Regulation of Detoxification Enzymes by Food Components in Intestinal Epithelial Cells

Abstract

The intestinal epithelial cells serve as a frontline to the intestinal tract. These cells serve various important functions and harbor the detoxification enzymes that function as an essential intestinal biological barrier system. The detoxification enzymes catalyze oxidation (phase I), conjugation (phase II), and excretion (phase III) reactions in three phases of metabolism to eliminate toxic compounds from the body. These detoxification enzymes are regulated by transcription factors, particularly nuclear receptors and drug receptors, such as the pregnane X receptor (PXR) and the aryl hydrocarbon receptor (AhR). On the other hand, because intestinal epithelial cells are routinely exposed to food, the activity of detoxification enzymes is thought to be regulated by food components. In this review, we highlight research from our group and others that have focused on understanding the role of food components in regulating the expression of detoxification enzymes via transcription factors in intestinal epithelial cells.

Introduction

The intestinal tract is an internal organ, but it is also subjected to a special environment due to its exposure to foreign substances such as food, microorganisms, and chemicals mixed in food, so it is also referred to as “an internal outer barrier.” The intestinal epithelial cells, located at the frontline of the intestinal tract, have many functions, including the digestion/absorption of dietary nutrients, as well as transduction/transformation (conversion) of signals that sense food components and secrete humoral factors such as cytokines. However, these cells are also known to serve an important function as a barrier to prevent invasion of foreign substances (Carriere et al., 2001). Consequently, intestinal epithelial cells can be located at the frontline of biological defense. These barrier functions are broadly classified as either physical or biological barriers. One physical barrier formed by intestinal epithelial cells is the existence of structures called tight junctions between the cells. Tight junctions bind the cells tightly together, forming a monolayer that acts as a physical barrier to invasion by foreign objects. There are two types of biological barriers. One of these is called the intestinal immune system, which uses the immune system as a barrier. In this system, inflammatory cytokines such as IL-6, IL-8, or TNF-α are secreted in response to foreign substances such as pathogenic bacteria that come in contact with or penetrate the intestinal epithelium, and these cytokines attract neutrophils and macrophages, thereby causing an inflammatory response to eliminate the foreign substance. The other barrier is called either the “detoxification and excretory system” or the “drug-metabolizing system” (Fig. 1). This system oxidizes/reduces/hydrolyzes highly lipophilic biological substances such as environmental pollutants that invade the intestinal epithelial cells (phase I). After these pollutants are conjugated (phase II), they are secreted from the cells via ATP binding cassette (ABC) transporters (phase III). This chain of events results in the detoxification/excretion system acting as a barrier. The enzymes involved in this biological barrier system are generally referred to as detoxification (drug-metabolizing) enzymes (Nakata et al., 2006).

Fig. 1.

Detoxification system in the intestinal epithelial cells

Detoxification enzymes are thought to catalyze synthesis or decomposition reactions of endogenous substances in the body (steroids, fatty acids, bile acid, etc.). It has been confirmed that these enzymes are largely expressed in the gastrointestinal tract, including the intestines, and in the liver. These enzymes also serve to detoxify invading xenobiotic substances that have entered the body orally and to excrete these substances from the body (Paine and Oberlies, 2007). Most of the phase I detoxification enzymes are comprised of the cytochrome P450 (CYP) superfamily; they are largely expressed in the liver and digestive tract. The substrate compounds for CYP range widely from biological substances such as steroids and cholesterol to drugs and carcinogens, which undergo oxidation reactions catalyzed by CYP (Xu et al., 2005). However, some of the lipophilic xenobiotics activated by oxidation and other reactions catalyzed by these phase I enzymes exhibit toxicity by binding to DNA and intracellular proteins. Phase II enzymes detoxify the reactants produced by phase I enzymes by adding glutathione (GSH) and glucuronic acid, converting the reactants into a hydrophilic form, and promoting excretion of the foreign substance via the intestine, bile, or urine. Phase II enzymes are involved in the detoxification of xenobiotics in this way (Talalay, 2000). Lastly, the metabolites produced by phase II enzymes or unreacted foreign substances are excreted out of the cells by a membrane transport system known as ABC transporters, as Phase III enzymes. Previous research has shown that inducers of phase I, II, and III detoxification enzymes share common transcription factors (Xu et al., 2005). The expression of detoxification enzymes is mainly regulated by aryl hydrocarbon receptors (AhR), a ligand-dependent transcription factor, and receptors in the nuclear receptor family. Transcription factors involved in the regulation of detoxification enzymes are broadly referred to as “drug receptors.” When a ligand binds to a drug receptor, the drug receptor migrates into the nucleus and binds to the histones of the responsive element of the drug receptor in the promoter region for the target drug-metabolizing enzyme gene. When this occurs, histone acetyltransferases (HATs) are recruited, and they change the structure of the non-acetylated heterochromatin to euchromatin via acetylation, making it possible to translate the DNA. This allows RNA polymerase II to bind to the promoter region and start transcribing the mRNA of the drug-metabolizing enzymes. NF-E2 related factor 2 (Nrf2) is also widely known as a transcription factor that regulates expression of detoxification enzymes, in addition to drug receptors (Surh et al., 2008).

Considering the background as presented above, this review introduces our research that various food components regulate/modulate expression of detoxification enzymes by transcription factors, using intestinal epithelial cell models.

Expression of UGT1A1 is Regulated by Flavonoids

As described previously, phase II detoxification enzymes convert lipophilic xenobiotics to their non-toxic hydrophilic forms through the addition of glucuronic acid. Human UDP-glucuronosyltransferase (UGT) are phase II enzymes that belong to a superfamily of 22 proteins (Rowland et al., 2013). UGTs catalyze the covalent linkage of glucuronic acid, derived from the cofactor UDP-glucuronic acid, to their substrate. It has been reported that UGT plays an essential role in the detoxification of the neurotoxin bilirubin by catalyzing its conjugation with glucuronic acid, and reduced activity of UGT1A1 causes unconjugated hyperbilirubinemia (Mackenzie et al., 1997; Sugatani, 2013). Therefore, identifying food components that can activate UGT1A1 to enhance its detoxification ability is of considerable interest.

We have investigated the effect of phytochemicals, particularly flavonoids, on the expression and regulation of UGT1A1 using the LS180 cell line as a model of human intestinal epithelial cells. The LS180 cell line was incubated with 23 different phytochemicals, and the mRNA levels of UGT1A1 was quantified by real-time PCR analysis. Among these phytochemicals, baicalein and 3-hydroxyflavone increased the mRNA expression of UGT1A1 in a dose- and time-dependent manner (Hiura et al., 2014). A similar upregulation of UGT1A1 mRNA levels was also observed in human intestinal Caco-2 and human hepatic HepG2 cell lines incubated with baicalein and 3-hydroxyflavone. Using western blot analysis increased UGT1A1 protein levels were confirmed which corresponded to significantly increased enzymatic activity of UGT1A1 in baicalein or 3-hydroxyflavone treated cell lines. These results suggested that baicalein and 3-hydroxyflavone increased protein levels and enzymatic activity of UGT1A1 through the upregulation of UGT1A1 mRNA expression.

To verify the regulatory mechanism of flavonoid-induced upregulation of UGT1A1, we cloned the promoter region of the human UGT1A1 gene into a reporter vector upstream of the luciferase reporter gene. Baicalein and 3-hydroxyflavone significantly induced promoter activity of the human UGT1A1 gene in a dose-dependent manner (Hiura et al., 2014). Human drug receptors, such as pregnane X receptor (PXR), constitutive androstane receptor (CAR), and aryl hydrocarbon receptor (AhR) have been reported to regulate the promoter activity of UGT1A1 (Hu et al., 2014). Therefore, expression vectors for the human NR1I2, NR1I3, and AHR genes coding for PXR, CAR, and AhR proteins, respectively, were constructed and co-transfected with the reporter vector in the LS180 cell line. The activation of the UGT1A1 gene promoter by baicalein was enhanced in the presence of PXR and AhR, while promoter activation by 3-hydroxyflavone was further enhanced only in the presence of PXR. Furthermore, using immunostaining the nuclear localization of AhR and PXR was confirmed in cell lines treated with baicalein and 3-hydroxyflavone, respectively. Taken together these data suggest that baicalein and 3-hydroxyflavone transcriptionally regulate the expression of UGT1A1 via drug receptors such as PXR and AhR in the intestinal epithelia (Fig. 2). Additionally, Sugatani et al. demonstrated that the flavonoid chrysin regulates the expression of UGT1A1 at the transcriptional level (Sugatani et al., 2004). Furthermore, bioactive terpenoids and flavonoids from Ginkgo biloba extract have been reported to induce the expression of hepatic drug-metabolizing enzymes, including UGT1A1 (Li et al., 2009). These reports strengthen and support our finding that phytochemicals regulate the expression of UGT1A1.

Fig. 2.

Putative regulatory mechanism of upregulation of UGT1A1 expression by baicalein and 3-hydroxyflavone

Expression of NQO1 is Regulated by Cysteine

We focused on the phase II detoxification enzyme NAD(P) H:quinone oxidoreductase 1 (NQO1), which localizes to the cytosol in mammalian cells (Jaiswal, et al., 1991; Nioi and Hayes, 2004; Williams, et al., 1986). In human, NQO1 is primarily expressed in the epithelial and endothelial cells of lung and intestine (Siegel and Ross, 2000), whereas in rats and mice NQO1 is highly expressed in the liver (Nioi and Hayes, 2004).

Quinones are commonly found in vehicle exhaust gases, cigarette smoke, and some foods. In the body, quinones are reduced to highly reactive free radicals called semiquinones by cytochrome P450 reductases. Semiquinones interact with proteins and DNA and can produce reactive oxygen species (ROS) in the cell. However, NQO1 can prevent semiquinone-induced oxidative stress by catalyzing a single-step two-electron reduction reaction of quinones and converting them to hydroquinones (Ross and Siegel, 2004). It has been reported that the expression of NQO1 is regulated by food components, including polyphenols and isothiocyanates (Thimmulappa et al., 2002; Riedl et al., 2009; Tanigawa, et al., 2007). Because no other food component had been implicated in the regulation of NQO1 expression, we focused on amino acids in food and examined their effect on gene expression and protein activity.

We examined the effect of 20 amino acids on the NQO1 mRNA expression in the LS180 cell line. Among all amino acids, cysteine markedly increased mRNA expression level of NQO1 (Satsu et al., 2012). The mRNA expression of NQO1 was about 5-fold higher in cell lines treated with 10 mM cysteine for 24 hours. Cysteine also induced the mRNA expression of other phase II drug metabolizing enzymes, such as UGT1A1 and heme oxygenase-1 (HO-1) in the LS180 cell line. Furthermore, cysteine increased the protein level and enzyme activity of NQO1 in a dose- and time-dependent manner, suggesting that this amino acid activates NQO1 to increase its protein level and enzyme activity.

Because cysteine is imported into cells via amino acid transporters, the characteristics of cysteine uptake were examined in the LS180 cell line using ¹⁴C-labelled L-cysteine. Significant inhibition of cysteine uptake was observed in the presence of alanine, serine, and glutamate, suggesting that these amino acids use the same cellular transporters as cysteine. Additionally, cysteine-induced mRNA expression of NQO1 was significantly reduced in the presence of alanine, serine, or glutamate, suggesting that uptake of cysteine was essential for the observed activation of NQO1 in the LS180 cell line. It is well known that cysteine is the precursor of glutathione (GSH), a major antioxidant in mammalian cells. In the cysteine-treated cell line, an increase in the intracellular levels of GSH was observed. Therefore, the role of GSH in the cysteine-induced upregulation of NQO1 mRNA expression was examined. However, the induction of NQO1 mRNA expression by cysteine was unaffected when GSH synthesis was blocked with the inhibitor buthionine sulfoximine (BSO), which significantly decreased intracellular levels of GSH. This result showed that GSH is not involved in the induction of NQO1 mRNA expression by cysteine. Furthermore, a structure-activity relationship study was performed to understand cysteine-induced upregulation of NQO1. N-Acetylcysteine, methionine, cysteic acid, homocysteine, ethylcysteine, methylcysteine, were selected as cysteine structural analogues, to investigate the effect of these compounds on NQO1 mRNA expression. Like cysteine, only ethylcysteine and methylcysteine activated NQO1 mRNA expression, suggesting that the thiol- and amino-residues are essential for this gene activation.

To investigate the regulatory mechanism of NQO1 activation by cysteine, we cloned the promoter region (−797 - +17) of the human NQO1 gene in a reporter vector to measure promoter activity in the absence and presence of cysteine. The promoter activity of the NQO1 gene showed a dose-dependent response to cysteine concentration. However, when the antioxidant response element (ARE) in the promoter region of the NQO1 gene was mutated, the transcriptional activation of the reporter by cysteine was completely abolished. This result suggested that the transcription factor Nrf2 that binds to ARE could be involved in the activation of NQO1 by cysteine. To confirm the involvement of Nrf2 in this regulation, small interfering RNA (siRNA) was used to silence the NFE2L2 gene that encodes the Nrf2 protein in humans. The transcriptional activation of NQO1 by cysteine was significantly reduced by the siRNAs. Next, the effect of cysteine on the interaction of Nrf2 with Kelch-like ECH-associated protein 1 (Keap1), which is a cytosolic protein that binds and aids in the degradation of Nrf2, was investigated. Using western blot analysis the localization of Nrf2 and Keap1 was studied, and elevated levels of nuclear Nrf2 protein and reduced levels of cytosolic Keap1 protein were observed after cysteine treatment. These findings strongly suggest that cysteine induces transcriptional activity of NQO1 via Nrf2 protein. The effect of cysteine on the expression of NQO1 was also examined in vivo. C57BL/6 mice were administered water with or without cysteine at a dosage of 400 mg/kg body weight for 16 days. Total RNA extracted from the recovered intestinal mucosa of the mice were analyzed with real-time PCR. Administration of cysteine significantly increased the expression level of NQO1 mRNA in the mouse intestinal mucosa. Thus, it was shown that NQO1 is activated by cysteine in the mouse intestinal mucosa as well as in the intestinal epithelial LS180 cell line (Satsu et al., 2012).

Based on these data, it can be concluded that the expression of NQO1 is not only regulated by phytochemicals, but also by the amino acid cysteine (Fig. 3). Because cysteine is integral in foods consumed daily, it is thought that the expression of NQO1 is constitutively induced by cysteine. However, the Standard Tables of Food Composition in Japan — 2015 - (Seventh Revised Version; http://www.mext.go.jp/en/policy/science_technology/policy/title01/detail01/sdetail01/1388553.htm) reports that the cysteine content in most foods consumed daily is relatively low compared to other amino acids. Therefore, it is assumed that NQO1 is not induced by cysteine in daily foods, but induction by phytochemicals is expected. However, when a high concentration of cysteine is administered in the form of supplements, it is likely that NQO1 expression is induced by cysteine. Under this condition, however, it is uncertain whether phytochemicals can further induce NQO1 expression or conversely suppress NQO1 induction by cysteine. Further studies are needed to explore the relationship between cysteine and phytochemicals, and whether they synergistically induce or offset the effects of both.

Fig. 3.

Putative regulatory mechanism of cysteine-induced upregulation of NQO1 expression

Phytochemicals Activate PXR and Its Target Genes

As described above, activation of various transcription factors, such as drug receptors, are involved in the regulation of drug metabolizing enzymes by food components. Conversely, we screened for food components that regulate the drug receptor PXR and investigated whether the food could regulate the expression of drug metabolizing enzymes.

PXR is highly expressed in the liver and small intestine (Giguere, 1999). It recognizes various substances including steroids, bile acids, and drugs, and metabolizes them by regulating the expression of genes encoding drug metabolizing enzymes. PXR is also reported to sense and adapt to various chemical compounds with diverse chemical structures. The X-ray crystal structure of the PXR ligand-binding domain shows a large, flexible, hydrophobic ligand-binding cavity that can recognize a variety of chemical structures in multiple binding modes (Watkins et al., 2001; Watkins et al., 2003).

In the absence of ligands, PXR localizes to the cytosol. When PXR binds a ligand, it translocates to the nucleus and forms a heterodimer with the nuclear retinoid X receptor (RXR) protein. The PXR/RXR heterodimer binds the PXR response element (PXRE) in the promoter region of target genes and facilitates the binding of the steroid receptor coactivator 1 (SRC-1) protein resulting in the transcriptional activation of the target genes (Camahan and Redinbo, 2005). An everted repeat with a spacer of 6 bp (ER-6) in the upstream region of the cytochrome P450 enzyme CYP3A4, and a direct repeat with a 4 bp spacer (DR-4) in the upstream region of the multidrug resistance protein 1 (MDR1) have been described as PXRE (Nakata et al., 2006). In addition to CYP3A4 and MDR1, the PXR protein is also known to regulate several other drug metabolizing enzymes, such as the phase I enzymes CYP2B6 and CYP2C9, the phase II enzyme UGT1A1, and the phase III enzyme MRP2 (Giguere, 1999; Kliewer et al., 2002). PXR plays a major role as a xenosensor that recognizes xenobiotics and functions as a master regulator of detoxification enzymes. Therefore, we screened food components that can enhance the biological barrier function via PXR-dependent activation of genes encoding detoxification enzymes.

First, an assay was developed to screen for food components that induce PXR-dependent transcriptional activity. A reporter vector for luciferase activity containing four tandem repeats of DR-4, and an expression vector for the human NR1I2 gene encoding PXR were constructed and transiently transfected into the LS180 cell line. Phytochemicals were added to the transfected cells and incubated for 24 h, prior to reporter activity assay. Of the 39 different phytochemicals tested, tangeretin, ginkgolide A and ginkgolide B showed significant increase in PXR-dependent transcriptional activity (Satsu et al., 2008 and Fig. 4). The induction by these phytochemicals was dose-dependent, and they also increased the activity of the human MDR1 gene promoter, which contains the DR-4 sequence. Next, the effect of tangeretin and ginkgolide A/B on mRNA levels of CYP3A4 and ABCB1 that encodes MDR1 protein were examined and upregulation of transcription of both genes by all three phytochemicals was observed. Furthermore, the increased protein levels and transporter activity of MDR1 by all three phytochemicals were confirmed. Together these results suggest that tangeretin and ginkgolide A/B can transcriptionally activate the expression of ABCB1, thereby increasing the level of MDR1 protein and enhancing its transporter activity.

Fig. 4.

Effect of phytochemicals on PXR-dependent luciferase activity

6-Shogaol Induces AhR-mediated Transcriptional Activation

Like PXR, AhR is also a drug receptor that regulates the expression of drug metabolizing enzymes (Hahn, 2002). The AhR protein binds to the xenobiotic response element (XRE) in the promoter region of its target genes to bring about their transcriptional activation. AhR was initially characterized as a dioxin receptor. Dioxin bound AhR activates the expression of the phase I drug metabolizing enzyme CYP1A1, which in turn augments the carcinogenicity of a carcinogenic substance. Therefore, AhR has been reported to be involved in dioxin toxicity (Mimura and Fujii-Kuriyama, 2003). We have previously screened for flavonoids that could suppress 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced AhR-dependent transcriptional activity and investigated their properties (Hamada et al., 2006; Hamada et al., 2010). In recent years, new physiological functions have been revealed for AhR (Abel and Haarmann-Stemmann, 2010). For instance, AhR has E3 ubiquitin ligase activity and is involved in the suppression of colon carcinogenesis by degrading β-catenin (Kawajiri et al., 2009). Therefore, an assay was developed to screen for food components that could regulate AhR-dependent transcriptional activity.

To develop this assay, an expression vector for the human AHR gene was constructed by PCR, and the AhR-responsive vector was generated by ligating annealed oligonucleotides, consisting of three tandem repeats of XRE, in the pGL3 promoter of the reporter vector. Both vectors were stably co-transfected into the HepG2 cell line, and the clone that showed the highest response to the carcinogen 3-methylcholanthrene (3-MC) was selected after limiting dilution method. The selected clone exhibited increased luciferase activity with 3-MC treatment in a concentration-dependent manner (Satsu et al., 2015).

Using this clone, 23 different hot water extracts (HWEs) of vegetables were screened for enhanced AhR-dependent transcriptional activation of the reporter gene. The HWE of ginger significantly increased reporter activity (Yoshida et al., 2014). An ethyl-acetate fraction (EAF) from ginger showed greater AhR-mediated transcriptional activity than the HWE of ginger. HPLC analysis of the EAF from ginger contained two peaks that were identified as 6-shogaol and 6-gingerol. When the effect of 6-shogaol and 6-gingerol on AhR-dependent transcriptional activation of the reporter was observed, only 6-shogaol showed increased transcriptional activity, and 6-gingerol had no effect. Also the effect of 6-shogaol on the mRNA expression levels of CYP1A1, UGT1A1, and ABCG2, the gene that encodes the breast cancer resistant protein (BCRP), which are target genes of AhR in the HepG2 cell line was examined. 6-Shogaol significantly upregulated the mRNA levels of these target genes and increased the protein levels of CYP1A1, suggesting that 6-shogaol is a major compound in ginger that activates AhR and the expression of AhR-dependent target genes (Fig. 5).

Fig. 5.

6-shogaol activates AhR and its target genes

Conclusion

In this review, we have highlighted that various food components, such as phytochemicals and amino acids regulate the expression of genes coding for drug metabolizing enzymes through transcription factors such as PXR, AhR, and Nrf2 in human intestinal epithelial cells. Conservation of the regulation of gene expression by food components was also observed in an in vivo mouse model. These findings highlight the positive effects of enhancing the biological barrier function in the intestinal epithelia. However, these food components could also affect drug metabolism and their side effects should be considered prior to use.

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