Species and Tissue Differences in β-Estradiol 17-Glucuronidation

Abstract

Uridine 5′-diphosphate-glucuronosyltransferase (UGT) is expressed in the liver and extrahepatic tissues. One of the major metabolic pathways of β-estradiol (E2) is glucuronidation at the 17-hydroxy position by UGTs. This study was performed to determine E2 17-glucuronidation kinetics in human and rodent liver, small intestine, and kidney microsomes and to clarify the species and tissue differences. In the human liver and small intestine, Eadie–Hofstee plots exhibited biphasic kinetics, suggesting that E2 17-glucuronide (E17G) formation was catalyzed by more than two UGT isoforms in both tissues. The K_m values for E17G formation by the high-affinity enzymes in the human liver and small intestine were 1.79 and 1.12 µM, respectively, and corresponding values for the low-affinity enzymes were 3.72 and 11.36 µM, respectively. Meanwhile, E17G formation in the human kidney was fitted to the Hill equation (S₅₀=1.73 µM, n=1.63), implying that the UGT isoform catalyzing E17G formation in the kidney differed from that in the liver and small intestine. The maximum clearance for E17G formation in the human kidney was higher than the intrinsic clearance in the liver. E17G formation in the rat liver and kidney exhibited biphasic kinetics, whereas that in the small intestine was fitted to the Hill equation. In mice, all 3 tissues exhibited biphasic kinetics. In conclusion, we reported species and tissue differences in E2 17-glucuronidation, which occurred not only in the human liver but also in the extrahepatic tissues particularly the kidney.

Uridine 5′-diphosphate-glucuronosyltransferase (UGT) is one of the major phase II drug-metabolizing enzymes. UGT conjugates endogenous compounds including steroid hormones with glucuronic acid from the cofactor uridine 5′-diphosphate-glucuronic acid (UDPGA). Glucuronides have enhanced hydrophilicity compared with their parent compounds and, therefore, they are easily excreted in the urine and bile.¹⁾ A recent study reported the presence of UGTs in extrahepatic tissues such as the small intestine and kidney,²⁾ implying that UGT may contribute to modulating the concentration of endogenous compounds in the body.

β-Estradiol (E2) is an important endogenous estrogen that has a wide spectrum of pharmacological effects. E2 has the potential to protect the liver, small intestine, and kidney against oxidative stress.^3,4) The expression of glutathione S-transferase⁵⁾ and superoxide dismutase⁶⁾ has been reported to be increased by E2, suggesting a possible antioxidant role mediated by the upregulation of antioxidant enzymes. Glucuronidation of E2 at the 17-hydroxy position is one of the major metabolic pathways, which functionally inactivates E2.⁷⁾ However, a previous study showed that E2 17-glucuronide (E17G) caused cholestasis by inhibiting multidrug resistance-associated protein 2.⁸⁾ Therefore, it is essential to account for the kinetics of E2 17-glucuronidation in the liver and extrahepatic tissues when elucidating the pharmacological effects of E2. Numerous studies have evaluated the in vivo effects of E2 using experimental animal models such as rats and mice, and many researchers have attempted to extrapolate the animal data to humans.^9–11) Since species differences in the kinetics of serotonin¹²⁾ and propofol glucuronidation¹³⁾ have been reported between human and rodents, it would be expedient to clarify whether similar differences occur in E17G formation.

Human UGT1A3, UGT1A4, UGT1A10, UGT2A1, UGT2B7, and UGT2B17 have been shown to catalyze E17G formation.¹⁴⁾ UGT1A4 and UGT2B7 mRNAs are expressed at high levels among the hepatic UGT isoforms involved in E17G formation, whereas UGT1A10 has been found predominantly in the extrahepatic tissues.²⁾ In rats, E17G is formed by Ugt1a2, Ugt2b2, and Ugt2b3¹⁵⁾ and there is a tissue difference in the mRNA expression of these Ugt isoforms.¹⁶⁾ The various expression patterns of UGT isoforms may contribute to the differences in E2 17-glucuronidation between tissues and species. Therefore, the purpose of the present study was to investigate E17G formation in the liver and extrahepatic tissues of humans and rodents and clarify the possible species and tissue differences in E2 17-glucuronidation kinetics.

MATERIALS AND METHODS

Materials

Alamethicin, E2, E17G, and UDPGA trisodium salt were purchased from Sigma-Aldrich (St. Louis, MO, U.S.A.). All other chemicals and solvents were of the highest grade commercially available.

Preparation of Liver, Small Intestine, and Kidney Microsomes

The present study was approved by the Institution Animal Care and Use Committee of Meijo University. The human liver and small intestine microsomes (pooled from 50 and 7 donors, respectively) were purchased from Corning (Corning, NY, U.S.A.). The human kidney microsomes (pooled from 4 donors) were obtained from KAC (Kyoto, Japan). The liver, small intestine, and kidney were removed from 8-week-old male Sprague–Dawley rats or C57BL/6N mice (Japan SLC, Hamamatsu, Japan). Pooled rat and mouse (n=5 each) liver, small intestine, and kidney microsomes were prepared as described previously,¹²⁾ and were stored at −80°C until used. Protein concentration was measured using the Lowry method with a Bio-Rad protein assay kit (Bio-Rad, Hercules, CA, U.S.A.).

E2 17-Glucuronidation

E2 17-glucuronidation was determined according to the method of Jantti et al.¹⁷⁾ with slight modifications. The incubation mixture contained 50 mM Tris (hydroxymethyl) aminomethane–HCl buffer (pH 7.4), 5 mM MgCl₂, 25 µg/mL alamethicin, 0.2–100 µM E2, and the microsomal proteins. The microsomal liver, small intestine, and kidney protein concentrations were as follows: human, 0.01, 0.05, and 0.2 mg/mL, respectively; rat, 0.01, 0.05, and 0.5 mg/mL, respectively; and mouse, 0.01, 0.5, and 0.5 mg/mL, respectively. The reaction was initiated by adding UDPGA (final concentration, 3 mM) and incubating at 37°C, and subsequently terminated by boiling at 100°C for 3 min. E17G was quantified using LC-MS/MS. LC was performed using a prominence apparatus (Shimadzu, Kyoto, Japan), which was equipped with an InertSustain C₁₈ (3 µm, 2.1×150 mm, GL Science, Tokyo, Japan) and the column temperature was 40°C. The mobile phase was 0.15% ammonium–methanol (70 : 30, v/v), which was run at a flow rate of 0.2 mL/min, while the detection was performed using an API4000 MS/MS spectrometer (Applied Biosystems, Foster City, CA, U.S.A.) operated in the positive ion mode with electrospray ionization. The mass to charge ratio was 447.4 and 75.1 m/z, respectively for E17G, which had a retention time of 6.2 min. The detection limit for E17G was 10.0 fmol while the quantification limit of the reaction mixture was 1.0 nM with a coefficient of variation of <10%.

Enzyme Kinetic Analysis

The kinetics were analyzed using the KaleidaGraph computer program (Synergy Software, Reading, PA, U.S.A.). The following Michaelis–Menten and Hill equations were used for the analysis: V=V_max×S/(K_m+S) and, V=V_max×Sⁿ/(Sⁿ₅₀+Sⁿ), respectively, where V is the velocity of the reaction, V_max is the maximum velocity, S is the substrate concentration, K_m or S₅₀ is the substrate concentration showing the half-V_max, and n is the Hill coefficient. Intrinsic clearance (CL_int) was calculated as V_max/K_m for the Michaelis–Menten model or sigmoidal kinetics while the maximum clearance (CL_max) was calculated as V_max×(n−1)/(S₅₀×n (n−1)^1/n).

RESULTS

Kinetic Analyses of E2 17-Glucuronidation in Human Microsomes

E17G formation in the human liver and small intestine was fitted to the Michaelis–Menten model, and the Eadie–Hofstee plots exhibited biphasic kinetics (Fig. 1). The K_m values of the high-affinity enzymes were similar between the liver (1.79 µM) and small intestine (1.12 µM). In contrast, E17G formation in the human kidney was fitted to the Hill equation, and the S₅₀ value and Hill coefficient were 1.73 µM and 1.63, respectively. The V_max and CL_max values in the human kidney were higher than those in the liver.

Fig. 1. Kinetic Study of E17G Formation in Humans

E17G formation was measured in human liver (A), small intestine (B), and kidney (C) microsomes treated with E2 concentrations ranging from 0.2 to 100 µM. Eadie–Hofstee plots are shown as insets in each graph.

Kinetic Analyses of E2 17-Glucuronidation in Rat Microsomes

E17G formation in the rat liver and kidney was fitted to the Michaelis–Menten model, and the Eadie–Hofstee plots exhibited biphasic kinetics (Fig. 2). However, the K_m values differed between the liver and kidney. Compared to that reported in the liver, the K_m values in the kidney were approximately 4-fold higher for both the high- and low-affinity enzymes. In contrast, E17G formation in the rat small intestine was fitted to the Hill equation with a coefficient of 2.0. The V_max and CL_int values in the liver were the highest of all the 3 tissues examined.

Fig. 2. Kinetic Study of E17G Formation in Rats

E17G formation was measured in rat liver (A), small intestine (B), and kidney (C) microsomes treated with E2 concentrations ranging from 0.2 to 100 µM. Eadie–Hofstee plots are shown as insets in each graph.

Kinetic Analyses of E2 17-Glucuronidation in Mouse Microsomes

The E17G formation in all 3 tissues investigated was fitted to the Michaelis–Menten model, and the Eadie–Hofstee plots exhibited biphasic kinetics (Fig. 3). The K_m value of high-affinity enzymes in the mouse liver was calculated to be 0.48 µM, which was lower than that in the small intestine but similar to that in the kidney. The V_max and CL_int values in the liver were the highest of all the mouse tissues examined in this study.

Fig. 3. Kinetic Study of E17G Formation in Mice

E17G formation was measured in mouse liver (A), small intestine (B), and kidney (C) microsomes treated with E2 concentrations ranging from 0.2 to 100 µM. Eadie–Hofstee plots are shown as insets in each graph.

Comparison of E2 17-Glucuronidation in Liver, Small Intestine, and Kidney Microsomes from Humans, Rats, and Mice

E2 17-glucuronidation was determined at 1 µM E2, which was below or near the K_m or S₅₀ values of all species and tissues examined in the present study. As shown in Fig. 4, E2 17-glucuronidation in the rat and mouse liver was 6.6- and 2.2-fold higher than that in the human liver, respectively. In contrast, glucuronidation in the human small intestine was 148- and 128-fold higher than that in the rat and mouse small intestine, respectively. Similar to the small intestine, the activity in the human kidney was remarkably higher than that in the rat and mouse kidney.

Fig. 4. E2 17-Glucuronidation in Liver, Small Intestine, and Kidney Microsomes from Humans, Rats, and Mice

The concentration of E2 was 1 µM. Each column represents the mean±S.D. of 3 independent determinations.

DISCUSSION

In humans, CL_int values suggest that E2 17-glucuronidation can occur in both the liver and extrahepatic tissues, particularly the kidney. Similarly, it was reported that the CL_int value for propofol glucuronidation in the human kidney microsomes was 1.4-fold higher than that in the liver microsomes.¹⁸⁾ Therefore, the kidney is likely the important organ where some UGT substrates are conjugated. In contrast, E2 17-glucuronidation in rodents mainly occurred in the liver (Table 1).

Table 1. Kinetics Parameters of E17G Formation in the Humans, Rats, and Mice

Species	Tissue	Affinity	Km (S50) (µM)	Vmax (pmol/min/mg protein)	CLint (CLmax) (µL/min/mg protein)	Model	na)
Human	Liver	High	1.79±0.17	242.55±24.11	135.57±1.20	M–M
		Low	3.72±0.11	372.76±2.93	100.16±3.59	M–M
	Small intestine	High	1.12±0.04	57.37±0.83	48.69±1.00	M–M
		Low	11.36±0.10	355.38±7.17	31.29±0.46	M–M
	Kidney		1.73±0.09b)	288.02±4.50	161.86±1.13c)	Hill	1.63
Rat	Liver	High	0.91±0.22	1239.55±233.09	1372.80±110.76	M–M
		Low	8.10±1.34	6182.77±410.71	771.32±77.53	M–M
	Small intestine		19.33±3.51b)	13.67±1.15	1.44±0.06c)	Hill	2.00
	Kidney	High	4.81±1.81	2.03±0.75	0.42±0.01	M–M
		Low	32.46±2.75	11.85±0.37	0.37±0.02	M–M
Mouse	Liver	High	0.48±0.03	286.34±8.02	593.98±18.18	M–M
		Low	15.04±0.16	2750.43±28.13	182.89±2.33	M–M
	Small intestine	High	4.53±1.72	1.27±0.40	0.29±0.03	M–M
		Low	165.80±36.33	33.13±5.12	4.95±0.56	M–M
	Kidney	High	0.72±0.23	1.95±0.34	2.79±0.37	M–M
		Low	28.91±4.01	31.56±3.91	1.12±0.27	M–M

Each value represents the mean±S.D. of 3 independent determinations. M–M: Michaelis–Menten model. a) Hill coefficient. b) S₅₀. c) CL_max.

There were remarkable species differences between human and rodents in the tissues where E2 17-glulcuronidation mainly occurs (Fig. 4). The E2 levels in the plasma have been reported to be 4–6 pg/mL in male rats¹⁹⁾ and mice,²⁰⁾ whereas it was 30 pg/mL in men.²¹⁾ In humans, E2 17-glucuronidation in the small intestine and kidney might contribute to modulating E2 concentrations in the body compared with rodents. Propofol glucuronidation in rat liver microsomes is higher than it is in the intestine²²⁾ and kidney²³⁾ microsomes. Even if the CL_int value of glucuronidation of the UGT substrate was low in the rodent kidneys, the substrate might be glucuronidated in the human kidney to some degree.

In humans, the Eadie–Hofstee plots for E17G formation exhibited biphasic kinetics in the liver and small intestine (Fig. 1). The results suggest that E17G formation is catalyzed by more than two UGT isoforms in the human liver and small intestine. Since E2 17-glucuronidation was fitted to the Hill equation in the human kidney, the UGT isoform catalyzing E17G formation there likely differs from those in the liver and small intestine. Human UGT1A3, UGT1A4, UGT1A10, UGT2A1, UGT2B7, and UGT2B17 have been reported to catalyze E17G formation.¹⁴⁾

Recently, several studies have estimated the protein content of each UGT isoform in tissues using LC-MS/MS.^24–26) In the human liver, UGT2B7 and UGT1A4 (80.7 and 54.3 pmol/mg, 27 and 18% of hepatic UGT proteins, respectively) were highly expressed, whereas both UGT1A3 and UGT2B17 were 8.0 pmol/mg (each 2.7% of total hepatic UGT proteins),²⁴⁾ and the expression of other isoforms involved in E17G formation were below detectable levels in the human liver.²⁴⁾ Furthermore, for UGT2B7, UGT1A4, UGT1A3, and UGT2B17, the K_m values of E2 17-glucuronidation have been reported to be 5.96±0.93, 10.6±1.85, 66.7±14.2, and 11.4±2.46 µM, respectively,²⁷⁾ which were higher than those of the high- and low-affinity human liver enzymes in this study.

The CL_int values for E17G formation in the recombinant UGTs was calculated using the enzyme kinetic parameters reported by Itäaho et al.,²⁷⁾ where the relative protein expression levels of UGT isoforms were determined using anti-histidine (His)-tag antibodies. The CL_int values of the recombinant UGT2B7, UGT2B17, UGT1A4, and UGT1A3 were 105.9, 28.7, 1.32, and 1.29 µL/min/mg protein, respectively. Concerning the catalytic efficiency, it is speculated that E17G was formed mainly by UGT2B7 and slightly by UGT1A4 and UGT2B17. However, the UGT isoforms catalyzing E17G formation in the human liver could not be conclusively determined in the present study. In the human small intestine, the UGT2B17 content was the highest (112 pmol/mg, 60% of intestinal UGT proteins), followed by UGT1A10 and UGT2B7 (17.9 and 15.7 pmol/mg, 9 and 8%, respectively).²⁵⁾ The K_m values of E17G formation by the high- and low-affinity enzymes in the human small intestine were similar to those of the recombinant UGT1A10 and UGT2B17 (2.63±1.08 and 11.4±2.46 µM, respectively).²⁷⁾ Although there was a slight difference in the K_m values between the present and previous²⁷⁾ reports, it is possible that UGT1A10, UGT2B7, and UGT2B17 may be responsible for E17G formation in the human small intestine.

Furthermore, these results indicate that the tissue-specific expression profile of UGT isoforms caused the kinetic differences observed between the liver and small intestine. A previous study reported that only 3 UGT isoforms were detected at the protein level in the kidney, and their expression was in the following descending order of magnitude: UGT1A9>UGT2B7>UGT1A6.²⁵⁾ Recombinant UGT1A6 and UGT1A9 did not exhibit detectable E2 17-glucuronidation activities.²⁷⁾ The K_m value in a previous study of E17G formation by recombinant UGT2B7²⁷⁾ was 3.4-fold higher than the S₅₀ value of the human kidney microsomes in the present study. Moreover, E17G formation by the recombinant UGT2B7 did not fit allosteric kinetics.²⁷⁾ These results suggest that the isoform involved in E17G formation in the kidney may not be UGT1A9, UGT2B7, or UGT1A6.

In rats, Ugt1a2, Ugt2b2, and Ugt2b3 have been reported to be responsible for E17G formation.¹⁵⁾ Ugt2b2 and Ugt2b3 have been reported to be highly expressed in the liver,¹⁶⁾ suggesting that these isoforms may be associated with E17G formation in rat liver. Ugt1a2 mRNA was not expressed in the liver or kidney but was found predominantly in the small intestine.¹⁶⁾ This observation indicates that Ugt1a2 participates in E17G formation in the small intestine.

In mice, the Eadie–Hofstee plots for E17G formation exhibited biphasic kinetics in the 3 investigated tissues (Fig. 3), suggesting that E17G formation was catalyzed by more than two Ugt isoforms in the mouse liver, small intestine, and kidney. It has been reported that Ugt2b1 and Ugt2b5 are the major isoforms responsible for E17G formation in mice,²⁸⁾ and that these isoforms are highly and exclusively expressed in mouse liver,^29,30) indicating that Ugt2b1 and Ugt2b5 may catalyze E2 17-glucuronidation in mouse liver.

In conclusion, the present study demonstrated that species and tissue differences exist in E2 17-glucuronidation. Furthermore, our results suggest that E2 17-glucuronidation in humans occurs not only in the liver but also in the extrahepatic tissues and shows a distinct profile compared to that of rats and mice. Moreover, the E2 concentration and its pharmacological effect in extrahepatic tissues may be overestimated in attempts to extrapolate animal data to humans. Finally, these findings have contributed valuable information in elucidating the pharmacokinetics and pharmacological effects of E2 in extrahepatic tissues.

Acknowledgments

This work was supported in part by the Japan Society for the Promotion of Science (JSPS) KAKENHI Grant Numbers 25460200 and 16K08385.

Conflict of Interest

The authors declare no conflict of interest.

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