**2. Circadian rhythms affect Phase-I, Phase-II, and transporter gene expressions in the liver**

Liver is the major site of xenobiotics metabolism and disposition. Accumulating evidence clearly indicates that circadian rhythms affect the gene/protein expression encoding xenobiotics uptake (Oatps and Ntcp), Phase-I metabolism (P450) and detoxication (Nrf2, MT-1, and GSH systems), Phase-II conjugation (glutathione S-transferases, UDP-glucuronosyltransferases, and sulfotransferases), and efflux transporters (Mrps and MDR) (**Figure 1**).

*Diurnal variation of hepatic uptake transporters*. In the liver, the major uptake transporters are organic anion transporting polypeptides (Oatp1a1, Oatp1a4, Oatp1b2, and Oatp2b1), organic cation transporter (Oct1), organic anion transporters (Oat2 and Oat6), and others [19]. The expressions of Oatp1a1, Oatp1a4, Oatp1b2, Oct1, and Oat2 display diurnal oscillations, with higher expression in the morning, while Oatp2b1 did not show circadian variation [20]. Na<sup>+</sup> -taurocholate cotransporting polypeptide (Ntcp and Slc10a1) is a major bile acid uptake transporter that localizes to the basolateral membrane of hepatocytes, and displays apparent circadian rhythm, with higher expression in the afternoon [20–22].

*Diurnal variation of hepatic Phase-I P450 metabolism enzyme genes*. Hepatic cytochrome P450 is the major enzyme catalyzing the Phase-I drug metabolism. Most drugs are metabolized by

reproductive system [3, 4]. Disruption of biological rhythms produces negative effects in the short and long terms leading to various diseases [4]. For example, clock dysfunction accelerates the development of liver diseases such as fatty liver diseases, hepatitis, cirrhosis,

Circadian oscillations are generated by a set of genes forming a transcriptional autoregulatory feedback loop. In mammals, these include the core clock regulators (Clock, Bmal1, and Npsa2), the clock feedback loop regulator genes (Per1, Per2, Per3, Cry1, and Cry2), and the clock target genes (DBP, Rev-erbα (Nr1d1), RORα, Tef, CK1δ, etc.) [6, 7]. The central clock is located in the suprachiasmatic nucleus in the hypothalamus and peripheral clocks in all tissues. Peripheral clocks in the liver have fundamental roles in maintaining liver homeostasis, including the regulation of energy metabolism and the expression of enzymes controlling the absorption and metabolism of xenobiotics [8]. Over the past three decades, researchers have investigated the molecular mechanisms using global clock-gene knockout mice, or clock gene mutant mice, or other genetic and molecular biology tools to elucidate molecular architecture of circadian clock in mammals [9]. Chronopharmacology and chronotoxicology is a new interdisciplinary science aimed at studying the influence of circadian system on drug disposition, efficacy, and toxicity. Xenobiotics absorption, distribution, metabolism, especially by P450, and excretion [10–14], all under circadian regulation. Circadian variations on these hepatic drug processing genes [15] greatly influence therapeutic effects and toxicity of drugs [10, 16–18]. The chronotherapy of anticancer drugs gives an excellent example [18]. This chapter will focus on the general aspects of circadian rhythms on drug/toxicant disposition and biological effects, and will also discuss the effects of drugs/ toxicants on circadian clock gene expression as a novel target of chronopharmacology and chronotoxicology. A dozen of our publications in recent 5 years were also included for discussion.

and liver cancer. Liver disorders also, in turn, disrupt circadian clock function [5].

**2. Circadian rhythms affect Phase-I, Phase-II, and transporter gene** 

and sulfotransferases), and efflux transporters (Mrps and MDR) (**Figure 1**).

circadian rhythm, with higher expression in the afternoon [20–22].

Liver is the major site of xenobiotics metabolism and disposition. Accumulating evidence clearly indicates that circadian rhythms affect the gene/protein expression encoding xenobiotics uptake (Oatps and Ntcp), Phase-I metabolism (P450) and detoxication (Nrf2, MT-1, and GSH systems), Phase-II conjugation (glutathione S-transferases, UDP-glucuronosyltransferases,

*Diurnal variation of hepatic uptake transporters*. In the liver, the major uptake transporters are organic anion transporting polypeptides (Oatp1a1, Oatp1a4, Oatp1b2, and Oatp2b1), organic cation transporter (Oct1), organic anion transporters (Oat2 and Oat6), and others [19]. The expressions of Oatp1a1, Oatp1a4, Oatp1b2, Oct1, and Oat2 display diurnal oscillations, with higher expression in the morning, while Oatp2b1 did not show circadian variation [20].


*Diurnal variation of hepatic Phase-I P450 metabolism enzyme genes*. Hepatic cytochrome P450 is the major enzyme catalyzing the Phase-I drug metabolism. Most drugs are metabolized by

**expressions in the liver**

16 Circadian Rhythm - Cellular and Molecular Mechanisms

Na<sup>+</sup>

**Figure 1.** Drug metabolism (Phase-I, Phase-II, transporter) and detoxication (GSH, Nrf2, MT-1) gene expression show circadian oscillations.

P450 1–4 family enzymes. P450 enzyme genes and corresponding nuclear receptors display diurnal oscillations: AhR and Cyp1a1, 1a2 are higher in the morning; CAR and Cyp2b10 are higher in the afternoon and evening; PXR is higher in the afternoon but Cyp3a11 and Cyp3a25 are higher in the morning; PPARα is higher in the morning but Cyp4a10 is higher in the evening [23]. Cyp7a1 is a rate-limit enzyme gene for bile acid synthesis, displays a typical circadian rhyme, with the peak around 18:00 [21–24]. Bile acid synthesis is controlled by the circadian clock and Rev-erbα is a major clock gene controlling bile acid homeostasis [25].

In the liver, circadian rhythm serves to synchronize the metabolism of bile acid, glucose, and lipid, and their disruption could lead to diseases and affect chronotherapy [26]. Indeed, the liver is the key organ to maintain energy metabolism which is greatly influenced by feeding, diets, and diurnal variation [5]. For example, Peroxisome proliferator-activated receptorgamma coactivator (PGC1α) stimulates the expression of clock genes, notably Bmal1 (also called Arntl) and Rev-erbα (also called Nr1d1), through coactivation of the ROR family of orphan nuclear receptors. Mice lacking PGC-1α show abnormal diurnal rhythms of activity, body temperature and metabolic rate [27]. Circadian clocks regulate metabolic processes not only by simply in response to daily environmental/behavioral influences but also by synchronizing the cell with its environment to modulate a host of metabolic processes [27–29].

*Diurnal variation of hepatic detoxification enzyme genes*. Many antioxidant enzyme genes display diurnal variations, such as the Nrf2 detoxication pathway genes [30], enzymatic detoxication components such as superoxide dismutase (SOD), catalase, glutathione peroxidase (GSH-Px1) and non-enzymatic protein such as metallothionein (MT) [31]. GSH is low in the afternoon which is partially responsible for acetaminophen hepatotoxicity when given in the afternoon [23].

*Diurnal variation of hepatic Phase-II metabolism gene/proteins*. Glucuronide and sulfate conjugations are major Phase-II pathways in the biotransformation and elimination of a wide variety of endogenous compounds, drugs, and other xenobiotics. Diurnal variations of these Phase-II reactions were reported in the 1980s [32]. Consistent to the variation in the conjugation reactions, the expression of Ugt1a5, 2a3, 2b34, 2b36 and UDP-gpb, as well as Sult1a1, 1a5, and Sult5a1, all show diurnal oscillations [20]. Hepatic GSH has the trough at dusk [30], and the activities of GSH S-transferase [33] were lower at the dark phase and the expression of Gst1a1/1, Gst1a4, Gstm2, and Gstt1/2 display diurnal rhythms which are generally lower in the dark phase [20].

*Diurnal variation of hepatic Phase-III efflux transporters*. P-glycoprotein is the major efflux pump in the liver, and its expression shows circadian variation together with the diurnal expression of Abcb1 [34]. In addition to P-glycoprotein, hepatic multidrug-resistant protein 2 (MRP2), breast cancer resistant protein (BCRP) also show circadian oscillations [35]. Diurnal variations in hepatic mRNA expression of multidrug-resistant gene 1a (Mdr1a), Mrp2, and Bcrp were also evident [20, 35].

Diurnal variation of hepatic Phase-I, Phase-II, Phase-III, and the nuclear transcription factors would affect the xenobiotic metabolism when administered at the different times of the day to impact their efficacy and toxicity, the time really matters [15].
