Hepatic GSTP1/2 Exhibits High Sensitivity to Testosterone-Mediated Regulation in Mice

Abstract

Glutathione S-transferases (GSTs) are essential phase II detoxification enzymes encoded by a diverse gene superfamily. Among them, the pi class (GSTP) includes 2 isozymes, GSTP1 and GSTP2, which share a high degree of sequence similarity. In mice, hepatic GSTP1/2 expression is higher in males than in females. To investigate the regulatory mechanisms underlying this sex difference, we examined orchiectomized mice treated with testosterone. Orchiectomy reduced hepatic GSTP1/2 expression and associated enzyme activity, both of which were restored following testosterone administration. To assess the sensitivity of GSTP1/2 to testosterone fluctuations, we compared mice experiencing a serum testosterone surge with those maintaining baseline levels. Mice with elevated testosterone exhibited increased hepatic GSTP1/2 protein expression and enzyme activity, demonstrating the high responsiveness of GSTP1/2 to testosterone. To our knowledge, this is the first study to demonstrate that testosterone surges regulate both the expression and enzymatic function of a specific protein. These findings underscore testosterone’s critical role in the male-dominant expression of GSTP1/2 and highlight its sensitivity to physiological fluctuations in testosterone levels. Further studies are warranted to elucidate the molecular mechanisms by which testosterone surges influence gene expression.

INTRODUCTION

In mice, cytosolic glutathione S-transferases (GSTs) are encoded by a gene superfamily that is divided into 7 classes, including the pi class.¹⁾ This class consists of 2 isoforms, GSTP1 and GSTP2, which share a high degree of sequence similarity, differing by only 6 amino acids out of 210. The hepatic GSTP1/2 expression is sexually dimorphic, with significantly higher levels in males than in females.²⁾ Although both sexes exhibit low GSTP1/2 expression during early development, a male-biased expression pattern emerges at puberty.³⁾ The sexually dimorphic expression is known to be mediated by testosterone⁴⁾ as well as by the sex-specific release pattern of growth hormone (GH).³⁾ In male mice, GH secretion is intermittent, whereas in females, it occurs in a nearly continuous manner.

GSTPs play key roles in cellular defense and signaling regulation. They are particularly effective at detoxifying electrophilic α, β-unsaturated aldehydes, which are reactive byproducts of oxidative stress and lipid peroxidation.⁵⁾ Beyond detoxification, GSTP1 also regulates cell growth and apoptosis through its interactions with key signaling pathways involving c-Jun N-terminal kinase⁶⁾ and apoptosis signal-regulating kinase 1.⁷⁾

GST activity in animal tissues is typically assessed using substrates that react with glutathione (GSH) in the presence of GST enzymes. Among these, 1-chloro-2,4-dinitrobenzene (CDNB) is widely used because it detects activity across multiple GST isoforms, while ethacrynic acid (EA) serves as a GSTP-specific substrate.⁸⁾

Testosterone is the primary male sex hormone that is essential for sexual development, masculinization, and fertility. It exerts its effects through the androgen receptor (AR), a ligand-dependent nuclear transcription factor. Testosterone is released episodically in mammalian males, with highly variable intervals between surges.^9–12) Notably, these fluctuations are more pronounced in mice than in other species, including humans, bulls, rabbits, and rats.¹⁰⁾ In mice, testosterone remains at basal levels (approximately 1 ng/mL) for approximately 75% of the time, with spontaneous peaks occurring about 4 times daily, each lasting for approximately 1.5 h.¹³⁾ Despite these fluctuations, studies comparing gene expression between basal and peak testosterone levels remain limited, thereby leaving a gap in understanding the regulatory effects of testosterone dynamics.

This study investigates the regulation of male-preferential hepatic GSTP1/2 expression by testosterone. Specifically, we examined hepatic GSTP1/2 expression under 2 conditions: (1) in orchiectomized mice with (ORT) and without (OR) testosterone supplementation, and (2) in mice experiencing a testosterone surge versus those at basal levels. Our findings highlight the critical role of testosterone in driving male-dominant GSTP1/2 expression and reveal its sensitivity to physiological testosterone fluctuations.

MATERIALS AND METHODS

Mice

This study followed the NIH Guide for the Care and Use of Laboratory Animals and was approved by the Seoul National University IACUC (Approval No. SNU-220619-1-3). C57BL/6 mice (7–8 weeks old) were individually housed in pathogen-free cages with ad libitum access to food and water, and were acclimated for 2 weeks prior to experimentation.

Orchiectomy and Testosterone Administration

Surgical orchiectomy was performed following the procedure described by Valkenburg et al.¹⁴⁾ Anesthesia was induced via intraperitoneal injection of alfaxalone–xylazine–HCl (20 : 1; 5 mL/kg). Sham-operated controls (Sham) underwent the same surgical procedure without organ removal. Two weeks after surgery, OR were subcutaneously administered testosterone (20 μg/g; Wako, Osaka, Japan) or corn oil (vehicle; Otoki, Seoul, Korea) based on previous studies.^15,16) Injections were given 3 times at 2-d intervals, and mice were sacrificed 2 d after the final dose.

Serum Testosterone

Mice were sacrificed between 1:00 and 3:00 p.m. using an overdose of alfaxalone/xylazine–HCl (20 : 1). Blood was collected via cardiac puncture, stored at 4°C overnight, and centrifuged at 800 × g for 20 min at 4°C to obtain serum. Serum testosterone levels were measured using a rat/mouse testosterone assay kit (Immuno-Biological Laboratories, Minneapolis, MN, U.S.A.).

Western Blot

Western blot was performed as described previously.¹⁷⁾ Primary antibodies against GSTP1 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Proteintech, Rosemont, IL, U.S.A.) and horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin G secondary antibody (GenDEPOT, Katy, TX, U.S.A.) were used. Protein bands were visualized with a chemiluminescent detection kit (AbFrontier, Pohang, Korea), and densitometric analysis was conducted using ImageJ (v1.54, https://imagej.net/).

Quantitative Reverse Transcription PCR (RT-qPCR)

RT-qPCR was performed as described previously.¹⁷⁾ The expression levels of GSTP1/2 and GAPDH mRNA were quantified using specific primers: GSTP1/2: forward, 5′-TGGAAGGAGGAGGTGGTTACCA-3′ and reverse, 5′-GGTAAAGGGTGAGGTCTCCATC-3′; and GAPDH: forward, 5′-CATCACTGCCACCCAGAAGACTG-3′ and reverse, 5′-ATGCCAGTGAGCTTCCCGTTCAG-3′. The expression level of GSTP1/2 mRNA relative to GAPDH was determined using the ΔΔCt method.

GST Activities

GST enzyme activities were analyzed using CDNB and EA as substrates.¹⁸⁾ Liver tissues were homogenized in cold potassium phosphate buffer (100 mM, pH 6.8, with 2 mM ethylenediaminetetraacetic acid) and centrifuged at 10000 × g for 15 min at 4°C. The resulting supernatant was used for activity assays in a 200 μL reaction mixture containing 100 mM potassium phosphate buffer (pH 6.8), 1 mM GSH, and tissue homogenate protein. For CDNB, 5 μg of protein was used with absorbance measured at 340 nm, while for EA, 20 μg of protein was used with absorbance measured at 270 nm.

Statistics

Statistical analyses were performed using IBM SPSS Statistics version 26 (IBM, Armonk, NY, U.S.A.). Significance was evaluated using the Mann–Whitney U test, with a p-value of less than 0.05 considered significant.

RESULTS

Reduced Hepatic GSTP1/2 Expression Following Orchiectomy

To examine the impact of testosterone deficiency and supplementation on hepatic GSTP1/2 expression, we compared Sham, OR, and ORT (Fig. 1). Hepatic GSTP1/2 protein levels declined following orchiectomy, but were restored with testosterone supplementation (Fig. 1A). Similarly, GSTP1/2 mRNA levels decreased by 77.4% in OR compared to Sham, and were restored by 134% in ORT (p < 0.05; Fig. 1B). GST activity toward EA decreased by 19.8% in OR relative to Sham, and increased by 18.6% with ORT (p < 0.05; Fig. 1C). Total GST activity, assessed with CDNB, was reduced by 48.5% in OR compared to Sham, and increased by 44.2% in ORT relative to OR (p < 0.05; Fig. 1D). These findings underscore the essential role of testosterone in maintaining hepatic GSTP1/2 expression and activity.

Fig. 1. Reduced Hepatic GSTP1/2 Expression in OR Mice, Restored by ORT

(A) Western blot. (B) RT-qPCR, with GSTP1/2 mRNA levels normalized to GAPDH and expressed relative to the Sham group, set at 1.0. (C) GST activity toward EA. (D) GST activity toward CDNB. Bars represent the mean ± S.D. of independent samples (n = 4), with each circle indicating an individual data point. Asterisks (*) denote significant differences (p < 0.05) between the 2 treatment groups at either end of the horizontal lines. OR: orchiectomized mice; ORT: testosterone replacement; S.D.: standard deviation.

Variability in Serum Testosterone Levels

Serum samples were collected from 74 male mice to measure testosterone levels (Fig. 2). To minimize the effects of diurnal fluctuations, blood samples were collected within a narrow time window. The testosterone levels varied widely, ranging from 0.13 to 93.99 ng/mL, with a median of 5.44 ng/mL. Notably, 37% of the mice had testosterone levels below 1 ng/mL, indicating a notable subpopulation with low circulating hormone levels. For GSTP1/2 expression analyses, the 9 mice with the highest serum testosterone levels, ranging from 42.9 to 93.99 ng/mL, were designated as the high-testosterone (HT) group, while the 9 with the lowest levels, ranging from 0.13 to 0.43 ng/mL, comprised the low-testosterone (LT) group.

Fig. 2. High Individual Variation in Serum Testosterone Concentrations in Male Mice (n = 74)

The Y-axis is presented on a logarithmic scale. Each dot represents an individual measurement, and whiskers extend to the minimum and maximum observed values.

Elevated Hepatic GSTP1/2 Expression in HT Mice

We assessed the impact of serum testosterone on hepatic GSTP1/2 by comparing the HT and LT groups (Fig. 3). GSTP1/2 protein levels were 113% higher in HT mice compared to LT mice (p < 0.05; Figs. 3A, 3B), accompanied by a 114% increase in GSTP1/2-associated enzymatic activity (p < 0.05; Fig. 3C). Consistently, serum testosterone levels showed a moderate positive correlation with hepatic GST activity toward EA in the HT and LT groups (Spearman’s coefficient = 0.497, p < 0.05; Supplementary Fig. S1). In contrast, GSTP1/2 mRNA levels and the total GST activity did not differ significantly between the groups (Figs. 3D, 3E).

Fig. 3. Hepatic GSTP1/2 Expression in Male Mice with HT and LT Levels

(A) Western blot. (B) Densitometric quantification of Western blot signals for GSTP1/2 protein, normalized to GAPDH and expressed relative to the HT group, set at 1.0. (C) GST activity toward EA. (D) RT-qPCR, with GSTP1/2 mRNA levels normalized to GAPDH and expressed relative to the HT group, set at 1.0. (E) GST activity toward CDNB. Serum testosterone levels in the HT and LT groups ranged from 42.9 to 93.99L and 0.13 to 0.43 ng/mL, respectively, as shown in Fig. 2. Bars represent the mean ± S.D. of independent samples (n = 5–9), with each circle indicating an individual data point. Asterisks (*) denote significant differences (p < 0.05) between the 2 groups. HT: high serum testosterone; LT: low serum testosterone.

DISCUSSION

Previous studies have demonstrated the male-dominant hepatic expression of GSTP1/2 and its dependence on testosterone in mice.^2,3) The reduced hepatic GSTP1/2 expression observed in OR (Fig. 1) aligns with these findings.⁴⁾ The restoration of GSTP1/2 expression following the testosterone treatment further underscores the regulatory role of testosterone. Additionally, the decline in GSTP1/2-related enzyme activity and its recovery upon testosterone treatment may be linked to changes in the hepatic GSTP1/2 expression levels. We also observed that bicalutamide, an AR inhibitor, downregulates GSTP1/2 expression and its associated enzyme activity in mice (Supplementary Fig. S2), likely due to its inhibitory effects on AR.

To assess the GSTP1/2 sensitivity to testosterone fluctuations, we compared mice experiencing transient serum testosterone surges with those maintaining baseline levels (Fig. 3). The mice with elevated testosterone levels exhibited increased hepatic GSTP1/2 protein expression and enhanced enzyme activity. Consistently, serum testosterone levels showed a moderate positive correlation with hepatic GSTP1/2 activities in HT and LT groups (Supplementary Fig. S1). These findings suggest that GSTP1/2 expression in liver tissues is highly responsive to acute testosterone fluctuations. To our knowledge, this is the first study to demonstrate that transient testosterone surges can regulate both protein expression and enzymatic activity.

Notably, the observed changes in GSTP1/2 protein levels were not correlated with the mRNA expression in the HT and LT groups (Fig. 3), suggesting the involvement of post-transcriptional regulation. Steroid hormone-mediated post-transcriptional regulation has been previously documented. For example, testosterone enhances fibroblast growth factor-2 translation in mouse testes via an internal ribosome entry site-dependent mechanism.¹⁹⁾ Dihydrotestosterone increases AR protein levels in human granulosa-like KGN cells without affecting AR mRNA expression.²⁰⁾ Additionally, 17β-estradiol has been shown to reduce GSTP protein levels in rat hepatocytes without altering its mRNA expression.²¹⁾ These findings support the notion that transient testosterone surges may regulate GSTP1/2 expression primarily through post-transcriptional mechanisms, particularly given that each surge lasts for approximately 1.5 h.¹³⁾ In contrast, the upregulation of both GSTP1/2 mRNA and protein observed in testosterone-treated OR (Fig. 1) may reflect the longer-term effects of testosterone exposure.

Previous studies have reported substantial variability in serum or plasma testosterone levels among male mice maintained under similar conditions.^11,12) In agreement with this, we found considerable inter-individual variation in serum testosterone levels among singly-housed male mice sacrificed within a narrow time window (Fig. 2). These findings suggest that synchronizing testosterone oscillations cannot be achieved solely by optimizing housing conditions or accounting for diurnal variations.

Even under strictly-controlled husbandry conditions and experimental protocols, considerable variation in pharmacological and toxicological outcomes is frequently observed. If testosterone surges influence the expression of metabolic enzymes such as GSTPs, which in turn affect pharmacological and toxicological responses, this mechanism could account for some of the observed variability in animal studies.

GH regulates GSTP expression via the Janus kinase/signal transducer and activator of transcription (STAT) pathway, with STAT5 activation modulating gene transcription in a tissue-specific manner.²²⁾ In male mice, GH secretion is intermittent, whereas in females, it occurs in a nearly continuous manner. These distinct secretion patterns regulate sex-biased liver gene expressions, including that of GSTP1.²³⁾ The regulation of GH secretion is established by gonadal steroids during the neonatal period and becomes evident at puberty.³⁾ The continuous presence of testosterone is essential for maintaining low basal GH levels.²⁴⁾ Given these findings and the observed effects of the testosterone surge on GSTP1/2 expression, future research should explore the potential interplay between GH and testosterone in regulating GSTP1/2 expression.

Taken together, these findings highlight testosterone’s role in male-dominant hepatic GSTP1/2 expression and its sensitivity to testosterone fluctuations. Further investigations are warranted to elucidate how transient testosterone surges regulate genes involved in drug metabolism and toxicity, and understand the broader implications of these regulatory mechanisms.

Acknowledgments

This work was supported by the Research Institute for Veterinary Science, Seoul National University.

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

This article contains supplementary materials.

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