Involvement of Bmal1 and Clock in Bromobenzene Metabolite-Induced Diurnal Renal Toxicity

Abstract

Circadian rhythms are endogenous oscillators that regulate 24 h behavioral and physiological processes. Our previous investigation demonstrated that bromobenzene metabolite (4-bromocatechol: 4-BrCA) exhibited chronotoxicity (i.e., the nephrotoxicity induced by 4-BrCA was observed during the dark phase, while not observed at light phase in mice). However, the molecular mechanism is still unknown. The aim of the present study is to investigate the cellular molecule(s) involved in the 4-BrCA-induced nephrotoxicity using mouse renal cortex tubular cell lines (MuRTE61 cells). We found that 4-BrCA showed dose dependent (0.01–1 mM) cell proliferation defect in MuRTE61 cells. By treating with 0.03 mM 4-BrCA, we demonstrated that major clock genes (Bmal1, Clock, Cry1, Cry2, Per1, and Per2) were significantly downregulated. Interestingly, the expression levels of two genes, Bmal1 and Clock, continued to decrease after 3 h of treatment with 4-BrCA, while Cry1, Per1, and Per2 were unchanged until 24 h of treatment. Moreover, BMAL1 and CLOCK levels are higher at light phase. We speculated that BMAL1 and CLOCK might function defensively against 4-BrCA-induced nephrotoxicity since the expression levels of Bmal1 and Clock were rapidly decreased. Finally, overexpression of Bmal1 and Clock restored 4-BrCA-induced cell proliferation defect in MuRTE61 cells. Taken together, our results suggest that Bmal1 and Clock have protective roles against 4-BrCA-induced nephrotoxicity.

INTRODUCTION

Circadian rhythms are endogenous oscillators that regulates 24 h behavioral and physiological processes. For example, blood pressure is known to exhibit a circadian rhythm.¹⁾ Disease frequency is also known to affect circadian rhythm since there are higher occurrences of myocardial ischemia in the morning and asthma at night.²⁾ The circadian clock is thought to play a crucial role in these diurnal variations. At the molecular level, the expression of clock genes is controlled by two clock genes, muscle arnt-like1 (Bmal1) and circadian locomotor output cycles kaput (Clock). These two clock genes form a heterodimer and work as a set of transcription activators. Activation of BMAL1/CLOCK induces the protein levels of repressors such as PERIOD1, 2 (PER1, 2) and CRYPTOCHROME1, 2 (CRY1, 2), that in turn inhibit their own transcription by binding with the BMAL1/CLOCK heterodimer. It is well known that the pharmacokinetics of medications, such as anticancer drugs and hypoglycemic drugs, alter the treatment time.^3,4) In addition, acetaminophen-induced hepatic injury was modulated by several clock-gene knockout mice.^5–7) Therefore, it is important to consider treatment time and focus on the clock genes.

We have developed the chronotoxicology considering the relationship between injection time and the toxic severity of medicines or chemicals.^8–13) For example, we demonstrated that streptomycin, which induces renal toxicity, showed clear diurnal validation in mice.¹²⁾ Streptomycin induced renal injury during the dark phase, but did not induce any damage when administrated during the light phase. We also reported that cadmium-induced chrono-toxic effects were drastically different.⁸⁾ Cadmium showed strong hepatic injury during light-phase administration, but did not induce hepatotoxicity when administrated during the night; the difference in the severity of hepatotoxicity was “all or none.” Since the toxic peaks of streptomycin and cadmium are opposite times, it is important to understand the circadian sensitivity in each medicine or chemical.

We previously showed bromobenzene (BB), which is known to induce hepatic and renal toxicity,^14,15) exhibited severe toxicity during the light phase than the dark phase.⁹⁾ Additionally, injection of BB-metabolites such as 4-bromocatechol (4-BrCA) confirmed renal dysfunction during the dark phase.¹¹⁾ Although our previous studies have shown that BB and BB-metabolites exhibited diurnal variation, the cellular molecule(s) involving this chronotoxicity have not been evaluated. Therefore, in the present study, we investigated the diurnal mechanism of 4-BrCA-induced renal toxicity in MuRTE61 cells, a murine renal cortex tubular cell line.

MATERIALS AND METHODS

Cell Proliferation Assay

MuRTE61 cells were obtained from p53 knockout mice according to our previous experiment¹⁶⁾ and maintained in a Renal Epithelial Cell Growth Medium (REGM) BulletKit (CC-3190: Lonza, Visp, Swiss) at 37 °C in a humidified atmosphere with 5% CO₂. MuRTE61 cells were plated in 96-well plates at a density of 10000 cells per well. After 24 h of seeding, various concentrations (0.01–1 mM) of 4-BrCA (Tokyo Kasei Co., Ltd., Tokyo, Japan) were treated. After 24 h of the 4-BrCA treatment, the cell viability was evaluated using Alamar Blue (Bio-Rad Laboratories, Hercules, CA, U.S.A.).

Plasmid Construction

The progress of plasmid construction is described in Supplementary Fig. S1. The cDNA of the coding region of the Bmal1 (NM_007489) gene was amplified from the liver of male C57BL/6J mice using the Expand High Fidelity PCR System (Roche, Basel, Switzerland), and then subcloned into a pANT vector (NIPPON Gene, Tokyo, Japan) and named pANT-Bmal1. The Bmal1 fragment was amplified with specific primer sets (forward primer [5′-ATGGCGGACCAGAGAATGGAC-3′] and M13 reverse primer [5′-GGAAACAGCTATGACCATGA-3′] using KOD-one (Toyobo, Osaka, Japan). The amplified Bmal1 fragment was restricted with XbaI at 37 °C for 1 h. pcDNA3.1-neo was digested with EcoRV and XbaI at 37 °C for 1 h. The Bmal1 fragment was subcloned into the pcDNA3.1-neo fragment using Ligation high Ver.2 (Toyobo); this construct was named pcDNA3.1-neo-Bmal1. The cDNA of the coding region of the Clock (NM_007715) gene was amplified from the liver of male C57BL/6J mice using the Expand High Fidelity PCR System, and then subcloned into a pANT vector and named pANT-Clock-F1 (389-1986) and pANT-Clock-F2 (1967-2956). Two fragments of Clock were amplified with specific primer sets (F1:forward primer [5′-GCGTTTAAACTTAAGATGGTGTTTACCGTAAGCTG-3′] and reverse primer [5′-GTCATCTTTTCATGAGCTGGCA-3′]; F2: forward primer [5′-AAAGACCAGCTAGAGCAGCG-3′] and reverse primer [5′-AACGGGCCCTCTAGA CTACTGTGGCTGGACCTTGG-3′]) using KOD-one. The pcDNA3.1 inverse PCR fragment was amplified using a specific forward primer (5′-TCTAGAGGGCCCGTTTAAAC-3′) and reverse primer (5′-CTTAAGTTTAAACGCTAGCCAG-3′). The Clock fragments were subcloned into the pcDNA3.1-neo fragment using the In-Fusion HD Cloning Kit (TaKaRa Bio, Shiga, Japan); this construct was named pcDNA3.1-neo-Clock.

Rescue Experiment

MuRTE61 cells were plated onto 96-well plates at a density of 10000 cells per well and transfected with 100 ng pcDNA3.1-neo, 50 ng pcDNA3.1-neo and 50 ng pcDNA3.1-neo-Bmal1, 50 ng pcDNA3.1-neo and 50 ng pcDNA3.1-neo-Clock, or 50 ng pcDNA3.1-neo-Bmal1 and 50 ng pcDNA3.1-neo-Clock after 3 h of seeding. After 24 h of transfection, cells were treated with 0.03 mM 4-BrCA followed by evaluation of cell numbers after 24 h of 4-BrCA treatment. The construction of plasmids is described above.

Quantitative RT-PCR

MuRTE61 cells were plated at a density of 300000 cells per 35-mm dish. After 24 h of cell seed, they were treated with 0.03 mM 4-BrCA or dimethyl sulfoxide (DMSO). After 3, 6, 12, or 24 h of treatment, total RNA was extracted using the RNA Basic Kit (NIPPON Genetics, Tokyo, Japan), according to the manufacturer’s protocol. The procedure for reverse transcription, PCR conditions, and primer sequences have been described previously.⁴⁾

Western Blot Analysis

MuRTE61 cells were plated at a density of 150000 cells per 35 mm dish and transfected with 2 µg pcDNA3.1-neo, 1 µg pcDNA3.1-neo-Bmal1 and 1 µg pcDNA3.1-neo, 1 µg pcDNA3.1-neo-Clock and 1 µg pcDNA3.1-neo, or 1 µg pcDNA3.1-neo-Bmal1 and 1 µg pcDNA3.1-neo-Clock using TransIT-LT1 Transfection Reagent (TaKaRa Bio) after 24 h of seeding. Twenty-four hours after the transfection, cells were treated with 0.03 mM 4-BrCA or DMSO. After 24 h, cells were homogenized with 100 µL ice-cold radio immunoprecipitation assay (RIPA) buffer (Nacalai Tesque, Kyoto, Japan) containing a protease inhibitor and centrifuged (18000 × g for 20 min at 4 °C). Protein samples (10 µg) were separated by 10% sodium dodecyl sulfate (SDS)- polyacrylamide gel electrophoresis (PAGE), and transferred to polyvinylidene difluoride (PVDF) membrane. The primary antibodies used were as follows: mouse BMAL1 monoclonal antibody, mouse CLOCK monoclonal antibody, and mouse glutathione peroxidase 4 (GPX4) monoclonal antibody (Santa Cruz Biotechnology, Dallas, TX, U.S.A.), and mouse β-actin monoclonal antibody (MBL, Aichi, Japan). A peroxidase-conjugated anti-mouse immunoglobulin G (IgG) was used as a secondary antibody (1 : 5000 dilution; Cell Signaling Technology, Beverly, MA, U.S.A.).

Measurement of Total Glutathione (GSH) Levels

MuRTE61 cells were plated at a density of 150000 cells per 35 mm dish and transfected with 2 µg pcDNA3.1-neo, 1 µg pcDNA3.1-neo-Bmal1 and 1 µg pcDNA3.1-neo, 1 µg pcDNA3.1-neo-Clock and 1 µg pcDNA3.1-neo, or 1 µg pcDNA3.1-neo-Bmal1 and 1 µg pcDNA3.1-neo-Clock using TransIT-LT1 Transfection Reagent after 24 h of seeding. Twenty-four hours after the transfection, cells were treated with 0.03 mM 4-BrCA or DMSO. After 24 h, cells, MuRTE61 cells were scraped in PBS and measured cell numbers. We added 10 mM hydrochloric acid to cell palette and stored −80 °C. After we repeated freezing and thawing twice, we added 5% sulfosalicylic acid and centrifuged at 8000 × g for 10 min at 4 °C. Supernatants were assayed for the total GSH using the Total Glutathione Quantification Kit (Dojindo Laboratories, Kumamoto, Japan) according to the manufacture’s instruction and previously described.^17–19)

Statistical Analysis

Multiple comparisons were made using the post-hoc Tukey’s test. All statistical analyses were performed using SPSS 24.0 software (IBM, Chicago, IL, U.S.A.). Values of p < 0.05 were considered statistically significant.

RESULTS

First, we performed the cell proliferation assay using MuRTE61 cells. Treatment with 4-BrCA for 24 h exhibited a dose-dependent reduction in the proliferation of MuRTE61 cells (Fig. 1). Since significant inhibition was observed at least 0.03 mM 4-BrCA treatment, we selected 0.03 mM 4-BrCA in the following experiment. To explore the mechanism of 4-BrCA-induced diurnal toxicity, we measured six kinds of clock gene (Bmal1, Clock, Cry1, Cry2, Per1, and Per2) expression levels after 4-BrCA treatment. We found that the expression levels of all the clock genes were downregulated following the treatment of MuRTE61 cells with 4-BrCA at 24 h (Fig. 2). Among them, Bmal1 and Clock expression levels continued to decrease from the 3 h treatment with 4-BrCA (Figs. 2A, B), while Cry1, Per1, and Per2 were not changed until the 24 h treatment (Figs. 2C, E, F). The expression level of Cry2 was downregulated from 12 h of treatment (Fig. 2D). A recent study reported that clock gene disruption was associated with diseases such as chronic obstructive pulmonary disease and obstructive sleep apnea.²⁰⁾ Alifu et al. showed that Cry1a, Cry2a, and Per2 regulate the oxidative status in zebrafish.²¹⁾ Moreover, we also demonstrated that zinc-induced toxicity was protected by overexpression of Per2 in Hepa1–6 cells.¹³⁾ These data suggest that clock genes act as antioxidant proteins^22,23) and Bmal1/Clock expression levels affect 4-BrCA-induced diurnal renal toxicity.

Fig. 1. Influence of 4-BrCA Treatment on Cell Proliferation of MuRTE61 Cells

MuRTE61 cells were plated at a density of 10000 cells onto 96-well plates. After 24 h of cell seeding, cells were treated with 4-BrCA (0.01–1 mM). The proliferation of cells was measured 24 h after the 4-BrCA treatment. Data are plotted as mean ± standard deviation (S.D.) of groups. ** p < 0.01 and *** p < 0.001 versus control (n = 6).

Fig. 2. Influence of 4-BrCA on Expression Levels of Six Clock Genes in MuRTE61 Cells

MuRTE61 cells were plated at a density of 300000 cells per 35 mm dish. After 24 h of cell seeding, cells were treated with 4-BrCA (0.03 mM). After 3, 6, 12, and 24 h of the 4-BrCA treatment, the expression levels of clock genes (Bmal1, Clock, Cry1, Cry2, Per1, or Per2) were measured by RT-PCR. Data are plotted as mean ± S.D. of groups. ** p < 0.01 and *** p < 0.001 versus control at same time (n = 4).

To investigate this hypothesis, we investigated the protective effects of BMAL1 and CLOCK against 4-BrCA-induced toxicity in MuRTE61 cells. We showed that transfection of Bmal1 or Clock upregulated each gene (Figs. 3A, B). As shown in Fig. 4A, 4-BrCA treatment markedly reduced the proliferation of MuRTE61 cells, whereas overexpression of Bmal1 or Clock partially reversed the 4-BrCA-induced toxicity. Moreover, combination of Bmal1 and Clock transfection completely rescued the suppressed cell proliferation under 4-BrCA conditions (Fig. 4A). These results indicated that overexpression of Bmal1 and Clock modulated 4-BrCA-induced nephrotoxicity by protecting the disruption of Bmal1 and Clock. Since 4-BrCA was reported to deplete GSH levels in the mouse kidney,²⁴⁾ we also estimated the GSH levels in MuRTE61 cells. We found that treatment with 4-BrCA significantly decreased the total GSH levels in the MuRTE61 cells (Fig. 4B). In addition, we demonstrated that overexpression of Bmal1 and Clock protected glutathione downregulation against 4-BrCA treatment (Fig. 4B). Finally, since our previous study demonstrated the GPX4 downregulation by 4-BrCA injection,¹¹⁾ we tested the participation of GPX4, an indicator of ferroptosis. We found that the treatment with 4-BrCA decreased GPX4 level in MuRTE61 cells. In contrast, overexpression of Bmal1 or Clock partially sustained the GPX4 level (Fig. 4C). Moreover, GPX4 level was not changed by overexpression of Bmal1 and Clock against 4-BrCA treatment (Fig. 4C). Taken together, Bmal1 and Clock may involve in protect the 4-BrCA-induced ferroptosis.

Fig. 3. Transfection of Bmal1 and Clock in MuRTE61 Cells

(A, B) MuRTE61 cells were plated at a density of 300000 cells per 35-mm dish. After 24 h of cell seeding, cells were transfected with 2 µg of plasmids (control, Bmal1, or Clock). The expression levels of Bmal1 (A) and Clock (B) were measured by RT-PCR or Western blotting at 24 h after the transfection.

Fig. 4. Protective Effect of Bmal1 and Clock against 4-BrCA-Induced Cell Proliferation Inhibition in MuRTE61 Cells

(A) MuRTE61 cells were plated at a density of 10000 cells onto 96-well plates and transfected with 50–100 ng plasmids (control, Bmal1, Clock, or Bmal1 and Clock) after 3 h of seeding. After 24 h of transfection, each cell was treated with 0.03 mM of 4-BrCA, followed by the measurement of the proliferation of MuRTE61 cells at 24 h after the treatment. Data are plotted as mean ± S.D. of groups. ** p < 0.01 and *** p < 0.001 (n = 6). (B) MuRTE61 cells were plated at a density of 150000 cells per 35 mm dish and transfected 2 µg plasmids (control, Bmal1, Clock, or Bmal1 and Clock) after 24 h of seeding. After 24 h of cell transfection, each cell was treated with 0.03 mM 4-BrCA or DMSO, followed by measurement of the total GSH levels of MuTE61 cells for 24 h after treatment. Data are plotted as mean ± S.D. of groups. * p < 0.05 and ** p < 0.01 (n = 3). (C) MuRTE61 cells were plated at a density of 150000 cells per 35 mm dish and transfected 2 µg plasmids (control, Bmal1, Clock, or Bmal1 and Clock) after 24 h of seeding. After 24 h of cell transfection, they were treated with 0.03 mM 4-BrCA or DMSO for 24 h. We extracted each protein using RIPA buffer and detected GPX and β-actin by Western blot analysis.

DISCUSSION

BB is an industrial solvent that causes necrosis of the liver and kidneys. BB metabolites bioactivated by CYP1A2, CYP1B1, and CYP2E1 are thought to induce toxicity.²⁵⁾ Among the BB metabolites, 4-BrCA, 3-bromophenol, and bromohydroquinone were reported to induce nephrotoxicity.^24,26) We previously demonstrated that 4-BrCA administration during the dark phase downregulated GPX4 levels, as an indicator of ferroptosis, in mice.¹¹⁾ Ferroptosis was identified as a form of cell death, which inhibits the import of cystine, leading to GSH depletion and inactivation of GPX4.²⁷⁾ GSH plays a crucial role in maintaining health by protecting against toxic compounds. Many biological factors including GSH are known to show the 24 h rhythmicity.²⁸⁾ 4-BrCA was reported to deplete renal GSH levels.²⁴⁾ We also demonstrated that GSH levels in the kidneys were different during the entire 24 h period (Supplementary Fig. S2). Since GSH has a crucial role for antioxidant status, it is feasible that GSH-related proteins or genes may be associated with diurnal toxicity.

In addition to GSH, clock genes such as Bmal1, Cry1, Cry2, and Per2 have been suggested to have antioxidant properties.^21,29,30) Moreover, Li et al. reported dose-dependent suppression of BMAL1 and CLOCK by treatment with cigarette smoke extract in human bronchial epithelium cells and overexpression of BMAL1 and CLOCK suppressed the influence of cigarette smoke extract.²⁰⁾ Since we and other researchers showed Bmal1 and Clock at ZT2 is much higher than that of ZT14^13,31) (Supplementary Fig. S3), we hypothesized that diurnal variation in the antioxidant or Bmal1/Clock genes might have the potential to display our diurnal variation. Our results demonstrated that the expression levels of six clock genes were repressed by treatment with 4-BrCA at 24 h. The transcription factors, CLOCK and BMAL1, form the positive limb of the feedback loop, act in the form of a heterodimer, and activate the transcription of core clock genes and clock-controlled genes, harboring E-box elements within their promoters. The core clock genes PER and CRY1/2, which are transcriptional repressors, form the negative limb of the feedback loop. PER/CRY heterodimers interact with the CLOCK/BMAL1 heterodimers, and inhibit their activity, thereby negatively regulate their expressions. Notably, 4-BrCA rapidly downregulated the expression levels of transcription factors (Bmal1 and Clock) followed by downregulation of the expression levels of transcriptional repressors (Cry1, Cry2, Per1, and Per2). Since recent study reported that disruption of the clock gene is an initial manifestation of several diseases such as hypertension and inflammatory bowel diseases,^32,33) we hypothesized that disruption of clock gens expressions might lead to renal injury. We demonstrated that overexpression of Bmal1 or Clock partially protected 4-BrCA-induced toxicity. Moreover, overexpression of both Bmal1 and Clock completely protected 4-BrCA-induced cell proliferation inhibition. These results indicated that heterodimer of BMAL1 and CLOCK or homodimer of BMAL1 and CLOCK affected the 4-BrC-induced toxicity. Although it is still unclear how BMAL1 and/or CLOCK protects 4-BrC-induced toxicity, we have newly demonstrated that disruption of BMAL1 and CLOCK may induce 4-BrCA-induced toxicity. Since the data are derived from an in vitro test, our present study has some limitations. In the future, we need to monitor Bmal1 and Clock expression levels in vivo and use Bmal1- and Clock-knockout mice.

In conclusion, our present study demonstrated that 4-BrCA-induced renal toxicity might be downregulated with the expression levels of clock genes, Bmal1 and Clock, in MuRTE61 cells. Our present research provides valuable information related to chronotoxicology.

Acknowledgments

This work was supported by JSPS KAKENHI Grant Numbers JP22K15328, JP20K10416, and JP20K21733.

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

This article contains supplementary materials.

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