Copper-induced diurnal hepatic toxicity is associated with Cry2 and Per1 in mice.

BACKGROUND: This study aimed to investigate diurnal variations in copper-induced hepatic toxicity and the molecular mechanisms underlying this chronotoxicity. METHODS: Male C57BL/6J mice were intraperitoneally injected with copper chloride (CuCl2) at zeitgeber time 2 (ZT2) or 14 (ZT14), twice per week for 5 or 8 weeks. Seventy-two hours after the final CuCl2 injection, the mice were euthanized, and plasma samples were collected. The livers and kidneys were collected and weighed. In vitro experiments were performed to assess cell viability and fluctuations in clock gene expression levels in Hepa1-6 cells after CuCl2 treatment. We examined copper homeostasis- and apoptosis-related genes under clock genes overexpression. RESULTS: Repeated CuCl2 administration for 8 weeks resulted in more severe toxicity at ZT14 compared to ZT2. CuCl2 administration at ZT14 elevated plasma aspartate aminotransferase, hepatic tumor necrosis factor-α, and interleukin-6 for 5 weeks, whereas the toxic effects of CuCl2 administration at ZT2 were weaker. Moreover, CuCl2 treatment inhibited Hepa1-6 cell viability in a dose-dependent manner. We observed increased expression of three clock genes (Ciart, Cry2, and Per1) after CuCl2 treatment. Among them, overexpression of Cry2 and Per1 accelerated CuCl2-induced inhibition of Hepa1-6 cell viability. Moreover, we found that the overexpression of Cry2 and Per1 regulates cleaved caspase-3 by modulating the copper transporter genes ATP7B and CTR1. CONCLUSION: These results suggest that CuCl2-induced diurnal toxicity is associated with Cry2 and Per1 expression through the regulation of copper transporter genes in mice.

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

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.

Involvement of Npas2 and Per2 modifications in zinc-induced acute diurnal toxicity in mice.

Zinc (Zn) is one of the most essential trace elements in the body and an integral part of many enzyme systems. Zn deficiency is characterized by growth retardation, loss of appetite, and impaired immune function. In contrast, Zn overdoses can be associated with liver, kidney, and stomach damage. We focused on the "chronotoxicity," or the relationship between injection time and severity of chemical toxicity. The aim of this study was to investigate the chronotoxicity of Zn and the in vivo factors involved. Seven-week-old male ICR mice were administered Zn at six different time points per day (zeitgeber time [ZT]: ZT2, ZT6, ZT10, ZT14, ZT18, and ZT22). Mortality was monitored for 7-days after administration. The mice were tolerant to Zn administered at ZT2 and ZT6, and were highly sensitive at ZT14 and ZT18. Furthermore, when mice were administered a non-lethal dose of Zn, the levels of hepatic injury indicators (AST and ALT) were much higher at ZT14 than at ZT2. To explore the mechanism of Zn-induced diurnal hepatotoxicity, we performed an in vitro experiment, focusing on the clock genes. We found that Zn downregulated the expression levels of several clock genes, neuronal PAS domain protein 2 (Npas2) and Peroid2 (Per2), in Hepa1-6 cells. Interestingly, overexpression of both Npas2 and Per2 restored Zn-induced toxicity in Hepa1-6 cells. Since NPAS2 and PER2 are known to modulate the hepatic injury induced by carbon tetrachrolide or acetaminophen, our results suggest that Zn-induced diurnal toxicity may be associated with modulation of Npas2 and Per2 gene expression.

Different Renal Chronotoxicity of Bromobenzene and Its Intermediate Metabolites in Mice.

Bromobenzene (BB) is known to pose a serious threat to human health. We previously demonstrated that BB showed chronotoxicity, that is, daily fluctuations in the severity of hepatotoxicity induced in mice. Although BB showed mild nephrotoxicity, a daily fluctuation was not observed in this toxicity. This might be attributed to the fact that BB-induced chronotoxicity is observed only in the liver and not in the kidneys and that the damage caused by BB is prominent in the liver, masking the daily fluctuation in nephrotoxicity. To confirm these two possibilities, we examined the daily fluctuations in nephrotoxicity due to BB intermediate metabolites that target the kidneys: 3-bromophenol, bromohydroquinone, and 4-bromocatechol. Mice were injected with 3-bromophenol, bromohydroquinone, or 4-bromocatechol intraperitoneally at six different time points in a day (zeitgeber time (ZT): ZT2, ZT6, ZT10, ZT14, ZT18, or ZT22). Mortality was monitored for 7 d post-injection. Mice were more sensitive to the acute toxicity of these metabolites around at ZT14 (dark-phase) exposure than around at ZT2 (light-phase) exposure. Furthermore, mice administered with a non-lethal dose of 4-bromocatechol showed significant increases in the levels of plasma blood urea nitrogen and renal malondialdehyde at ZT14 exposure. Moreover, glutathione peroxidase-4, a ferroptosis indicator, was attenuated at ZT14 exposure. These results indicate the toxicity of BB metabolites was higher during the dark-phase exposure, and demonstrate the reason why the diurnal variation of nephrotoxicity by BB was not observed in our previous report is that renal damage was masked due to severe hepatic damage.

Sasa veitchii extract induces anticancer effects via inhibition of cyclin D1 expression in MCF-7 cells.

Sasa veitchii and other Sasa species are traditional medicinal herbs belonging to a group of Japanese bamboos collectively called Kumazasa, and these species possess the potential for a wide variety of uses. The present study aimed to elucidate the anticancer mechanisms exerted by S. veitchii extract (SE) against a human breast cancer cell line, MCF-7 cells. Freeze-dried Sunchlon® was used as the SE, and cell proliferation activity was measured using the [3H]-thymidine incorporation assay. Induction of apoptosis was assessed via Annexin V and caspase-3 fluorescent staining, the induction of necrosis was measured via propidium iodide staining, and cell cycle-related protein expression was determined using western blotting. The IC50 value of the SE was 7.7 µg/mL in MCF-7 cells. Although the primary active ingredient in Sunchlon® is sodium copper chlorophyllin (0.25%), the present results indicated that ingredients other than SCC exert anti-cancer activities (the IC50 value of SCC was 715 µg/mL), and late apoptosis or necrosis was induced in an SE dose-dependent manner. The expression levels of cyclin D1 and Cdk6 were decreased after SE treatment, and there was no change in the Cdk1/2 expression levels. Additionally, the expression of the necrosis-related cell death indicators RIP1 and RIP3 was increased in response to high-dose SE treatments, and this was indicative of cells preparing for programmed cell death. SE induces cell death in MCF-7 cells via the inhibition of cyclin D1 expression at low concentrations, and this extract induces programmed necrosis (necroptosis) by potentiating RIP1/RIP3 expression.

Chronotoxicity of Streptomycin-Induced Renal Injury in Mice.

The aim of the present study was to investigate the "chronotoxicity" of streptomycin (SM) in relation to its circadian periodicity. Male ICR mice were injected intraperitoneally with SM (780 mg/kg, one shot) one of six time points throughout the day. Mortality was monitored until 14 d after the injection and clearly differed depending on the timing of the injection (i.e., mice were more sensitive to injection during the dark phase). Moreover, when mice were administered with non-lethal doses of SM (550 mg/kg, every 24 h for 3 d, in the light phase or dark phase), the levels of nephrotoxicity indicators (blood urea nitrogen and renal levels of malondialdehyde and cyclooxygenase-2) were significantly increased by the injection in the dark phase, but not in the light phase. These results suggested that SM showed clear chronotoxicity. Our current data indicated that chronotoxicology may provide valuable information on the importance of injection timings for evaluations of toxicity and undesirable side effects.

Diurnal Variation of Sitagliptin-Induced Pharmacological Effects in C57BL/6J Mice.

Chronopharmacology is the study of the varying responses of drugs to changes in biological timing and endogenous periodicities. The dipeptidyl peptidase-4 inhibitor sitagliptin is a globally prescribed anti-hyperglycemic drug. Although dipeptidyl peptidase-4 inhibitors are usually administered once, the specific intake time is generally not mentioned. Therefore, this study aimed at investigating the diurnal effects of sitagliptin-induced anti-hyperglycemia in high-fat diet (HFD)-induced obesity in mice. Five-week-old male C57BL/6J mice were fed normal (control) diet or HFD for 10 weeks. During the last 2 weeks, the mice were administered saline or sitagliptin (10 mg/kg, per os) in the light or dark phase, respectively. At the end of the experiment, the mice were euthanized after an 18 h fasting period, and plasma and tissue samples (liver, kidney, and epididymal white adipose tissues) were collected, or the oral glucose tolerance test was performed. Sitagliptin administration in the light phase significantly decreased plasma glucose levels, insulin levels, hepatic steatosis, and restored the glucose tolerance compared with the HFD group. In contrast, these parameters remained unchanged in the dark phase-treated mice. Our data therefore suggests that sitagliptin portrays definite chronopharmacology, which may provide valuable information on the importance of drug administration timing for maximum pharmacological effects.