TOXICOLOGY AND APPLIED PHARMACOLOGY 143, 380–387 (1997) ARTICLE NO. TO968089 Vitamin E Modulation of Hepatic Focal Lesion Growth in Mice 1 KYLE L. KOLAJA AND JAMES E. KLAUNIG 2 Division of Toxicology, Department of Pharmacology and Toxicology, Indiana University School of Medicine, Indianapolis, Indiana Received May 10, 1996; accepted November 25, 1996 precursor to hepatic cancer (Pitot, 1990). Investigations into Vitamin E Modulation of Hepatic Focal Lesion Growth in Mice. the mechanisms of the growth and death of early focal hepa- KOLAJA, K. L., AND KLAUNIG, J. E. (1997). Toxicol. Appl. Pharma- tocytes may help further to elucidate how these lesions de- col. 143, 380–387. velop into cancer. The effect of DL-a-tocopherol acetate (vitamin E) on hepatic Oxidative stress, an imbalance in the prooxidant/antioxi- focal lesion growth in male B6C3F1 mice previously treated with dant status of the cell (Sies, 1991), can influence the hepato- diethylnitrosamine (DEN) was investigated. After hepatic focal carcinogenic process (Clayson et al., 1994; Klaunig et al., lesions were formed, mice were placed into one of the following 1995). The generation of reactive oxygen species (ROS) dose groups: 0 mg vitamin E/kg NIH-07 diet, 50 mg vitamin E/ has been a proposed mechanism by which some xenobiotics kg NIH-07 diet (control diet), 250 mg vitamin E/kg NIH-07 diet, produce cancer (Sun, 1990; Borek, 1991; Cerutti and Trump, and 450 mg vitamin E/kg NIH-07 diet. Mice were euthanized after 1991; Trush and Kensler, 1991). Compounds can produce either 30 or 60 days of dietary treatment. In normal (nonlesion) ROS through biotransformation into reactive intermediates liver, vitamin E deficiency (0 mg/kg diet) increased hepatic DNA and lead to genotoxic events such as DNA strand breaks synthesis. In addition, vitamin E supplementation (450 mg/kg diet) or oxidative DNA damage (e.g., 8-OH deoxyguanosine). In decreased the incidence of hepatic apoptosis, while vitamin E de- ficiency (0 mg/kg diet) increased the incidence of hepatic apoptosis. addition to reacting with important cellular macromolecules The effect of vitamin E-induced lesion growth was examined by (lipids, DNA, and proteins), oxidative free radicals can stim- measuring the number of focal lesions per liver and the relative ulate ‘‘immediate early genes’’ such as c-myc, c-jun, and c- focal lesion volume. High-dose vitamin E supplementation (450 fos (Crawford, 1990). Transcription factors such as AP-1 mg/kg diet) appeared to enhance the growth of hepatic focal le- (composed of a jun and fos heterodimer) and NFk-B are sions. In particular, basophilic lesions appeared to be the most stimulated by oxidative stress and can lead to enhanced cell sensitive to high-dose vitamin E modulation (450 mg/kg diet) as replication (Maki, 1992; Pahl and Baeuerle, 1994). ROS also evidenced by increased number, volume, and labeling index of can stimulate protein kinase C (Gopalakrishna and Ander- hepatic focal lesions. Vitamin E deficiency also appeared to en- son, 1989), another regulator of hepatocyte proliferation. In hance the growth of hepatic focal lesions, though to a lesser extent addition, oxidative stress can block gap junctional intercellu- than vitamin E supplementation (450 mg/kg diet). In the present lar communication, an important mechanism of tumor pro- study, both vitamin E supplementation (450 mg/kg diet) and defi- ciency (0 mg/kg diet) appeared to enhance focal lesion growth motion and cell proliferation (Ruch and Klaunig, 1988). Oxi- albeit neither treatment enhanced lesion growth as dramatically as dative stress has also been suggested to be a stimulator of known nongenotoxic hepatocarcinogens (e.g., phenobarbital and apoptosis (programmed cell death) (Buttke and Sandstrom, dieldrin). The data presented here suggest that oxidative stress in 1991). Because several studies have indicated that hepatic focal hepatocytes may be a component of the liver tumor promo- tumor promotion is an imbalance in cell proliferation and tion process.  1997 Academic Press apoptosis (Schulte-Hermann et al., 1990; Kolaja et al., 1996a,b), oxidative stress may contribute to the growth of selected focal lesions into neoplasms. Hepatocarcinogenesis is a multistage process that contains Vitamin E, the major lipid-soluble antioxidant in liver, a reversible intermediate stage represented by hepatic focal can scavenge free radicals and preserve membrane integrity lesions (Foulds, 1954; Schulte-Hermann, 1987; Pitot, 1990). (Sies, 1991). Modulation of dietary intake of vitamin E, by A small percentage of these ‘‘early’’ lesions appear to be a either supplementation or deficiency, can lead to an imbal- ance in the oxidative status of a cell. Supplementation of 1 This work was submitted in partial fulfillment of the Ph.D. in Toxicol- vitamin E increases the ability to cope with oxidative stress ogy at Indiana University School of Medicine. (Bachowski et al., 1995). Conversely, vitamin E deficiency 2 To whom correspondence and reprint requests should be addressed can lead to increased production of oxidative damage such at Division of Toxicology, Department of Pharmacology and Toxicology, as lipid peroxidation (Peterson et al., 1992). Vitamin E sup- Indiana University School of Medicine, 1001 Walnut Street MRF 003, Indianapolis, IN 46202. Fax: (317) 274-7787. plementation and deficiency appears to be a method to modu- 380 0041-008X/97 $25.00 Copyright  1997 by Academic Press All rights of reproduction in any form reserved.