**3. Circadian rhythm disruption affects therapeutic effects and toxicity of xenobiotics in the liver**

**Table 1** gives a few examples of how the disruption of circadian clock could affect drug effect and toxicity. Most of the examples used genetic models with disruption of circadian clock genes or administration of drugs at different times.

Carbon tetrachloride is a commonly used hepatotoxicant. In SD rats, administration of CCl<sup>4</sup> in the afternoon showed more toxicity than administrated in the morning, the increased toxicity was accompanied by the lowest hepatic GSH levels in the afternoon [36]. Acute CCl<sup>4</sup> toxicity was increased in Per2−/− mice. At the 12-h time point after CCl<sup>4</sup> treatment, more vacuolations were observed in the liver tissues of Per2-null mice as compared to wild-type (WT) mice, and at 24 h after CCl<sup>4</sup> treatment, more severe hepatic necrosis was evident than that occurred in WT mice. A deficit of the Per2 gene enhanced Ucp2 gene expression levels in the liver leading to reduced ATP and increased production of toxic CCl<sup>4</sup> derivatives. The absence of Per2 also caused an increased expression of Clock gene [37]. Per2-null mice were not only sensitive to CCl4 -induced acute hepatotoxicity, but also to CCl<sup>4</sup> -induced chronic toxicity and fibrosis. CCl<sup>4</sup> caused much more severe liver fibrosis and activated hepatic stellate cell (HSC) in mPer2 null mice as compared to WT mice. Per2-null mice exhibited less efficiency in fibrosis resolution and apoptosis resistance in HSC. Transfection of Per2 cDNA into CCl4 -exposed HSC restored apoptosis sensitivity with up-regulation of the TRAIL-R2/ DR5 signaling pathway [38].

was no difference of acetaminophen toxicity between Per2-null and WT mice, but at 20:00 when the Per2 expression is highest, Per2-null mice had less liver injury, with less Cyp1a2 expression to bio-activate acetaminophen [40]. In another study, acetaminophen toxicity is greater at Zeitgeber time (ZT)14 than at ZT2, and clock-deficient mice are resistant to the toxicity at ZT14, with prolonged pentobarbital sleep time (PBST), indicating the reduced activation of acetaminophen [41]. Use Bmal1 mutant mice (Bmal1fx/fxCreAlb), the acetaminophen toxicity at ZT12 was decreased, along with decreased APAP protein adducts and altered acetaminophen metabolism kinetics (increased AA-Gluc), possibly due to decreased NADPH-cytochrome P450 oxidoreductase gene expression and activity at ZT12, as compared to WT mice [42].

**Drug/toxicant Animal models Chronotoxicology References**

Carbon tetrachloride Per2−/− mice Acute toxicity increased in Per2−/− mice [37]

Acetaminophen KM mice 18:00 toxicity >6:00 [23] Acetaminophen Per2−/− mice Toxicity decreased in Per2−/− mice [40] Acetaminophen Clock−/− mice Toxicity decreased in Per2−/− mice, with

levels

Per2−/− mice

prolonged PBST

induction of Cyp1a1

induction of Cyp1a1

metabolic enzyme genes

apoptosis tolerance

to low level of GSH at ZT8

Cyp1b1

fibrosis

Per1−/−/Per2−/− mice Lost diurnal variation in bile acid

ZT17

half-life

adducts, altered APAP metabolism

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

Abolished diurnal variation of TCDD

Abolished diurnal variation of B[a]P

[36]

19

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

[38]

[41]

[42]

[43]

[44]

[45]

[46]

[47]

[48]

[49]

[51]

[52]

Carbon tetrachloride SD rats 18:00 toxicity >6:00, with lowest GSH

Carbon tetrachloride Per2−/− mice Chronic toxicity, fibrosis increased in

Acetaminophen Bmal1fx/fxCreAlb mice Reduced toxicity, reduced protein

Dixon (TCDD) Per1Idc,Per2Idc, Per1/Per2Idc mice

Benzo[a]pyrene Clock mutant (Clk/Clk)

Cholestyramine diet restricted feeding

mice

**Table 1.** Circadian clock gene expression as novel targets in toxicology.

Dixon (TCDD) Per1Idc,Per2Idc mice, cells Increased TCDD induction of Cyp1a1,

Bile duct ligation Per2−/− mice Increased BDL-induced liver injury and

Isoniazide Swiss mice Isoniazid hepatotoxicity at ZT1 > ZT9,

Chlorozoxazone Wistar rats Diurnal variation in CYP2E1 affect its

Diethylnitrosamine (DEN) Clockmut mouse hepatocytes Decreased DEN metabolism and

Cadmium ICR mice Toxicity at ZT 8 > ZT 20, corresponding

Alcohol Per1−/−, Per2−/− mice Less susceptible to alcohol toxicity [50]

Acetaminophen hepatotoxicity also displays diurnal variations. When given acetaminophen in the afternoon, toxicity was greater than that given in the early morning [23, 39]. At 8:00, there


**Table 1.** Circadian clock gene expression as novel targets in toxicology.

*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

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

**Table 1** gives a few examples of how the disruption of circadian clock could affect drug effect and toxicity. Most of the examples used genetic models with disruption of circadian clock

Carbon tetrachloride is a commonly used hepatotoxicant. In SD rats, administration of CCl<sup>4</sup> in the afternoon showed more toxicity than administrated in the morning, the increased toxicity was accompanied by the lowest hepatic GSH levels in the afternoon [36]. Acute CCl<sup>4</sup>

vacuolations were observed in the liver tissues of Per2-null mice as compared to wild-type

that occurred in WT mice. A deficit of the Per2 gene enhanced Ucp2 gene expression levels

absence of Per2 also caused an increased expression of Clock gene [37]. Per2-null mice were

late cell (HSC) in mPer2 null mice as compared to WT mice. Per2-null mice exhibited less efficiency in fibrosis resolution and apoptosis resistance in HSC. Transfection of Per2 cDNA

Acetaminophen hepatotoxicity also displays diurnal variations. When given acetaminophen in the afternoon, toxicity was greater than that given in the early morning [23, 39]. At 8:00, there



treatment, more severe hepatic necrosis was evident than

caused much more severe liver fibrosis and activated hepatic stel-

treatment, more

derivatives. The


to impact their efficacy and toxicity, the time really matters [15].

**toxicity of xenobiotics in the liver**

18 Circadian Rhythm - Cellular and Molecular Mechanisms

(WT) mice, and at 24 h after CCl<sup>4</sup>

not only sensitive to CCl4

toxicity and fibrosis. CCl<sup>4</sup>

DR5 signaling pathway [38].

into CCl4

genes or administration of drugs at different times.

**3. Circadian rhythm disruption affects therapeutic effects and** 

toxicity was increased in Per2−/− mice. At the 12-h time point after CCl<sup>4</sup>

in the liver leading to reduced ATP and increased production of toxic CCl<sup>4</sup>

also evident [20, 35].

was no difference of acetaminophen toxicity between Per2-null and WT mice, but at 20:00 when the Per2 expression is highest, Per2-null mice had less liver injury, with less Cyp1a2 expression to bio-activate acetaminophen [40]. In another study, acetaminophen toxicity is greater at Zeitgeber time (ZT)14 than at ZT2, and clock-deficient mice are resistant to the toxicity at ZT14, with prolonged pentobarbital sleep time (PBST), indicating the reduced activation of acetaminophen [41]. Use Bmal1 mutant mice (Bmal1fx/fxCreAlb), the acetaminophen toxicity at ZT12 was decreased, along with decreased APAP protein adducts and altered acetaminophen metabolism kinetics (increased AA-Gluc), possibly due to decreased NADPH-cytochrome P450 oxidoreductase gene expression and activity at ZT12, as compared to WT mice [42].

In Per1, Per2-deficient mice, the ability of AhR ligand dioxin (TCDD) to induce the Cyp1a1 and Cyp1b1 was enhanced, especially with targeted interruption of Per1 [43]. TCDD induction of Cyp1a1 was 23–43 fold greater during the night time (ZT18) than at the day time (ZT6) in WT mice. However, the diurnal variation in the TCDD induction of Cyp1a1 expression was abolished in Per1ldc, Per2ldc, and Per1ldc/Per2ldc mutant mice, suggesting that Per1, Per2 and their timekeeping function in the circadian clockworks mediate the diurnal variation in TCDD induction of Cyp1a1 [44]. Clock mutant Clk/Clk mice failed to show typical oscillation of AhR expression, and BaP (an AhR ligand) induction of Cyp1a1 was disrupted [45].

In Per2−/− mice, bile duct ligation (BDL)-induced liver injury and fibrosis was increased, along with increases in TNFα, TGFβ1, Col1α, and TIMP1 in livers of Per2-null mice as compared to WT mice [46]. In Per1−/− and Per2−/− mice fed on 2% cholestyramine diet, and/or restricted feeding (phase-shift peripheral clock), liver bile acid levels were increased, and the nuclear receptors CAR and PXR were activated, together with the increased serum enzyme AST levels, indicative of liver damage. In these Per1−/− and Per2−/− mice, the circadian expression of key bile acid synthesis and transport genes, including Cyp7a1 and Ntcp, was lost [47].

The hepatotoxic potential of antituberculosis drug isoniazid varied when it was administered at ZT1, ZT9, and ZT17, and the toxicity was highest when isoniazid was given at ZT1 [48]. Chlorzoxazone is a CYP2E1 metabolized drug, and its kinetics and half-life were altered with the diurnal variation of CYP2E1 activity. The value of chlorzoxazone half-life in plasma of the light phase group was significantly longer than the dark phase group, with an increase of 6-hydroxychlorzoxazone production [49]. Acute alcohol-induced higher toxicity at ZT13 than ZT1 when Per1 and Per2 were highly expressed. Per1−/− and Per2−/− mice were less susceptible to alcohol hepatotoxicity, especially in Per1 null mice. Per1 null mice had decreased expression of peroxisome proliferators-activated receptor-gamma and its target genes related to lipid metabolism such as Srebp1, fatty acid synthase (Fas), CD36, diacylglycerol O-acyltransferase 2 (Dgat2), AP2, and adipsin [50]. In primary hepatocytes isolated from Clock mutant Clk/Clk mice and WT mice, diethylnitrosamine (DEN) induced apoptosis and cell death were reduced in Clock-deficient mice, probably due to decreased DEN metabolism [51]. Cadmium hepatotoxicity is independent of metabolic activation; while its mortality was high at ZT8 than ZT20 when the hepatic GSH level was lowest [52].

producing a Parkinson-like syndrome, but it also produces liver injury. In an attempt to examine the effect of Mn on the central and peripheral clocks, rats were given Mn 1 and 5 mg/kg, ip, every 2 days for 1 month, and the hypothalamus and liver were removed to examine the clock gene expression (**Figure 2**). The results showed that Mn-induced aberrant expression of circadian clock genes in both hypothalamus and liver, and liver was more sensitive to Mn-induced decreases in clock gene Bmal1, Per1, and increase in Dbp, indicating that both central and peripheral clocks could be disrupted by drugs/toxicants [53]. Another example is chronic alcohol administration. Chronic alcohol consumption produced disruption of circadian clock gene expression in both central (hypothalamus) and peripheral tissues (liver and colon) [54], and the liver appeared to be more susceptible than brain in alterations of metabolic genes and core molecular clock disruption. In addition to the fatty liver and affected the diurnal oscillations of metabolic genes (alcohol dehydrogenase 1, carnitine palmitoyltransferase 1a, Cyp2e1, Phosphoenolpyruvate carboxykinase 1, pyruvate dehydrogenase kinase 4, Ppargc1a, Ppargc1b and Srebp1c), the diurnal oscillations of core clock genes (Bmal1, Clock, Cry1, Cry2, Per1, and Per2) and clock-controlled genes (Dbp, Hlf, Nocturnin, Npas2, Rev-erbα, and Tef) were altered in livers from ethanol-fed mice. In contrast, ethanol had only minor effects on the expression

**Figure 2.** Neurotoxicant manganese intoxication produced aberrant expression of circadian clock genes in both central

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

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

21

of core clock genes in the suprachiasmatic nucleus (SCN) [55].

(hypothalamus) and peripheral (liver). Adapted from Li et al. [53].

**chronopharmacology**

examples including our work in the field.

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

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

Thus, alterations of diurnal oscillations would affect drug metabolism, efficacy, and toxicity. On the other hand, drugs could target circadian clock gene expressions to produce biological effects, which will be discussed below.
