Glucocorticoid-Dependent Expression ofO⁶-Methylguanine-DNA Methyltransferase Gene Modulates Dacarbazine-Induced Hepatotoxicity in Mice

Abstract

O 6-methylguanine-DNA methyltransferase (MGMT) plays a crucial role in the defense against the alkylating agent-induced cytotoxic lesion O 6 -alkylguanine in DNA. Although a significant circadian variation in MGMT activity has been found in the liver of mice, the exact mechanism of the variation remains poorly understood. In this study, we present evidence that glucocorticoids were required for the 24-h oscillation of MGMT expression in mouse liver. The exposure of mouse hepatic cells (Hepa1-6) to dexamethasone (DEX) significantly increased the mRNA levels of MGMT in a dose-dependent manner. The DEX-induced increase in MGMT expression was reversed by concomitant treatment with RU486 [11␤- [p-(dimethylamino) phenyl]-17␤-hydroxy-17-(1-propynyl)estra-4,9-dien-3-one], a glucocorticoid receptor antagonist. The mRNA levels of MGMT and its enzymatic activity in the liver of mice showed significant 24-h oscillations, which were not observed in adrenalectomized mice. A single administration of DEX to adrenalectomized mice significantly increased the mRNA levels of MGMT in the liver. These findings suggest that the 24-h oscillation in the hepatic expression of MGMT is caused by the endogenous rhythm of glucocorticoid secretion. Dacarbazine (DTIC), a potent O 6 -guanine-alkylating agent, causes serious hepatotoxicity accompanied by hepatocellular necrosis and hepatic vein thrombosis. DTIC-induced hepatotoxicity in mice was attenuated by administering the drug at the time of day when MGMT expression was abundant. The present findings suggest that glucocorticoid-regulated oscillation in the hepatic MGMT expression is the underlying cause of dosing timedependent changes in DTIC-induced hepatotoxicity.Cytotoxic alkylating agents, cystemustine, dacarbazine (DTIC), and temozolomide, are highly effective against lymphatic and several types of solid tumor (Marchesi et al., 2007). They damage DNA by forming adducts with bases, the most common of which occur at the N 7 position of guanine and the N 3 position of adenine, which are repaired by the base excision repair mechanism. On the other hand, another important lesion formed, in terms of mutagenesis and cytotoxicity, is O 6 -alkylguanine (Marchesi et al., 2007). This adduct can be removed by O 6 -methylguanine-DNA methyltransferase (MGMT), which thus plays a key role in DNA repair from O 6 -guanine-alkylating agent exposure (Kaina et al., 2007). In fact, the lethal effect of DTIC in homozygous MGMT knockout mice was more severe than it was in wildtype mice (Shiraishi et al., 2000).It has been reported that MGMT activity in the liver of mice shows significant circadian variation, and this rhythmicity could account for the dosing time-dependent change in cystemustine-induced toxicity (mortality and myelosuppression) in mice (Martineau-Pivoteau et al., 1996a,b). Time-

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“…1D) Table S2). The functional analysis of the genes, done using KEGG database (6), showed that 12 biological pathways were enriched in a statistically significant manner (P < 0.05; Supplementary Table S3). Among these pathways, we further focused on the cell-cycle pathway because Atf4 À/À cells introduced with oncogenes resulted in G 0 -G 1 phase arrest (Fig.…”

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