The Role of Diet in Modulating Gut Detoxification Mechanisms

2004 Poultry Science Association, Inc.

The Role of Diet in Modulating Gut Detoxification Mechanisms D. M. Barnes1 Department of Animal Sciences, University of Wisconsin, Madison, Wisconsin 53706

SUMMARY Livestock ingest numerous potentially toxic compounds, including natural plant and fungal metabolites, insect chemical defenses, human-made environmental pollutants, and therapeutic drugs. In turn, animals evolved physiological mechanisms to protect themselves from these ingested toxins. Enzymes have been described that first activate toxins cytochrome P-450 and then conjugate the compounds (glutathione-S-transferase, GST) to detoxify, enhance elimination, or both. Two additional members of this defense have recently been described. P-glycoprotein (Pgp) and multidrug resistance associated protein (MRP) differ from the detoxifying enzymes in that they are membrane-bound efflux transport proteins that mediate a reduction in intracellular toxin accumulation. The relationship between phase I and II metabolism of dietary toxins and the efflux transport proteins has not been examined for any livestock species. In this report the role of dietary constituents in the regulation of detoxification pathways and the importance of this regulation to animal agriculture are discussed. Key words: phase I and phase II enzymes, P-glycoprotein, multidrug resistance protein, diet, regulation 2004 J. Appl. Poult. Res. 13:120–126

DESCRIPTION OF PROBLEM Animals that consume plant parts (fruits, seeds) ingest numerous biologically active compounds produced by the plant itself for the purpose of defense against damage from the environment, pathogens, herbivores (natural secondary compounds), or as a contaminant (products of microbial or fungal pathogens, pesticides, and herbicides). In turn, animals have evolved physiological mechanisms to protect themselves against these ingested toxins. At the level of the cell, numerous enzymes have been described that first activate a parent compound (phase I metabolism, e.g., cytochromes P-450) and then conjugate the com1

pound [phase II, e.g., glutathione-S-transferase (GST)] to detoxify it, enhance elimination or both. In some cases this system fails because phase I metabolism creates a metabolite that is more toxic than the original compound (e.g., aflatoxin B1). More recently, 2 additional members of this defense system have been identified. P-glycoprotein (Pgp) and multidrug resistance associated protein (MRP) differ from the detoxifying enzymes in that they are membranebound efflux transport proteins that effectively lower intracellular concentrations of toxins [1, 2, 3]. Regulation of these detoxification systems is complex; however, recent literature shows that dietary constituents have a significant role in the detoxification process. This re-

To whom correspondence should be addressed: [email protected].

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Primary Audience: Research Nutritionists, Physiologists

BARNES: INFORMAL NUTRITION SYMPOSIUM port examines the role of the small intestine in toxin metabolism and the interaction between diet and detoxification pathways.

SMALL INTESTINE

Thus, the increase in dietary levels of a substrate is positively correlated with P-450 expression [1]. The P-450 act on a wide range of substrates and convert lipophilic insoluble compounds into more soluble forms for their eventual excretion. This conversion, or biotransformation, involves the hydrolysis, reduction, or oxidation of a substrate, resulting in the introduction or exposure of a functional group [4]. The P-450 enzymes constitute a superfamily of proteins that are responsible for drug and toxin metabolism, steroid synthesis, and steroid modification. Fourteen P-450 gene families have been identified in mammals; only cytochrome P-450 subfamilies 1, 2, and 3 have been linked to drug/toxin metabolism. Expression of cytochrome P-450 (predominantly the 2C and 3A subfamilies) varies along the length of the intestine and along the villus axis [6]. Cytochrome P-450 activity declines along the length of the intestine and from the villus tip to the crypt. Thus, as the absorptive function of the enterocyte and intestine decline, so does the expression of P-450. Levels of intestinal cytochrome P-450 are reported to be less than those found in the liver (100 pmol/mg microsomal protein and 300 pmol/mg microsomal protein, respectively) although this comparison is confounded by the homogenous expression of P450 in the liver. This difference between intestine and liver P-450 activity is often used as the basis for the idea that the intestine is of little importance in toxin metabolism. If, however, care is taken to examine only the columnar epithelium of the intestine, the difference between liver and intestine are greatly reduced [4]. It is important to note that this mechanism of detoxification requires that potential toxins enter the cell and travel through the blood in order to be metabolized. Thus, with P-450 alone, the organism remains at risk for the detrimental effects of xenobiotics. Conjugation enzymes [e.g., uridine diphosphate (UDP)-glucuronosyltransferase and GST] are another important component in the biotransformation of many xenobiotics. These enzymes catalyze the glucuronidation, methylation, sulfation, or acetylation of lipophilic compounds, effectively increasing their water solubility and, presumably, their excretion. Dis-

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The small intestine functions predominantly as an absorptive organ extracting nutrients from luminal contents. Nutrient absorption, accomplished by absorptive cells in the mucosa, is a relatively efficient process resulting in only 10 to 20% of the original digesta passing to the colon. Often overlooked, due to the significance of its primary role in nutrition, is the importance of intestinal metabolism of nonnutritive dietary constituents. The small intestine is the first site of toxin exposure, the first barrier to systemic exposure to dietary toxicants, and an important route of secretion of endogenous metabolites. Just like the liver, the small intestine metabolizes many dietary constituents using a combination of phase I and phase II enzymes and efflux transport proteins. However, the importance of intestinal metabolism in the overall detoxification process remains a contentious issue. For example, intestinal metabolism of a number of important drugs (cyclosporin, verapamil, nifedipine) significantly reduces bioavailability, in some instances to as low as 30% [4], indicating that the intestine is a significant barrier to toxin entry. Others conclude that the lower expression of some cytochrome P-450 in the gut supports a limited role for the intestine in first pass metabolism. Information about intestinal toxin metabolism in birds is limited; this report outlines the concept and explores the potential interaction between diet and intestinal efflux proteins in toxin metabolism. The most extensively studied mechanism responsible for the metabolism of xenobiotics is the action of the cytochrome P-450. These enzymes are widely distributed throughout the body with the greatest concentrations in the liver and in tissues exposed to the external environment (e.g., intestine, lung, eye). This tissue distribution and efficient extraction from blood results in limited systemic exposure to many xenobiotics. Another important characteristic of cytochrome P-450 in the detoxification process is their induction by substrates, providing an adaptive response to substrate exposure [5].

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made substrates? Can transporter function be modulated to improve protection against specific toxins (e.g., mycotoxins)?

MECHANISMS OF MODULATION Regulation of Pgp, MRP, and cytochrome P-450 function appears to occur at several levels. An important question that remains unanswered is whether or not they are coordinately regulated in response to exogenous stimuli (i.e., dietary constituents, antibiotics, mycotoxins). Substrates, such as chemotherapeutic drugs, environmental pollutants, and cytotoxic compounds, are well-known regulators of Pgp, MRP, P-450 mRNA, and protein levels; however, the specific mechanisms for many of these changes are not well established [10, 9]. Increases in membrane Pgp protein have been associated with increased Pgp mRNA [14, 15, 16] and increased mRNA stability [17]. A role in the direct regulation of Pgp by posttranslational modification has been proposed for protein kinase C specifically and Pgp phosphorylation in general [18, 19]. Other modulators of Pgp mRNA and protein include reactive oxygen species, which increase both Pgp mRNA and protein in hepatocyte cultures [16], cAMP-dependent protein kinase, which appears to be required for expression of Pgp mRNA [20], and metals, such as arsenic [15, 21]. The induction of P-450 activity by many environmental contaminants and drugs is well established in both mammals and birds. The mechanism of P-450 induction involves the binding of ligand-activated receptors that interact with other proteins to activate P-450 transcription. Recently, the coinvolvement of the pregnane X receptor (PXR) in the induction of Pgp and P-450 3A has been described [5], providing a direct link between the regulation of Pgp and P-450. These investigators hypothesized that substrate overlap, common tissue expression, and coinduction with some substrates may indicate a common regulatory mechanism between Pgp and P-450. Geick et al. [5] demonstrated that oral administration of the antibiotic rifampin activated PXR and induced the expression of P-450 3A and Pgp, resulting in lower digoxin absorption in vivo. Schuetz et al. [22] reported similar findings for Pgp and indicated that PXR also regulated MRP3 and MRP4 expression.

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tribution of UDP-glucuronosyltransferase and GST, 2 enzymes whose actions produce substrates for MRP efflux, does not appear to be uniform along the intestine nor along the cryptvillus axis. Enzyme activity is generally greatest in the proximal intestine and toward the villus tip [4]. Information about enzyme levels is limited, in part, by the lack of isozyme-specific antibodies. As with Pgp, MRP, and P-450, these conjugation enzymes are inducible by dietary substrates [7, 8]. The permeability (P) glycoprotein and MRP are transmembrane proteins of about 170 and 190 kDa, respectively, that act as adenosine diphosphate- dependent efflux pumps to exclude compounds from cells [9]. These so called multidrug or multixenobiotic transporters are part of a superfamily of adenosine diphosphate-binding cassette proteins found in many vertebrates and invertebrates that transport a wide range of substrates, including peptides, polysaccharides, steroids, and a wide range of drugs [10, 11, 2, 12]. Both proteins decrease the cellular accumulation of substrates and increase their excretion from the intestine and liver. Investigators have hypothesized that due to their broad substrate specificity and specific tissue expression, transport (efflux) by Pgp and MRP protects cells (as well as the whole organism) from the entry of drugs and toxins [13]. Their role as a barrier to toxin absorption is supported, in part, by their specific expression in tissues acting as physiological barriers (e.g., choroid plexus, blood testis barrier, intestinal epithelium) and in tissues responsible for the detoxification/metabolism of endogenous and exogenous xenobiotics (e.g., small intestine, kidney, and liver) [13, 11]. Thus, the modulation of Pgp or MRP transport activity or both, in coordination with the phase I and phase II toxin-metabolizing enzymes, may play an important role in animal adaptation to toxic dietary constituents. Several important questions related to these proteins remain unresolved. For example, which natural and manmade compounds found in animal diets are substrates for export by these transporters? Are the transporters coordinately regulated with phase I and phase II detoxification enzymes in response to dietary toxins? Are the transporters induced following exposure to natural and man-

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EVIDENCE OF DIETARY MODULATION/REGULATION OF PGP, MRP, AND CYTOCHROME P-450

ROLE OF PGP AND MRP IN TOXIN METABOLISM There is little evidence that Pgp expressed in excretory organs is essential for the elimination of endogenous toxic metabolites, because knockout mice (mdr1a, mdr1b, or the double knockout) have a normal life span, reproduce, and otherwise appear normal under laboratory conditions [30, 31]. However, when treated with exogenous Pgp substrates (vinblastine, ivermectin [31]), these knockout mice exhibit increased sensitivity to the toxic effects of the compounds compared with wild-type animals. Similar sensitivity has been observed in Pgp and MRP knockout C. elegans. [32, 33]. Deletion of the Pgp gene increased nematode sensitivity to colchine and chloroquine, whereas the MRP knockout showed increased sensitivity to heavy metals [32, 33]. The increased sensitivity to toxins is consistent with the previously reviewed lines of evidence that Pgp and MRP play an important role in the gut and liver in mediating exogenous xenobiotic absorption and excretion. The clearest link between Pgp or MRP expression and toxin defense comes from literature describing the adaptation of aquatic organisms to environmental pollutants. Three types of observations support Pgp induction due to environmental toxin exposure [reviewed in 10]. First, Kurelec [34] showed a greater accumulation of the Pgp substrate vincristine in the gill tissue of snails from unpolluted sites compared with snails from polluted areas (less Pgp protein resulted in greater vincristine accumulation). Second, Smital and Kurelec [35] demonstrated that mussels transplanted from clean to polluted sites very rapidly increased expression of Pgp to levels equal to resident animals. Third, laboratory exposure to diesel-2 oil, polyaromatic hydrocarbons, and pesticides (Arochlor 1254, chlorbenside) increase the Pgp expression in aquatic organisms [35]. In addition to the anthropogenic substrates described above, Pgp expression is induced by natural products and changes in environmental conditions (temperature, algal blooms [10]). Many Pgp substrates have been isolated from aquatic organisms, including okadaic acid from Dinophysis, and calyculin A from Acetabularia, both marine alga

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Lo and Huang [23] compared the effects of diet on etoposide (a Pgp/MRP substrate) absorption using rats that were fed both natural and artificial rodent diets. They found that in vitro exposure of the intestine to the natural diet increased etoposide absorption, presumably by inhibition of Pgp-mediated etoposide efflux. This effect was much less pronounced after the diets were consumed for 7 d. Walker [24] has postulated that cytochrome P-450 expression in birds corresponds to their evolutionary exposure to substrates/toxins. That is, fish-eating seabirds would have lower P-450 activity than foraging birds, due to the lack of toxin in their prey compared with the phytochemicals in fruits and seeds [24]. We examined whether the addition of potential Pgp substrates to the diet could affect the levels of Pgp expression in chickens [25]. We determined the relative amount of hepatic Pgp protein in chickens fed either a control diet or a diet supplemented with 1 of 2 common antibiotics, bacitracin or monensin (monensin can directly interact with Pgp [26, 27]). Monensin increased the level of Pgp protein found in the liver canalicular membranes by 26%. However, bacitracin decreased the level of Pgp by 92%, which was unexpected, since we are not aware of any reports of decreased Pgp expression. In other experiments, chickens fed diets containing St. John’s wort (SJW) decreased both Pgp and MRP expression in the liver (unpublished observations). Saint John’s wort contains several phytochemicals, including hypericin, reported to interact with Pgp [28]. This result was again unexpected, as the decreased Pgp expression is in contrast to the effects of SJW in humans, in which chronic consumption increased Pgp, MRP, and P-450 3A levels [29, 28]. Durr et al. [29] reported that dietary SJW extract increased Pgp and P450 3A expression 3.8- and 2.5-fold, respectively, in rat liver. Thus, while dietary modulation of Pgp and MRP occurs in the chicken, the effects of substrates appear to be different than those observed in mammals.

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124 [10]. These data support the hypothesis that efflux transporters (Pgp and MRP) provide a first line of defense against nonnutritive, potentially toxic dietary constituents and may show modulation in species that consume specialized foods rich in potential toxins [13].

INTESTINAL ADAPTATION TO DIET COMPOSITION

POTENTIAL IMPORTANCE TO ANIMAL AGRICULTURE Mycotoxin Metabolism Animal diets contain a number of toxins, including mycotoxins and antibiotics. The mycotoxins produced by Aspergillus, Fusarium, and Penicillum species in particular have been well studied for their impact on both human and animal health. Mycotoxin contamination common in many agricultural crops can cause acute, or chronic disease and results in significant economic loss. The major mycotoxins contributing to this loss are the aflatoxins, trichothecenes, fumonisins, and metabolites from Penicillum.

Disease Resistance In addition to its important role in drug metabolism, Pgp appears to be an important component of the immune system that provides protection against viral infection and locally high concentrations of toxins produced during

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The ecological significance of feeding behavior and diet composition has been examined for a number of species. The gastrointestinal tract of some avian species exhibit considerable flexibility in the face of altered food composition and levels of intake. For example, chronically increased feeding rates result in an enlarged gut that effectively increases both gut volume and the total capacity for breakdown and absorption of nutrients, without responses in tissue-specific enzyme and transport activity [36, 37]. Similarly, chronically increased dietary substrate level (i.e., higher carbohydrate, fat, or protein in the diet) increases tissue-specific rates of chemical breakdown [38, 39, 40]. Thus, regulation is found for both digestive enzymes and absorptive transporters. For nontoxic nutrients at least, transport tends to be correlated with dietary levels. Based on these findings, we can reasonably hypothesize that the considerable flexibility in intestinal adaptation to nutrient composition would provide a similar adaptive advantage if extended to intestinal detoxification systems and, specifically, those targeting lipophilic substrates.

Mycotoxins are metabolized by hepatic phase I and phase II enzymes; however their metabolism by enterocytes and interaction with epithelial efflux transport proteins is poorly understood. Understanding the fate of mycotoxins in the intestinal mucosa is important, since there is growing recognition that the intestine is a major metabolic organ for toxins [12]. Loe et al. [41] showed that aflatoxin B1 was transported by MRP but not Pgp. The most extensively studied mechanisms responsible for the metabolism of mycotoxins are the actions of the cytochrome P450s and the transferase enzymes (UDP-glucuronosyltransferase, GST). These enzyme systems act on mycotoxins (as well as a wide range of other substrates) to increase solubility and enhance excretion. The specific interaction of mycotoxins with gut detoxification pathways and the efflux transporters in particular has not been documented in any livestock species. Understanding to what degree mycotoxins interact with Pgp and MRP has important implications in animal health because many antibiotics are also substrates for these proteins. If the capacity of Pgp and MRP is saturated or if antibiotic substrates are preferred, the natural protection against mycotoxins provided by Pgp, MRP, or both would be limited. That is, Pgp/ MRP would be occupied by the transport of antibiotics, permitting increased absorption of mycotoxins. In addition, if Pgp, MRP, or both protect against mycotoxins, selection for these proteins would improve animal production on diets containing mycotoxins. It is well known that bird species differ in their sensitivity to mycotoxins and that genetic selection can increase resistance, and the increased resistance is partially due to increased expression of P450. The role of Pgp and MRP in mycotoxin resistance was not determined; however, differences in drug sensitivity within a species have been explained by differences in Pgp and MRP expression.

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cells from programmed cell death (apoptosis) induced by activation of the immune system and may be involved in antigen presentation. Bezombes et al. [46] showed that apoptosis induced by tumor necrosis factor-α (1 of several cytokines produced during an acute-phase immune response) increased in the presence of PSC-833, a potent inhibitor of Pgp function. Masci et al. [42] interpreted their data to suggest that Pgp altered the major histocompatibility complex class 1 forms expressed on the cell surface and, thus, may be involved in transport of antigenic peptides. We have previously shown that Pgp expression in the liver and spleen increases rapidly during an immune challenge [25].

CONCLUSIONS AND APPLICATIONS 1. Diet impacts the function of detoxification systems in the intestine and liver. 2. Two recently described efflux transport proteins, Pgp and MRP, contribute to the metabolism of endogenous and exogenous toxins. 3. Pgp and MRP may have significant impact on disease resistance and sensitivity to dietary toxins such as mycotoxins. 4. The relationship between phase I and II metabolism and the efflux transport proteins has not been examined in livestock and represents an opportunity for improving animal health and productivity.

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