, especially if some form of FR is induced. Quantitatively comparing in vitro tests to in vivo dose response, in attempts to find good biomarkers, can be confounded when diet-induced modulation affects exposure at the interface.

Table 2 Blood lipids and diet in F-344 rats

3.2. Pharmacokinetics

AL is ad libitum, Rest. is restricted. M is male, F is female, number is 12 per group. Chol. is mean :i: SD of cholesterol (mg/dl). Trig. is mean _+SD triglycerides (mg/dl). Ages are in mouths. Animals are same rats as used for Witt et al., 1991.

In evaluating the effects of diet on agent pharmacokinetics it is useful, despite their interaction, to consider the effects of food consumption on agent disposition and metabolism separately. Disposition. As shown in Fig. 4, FR alters body composition. This figure shows the relationship between a graded FR and fat loss. In female Sprague-Dawley rats, there is a disproportionate loss of body fat with restriction. Using the techniques detailed in Dully et al. (1989) to measure body lipid in the male F-344 rat, a 40% FR results in a loss of approximately 25% of the lean body mass, but over 80% of the body fat. The enzymes of fat metabolism are altered by FR (Feuers et al., 1989) with a significant change in the blood lipids (Table 2). It can be seen that total cholesterol levels do not clearly correspond

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Sex

Diet

Age

Chol.

Trig.

M

AL

M

Rest.

F

AL

F

Rest.

12 24 12 24 12 24 12 24

131 151 142 108 170 167 106 113

284 187 108 90 144 174 69 65

to the intensity of FR. This lack of concordance may result from the use of a single time point during the day to assess the effects of FR, which is associated with complex diurnal variations (Duffy et al., 1989). Despite this single sample methodology, lowered triglyceride (TRIG) level has been consistent found with FR across a number of genotypes of rats and mice. This suggests that organismic fat metabolism is significantly modified by FR. PR, FtR and CbR appear to affect these same metabolic processes (Herzberg, 1991). The changes in TRIG also affect cells. In hepatocytes isolated from animals fed high fat, normal or FR diets, it was found that the amount of cellular fat microdroplets was directly correlated with the cellular TRIG levels, which were directly related to the blood TRIG level. Increased levels of cellular fat microdroplets resuited in increased sequestering of exogenously administered BP, which decreased BP metabolism (Zaleski et al., 1991). Thus, added to the changes in the organism's agent disposition that result from dietary modulation, there can be changes in cellular 'disposition' (or microdisposition). These changes can impact agent metabolism, and can be seen to be relevant to cells isolated for use in in vitro tests, e.g., as isolated hepatocytes are often used. Changes in other macronutrients will also change body composition. High fat diets are associated with obesity (Herman and Polivy, 1984)

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(i.e., a high body fat component), while FE diets are associated with low body fat (Heaton et al., 1983). Thus, diet appears to alter the body composition and, consequently, the disposition of xenobiotics. The separation of the effects of obesity and FR was first accomplished by Harrison et al. (1984). They studied the effects of CR on genetically obese animals and found that, although obese animals that were restricted still had significantly higher than normal body fat, FR improved their lifespan as much as it did animals with normal body fat levels. The components of fat can also change agent disposition. For instance, when fish oil is fed to rabbits, the half-life of perfused propafenone, a cardiac drug, in isolated heart is doubled compared to its half-life when lard or safflower oil is used (Gillis et al., 1992). Again, the nutritional provenance of a tissue significantly altered its in vitro response. Other effects on disposition include effects on renal clearance (excretion in Fig. 1). As noted above, with FR there is an increase in water intake, which will alter clearance by increasing urinary output. Increasing protein and fiber content in diet will often also increase water consumption, with a similar change in urinary output (Rao et al., 1993). In addition, FR inhibits the onset of renal nephropathy. For example, the effect of a 40% FR, as a function of age, on the incidence and severity of nephropathy for F-344 males is shown in Table 3. Since renal pathology influences clearance, diet can influence agent disposition through affecting the kidney. PR has Table 3 Nephropathy and diet in F-344 male rats Age

12 18 24 30

AL

Rest.

Inc.

Sev.

Inc.

Sev.

58 100 100 I00

1.0 1.9 2.6 3.4

8 75 83 100

1.0 1.0 1.1 1.7

A L is ad libitum, Rest. is restricted. Ages are in months. Incidences (Inc.) are in percent, severity (Sev.) is m e a n of 12 animals on a scale of 1 (least) to 4 (most severe). Adapted from Witt et al., 1991.

some of the same effects, with a 15% protein diet compared to 23% protein diet significantly inhibiting the onset of renal disease at approximately 25 months of age (Rao et al., 1993). The mechanisms of this effect are unclear, but may be related to the generation of lower levels of toxic protein waste products, such as ammonia, resulting in reduction of damage to the kidney. In addition, the source of the protein is important as soy seems to be less pathogenic than casein or lactalbumin to the kidney in long-term studies (Shimokawa et al., 1993). The mechanism of this effect is unknown. However, dietary soy protein changes the excretion of magnesium (Mg) that occurs when using casein as a protein source (Brink et al., 1992). Fructose is also more damaging to the kidney than glucose, and the effect seems to result from altered calcium, Mg and phosphate transport and retention (Bergstra et al., 1993), suggesting the importance of the role of mineral metabolism in many of the nutritional effects seen in vivo. Mineral metabolism is also altered with the type of dietary fiber used because of their different physical properties, such as the ability to perform cation exchange. The changes in metal ion transport are especially relevant to those changes seen in the toxicity of heavy metals. For instance, a 50% PR results in an accelerated accumulation of methylmercury in the kidney and a depressed excretion of the ion (Adachi et al., 1992), probably through increased resorption of the metal. Urinary ion transport appears to be especially sensitive to nutritional modulation because of the complex process of water and electrolyte resorption occurring in the nephron. As noted above, fecal composition, transit time and stool size are modulated by various dietary components, but especially fiber type and FE. Since both urinary and fecal excretion can be altered by diet, it is reasonable to assume that elimination of toxic agents will be altered. This is especially important for agents involving enterohepatic circulation, such as noted above for the estrogens. Enterohepatic circulation is often important in the disposition of larger (> 300 M r) and more lipophilic molecules. This circulation can be altered by FE through the physical prop-

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erties mentioned above, as well as by the fiber's constituents. Metabolism. Changes in macronutrients can significantly alter agent metabolism. Much of the older literature concerning the effect of macronutrients on the cytochrome P450 system has been reviewed recently (Anderson and Kappas, 1991). Much of this work was done before the characterization of the isoforms important to the action of this enzyme super-family was known. In addition, much of the older work used diets that were nutritionally deficient. As noted above, a 40% FR will reverse the sex-specific alterations seen in the livers of male F-344 rats, i.e., to demasculinize males, consistent with a significant decrease in the testosterone/ estradiol ratio seen in these animals (Manjgaladze et al., 1993). In other experiments, FR has been shown to result in loss of cyclicity in female mice (Nelson et al., 1985), decreases in fertility in male mice (Chapin et al., 1993) and fertility problems in Sprague-Dawley (S-D) rats (Merry and Holehan, 1991). One consequence of these hormonal changes is the alteration of the sex-specific isoforms important to steroid metabolism in the liver that is important in agent metabolism. For instance, CYP2Cll, which is a male-specific isoform, decreases in activity with FR (Manjgaladze et al., 1993). Similar alterations were also seen in the isoforms of the conjugating enzymes, such as glutathione S-transferase (GST), with consequent changes in phase II metabolism. For AFB1 activation, there is a decrease in the male-specific CYP2Cll, as noted above, after 40% FR with consequent decreases in AFB1 activation. On the other hand, there is an increase in CYP1A1 activity, important to the metabolism of the polyaromatic hydrocarbons and, consequently, BP activation (Chou et al., 1993), similar to that seen by Jagadeesan and Krishnaswamy (1989) in Wistar rats. However, 35% FR in young S-D rats had little effect on aryl hydrocarbon hydroxylase (AHH) activity (Zaleski et al., 1991). There is little information on the alterations of agent metabolism by restriction of other macronutrients using nutritionally adequate diets, without the complications that occur by altering

caloric consumption. PR of 50% (20% of calories to 10%) in a host mediated assay leads to approximately a 15% lower activation of DMH (Kari et al., 1983). Microsomes from a 67% PR diet showed a 44% decrease in the ability to N-demethylate aminopyrine, a 24% decrease in BP hydroxylation, and no change in aniline hydroxylation (Sonawane and Yaffe, 1983) while UDPglucuronyltransferase activity was elevated 123%. Protein deficiency rather than simple nutritionally adequate restriction, can increase oxidative damage, which will alter isoform activity (Huang and Fwu, 1992). FtR studies are often complicated by extensive use of fat-free diets, which are missing essential fatty acids, as a control. Fat source may also be important. Tallow was twice as effective as corn oil in the ethanol induction of CYP2E1 in rats, whereas CYP2B1 induction was similar with the two fats (Takahashi et al., 1992). GST activity was lower with corn oil as a fat than fish oil or beef tallow, after exposure to DMH as a toxicant (Kuratko and Pence, 1992). Microsomes derived from animals eating corn oil were approximately 10-20% more effective in metabolizing aminopyrine than microsomes from animals eating lard (Rowe and Willis, 1976). Relevant to the extra-hepatic metabolism noted in Fig. 1, FR increased, and high fat decreased, the AHH activity in lung, while both treatments increased AHH activity in the kidney in a short-term experiment in S-D rats (Kwei et al., 1991). Lowered AHH activity indicates lower local activation of agent, although the level of activity was two orders of magnitude less than in the liver (which showed no significant changes with dietary manipulation under these conditions). Both treatments stimulated the UDPglucuronosyltransferase activity of liver, suggesting better conjugation of the compound. The effect of restriction on metabolism is not a simple one. Although there is a .good deal of work done on the effect of fat and fat components on metabolism, with some work on protein components, many of the studies are confounded by the factors mentioned above. Few efforts have been directed at understanding the effects of PR and FtR. CbR and FE also remain relatively uncharacterized. FR appears to work through

160

hormonal mechanisms, resulting in changes that are specific for various isoforms of the phase I and phase II enzymes (Manjgaladze et al., 1993). When PR, FtR, and FE are characterized, it will not be surprising if some (or most) of their effects have similar mechanisms as FR. Although often FR reduces the toxic expression of compounds, it can be seen that it occasionally results in increased activation of agents (e.g., BP; Chou et al., 1993). By characterizing the various changes in the isoforms seen with restriction, especially for the human isoforms, the effect of restriction on agent metabolism can be evaluated. 4. Effects on the genome

In addition to the equivalent changes in agent exposure that occur with nutritional modulation in the organism, there is also an effect at the level of macromolecular adduction. A 40% FR on the activating isoforms of the agent AFB1, as well as the stimulation of the appropriate phase II isoforms, result in less unscheduled DNA synthesis (UDS) as a consequence of exposure to the agent (Chou et al., 1993). In addition, there have been a number of studies which show an increase in excision DNA repair, using genotoxic agents, with FR (Fig. 1) (Haley-Zitlin and Richardson, 1993). This change in excision DNA repair can be considered as equivalent to decreasing the effective biological dose resulting from exposure to the compound. The effect of FR on repair may also be important for the life extension seen with FR (Hart and Setlow, 1972; Hart and Turturro, 1981; Turturro and Hart, 1984, 1991a), impacting on the incidence of spontaneous disease considered as background in a test. PR, FtR and FE will also affect background tumor incidences, and, related to that observation, there have been some efforts to show effects on oxidative levels of DNA damage (Djuric and Kritchevsky, 1993; Youngman, 1993), but effects on DNA repair have not been addressed. 5. Relevance to toxicity tests

It is clear that macronutrient changes, and the behavioral, physiological, and hormonal changes

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B W 1 2 (g) Fig. 5. Relationship of liver tumors at 24 months on test to body weight at 12 months on test for male B6C3F1 mice. BW at 12 months on test (13.5 months of age) and liver tumor incidence at 24 months on test (25.5 months of age) for the last 16 National Toxicology Program (NTP) bioassays. Correlation coefficient (r = 0.82) accounts for a significant percent of variability in liver tumor incidence. Data are derived from the latest NTP Technical Reports (Carcinogenicity Bioassays) Series, i.e., TR434, TR419, TR412, TR410, TR407, TR406, TR403, TR401, TR396, TR392, TR391, TR387, TR385, TR 366, TR365, and TR363.

in the organism which accompany them, cause significant alterations in agent absorption, disposition and metabolism. These changes are equivalent to modulation of agent exposure or biologically available dose. Although, given the cumulative nature of dietary effects, this modulation is most likely to be significant for long-term tests, acute toxicity can also be significantly modulated. For instance, there is a 22-fold decrease in the acute toxicity of Ganciclovir induced by a 40% FR (Berg et al., 1993). These complex modulations occur when nutrition is altered intentionally (as when a FR or PR diet is chosen) or unintentionally. An example of the latter results from the uncontrolled nature of ad libitum feeding used in chronic tests. Fig. 5 illustrates the relationship of liver tumors in B6C3F1 mice and body weight at 12 months on test, using the controls (no agent), for the last 16 reported NTP chronic bioassays. Because food consumption is often not measured in these tests, body weight is used as a marker since the diets are the same across the tests. The body weight at 12 months on test (approximately 13.5 months of age) is used because younger

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B W l 2 (g) Fig. 6. Relationship of liver tumors at 24 m o n t h s on test to body weight at 12 m o n t h s on test for female B6C3F1 mice. BW at 12 m o n t h s on test (13.5 m o n t h s of age) and liver tumor incidence at 24 m o n t h s on test (25.5 m o n t h s of age) for the last 16 National Toxicology Program (NTP) bioassays. Correlation coefficient (r = 0.78) accounts for a significant percent of variability in liver tumor incidence. Data from same studies as in Fig. 5.

animals have not integrated the effects of diet as long as the 12 month animals, and older animals' weights may be complicated by the effects of pathology. Note the variability, suggesting that there are large differences between experiments in food consumption. Given the high correlation of body weight and tumor incidence, it can be seen, for males (Fig. 5) and females (Fig. 6), that this analysis can account for much of the variability in the historical control liver tumor incidence in the bioassay. Males seem to have a greater propensity for these tumors (and generally weigh more). Interestingly, a practical threshold for observable liver tumor incidence seems to exist at approximately 30 g body weight at 12 months on test for females. It is also interesting that some of the chronic bioassays have animals with body weights so different from the average that either significant FR or overeating may have occurred, complicating the interpretation of test results. Besides this variability in food consumption occurring in each experiment, exposure to toxic agents in toxicity tests often result in weight alterations at some dose levels. In fact, a significant decrement in body weight gain in young growing

animals is often used to define the mafimum dose that the animal can tolerate (Interagency Staff Group, 1986). Agent exposure can affect food consumption. For example, there can be agent effects on palatability, which could result in FR (or overeating). In addition, chronic exposure to toxicants can induce nutritional deficiencies. For instance, exposure to prednisone or chlordiazepoxide will induce vitamin C deficiencies, and folate deficiencies can be induced by a number of different drugs, because metabolism of the drugs raises the requirements for folate (Parke, 1978). These dietary deficiencies often manifest themselves in weight loss. Thus, careful analysis of chronic toxicity studies involves consideration of the effects of dietary consumption on agent exposure, as well as the effects of agent-induced nutritional deficiencies. These considerations may be especially relevant to the often high (compared to human exposure) doses used in these studies.

6. Conclusion

Nutritional modulation using balanced diets, such as FR, can affect physiological, cellular, biochemical and molecular processes, and, consequently, all steps in the induction of toxicity. The focus above, on agent uptake, metabolism, disposition, etc., is fundamental in defining the doseresponse relationship for a toxicant, and thus, is relevant to the evaluation of almost every form of toxic insult. However, this focus should not obscure those studies which have shown the impact of dietary modulation on other steps in toxicogenesis. For instance, in carcinogenesis, FR has been shown to affect endocrine homeostasis, promotion, oncogene expression, progression and the immune response (Hart et al., 1992; Turturro and Hart, 1991b, 1993). For any toxic endpoint, the impacts of nutritional modulation on every step in the induction of the adverse effect, both facilitative and inhibitory, need to be considered when trying to estimate the impact of these dietary manipulations on toxicity. From the clinical standpoint, CbR has been used to treat diabetes for some time. PR has

162 r e c e n t l y b e e n shown to l o w e r p l a s m a a m i n o acid levels t h e r e b y a i d i n g in t h e d e l i v e r y o f c e r t a i n c a n c e r c h e m o t h e r a p e u t i c d r u g s to t h e i r t a r g e t s ( G r o o t h u i s et al., 1992). Effects o f a l t e r e d diets on d r u g r e s p o n s e have b e e n t h e o b j e c t o f clinical investigation ( M e l a n d e r et al., 1988). E.g., d r u g c o n j u g a t i o n a n d m e a n r e n a l c l e a r a n c e will b e significantly d e c r e a s e d w h e n a high c a r b o h y d r a t e low p r o t e i n d i e t is u s e d i n s t e a d o f a high p r o t e i n low c a r b o h y d r a t e o n e ( P a n t u c k et al., 1991). T h e high c a r b o h y d r a t e d i e t is actually a P R - F t R diet, while t h e high p r o t e i n d i e t is actually a C b R - F t R one. E v a l u a t i n g t h e l o n g - t e r m effects o f f o o d m o d i f i c a t i o n in m o d e l systems, especially for c o m p o u n d s w h i c h i n t e r a c t with m e t a b o l i c a n d p h a r m a c o k i n e t i c p a r a m e t e r s a f f e c t e d by d i e t m o d i f i c a t i o n , will b e very useful in a d d r e s s i n g t h e utility o f t h e s e practices. I n a d d i t i o n to t h e s e uses, it is c l e a r t h a t t h e i n t e r p r e t a t i o n o f o u r p r e s e n t in vivo tests, such as t h e c h r o n i c bioassay, as well as t h e i n t e r p r e t a t i o n o f in vitro tests using a n i m a l - d e r i v e d cells, dep e n d s on u n d e r s t a n d i n g t h e effects o f t h e nutritional m o d u l a t i o n in t h e animal. A s a result o f t h e s t u d i e s d i s c u s s e d above, a n d other, r e l a t e d , studies, it is r e a s o n a b l e to a s s u m e t h a t m u c h of w h a t has b e e n c h a r a c t e r i z e d as ' b i o l o g i c a l variability' is a result o f t h e n u t r i t i o n a l d i f f e r e n c e s b e t w e e n i n d i v i d u a l s a n d g r o u p s in t h e s e assays. Understanding the mechanisms and impact of t h e s e i n d u c e d c h a n g e s is i m p o r t a n t to b e t t e r apply t h e results o f in vitro to t h o s e o f in vivo tests, a n d to b e t t e r e x t r a p o l a t e to m a n t h e results o f toxicity tests in animals.

Acknowledgement W e wish to a c k n o w l e d g e t h e s u p p o r t p r o v i d e d by t h e c o l l a b o r a t i v e N I A / N C T R P r o j e c t on C a l o r i c R e s t r i c t i o n for this work.

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