The Journal of Toxicological Sciences
Online ISSN : 1880-3989
Print ISSN : 0388-1350
ISSN-L : 0388-1350
Original Article
Effects of indole and indoxyl on the intracellular oxidation level and phagocytic activity of differentiated HL-60 human macrophage cells
Shuhei TsutsumiYuki TokunagaShunsuke ShimizuHideki KinoshitaMasateru OnoKatsuhisa KurogiYoichi SakakibaraMasahito SuikoMing-Cheh LiuShin Yasuda
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2020 Volume 45 Issue 9 Pages 569-579

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Abstract

Indoxyl, a derivative of indole originating from tryptophan, may undergo phase-II sulfate-conjugation pathway, thereby forming indoxyl sulfate (IS) in vivo. We previously reported that IS, a well-known uremic toxin, can increase the intracellular oxidation level and decrease the phagocytic activity in a differentiated HL-60 human macrophage cell model. Using the same cell model, the current study aimed to investigate whether indole and indoxyl (the metabolic precursors of indoxyl and IS, respectively) may cause macrophage immune dysfunction. Results obtained indicated that intracellular oxidation level and cytotoxicity markedly increased upon treatment with indole and indoxyl, in comparison with IS. Incubation of the cells with indole and indoxyl also resulted in attenuated phagocytic activity. Human serum albumin (HSA)-binding assay confirmed that tryptophan and IS, but not indole and indoxyl, could selectively bind to the site II in HSA. Collectively, the results indicated that indole and indoxyl may strongly down-regulate the phagocytic immune function of macrophages, whereas IS, formed upon sulfate conjugation of indoxyl, may exhibit enhanced HSA-binding capability, thereby reducing the adverse effects of indoxyl.

INTRODUCTION

In human and other vertebrates, sulfate conjugation (sulfation) as catalyzed by the cytosolic sulfotransferases (SULTs) is one of the major pathways in the biotransformation and excretion of xenobiotics as well as the homeostasis of endogenous compounds such as catecholamines and steroid/thyroid hormones (Mulder and Jakoby, 1990; Falany and Roth, 1993; Weinshilboum and Otterness, 1994; Suiko et al., 2017). Sulfation in general leads to the inactivation of the substrate compounds containing a hydroxyl group or an amino group, thereby facilitating their removal from the body (Mulder and Jakoby, 1990; Falany and Roth, 1993; Weinshilboum and Otterness, 1994; Suiko et al., 2017). In some cases, however, sulfation of chemicals such as hyrdroxyarylamines may lead to their activation to form reactive carcinogens (DeBaun et al., 1970). Along the same line, we have previously reported that sulfation of naphthol isomers does not always result in the decrease of their antioxidant activity (Sugahara et al., 2018). Therefore, it is essential to investigate whether xenobiotic or endogenous compound(s) may become inactivated or activated via sulfation on a case-by-case basis.

Uremic toxins are biologically active molecules present in free or protein-bound form in the body fluids (Glassock, 2008). Due to impairment of kidney function, uremic toxins may accumulate and remain in patients with end-stage chronic kidney disease. Indoxyl sulfate (IS), a sulfate-conjugated form of indoxyl, is one of the uremic toxins that can promote renal and cardiovascular dysfunctions (Zhang and Davies, 2016). IS is originated from the metabolism of dietary tryptophan (Trp) (Niwa, 2010; Santana Machado et al., 2019). In the body, a portion of Trp is metabolized to form indole by tryptophanase of intestinal bacteria. Upon intestinal absorption, indole can be converted to 3-hyrdoxylindole, or called indoxyl, through Phase I reactions mediated by CYP2A6 or CYP2E1 in the liver (Gillam et al., 2000). Indoxyl may be further metabolized to IS by hepatic SULT1A1 (1A1*2) (Banoglu and King, 2002) or indoxyl β-glucuronide (IBG) under the action of UDP-glucuronosyltransferase. Figure 1 shows the metabolic conversion and chemical structures of Trp, indole, indoxyl, IS and IBG. To gain insight into the mechanisms of action of uremic toxins associated with disease risks, cytotoxic and physiological effects of IS have been subjected to a considerable amount of investigation (Zhang and Davies, 2016). Some studies have demonstrated that IS can generate free radicals in endothelial cells (Dou et al., 2007), stimulate endothelial release of micro particles (Faure et al., 2006), and enhance monocyte adhesion to vascular endothelium (Ito et al., 2010). It has been proposed that high serum IS level in patients suffering from chronic kidney disease may correlate with the disease progression and further complication resulting in related disorders such as impaired immune functions (Descamps-Latscha et al., 2002; Gao and Liu, 2017). Increased oxidative stress may lead to down-regulated immune system in uremic patients (Descamps-Latscha et al., 2002), while increased level of IS may promote monocyte differentiation into low-inflammatory, profibrotic macrophage cells (Barisione et al., 2016). We have recently reported that IS can increase the intracellular oxidation level and decrease the phagocytic activity in a differentiated HL-60 human macrophage cell model (Tsutsumi et al., 2020). To further understand the mechanism(s) of IS-induced immune dysfunction, it is important to find out whether sulfate conjugation may play a role in the activation, or inactivation, of indoxyl, a precursor of IS.

Fig. 1

The metabolic conversion and chemical structures of Trp, indole, indoxyl, IS and IBG.

IS is a well-known uremic toxin that accumulates especially in the end-stage of patients with chronic kidney diseases and it exerts toxic effects in vivo. A good number of studies have revealed a wide range of effects of uremic toxins, including IS (Otagiri, 2005; Glassock, 2008; Itoh et al., 2013; Zhang and Davies, 2016). In contrast, the effects of its precursor, indoxyl, are much less understood in similar pathological settings. It is conceivable that the intrinsically active detoxification process, particularly sulfation, may convert the bulk of indoxyl to IS as a biologically active end-product (Gryp et al., 2020). Sulfate-conjugation in general leads to the inactivation of target compounds (Mulder and Jakoby, 1990; Falany and Roth, 1993; Weinshilboum and Otterness, 1994; Suiko et al., 2017). For certain compounds such as N-hydroxy-2-acetylaminofluorene, however, sulfation may lead to their activation (DeBaun et al., 1970). We were interested in investigating whether sulfate conjugation may play a role in the activation or inactivation of indoxyl, a precursor of IS, using an in vitro phagocytic immune function model previously developed.

The current study aimed to investigate whether indole and indoxyl, as the metabolic precursors of IS and indoxyl, respectively, may cause the phagocytic immune dysfunction using a differentiated HL-60 human macrophage cell model. Furthermore, binding capability of these metabolic precursors, indole and indoxyl, toward human serum albumin (HSA) was determined in comparison with that of IS.

MATERIALS AND METHODS

Materials

Human serum albumin (HSA) type F-V, rifampicin, dimethyl sulfoxide (DMSO), L-tryptophan (Trp), and indole were products of Nacalai Tesque Inc. (Kyoto, Japan). 5-(Dimethylamino)-1-naphthalenesulfonamide (dansylamide) and indoxyl sulfate (IS) were purchased from Sigma-Aldrich Co. (Tokyo, Japan). BD140 was from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). 2,7-Dichlorodihydrofluorescein diacetate (DCFH-DA), indoxyl β-glucuronide (IBG), 2-acetylphenothiazine (2-APT), and Fluoresbrite fluorescent microspheres (1.0 μm) were products of Funakoshi Chemicals Co. (Tokyo, Japan). 3-Hyrdoxylindole (indoxyl) was from Cosmo Bio Co., Ltd. (Tokyo, Japan). Phorbol 12-myristate 13-acetate (PMA), RPMI-1640 with L-glutamine, Hank’s balanced salt solution (HBSS) without phenol red, and penicillin-streptomycin solution were from Fujifilm-Wako Pure Chemical Co. (Osaka, Japan). HL-60 human promyelocytic leukemia cells (JCRB0085) were obtained from Japanese Collection of Research Bioresources (Tokyo, Japan). Fetal bovine serum (FBS) was a product of Biowest (Nuaille, France). All other chemicals were of the highest grade commercially available.

Cell culture and preparation of differentiated macrophage cells

HL-60 human promyelocytic leukemia cells were routinely maintained under a 5% CO2 atmosphere humidified at 37°C in RPMI-1640 medium supplemented with 5% FBS, 100 U/mL penicillin, and 100 µg/mL streptomycin sulfate. For preparation of differentiated macrophage cells, HL-60 cells were seeded in a transparent or black 96-well plate at a density of 1 x105 cells/100 µL per well, and incubated for 48 hr in the presence of 10 nM PMA as described (Yasuda et al., 2012).

Measurement of viable cell number

Viable cell number was photometrically determined using a commercial Cell Counting Kit-8 (CCK-8) (Dojindo Labs, Kumamoto, Japan). Differentiated cells in individual wells of a transparent 96-well plate were incubated for 24 hr in the presence of varying concentrations of IS derivatives. Thereafter, CCK-8 reagent was added to each well, followed by a 3-hr incubation. Absorbance was measured at 450 nm using a microplate reader (SH-1000Lab, Corona Electric Co., Ltd., Ibaraki, Japan).

Measurement of intracellular oxidation level

Intracellular ROS formation was evaluated using DCFH-DA as a probe (LeBel et al., 1992). Briefly, differentiated macrophage cells per well were loaded with 0.1 mM DCFH-DA in HBSS (100 µL) at 37°C for 1 hr in a CO2 incubator. Afterwards, cells in each well were gently washed twice with fresh HBSS (100 µL), and incubated in the presence of individual IS derivatives (10-1,000 µM, 200 µL HBSS per well) at 37°C for 1 hr. In the experiments with the inhibitors, the cells were preincubated in the presence of individual chemicals in HBSS (200 µL) for 30 min, and then incubated for another 1 hr with indole or indoxyl (plus 10 µL, 1 mM or 100 μM final concentration, respectively). Afterwards, the fluorescence intensity (Ex. 490 nm, Em. 530 nm) was measured using a fluorescence microplate reader (MTP-601, Corona Electric Co., Ltd.).

Phagocytosis assay

Phagocytosis assay was performed using a previously established method (Monobe et al., 2007). Briefly, differentiated macrophage cells prepared in individual wells of a black 96-well plate were incubated in fresh RPMI medium (200 μL) supplemented with 5% FBS in the presence of varying concentrations of indole, indoxyl, or IS and 1% Fluoresbrite fluorescent microspheres for 24 hr. The cells were then gently washed twice with 200 μL fresh HBSS (200 μL). Afterwards, the fluorescence intensity of the fluorescent beads incorporated into the cells (Ex. 365 nm, Em. 492 nm) was measured using a fluorescent microplate reader.

Measurement of HSA-binding capability

HSA-binding capability was evaluated using dansylamide as a site-I-specific probe or BD140 as a site-II-specific probe, respectively (Er et al., 2013). A 150 µL reaction mixture, containing the tested sample (50 µL; in MilliQ water), 60 µM HSA (50 µL) in 10 mM phosphate buffer (pH 7.4), and 60 µM dansylamide or BD140 (50 µL) in 10 mM phosphate buffer (pH 7.4; with 1% DMSO), was incubated at 20-25°C for 30 min. Afterwards, the fluorescence intensity of the site-I-bound probe (Ex. 365 nm, Em. 492 nm) or the site II-bound probe (Ex. 365 nm, Em. 610 nm) was measured using a fluorescence microplate reader. The ratio of fluorescence intensity, F/F0 (where F is the fluorescence intensity measured in the presence of the test sample and F0 is the fluorescence intensity measured in the absence of the sample) was subsequently determined.

Statistical analysis

For statistical analysis, the values were expressed as mean ± standard deviation based on the data obtained from three or four independent experiments. Data in part were analyzed using statistical add-on software program (Statcel, OMS Co., Saitama, Japan) for Excel 2016 (Microsoft Co., Redmond, WA, USA). In the Dunnett test, the significant difference was considered at *P < 0.05 or **P < 0.01. A post-hoc Tukey-Kramer test was conducted for the multiple comparison and differences at P < 0.05 were considered significant.

RESULTS AND DISCUSSION

Indoxyl, a derivative of indole originating from tryptophan, may undergo phase-II sulfate-conjugation pathway, thereby forming indoxyl sulfate (IS) in vivo. We have recently discovered that IS can increase the intracellular oxidation level and decrease the phagocytic activity in a differentiated HL-60 human macrophage cell model (Tsutsumi et al., 2020). A number of studies have revealed the diverse effects of uremic toxins, including IS (Otagiri, 2005; Glassock, 2008; Itoh et al., 2013; Zhang and Davies, 2016). In contrast, few studies have been performed to investigate the effect of its precursor, indoxyl. Sulfate-conjugation in general leads to the inactivation of target compounds (Mulder and Jakoby, 1990; Falany and Roth, 1993; Weinshilboum and Otterness, 1994; Suiko et al., 2017). For certain compounds such as N-hydroxy-2-acetylaminofluorene, sulfation may lead to their activation (DeBaun et al., 1970). Whether sulfate conjugation can play a role in the activation or inactivation of indoxyl, a precursor of IS, remained unknown. The current study aimed to investigate whether indole and indoxyl (the metabolic precursors of indoxyl and IS, respectively) may cause the macrophage immune dysfunction.

Effects of indole, indoxyl, IS, and other metabolic derivatives of Trp on the cytotoxicity and intracellular oxidation level of human macrophage cells

We first attempted to determine the cytotoxic effects of a panel of metabolic derivatives of Trp, including Trp, indole, indoxyl, IS, and IBG, on the differentiated HL-60 macrophage cells. No significant effects of Trp (Fig. 2A), IS (Fig. 2D), and IBG (Fig. 2E) were observed at different concentrations ranging from 0, 100 to 1,000 μM, following a 24-hr incubation. A significant decrease in viable cell number was observed when the cells were incubated in the presence of indole at 250 μM and higher concentrations (e.g., 50.3% at 1,000 μM, cf. Fig. 2B), and that of indoxyl at 100 μM and higher concentrations (with an IC50 of 158 μM, cf. Fig. 2C), respectively. Upon this experimental setting, no cytotoxic effect of IS was found, which is in accord with our previous report (Tsutsumi et al., 2020). Indole has a widespread occurrence in the natural environment and has been shown to play important roles in bacterial physiology, pathogenesis and human diseases (Lee and Lee, 2010; Lee et al., 2015; Ma et al., 2018). Notably, indole at 1 mM concentration can increase epithelial cell tight-junction resistance and attenuate inflammation indicators in HCT-8 human enterocyte cells without causing any cytotoxicity (Bansal et al., 2010). In humans, cytochrome P450 monooxygenases can metabolize indole to form indoxyl (Gillam et al., 2000), a reactive product that may spontaneously produce indigo through radical oxidation (Stasiak et al., 2014; Ma et al., 2018). It is possible that the metabolic precursor of IS, indoxyl, may exert strong cytotoxicity due to its acute oxidizing capability.

Fig. 2

Effect of Trp (A), indole (B), indoxyl (C), IS (D) or IBG (E) on the viability of differentiated HL-60 macrophage cells. Cells were incubated in the presence of varying concentrations of IS derivatives for 24 hr. The viable cell number was photometrically determined using a commercial CCK-8 assay kit. Data shown represent mean ± S.D. derived from four independent experiments. Using Dunnett test, significant difference from control was considered at **P < 0.01. Trp; tryptophan, IS; indoxyl sulfate, IBG; indoxyl β-glucuronide.

We investigated whether a panel of metabolic derivatives of Trp, including indole and indoxyl, may modulate intracellular oxidation level in the differentiated HL-60 macrophage cells, using DCFH-DA as a probe. As shown in Fig. 3, the cellular oxidation level markedly increased when the cells were incubated for 1 hr in the presence of indole and indoxyl. Notably, a bell-shape curve of intracellular oxidation was observed at different concentrations of indoxyl. These results probably corresponded to the acute cytotoxicity of indoxyl at elevated concentrations (see Fig. 2C). It should be pointed out that in contrast to Trp and IBG, IS was indeed capable of enhancing intracellular oxidation in macrophage cells. Moreover, unlike IS, indole and indoxyl could strongly induce the cellular oxidation. Our previous study demonstrated that selective inhibitors for NADH oxidase (NOX) and organic anion transporting polypeptide2B1 (OATP2B1) could suppress the IS-induced oxidation (Tsutsumi et al., 2020). We next investigated whether indole and indoxyl can similarly enhance the intracellular oxidation level as does IS. Results indicated that incubation of the cells in the presence of 2-APT, an inhibitor for NOX, relieved the elevated cellular oxidation induced by indole in a concentration-dependent manner (Fig. 4A), but not by indoxyl (Fig. 4B). In parallel, incubation of the cells in the presence of rifampicin, an inhibitor for OATP2B1, also relieved the elevated cellular oxidation induced by indole (Fig. 4C), but not by indoxyl (Fig. 4D), in a concentration-dependent manner. It is possible that the mechanism underlying indole-induced increase in intracellular oxidation, similar to that of IS, involves the OPTP2B1 and NOX pathways. Therefore, it is conceivable that the metabolic precursor of IS, indoxyl, may also directly/indirectly cause cellular oxidation. The current results showing no effects of added 2-APT or rifampicin on the indoxyl-induced oxidation may be due to the intrinsic cytotoxicity of indoxyl.

Fig. 3

Effect of Trp, indole, indoxyl, IS or IBG on the intracellular oxidation level of differentiated HL-60 macrophage cells. DCF fluorescence intensity (Ex. 490 nm, Em. 530 nm) was measured to determine oxidation level. Data shown represent mean ± S.D. derived from four independent experiments. Tukey-Kramer’s test was conducted for the multiple comparison and values not sharing a common superscript letter are considered significantly different at P < 0.05. DCF; dichlorofluorescene, Trp; tryptophan, IS; indoxyl sulfate, IBG; indoxyl β-glucuronide.

Fig. 4

Effect of 2-APT (an NADH oxidase inhibitor) (A, B) and rifampicin (an OATP2B1 inhibitor) (C, D) on the indole- (A, C) or indoxyl-induced intracellular oxidation (B, D) in differentiated HL-60 macrophage cells. DCF fluorescence intensity (Ex. 490 nm, Em. 530 nm) was measured to determine oxidation level. Data shown represent mean ± S.D. derived from three independent experiments. Tukey-Kramer’s test was conducted for the multiple comparison and values not sharing a common superscript letter are considered significantly different at P < 0.05. 2-APT; 2-acetylphenothiazine, OATP2B1; organic anion transporting polypeptide2B1.

Previous studies demonstrated that IS can enhance ROS production, increase NOX activity, and decrease non-enzymatic antioxidant glutathione level in endothelial cells, resulting in the induction of oxidative stress (Dou et al., 2007). To investigate how indoxyl can produce ROS by a route other than NOX (e.g., xanthine oxidase, nitric oxide synthase, and mitochondrial ROS), thereby causing strong cytotoxicity, is another important issue (Dou et al., 2007). Additionally, the different concentration-dependent cytotoxic effects of indole and indoxyl (see Fig. 2) may be in part explained by their specific oxidation mechanisms (Ryter et al., 2007) underlying non-physiological (necrotic) or regulated (apoptotic) cell death pathway (Funakoshi et al., 2018). Indeed, it has been reported that oxidized indoxyl upon auto-oxidation exerted antiproliferative activity on leukemia cells (Hoessel et al., 1999) and cytotoxicity on thymic lymphocytes (Funakoshi et al., 2018). Notably, IS has been shown to bind to human serum albumin in blood circulation (Viaene et al., 2013; Vanholder et al., 2014), and act as an endogenous agonist for aryl hydrocarbon receptor to activate signaling cascades (Schroeder et al., 2010). Whether indoxyl, similar to IS, may bind to these proteins and/or other antioxidant proteins, thereby playing a role on cellular functions will be an interesting topic for future investigation. Moreover, it will be interesting to find out whether indole and indoxyl present in the body may exert stronger cytotoxic effects, compared with IS, on macrophages and other cells/organs in the patients with chronic kidney diseases. It is conceivable that sulfation may play an important role in the detoxification of indoxyl, generating IS. Further pharmacokinetic study may shed a light on this issue.

Effect of indole, indoxyl and IS, on the phagocytic activity of human macrophage cells

We investigated whether indole and indoxyl can interfere with the phagocytic activity of the macrophage cells using fluorescent beads. After a 24-hr incubation with the different concentrations of indole and indoxyl, respectively, the phagocytic activity of treated cells was found to decrease and reached 28.9% at 1,000 μM of indole (Fig. 5A) and 52.8% at 100 μM of indoxyl (Fig. 5B), in comparison with the control. Notably, indole showed stronger suppression of the phagocytic activity than did indoxyl, when tested at a concentration of 100 μM. Taking into consideration the cytotoxicity, it is possible that the decrease of viable cells (ref. Fig. 2) might have, at least partially, caused the indoxyl-induced suppression of the phagocytic activity. That IS moderately decreased the phagocytic activity (by 54.4% at 1,000 μM) (Fig. 5C) is in line with our previous study (Tsutsumi et al., 2020). Moreover, the inhibitory effect of the metabolic precursor of IS, indoxyl, on the phagocytosis of differentiated HL-60 macrophages was found to be stronger than that of IS. In this study, we investigated the effects of indole, indoxyl and IS on the phagocytic immune response. To understand the toxicity of uremic toxins in macrophages, further studies are needed in regard to different immune responses, including the production of inflammatory cytokines or nitric oxide.

Fig. 5

Effect of indole (A), indoxyl (B) or IS (C) on the phagocytic activity of differentiated HL-60 macrophage cells. Data shown represent mean ± S.D. derived from three independent experiments. Fluorescence intensity of beads incorporated (Ex. 365 nm, Em. 492 nm) was fluorometrically measured to determine phagocytic activity. Tukey-Kramer’s test was conducted for the multiple comparison and values not sharing a common superscript letter are considered significantly different at P < 0.05. DCF; dichlorofluorescene, IS; indoxyl sulfate.

HSA-binding capability of indole, indoxyl, IS and other metabolic derivatives of Trp

In the body, albumin is the most abundant protein in blood circulation. It is known that IS is present in both protein-bound form and free form in plasma (Viaene et al., 2013; Vanholder et al., 2014). We investigated the human serum albumin (HSA)-binding capacity of a panel of metabolic derivatives of Trp, including indole, indoxyl and IS using two different site-specific fluorescent probes. In a pilot experiment, upon incubation of HSA with 50 μM IS, the fluorescent intensity of dansylamide, an HSA-binding site-I specific probe, was only 79.2% in comparison with that of control, implying a 20.8% HSA-binding capacity (Fig. 6A). Under this experimental setting, 50 μM Trp, indole, and indoxyl showed, respectively, 19.0%, 7.75%, 10.5% HSA-binding, whereas IBG failed to show any binding. Upon incubation of HSA with 50 μM IS, the fluorescent intensity of BD140, an HSA-binding site-II specific probe, showed a level of 13.7% of that of the control, implying an 86.3% binding capacity of IS to HSA (Fig. 6B). Under the same experimental setting, 50 μM Trp, indole, and indoxyl showed, respectively, 59.8%, 16.8%, and 10.0% HSA-binding capacity, whereas IBG showed no HSA-binding capacity. That IS and Trp were capable of binding to HSA is in line with previously reported studies (Watanabe et al., 2001; Viaene et al., 2013; Vanholder et al., 2014; Tao et al., 2016). Serum albumin is known to bind and carry a great number of small molecules, including both endogenous (e.g., cholesterol, fatty acids, bilirubin, thyroxins) and xenobiotic compounds (e.g., drugs and toxins), thereby facilitating their solubilization, transportation, metabolism, and detoxification (Caraceni et al., 2013). It has been proposed that albumin may serve as a pool of chemically active compounds, including drugs and toxicants, that may circulate in non-active, albumin-bound form (Igisu, 1993; Olsen et al., 2004). It is worth pointing out that while being both Phase-II conjugated metabolites of indoxyl, IS but not IBG selectively displayed the HSA-binding capacity. These results raise the issue that the sulfate conjugation may play an important role in the detoxification of indoxyl, by enhancing HSA-binding affinity to allow the sulfate conjugate to be transported in inactivated state en route to excretion. In a preliminary study, we have confirmed that the IS-induced intracellular oxidation may be relieved when treated macrophage cells were incubated in the presence of additional HSA (data not shown). As the protein-bound form of uremic toxins are not easily eliminated from the body, previous studies have focused on the effective removal of IS and other uremic toxins in uremic patients by hemodialysis (Otagiri, 2005; Itoh et al., 2013). Therefore, it will be an important toxicological issue to clarify the pharmacokinetics as well as the chemical stability or biological activity of protein-bound form of IS. The current study has demonstrated that sulfate conjugation likely plays a role in the inactivation of indoxyl. Further studies are warranted in order to fully understand the mechanisms underlying IS-induced immune dysfunction.

Fig. 6

HSA-binding capacity of Trp, indole, indoxyl, IS or IBG. HSA-binding assays were performed using two different fluorescent probes specific for two distinct site of albumin. F is fluorescence intensity with the sample added and F0 is fluorescence intensity of control. F was determined at Ex. 365 nm and Em. 492 nm using dansylamide (a site-I specific probe) (A) or Em. 610 nm using BD140 (a site-II specific probe) (B). Data shown represent mean ± S.D. derived from four independent experiments. Tukey-Kramer’s test was conducted for the multiple comparison and values not sharing a common superscript letter are considered significantly different at P < 0.05. N.S. indicates no significant difference from control. Trp; tryptophan, IS; indoxyl sulfate, IBG; indoxyl β-glucuronide, HSA; human serum albumin.

In conclusion, we demonstrated here for the first time that indole and indoxyl (metabolic precursors of indoxyl and IS, respectively), but not IS, could strongly increase the intracellular oxidation level and decrease the phagocytic activity in differentiated HL-60 human macrophage cells. The HSA-binding capacity of IS was found to be higher than that of other metabolic derivatives of Trp, including indole and indoxyl. In contrast to IS, indoxyl could strongly down-regulate the phagocytic immune function of macrophages in terms of cytotoxicity. Importantly, the Phase-II sulfate conjugation, but not glucuronidation, could selectively increase the HSA-binding capacity of indoxyl, thereby reducing the toxic effects of indoxyl. More work will be needed in order to fully elucidate the biological functions of IS and other metabolic derivatives of Trp, particularly indoxyl, and to delineate the role of sulfate conjugation of uremic toxin(s).

ACKNOWLEDGMENTS

This work was supported in part by Grant-in-Aid for Young Scientists (B) (JSPS KAKENHI Grant Number 15K18700) and for Scientific Research (C) (JSPS KAKENHI Grant Number 20K05881), and Research and Study Program/Project of Tokai University General Research Organization (Kanagawa, Japan).

Conflict of interest

The authors declare that there is no conflict of interest.

REFERENCES
 
© 2020 The Japanese Society of Toxicology
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