**6. Toxicants affect circadian clock gene expression as a novel target of chronotoxicology**

**Table 3** lists some examples of known toxicants which disrupted circadian clock gene expression as a mechanism of their acute and chronic toxic effects to both brain and liver.

*Examples of hepatotoxicants*. Chronic carbon tetrachloride administration in C57 mice (0.6 mL/kg, IP, twice a week for 4 weeks) produced liver injury and fibrosis. The expression of clock genes and metabolic genes in fibrosis livers was altered. The amplitudes of circadian expressions of 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 and cytochrome P450 oxidoreductase (POR) was also lost [65].

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

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

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 phase-

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

shift, and in promoting time-of-day dependency in vitro as well as in vivo [63].

**6. Toxicants affect circadian clock gene expression as a novel target** 

sion as a mechanism of their acute and chronic toxic effects to both brain and liver.

**Table 3** lists some examples of known toxicants which disrupted circadian clock gene expres-

*Examples of hepatotoxicants*. Chronic carbon tetrachloride administration in C57 mice (0.6 mL/kg, IP, twice a week for 4 weeks) produced liver injury and fibrosis. The expression of clock genes and metabolic genes in fibrosis livers was altered. The amplitudes of circadian expressions of

of NF-κB mRNA expression in peripheral blood monocyte cells [64].

**of chronotoxicology**

metabolism-related genes (Sirt1, Ppara, Srebp-1c, Acc1, and Fas) [60].

24 Circadian Rhythm - Cellular and Molecular Mechanisms

at 18:00, but had little influence on Nr1d2 and RORα [62].

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

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

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 gene mutation in the hippocampus [54].

Thus, drugs/toxicants could affect central and peripheral circadian clock gene expression as targets of their therapeutic effects and/or toxicity [10].
