**5. Drugs affect circadian clock gene expression as a novel target of chronopharmacology**

Many drugs/toxicants could affect central and peripheral circadian clock gene expression as targets of chronopharmacology and chronotoxicology [10]. **Tables 2** and **3** provide some examples including our work in the field.


**Table 2.** Circadian clock gene expression as novel targets in pharmacology.

*Examples of drugs* Atorvastatin is an HMG-CoA reductase inhibitor used for hyperlipidemia. It is generally safe but may induce cholestasis. Repeated administration of Atorvastatin (10–100 mg/kg, po) to mice for 30 days produced hepatocyte swollen and feather-like degeneration, indicative of cholestatic injury, with increases of inflammation markers Egr1 and MT-1, and increased Cyp7a1, FXR, SHP, decreased bile acid transporters Ntco, Bsep, Oastα, and Ostβ. Since Cyp7a1 is a clock-driven gene, its effects on circadian clock gene expression were also examined. Atorvastatin increased the expression of Bmal1, Npas2, decreased the expression of Per2, Per3, Dbp, and Tef, but had no effect on Cry1 and Nr1d1 [56]. The similar effects on the circadian clock gene expression were also observed when atorvastatin was given at the low dose (10 mg/kg) but for a longer period of 90 days, although to a less extent [57].

resulting in the inhibition of acetyl CoA carboxylase (ACC), the rate-limiting enzyme in fatty acid synthesis. Metformin-activated liver casein kinase I α (CKIα) and muscle CKIε, known modulators of the positive loop of the circadian clock, thud resulting in phase advances in the

metabolism gene.

**Chronotoxicology References**

[65]

23

http://dx.doi.org/10.5772/intechopen.74597

[66]

[53]

[70]

[71]

[55]

[54]

 produced liver fibrosis, altered the amplitudes, meros, acrophases of clock gene expression; circadian rhythms of Cry2, PPARα and

Circadian Clock Gene Expression and Drug/Toxicant Interactions as Novel Targets…

Produced HCC, Markedly increased α-fetoprotein; at 10:00, expression of Bmal1 decreased, expressions of

Produced neuroinflammation and dopaminergic neuron loss; decreased expression of Bmal1, clock, Per1, Per2, while increased expression of Dbp and

Produced neuroinflammation and dopaminergic neuron loss; at the mRNA and protein levels, reduced expression of Bmal1, clock, Per1, Per2, Dbp,

Produced increases in serum TNFa, heart and liver apoptosis; Decrease Per1, Per2 2 h after dose at ZT4 in heart and liver; Increased Per2 8 and 26 h after

Produced steatosis, increased serum TG; diurnal oscillations of Bmal1, Clock, Cry1, Cry2, Per1, and Per2 and clock-controlled genes (Dbp, Hlf, Nocturnin, Npas2, Rev-erba, and Tef) were altered in

Altered the expression of circadian and metabolism genes in hippocampus, liver, and colon from array analysis; ClockΔ19 affect inflammation and

*Examples of active ingredients from herbal medicine*. Oleanolic acid is a triterpenoid used to reduce hyperlipidemia. Dietary oleanolic acid supplementation (0.01%) was provided to Apoe- and Apoa1-deficient mice and F344 rats. In Apoe-deficient mice, oleanolic acid supplementation increased hepatic lipid droplets, increased circadian clock genes, together with increases in lipid metabolism genes (fatty acid elongase 3, tubulin beta-2A chain, and claudin 1), while the expression of per3, amylase 2a5, ubiquitin-specific peptidase 2, and thyroid hormone-

Resveratrol is an active ingredient in grapes and red wine and shows beneficial effects in metabolic disorders. In HFD-fed mice, resveratrol restored high-fat diet-induced disorders about

liver and phase delays in the muscle for clock and metabolic gene expressions [58].

inducible hepatic protein (Thrsp) were decreased [59].

**Animal model (dose, route, time)**

0.6 ml/kg, ip, 2/week ×

DEN 100 mg/kg, IP+ CCl4 + EtOH × 16 weeks

1 and 5 mg/kg, IP, × 4

LPS 5 mg/kg, IP ×1, 200 days later, rotenone 0.5 mg/kg, sc × 20

IP at ZT4, 10, 16, 22 or at 2, 8, and 26 h after ZT 4

Lieber-DeCarli diet for

mice received Nanji liquid alcohol diet at ZT4 for 10 weeks

**Table 3.** Circadian clock gene expression as novel targets in toxicology.

Chronic CCl4

POR were lost.

Nr1d1

Dbp and Rev-erbα increased.

Nr1d1, while no effect on Cy1.

LPS in heart and liver

livers of ethanol-fed mice

BABL/C mice

4 weeks

weeks

LPS ICR mice, LPS 1 mg/kg,

Alcohol C57 mice, Per2Luc mice

Alcohol WT and ClockΔ19 mutant

injection

30–37 days

Diethylnitrosamine KM mice

Manganese SD rats

LPS + Rotenone SD rats

Carbon tetrachloride

Metformin is commonly used for type 2 diabetes. In C57 mice, metformin in the drinking water for 6 weeks led to increased serum leptin and decreased glucagon levels. The effect of metformin on liver and muscle metabolism was probably mediated through AMPK activation,


**Table 3.** Circadian clock gene expression as novel targets in toxicology.

*Examples of drugs* Atorvastatin is an HMG-CoA reductase inhibitor used for hyperlipidemia. It is generally safe but may induce cholestasis. Repeated administration of Atorvastatin (10–100 mg/kg, po) to mice for 30 days produced hepatocyte swollen and feather-like degeneration, indicative of cholestatic injury, with increases of inflammation markers Egr1 and MT-1, and increased Cyp7a1, FXR, SHP, decreased bile acid transporters Ntco, Bsep, Oastα, and Ostβ. Since Cyp7a1 is a clock-driven gene, its effects on circadian clock gene expression were also examined. Atorvastatin increased the expression of Bmal1, Npas2, decreased the expression of Per2, Per3, Dbp, and Tef, but had no effect on Cry1 and Nr1d1 [56]. The similar effects on the circadian clock gene expression were also observed when atorvastatin was given at the low dose (10 mg/kg) but for a longer period of 90 days,

in vitro as well as in vivo.

**Chronopharmacology References**

[56]

[58]

[59]

[60]

[61]

[62]

[63]

[64]

Swollen hepatocyte and feather-like degeneration; increased Cyp7a1, FXR, decreased bile acid transporters; increased expression of Bmal1, Npas2,

Increase in serum leptin and decreased glucagon levels. Increase in PGC1α, PPARα, AMPK; decrease in ACC in liver; Phase advance circadian clock and metabolic genes in liver and activation of liver casein

Increased lipid droplets with no change in oxidative stress; increased Bmal1, Clock, and Elovl3, Tubb2a, and Cldn1 decreased Per3, Amy2a5, Usp2, and Thrsp.

Ameliorated HFD-increased plasma leptin, lipids, and BW. Restored rhythmicity of Clock, Bmal1, and Per2; and clock-controlled lipid metabolism genes

Improve serum lipid profile; restore rhythmicity of PPARα, Srebp1, Cpt, and FAS; restore nighttime feeding-disrupted clock gene expression.

Decreased the amplitude of Clock, Npas2, Bmal1; increased Dbp, Nfil3 at 10:00, and increased Nr1d1 at

Polyporus and Bupleuri radix were effective in manipulating the peripheral circadian clock phase acutely, with stimulation time-of-day dependency

Increased total sleep time and slow wave sleep time; reversed model rat-induced inflammation markers; increased Cry1, Cry 2, and decreased NF-κB in PBMC.

(Sirt1, PPARa, Srebp-1, Acc1, and Fas).

18:00. No effect on Cry and Per genes.

decrease Per2, Dbp.

kinase Iα (CKIα)

Metformin is commonly used for type 2 diabetes. In C57 mice, metformin in the drinking water for 6 weeks led to increased serum leptin and decreased glucagon levels. The effect of metformin on liver and muscle metabolism was probably mediated through AMPK activation,

although to a less extent [57].

**Drugs Animal model (dose, route, time)**

Metformin C57 mice; 164 mg/kg

weeks

Oleanolic acid Apoe−/− mice on HFD,

weeks

Resveratrol C57 mice; fed normal or

Zuotai KM mice; 10 mg/kg, po × 7 days

dark

Jiao-Tai-Wan SD normal and model

Sea cucumber saponin (SCS)

Polyporus and Bupleuri radix

in drinking water for 6

F344 rats; 0.01% OA × 11

HFD; 0.1% Res × 11 weeks

ICR mice; 0.03% SCS diet night feeding × 2 weeks

ICR mice, Per2Luc mice 500 mg/kg, po × 3 days, at different ZT and light/

(HFD + PSD × 4 weeks) rats 2.2 g/kg, po × 4 weeks

**Table 2.** Circadian clock gene expression as novel targets in pharmacology.

Atorvastatin KM mice; 10–100 mg/kg, po × 30 days

22 Circadian Rhythm - Cellular and Molecular Mechanisms

resulting in the inhibition of acetyl CoA carboxylase (ACC), the rate-limiting enzyme in fatty acid synthesis. Metformin-activated liver casein kinase I α (CKIα) and muscle CKIε, known modulators of the positive loop of the circadian clock, thud resulting in phase advances in the liver and phase delays in the muscle for clock and metabolic gene expressions [58].

*Examples of active ingredients from herbal medicine*. Oleanolic acid is a triterpenoid used to reduce hyperlipidemia. Dietary oleanolic acid supplementation (0.01%) was provided to Apoe- and Apoa1-deficient mice and F344 rats. In Apoe-deficient mice, oleanolic acid supplementation increased hepatic lipid droplets, increased circadian clock genes, together with increases in lipid metabolism genes (fatty acid elongase 3, tubulin beta-2A chain, and claudin 1), while the expression of per3, amylase 2a5, ubiquitin-specific peptidase 2, and thyroid hormoneinducible hepatic protein (Thrsp) were decreased [59].

Resveratrol is an active ingredient in grapes and red wine and shows beneficial effects in metabolic disorders. In HFD-fed mice, resveratrol restored high-fat diet-induced disorders about the rhythmic expression of clock genes and clock-controlled lipid metabolism, ameliorated the rhythmites of plasma leptin, lipid profiles and whole body metabolic status (respiratory exchange ratio, locomotor activity, and heat production). Meanwhile, resveratrol modified the rhythmic expression of clock genes (Clock, Bmal1, and Per2) and clock-controlled lipid metabolism-related genes (Sirt1, Ppara, Srebp-1c, Acc1, and Fas) [60].

Bmal1 and Per1 were attenuated and the mesors in the expressions of Clock and Per1 were increased. Acrophases for the expressions of Clock, Per1 and Cry1 were significantly delayed. Circadian rhythm of Cry2 expression was lost in fibrosis group. The circadian rhythm of PPARα

Circadian Clock Gene Expression and Drug/Toxicant Interactions as Novel Targets…

http://dx.doi.org/10.5772/intechopen.74597

25

Chronic diethylenediamine (DEN) administration not only produce hepatocellular carcinoma and markedly enhanced expression of Afp, but also decreased the expression of Bmal1, increased the expression of Dbp and Rev-erbα (Nr1d1) [66]. Circadian disruption is well-known to promote carcinogenesis [67]. In the end-stage of human hepatocellular carcinoma, the expressions of the clock genes, including Bmal1, Per1, Per2, Cry1, and Cry2 were decreased, alone with decreases in clock targeted MT-1, MT-2, and MTF1 (which are considered as biomarkers of HCC). On the other hand, the expression of clock target genes Nr1d1 and Dbp was upregulated as compared with Peri-HCC and normal livers. Peri-HCC also had mild alterations in these gene expressions [68].

*Examples of neurotoxicants*. As mentioned in **Figure 2**, repeated Mn administration disrupted both central and peripheral liver circadian clock genes, with decreases in Bmal1, Clock, Npas2, Per1, Cry1, but increases in Dbp and Nr1d1. Mn-induced aberrant expression of these clock genes in the brain was consistent with that in the liver, and liver appeared to be more

Chronic neuroinflammation would aggregate neurotoxic effects of toxicants. Rats received a single injection of LPS at the dose of 5 mg/kg, and 200 days later given repeated injection of low dose of rotenone (0.5 mg/kg, sc, 5/week for 4 weeks), and produced neuroinflammation and loss of dopaminergic neurons in Substantia Nigra, replicate the model of Parkinson's disease [69]. In this PD model, aberrant expression of circadian clock genes in brain cortex was evident, as evidenced by decreases of core clock gene Bmal1, clock, and Naps2, decreases in circadian clock feedback gene Per1 and Per2, but had no effect on the expression of Cry1 and

LPS not only produces inflammation in the brain but also in the liver. ICR mice received LPS (1 mg/kg, IP) at ZT4, ZT10, ZT16, and ZT22, and liver and heart were harvested 2 h later for gene expression analysis. Hepatic expression of Per1 and Per2 was decreased after LPS injection at ZT6, but Per1 was increased 8 and 26 h after LPS injection. Heart speared to be more sensitive than the liver to these changes as at ZT4, both Per1 and Per 2 in the heart were decreased [71].

*Examples of chronic ethanol toxicity*. Alcoholic liver diseases are a major concern as it produced metabolic disruption. In C57 mice and Per2 mutant mice, ethanol administration altered the expression of clock genes in the liver, but not in the brain. Diurnal oscillations of core clock genes (Bmal1, Clock, Cry1, Cry2, Per1, and Per2) and clock-controlled genes (Dbp, Hlf, Nocturnin, Npas2, Rev-erba, and Tef) were altered in livers from ethanol-fed mice [55].

In clock mutant mice, altered clock and metabolism genes were evident in hippocampus, liver, and colon. Of particular interest was the finding that a high proportion of genes involved in inflammation and metabolism on the array was significantly affected by alcohol and the Clock

Thus, drugs/toxicants could affect central and peripheral circadian clock gene expression as

gene mutation in the hippocampus [54].

targets of their therapeutic effects and/or toxicity [10].

sensitive than hypothalamus to Mn-induced disruption of circadian clock [53].

Cry2, as well as the decreased expression of clock target gene Dbp and Nr1d1 [70].

and cytochrome P450 oxidoreductase (POR) was also lost [65].

Dietary sea cucumber saponin (SCS) has been shown to have beneficial effects on glucose and lipid metabolism, which is related to the circadian clock. Dietary SCS caused an alteration in rhythms and/or amplitudes of clock genes was more significant in the brain than in liver. In addition, the peroxisome proliferator-activated receptor (PPARα), sterol regulatory element binding protein-1c (SREBP-1c), together with their target genes carnitine palmitoyl transferase, and fatty acid synthase showed marked changes in rhythm and/or amplitude in SCS group mice [61].

*Examples of mixtures from traditional medicine*. Zuotai is an essential component of many popular Tibetan medicines. Mice were orally given Zuotai (10 mg/kg, 1.5-fold of clinical dose) daily for 7 days, and livers were collected every 4 h during the 24 h period to examine its effects on circadian clock gene expression. Zuotai decreased the oscillation amplitude of Clock, Npas2, Bmal1 at 10:00. For the clock feedback negative control genes, Zuotai had no effect on the oscillation of Cry1, Per1, Per2, and Per3. For the clock-driven target genes, Zuotai increased the oscillation amplitude of Dbp, decreased nuclear factor interleukin 3 (Nfil3) at 10:00, but had no effect on thyrotroph embryonic factor (Tef); Zuotai increased the expression of Nr1d1 at 18:00, but had little influence on Nr1d2 and RORα [62].

Polyporus and Bupleuri radix were popular traditional medicines. Polyporus (Zhulin) is used as a diuresis in the treatment edema, while Bupleuri radix (Chaihu) is used for chronic hepatitis. The Per2Luc mice were used to screen their effects on the circadian clock, and Polyporus was more effective than Bupleuri radix in manipulating the peripheral circadian clock phaseshift, and in promoting time-of-day dependency in vitro as well as in vivo [63].

Jiao-Tai-Wan (JTW), composed of Rhizome Coptidis and Cortex Cinnamomi, is a classical traditional Chinese prescription for insomnia. In obesity-resistant (OR) rats with chronic partial sleep deprivation (PSD) model, 4 weeks of administration of JTW increased total sleep time and total slow wave sleep (SWS) time in OR rats with PSD, and reversed the mode rats elevated serum markers of inflammation and insulin resistance, and these changes were also associated with the up-regulation of Cry1 mRNA and Cry 2 mRNA and the down-regulation of NF-κB mRNA expression in peripheral blood monocyte cells [64].
