Diurnal variation of cisplatin-induced renal toxicity in ICR mice.

Cisplatin (CDDP) is a platinum-based anticancer drug widely prescribed for its effectiveness in treating various forms of cancer. However, its major side effect is nephrotoxicity. Although several methods have been developed to mitigate CDDP-induced nephrotoxicity, an optimal approach has yet to be established. This study aimed to investigate the "chronotoxicity" of CDDP as a potential strategy to reduce its side effects. Male ICR mice were treated with CDDP (20 mg/kg, intraperitoneal injection, one shot) at zeitgeber time (ZT) 2 or ZT14 (light or dark phase). After 72 h, we collected plasma and kidney and evaluated several markers. We found that body weight change between ZT2 and ZT14 by CDDP was comparable. In contrast, many toxicological factors, such as plasma blood urine nitrogen, plasma creatinine, renal oxidative stress (malondialdehyde), DNA damage (γH2AX), acute kidney injury biomarker (KIM-1), and inflammation (Tnfα), were significantly induced at ZT14 compared to than that of ZT2. Our present data suggested that chronotoxicology might provide beneficial information on the importance of administration timings for toxic evaluations and unacceptable side effects.

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.

Chronotoxicity of Acrylamide in Mice Fed a High-Fat Diet: The Involvement of Liver CYP2E1 Upregulation and Gut Leakage.

Acrylamide (ACR) is produced under high-temperature cooking of carbohydrate-rich foods via the Maillard reaction. It has been reported that ACR has hepatic toxicity and can induce liver circadian disorder. A high fat diet (HFD) could dysregulate liver detoxification. The current study showed that administration of ACR (100 mg/kg) reduced the survival rate in HFD-fed mice, which was more pronounced when treated during the night phase than during the day phase. Furthermore, ACR (25 mg/kg) treatment could cause chronotoxicity in mice fed a high-fat diet, manifested as more severe mitochondrial damage of liver during the night phase than during the day phase. Interestingly, HFD induced a higher CYP2E1 expressions for those treated during the night phase, leading to more severe DNA damage. Meanwhile, the expression of gut tight junction proteins also significantly decreases at night phase, leading to the leakage of LPSs and exacerbating the inflammatory response at night phase. These results indicated that a HFD could induce the chronotoxicity of ACR in mice liver, which may be associated with increases in CYP2E1 expression in the liver and gut leak during the night phase.

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.

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.

Diurnal hepatic CYP3A11 contributes to chronotoxicity of the pyrrolizidine alkaloid retrorsine in mice.

1. Retrorsine (RTS) is a pyrrolizidine alkaloid (distributed in many medicinal plants) that has significant hepatotoxicity. Here, we aimed to determine the daily variations in RTS hepatotoxicity (chronotoxicity) in mice, and to investigate the role of metabolism in generating RTS chronotoxicity.2. Acute toxicity and pharmacokinetic studies were performed with mice after RTS administration at different times of the day. Hepatotoxicity was assessed by measuring plasma ALT (alanine aminotransferase) and AST (aspartate aminotransferase) levels. mRNA and proteins were determined by qPCR and Western blotting, respectively. Time-dependent in vitro metabolism of RTS was assessed by using mouse liver microsomes.3. We found that RTS toxicity was more severe in the dark phase (zeitgeber time 14 or ZT14 and ZT18) than in the light phase (ZT2 and ZT6). This chronotoxicity was associated with a dosing time difference in the systemic exposures of RTS and a pyrrolic ester metabolite (a cause of hepatotoxicity, measured by the levels of pyrrole-GSH conjugate and pyrrole-protein adducts due to a high chemical reactivity). Moreover, the CYP3A11 (a major enzyme for RTS bioactivation) inhibitor ketoconazole decreased the production of pyrrole-GSH conjugate and abrogated diurnal rhythm in RTS metabolism. In addition, E4bp4 (a circadian regulator of Cyp3a11) ablation abolished the rhythm of CYP3A11 expression and abrogated the dosing time-dependency of RTS toxicity.4. In conclusion, RTS chronotoxicity in mice was attributed to time-varying hepatic metabolism regulated by the circadian clock. Our findings have implications for reducing pyrrolizidine alkaloid-induced toxicity via a chronotherapeutic approach.

The Impact of the Timing of Dosing on the Severity of UNC569-Induced Ultrastructural Changes in the Mouse Retina.

Mer proto-oncogene tyrosine kinase (MerTK), expressed in the retinal pigment epithelium (RPE), regulates the phagocytosis of shed photoreceptor outer segments. To investigate the influence of dosing time on MerTK inhibitor UNC569-induced retinal toxicity, UNC569 at 100 mg/kg was orally administered to male mice at 2 different Zeitgeber times (ZT5.5 or ZT22) for 28 days. Electron microscopy was conducted at ZT2 after the final dosing. Additionally, the visual cycle components (11-cis-retinal, all-trans-retinal, all-trans-retinol, and 11-cis-retinol), which play an important role in maintaining retinal homeostasis, were quantified by liquid chromatography/mass spectrometry/mass spectrometry. Under electron microscopic examination, the number of phagosomes and phagolysosomes in the RPE increased in both the ZT5.5 and ZT22 administered groups, while endoplasmic reticulum dilatation in the RPE and chromatin aggregation of photoreceptor nuclei were observed only in the ZT22 administered group. No change was observed in any of the visual cycle components. These results suggest that the timing of the dosing in relation to the physiological MerTK phosphorylation affected the severity of changes in the RPE, leading to the apoptosis of the photoreceptor cells.

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.

[Chronotoxicity of 1.8 GHz radio-frequency radiation on plasma stress hormones and immune factors in mice].

OBJECTIVE: To study the chronotoxicity of radio-frequency radiation(RF) on the plasma stress hormones and immune factors in mice. METHODS: A total of 72 healthy C57 BL mice with circadian rhythm were divided into twelve groups: 6 Sham group and 6 RF groups. RF groups were exposed to 1.8 GHz RF at 226 µW/cm~2 for 60 days with 2 h/day respectively at corresponding zeitgeber time(ZT 0:00, ZT 4:00, ZT 8:00, ZT 12:00, ZT 16:00, ZT 20:00). The Sham group mice were exposed to the same condition without electromagnetic signal. At the end of last RF exposure, blood samples were collected from each animal. The concentrations of plasma stress hormones(ACTH, CORT) and immune factors(GM-CSF, TNF-α) were determined by enzyme linked immunosorbent assay(ELISA) method. RESULTS: The daily average levels of ACTH, CORT, GM-CSF and TNF-α were 84.12, 60.14, 1112.02 and 594.49 ng/L, which were decreased to 62.07, 41.21, 84.18 and 305.08 ng/L after 60 days of RF exposure. Compared to sham-exposed animals, the daily average levels of ACTH, CORT, GM-CSF and TNF-α were all significantly decreased(P<0.05). Circadian rhythms in the secreting of CORT, GM-CSF, TNF-α were disappeared(P>0.05), circadian rhythms of ACTH was shifted in RF-exposed mice, with the amplitude reduced from 12.45 to 4.88 and peak time postponed from 1:39 to 5:29. CONCLUSION: 1.8 GHz RF may weaken the function of stress and immune, and disturb their circadian rhythmicities.

Hepatocyte circadian clock controls acetaminophen bioactivation through NADPH-cytochrome P450 oxidoreductase.

The diurnal variation in acetaminophen (APAP) hepatotoxicity (chronotoxicity) reportedly is driven by oscillations in metabolism that are influenced by the circadian phases of feeding and fasting. To determine the relative contributions of the central clock and the hepatocyte circadian clock in modulating the chronotoxicity of APAP, we used a conditional null allele of brain and muscle Arnt-like 1 (Bmal1, aka Mop3 or Arntl) allowing deletion of the clock from hepatocytes while keeping the central and other peripheral clocks (e.g., the clocks controlling food intake) intact. We show that deletion of the hepatocyte clock dramatically reduces APAP bioactivation and toxicity in vivo and in vitro because of a reduction in NADPH-cytochrome P450 oxidoreductase gene expression, protein, and activity.

Chronotoxici-Plate Containing Droplet-Engineered Rhythmic Liver Organoids for Drug Toxicity Evaluation.

The circadian clock coordinates the daily rhythmicity of biological processes, and its dysregulation is associated with various human diseases. Despite the direct targeting of rhythmic genes by many prevalent and World Health Organization (WHO) essential drugs, traditional approaches can't satisfy the need of explore multi-timepoint drug administration strategies across a wide range of drugs. Here, droplet-engineered primary liver organoids (DPLOs) are generated with rhythmic characteristics in 4 days, and developed Chronotoxici-plate as an in vitro high-throughput automated rhythmic tool for chronotherapy assessment within 7 days. Cryptochrome 1 (Cry1) is identified as a rhythmic marker in DPLOs, providing insights for rapid assessment of organoid rhythmicity. Using oxaliplatin as a representative drug, time-dependent variations are demonstrated in toxicity on the Chronotoxici-plate, highlighting the importance of considering time-dependent effects. Additionally, the role of chronobiology is underscored in primary organoid modeling. This study may provide tools for both precision chronotherapy and chronotoxicity in drug development by optimizing administration timing.

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.

Recent advances in circadian-regulated pharmacokinetics and its implications for chronotherapy.

Dependence of pharmacokinetics and drug effects (efficacy and toxicity) on dosing time has long been recognized. However, significant progress has only recently been made in our understanding of circadian rhythms and their regulation on drug pharmacokinetics, efficacy and toxicity. This review will cover the relevant literature and a series of publications from our work summarizing the effects of circadian rhythms on drug pharmacokinetics, and propose that the influence of circadian rhythms on pharmacokinetics are ultimately translated into therapeutic effects and side effects of drugs. Evidence suggests that daily rhythmicity in expression of drug-metabolizing enzymes and transporters necessary for drug ADME (absorption, distribution, metabolism and excretion) are key factors determining circadian pharmacokinetics. Newly discovered mechanisms for circadian control of the enzymes and transporters are covered. We also discuss how the rhythms of drug-processing proteins are translated into circadian pharmacokinetics and drug chronoefficacy/chronotoxicity, which has direct implications for chronotherapy. More importantly, we will present perspectives on the challenges that are still needed for a breakthrough in translational research. In addition, knowledge of the circadian influence on drug disposition has provided new possibilities for novel pharmacological strategies. Careful application of pharmacokinetics-based chronotherapy strategies can improve efficacy and reduce toxicity. Circadian rhythm-mediated metabolic and transport strategies can also be implemented to design drugs.

Chronotoxicity of Semen Strychni is associated with circadian metabolism and transport in mice.

OBJECTIVES: We aimed to determine the circadian responses of mice to Semen Strychni and to investigate the role of pharmacokinetics in generating chronotoxicity. METHODS: Total extract of Semen Strychni was administered by oral gavage to wild-type (WT) and Bmal1-/- (a circadian clock-deficient model) mice at different circadian time points for toxicity (including survival) and pharmacokinetic characterization. Nephrotoxicity and neurotoxicity were evaluated by measuring plasma creatinine and creatine kinase BB (CK-BB), respectively. Drug metabolism and transport assays were performed using liver/intestine microsomes and everted gut sacs, respectively. KEY FINDINGS: Semen Strychni nephrotoxicity and neurotoxicity as well as animal survival displayed significant circadian rhythms (the highest level of toxicity was observed at ZT18 and the lowest level at ZT2 to ZT6). According to pharmacokinetic experiments, herb dosing at ZT18 generated higher plasma concentrations (and systemic exposure) of strychnine and brucine (two toxic constituents) compared with ZT6 dosing. This was accompanied by reduced formation of both dihydroxystrychnine and strychnine glucuronide (two strychnine metabolites) at ZT18. Bmal1 ablation sensitized mice to Semen Strychni-induced toxicity (with increased levels of plasma creatinine and CK-BB) and abolished the time dependency of toxicity. Metabolism of Semen Strychni (strychnine and brucine) in the liver and intestine microsomes of WT mice was more extensive at ZT6 than at ZT18. These time differences in hepatic and intestinal metabolism were lost in Bmal1-/- mice. Additionally, the intestinal efflux transport of Semen Strychni (strychnine and brucine) was more extensive at ZT6 than ZT18 in WT mice. However, the time-varying transport difference was abolished in Bmal1-/- mice. CONCLUSIONS: Circadian responses of mice to Semen Strychni are associated with time-varying efflux transport and metabolism regulated by the circadian clock (Bmal1). Our findings may have implications for optimizing phytotherapy with Semen Strychni via timed delivery.

Microarray profiling of LncRNA expression in the testis of pubertal mice following morning and evening exposure to 1800 MHz radiofrequency fields.

In this paper, the chronotoxicity of radiofrequency fields (RF) in the pubertal testis development and the involved molecular pathways were investigated by exposing four-week-old mice to RF (1800 MHz, SAR, 0.50 W/kg) in the morning and evening of each day for three weeks. Then, pathological changes and functional indices within the testis were determined. We also used a long non-coding RNA (lncRNA) microarray and GO/KEGG pathway analyses to determine lncRNA expression profiles and predict their potential functions. The cis and trans regulation of lncRNAs were investigated, and an interaction network was constructed using Cytoscape software. RF exposure led to a range of pathological changes in the testes of adolescent mice, as testicular weights and daily sperm productions decreased, and the testosterone secretion reduced. Furthermore, RF induced dysregulation in the expression of testicular lncRNAs. We identified 615 and 183 differentially expressed lncRNAs that were associated with morning and evening exposure to RF, respectively. From 15 differential expression lncRNAs both in morning RF group and evening RF group, we selected 6 lncRNAs to be validated by quantitative reverse transcription PCR (qRT-PCR). The differentially expressed lncRNAs induced by morning RF exposure were highly correlated with many different pathways, including Fanconi syndrome, metabolic processes, cell cycle, DNA damage, and DNA replication. Trans-regulation analyses further showed that differentially expressed lncRNAs were involved in multiple transcription factor-regulated pathways, such as TCFAP4, NFkB, HINFP, TFDP2, FoxN1, and PAX5. These transcription factors have all been shown to be involved in the modulation of testis development, cell cycle progression, and spermatogenesis. These findings suggest that the extent to which 1800 MHz RF induced toxicity in the testes and changed the expression of lncRNAs showed differences between morning exposure and evening exposure. These data indicate that differentially expressed lncRNAs play crucial roles in the RF exposure damage to the developing pubertal testis. Collectively, our findings provide a better understanding of the mechanisms underlying the toxic effects of RF exposure on testicular development.

Dosing-time dependent testicular toxicity of everolimus in mice.

The circadian timing system controls many biological functions in mammals including drug metabolism and detoxification, cell cycle events, and thus may affect pharmacokinetics, target organ toxicity and efficacy of medicines. Selective mTOR (mammalian target of rapamycin) inhibitor everolimus is an immunosuppressant and anticancer drug that is effective against several cancers. The aim of this study was to investigate dosing-time dependent testicular toxicity of subacute everolimus administration in mice. C57BL/6 J male mice were synchronized with Light-Dark (12h:12 h) cycle, with Light-onset at Zeitgeber Time (ZT)-0. Everolimus (5 mg/kg/day) was administered orally to mice at ZT1rest-span or ZT13activity-span for 4 weeks. Body weight loss, clinical signs, changes in testicular weights, testis histology, spermatogenesis and proliferative activity of germinal epithelium of seminiferous tubules were examined. Steady-state everolimus concentrations in testes were determined with validated HPLC method. Everolimus toxicity was less severe following dosing at ZT13 compared to ZT1, as shown with least body weight loss (p<0.001), least reductions in testes weights (p<0.001) and least histopathological findings. Everolimus-induced histological changes on testes included vacuolisation and atrophy of germinal epithelium, and loss of germinal cell attachment. The severity of everolimus-induced histological toxicity on testes was significantly more evident in mice treated at ZT1 than ZT13 (p<0.001). Spermatogenic cell population significantly decreased when everolimus administered at ZT1 compared to ZT13 (p<0.001). Proliferative activity of germinal epithelium was significantly decreased due to treatment at ZT1 compared to ZT13 (p<0.001). Everolimus concentrations in testes indicated a pronounced circadian variation, which was greater in mice treated at ZT1 compared to ZT13 (p<0.05). Our study revealed dosing-time dependent testicular toxicity of everolimus in mice, which was greater in severity when everolimus administered at early rest-span (daytime-ZT1) than early activity-span (nighttime-ZT13). These findings support the concept of everolimus chronotherapy for minimizing reproductive toxicity and increasing the tolerability of everolimus, as a clinical advantage.

Circadian clock regulates metabolism and toxicity of Fuzi(lateral root of Aconitum carmichaeli Debx) in mice.

BACKGROUND: Therapeutic applications of Fuzi (lateral root of Aconitum carmichaeli Debx) are seriously concerned with its toxic effects. Strategies and approaches to reducing toxicity are of great interest. PURPOSE: We aimed to characterize the diurnal rhythm of Fuzi toxicity, and to determine the role of metabolism and pharmacokinetics in generating toxicity rhythmicity. METHODS: Toxicity was determined based on assessment of heart injury and animal survival after dosing mice with Fuzi decoction at different circadian time points. Circadian clock control of pharmacokinetics and toxicity was investigated using Bmal1-deficient (Bmal1-/-) mice. RESULTS: Fuzi exhibited a diurnal rhythmicity in cardiotoxicity (reflected by plasma CK-MB and LDH levels). The highest level of toxicity was observed at ZT10 (5 PM), while the lowest level of toxicity occurred at ZT22 (5 AM). Also, a higher mortality rate was observed at ZT10 and lower mortality rates at other times of the day. ZT10 dosing of Fuzi generated higher systemic exposures of three toxic alkaloid ingredients aconitine (AC), hypaconitine (HA) and mesaconitine (MA) compared to ZT22. This was accompanied by reduced the formation of the metabolites (N-deethyl-AC, didemethyl-HA and 2­hydroxyl­MA) at ZT10. Bmal1 ablation resulted in an increased level of Fuzi toxicity at ZT22, while having no influences when drug was dosed at ZT10. As a consequence, circadian time-dependent toxicity of Fuzi was lost in Bmal1-deficient mice. In addition, Bmal1 ablation increased the plasma concentrations of AC, HA and MA in mice after oral gavage of Fuzi, and reduced formation of their metabolites (N-deethyl-AC, didemethyl-HA and 2­hydroxyl­MA). Moreover, Fuzi metabolism in wild-type liver microsomes was more extensive at ZT22 than at ZT10. Bmal1 ablation abrogated circadian time-dependency of hepatic Fuzi metabolism. CONCLUSIONS: Fuzi chronotoxicity in mice was attributed to time-varying hepatic metabolism and systemic exposure regulated by circadian clock. The findings may have implications in reducing Fuzi toxicity with a chronotherapeutic approach.

Circadian clock regulates hepatotoxicity of Tripterygium wilfordii through modulation of metabolism.

OBJECTIVES: We aimed to determine the diurnal rhythm of Tripterygium wilfordii (TW) hepatotoxicity and to investigate a potential role of metabolism and pharmacokinetics in generating chronotoxicity. METHODS: Hepatotoxicity was determined based on assessment of liver injury after dosing mice with TW at different circadian time points. Circadian clock control of metabolism, pharmacokinetics and hepatotoxicity was investigated using Clock-deficient (Clock-/- ) mice. KEY FINDINGS: Hepatotoxicity of TW displayed a significant circadian rhythm (the highest level of toxicity was observed at ZT2 and the lowest level at ZT14). Pharmacokinetic experiments showed that oral gavage of TW at ZT2 generated higher plasma concentrations (and systemic exposure) of triptolide (a toxic constituent) compared with ZT14 dosing. This was accompanied by reduced formation of triptolide metabolites at ZT2. Loss of Clock gene sensitized mice to TW-induced hepatotoxicity and abolished the time-dependency of toxicity that was well correlated with altered metabolism and pharmacokinetics of triptolide. Loss of Clock gene also decreased Cyp3a11 expression in mouse liver and blunted its diurnal rhythm. CONCLUSIONS: Tripterygium wilfordii chronotoxicity was associated with diurnal variations in triptolide pharmacokinetics and circadian expression of hepatic Cyp3a11 regulated by circadian clock. Our findings may have implications for improving TW treatment outcome with a chronotherapeutic approach.

Cyp3a11 metabolism-based chronotoxicity of brucine in mice.

Brucine is one of the main bioactive and toxic constituents of the herb drug Semen Strychni. Here we aimed to determine dosing time-dependent hepatotoxicity of brucine, and to investigate the role of metabolism in generation of brucine chronotoxicity. Brucine was administered to wild-type or Npas2-/- (a clock disrupted model) mice at different circadian time points for toxicity and pharmacokinetic characterization. The hepatotoxicity was evaluated by plasma alanine aminotransferase and aspartate aminotransferase measurements and histopathological analysis. The role of Cyp3a11 in brucine metabolism was determined by chemical inhibition assays and Cyp3a11-overexpressing HEK293 cells. Hepatic circadian Cyp3a11 mRNA and protein levels were determined by qPCR and Western blotting, respectively. The toxicity of brucine was more severe in the light phase [Zeitgeber time (ZT) 2 and ZT8] than in the dark phase (ZT14 and ZT20). Chemical inhibition and substrate metabolism assays suggested Cyp3a11 as a significant contributor to brucine metabolism. The Cyp3a11 mRNA, protein and activity in the livers of wild-type mice displayed significant circadian fluctuations. Npas2 ablation markedly down-regulated Cyp3a11 mRNA, protein and activity, and abrogated their circadian rhythms. The circadian time differences in brucine pharmacokinetics and liver distribution were lost in Npas2-/- mice, so were the time differences in brucine hepatotoxicity. In conclusion, chronotoxicity of brucine was determined by circadian variations in Cyp3a11 metabolism. The findings have implications in improving brucine (and possibly Semen Strychni) efficacy via dosing time optimization.

Circadian sensitivity to the cardiac glycoside oleandrin is associated with diurnal intestinal P-glycoprotein expression.

The cardiac glycoside oleandrin is a main active constituent of the botanical anti-cancer drug candidate PBI-05204, an extract of Nerium oleander. Here, we aimed to determine the circadian sensitivity of mice to oleandrin, and to investigate the role of intestinal P-gp in generating rhythmic drug toxicity. Toxicity and pharmacokinetic experiments were performed with wild-type, Bmal1iKO (intestine-specific Bmal1 knockout) and Bmal1fl/fl (control littermates of Bmal1iKO) mice. The cardiac toxicity (reflected by plasma CK-MB, LDH and cTn-I levels) varied significantly with the times of drug dosing in wild-type mice (a lower toxicity at ZT10 and more severe at ZT2/22). Dosing at ZT2 generated a higher drug exposure than ZT10, supporting a lower toxicity at ZT10. Intracellular accumulation of oleandrin (2.5-10 µM) was reduced in MDCKⅡ-MDR1 than in parental cells. MDR1 overexpression decreased the cell sensitivity to oleandrin toxicity. The net flux ratio (MDCKⅡ-MDR1 versus parental cells) was 2.9 for oleandrin. These data indicated oleandrin as a P-gp substrate. Both mdr1a mRNA and P-gp protein oscillated with the times of the day in small intestine of Bmal1fl/fl mice. Intestinal ablation of Bmal1 down-regulated mdr1a mRNA and P-gp protein, and abrogated their rhythms. Likewise, Bmal1 silencing led to down-regulated mdr1a mRNA and to a loss of its rhythmicity in serum-shocked CT26 cells. Based on luciferase reporter assays, Bmal1 regulated rhythmic mdr1a transcription through the clock output genes Hlf and E4bp4. Intestinal ablation of Bmal1 exacerbated oleandrin toxicity and enhanced drug exposure. Moreover, time dependency of toxicity and drug exposure were lost in Bmal1iKO mice. In conclusion, diurnal intestinal P-gp is a critical factor influencing daily oleandrin exposure and toxicity. Our findings have implications in minimizing oleandrin (and possibly Nerium oleander) toxicity and improving drug efficacy via dosing time optimization.