Short Communication Lack of Cytochrome P450 2E1 (CYP2E1) Induction in the Rat Liver by Starvation without Coprophagy

Starvation potentiates the hepatotoxicity of a variety of small molecules, including chlorinated hydrocarbons and nitrosamines, through the induction of CYP2E1. A change in CYP2E1 expression during starvation may also alter the pharmacokinetic profiles of xenobiotics. Northern blot and Western blot analyses revealed that hepatic CYP2E1 was not induced during starvation in rats placed in metabolic or wire-bottom cages in contrast to the induction of CYP2E1 in animals housed in solid-bottom cages. We studied the effect of coprophagy on the expression of hepatic CYP2E1 during starvation. The extent of coprophagy was 24% in fed rats. Fecal matter of starving rats was reduced to 14% of control and starving rats re-ingested ;1.6 g of feces per day. The effect of fecal matter on CYP2E1 expression (i.e., 1.6 g/kg/day for 3 days) was assessed in fed or starving rats. Starving rats gavaged with fecal matter for 3 days resulted in a 3.5-fold increase in the level of CYP2E1 mRNA, while fed rats gavaged with feces failed to show an increase in the mRNA. The increase in the CYP2E1 mRNA level accompanied the induction of CYP2E1. Starving rats gavaged with methanol extract of feces (500 mg/kg/day for 3 days) showed a 3.3-fold increase in CYP2E1 mRNA level in the liver. These results provide evidence that CYP2E1 is not induced by starvation without coprophagy, raising the contention that the mechanistic basis for CYP2E1 induction by starvation should be reevaluated. Cytochrome CYP2E1 (CYP2E1) is an ethanol-inducible form of P450 with broad substrate specificity. The substrates for CYP2E1 include organic solvents, carcinogens, and hepatotoxicants (Hu et al., 1995). CYP2E1 expression is affected by a variety of pathophysiological situations including starvation and diabetes (Hong et al., 1987; Yamazoe et al., 1989; Chen et al., 1999). Diets with high fat/carbohydrate ratios also increase CYP2E1 levels (Yoo et al., 1991; Yun et al., 1992). Starvation triggers a series of adaptive energy-metabolic responses, which include utilization of fatty acids and ketones. Acetone at large dose induces CYP2E1 (Tu et al., 1983; Johansson et al., 1988). Besides the induction of CYP2E1, dietary restriction and starvation induce other hepatic CYPs in rats (e.g., CYP3A2, CYP2B1, and CYP4A) (Longo et al., 2000). Starvation could potentiate hepatotoxicity of a variety of small molecules including chlorinated hydrocarbons and nitrosamines (Koop and Tierney, 1990) through the induction of CYP2E1. It has been proposed that starvation may induce CYP2E1 partly from production of ketone bodies such as acetone (Tu et al., 1983; Lieber, 1997). Other studies showed that diabetes induced CYP2E1 through mRNA stabilization (Song et al., 1987). In the present study, we report that starvation alone did not induce CYP2E1 and that induction of CYP2E1 was greatly affected by coprophagy in starving rats. Experimental Procedures Materials. Alkaline phosphatase-conjugated donkey anti-goat IgG was supplied from Life Technologies (Gaithersburg, MD). [a-P]dCTP (3000 mCi/mmol) was purchased from PerkinElmer Life Sciences (Arlington Heights, IL). Random prime-labeling kit was purchased from Promega (Madison, WI). Most of the other reagents were from Sigma Chemical Co. (St. Louis, MO). Animal Treatment. Fourto 5-week-old male Sprague-Dawley rats were purchased from Daehan Laboratory Animals (Eumsung, Korea). The animals were maintained in a clean room at the Animal Center for Pharmaceutical Research, College of Pharmacy, Seoul National University, at 20 to 23°C with 12-h light and dark cycles and relative humidity of 50%. Rats were supplied with filtered pathogen-free air and water ad libitum and fed conventional laboratory-autoclavable rodent diet 5055 from Purina Laboratory Chow (Kunsan, Korea). The diet contained crude protein (.23.5%), crude fat (.5.1%), crude fiber (3.9% or less), ash (8.0% or less), and added minerals (3.0% or less). Food intake and body weight were recorded every day during experiments. Animals were starved for 1 to 3 days after acclimatization. Livers were used for Western and Northern blot analyses. Animals were housed in metabolic cages (Tecniplast, Varese, Italy) or cages on wire-bottom floors. To completely avoid coprophagy, animals were individually caged in metabolic cages to allow complete separation of feces as animals housed in wire cages sometimes eat feces directly from evacuation. Groups of starving rats were administered feces, suspended in normal saline (1.6 g/kg/day for 3 days). Data points represent at least four independent experiments. Preparation of Methanol and Water Extracts of Feces. Dried feces (42 g) were sequentially extracted three times with methanol in an ultrasonic apparatus and extracted successively with boiling water. Removal of solvent from each preparation in vacuo yielded 1.2 g of methanol extract and 3.5 g of This work was supported by a Korea Research Foundation research fund and a research fund from Brain Korea 21. 1 Abbreviations used are: CYP, cytochrome P450; PNP, p-nitrophenol; SSC, standard saline citrate. Send reprint requests to: Sang Geon Kim, Ph.D., College of Pharmacy, Seoul National University, Sillim-dong, Kwanak-gu, Seoul 151-742, Korea. E-mail: [email protected] 0090-9556/01/2903-213–216$3.00 DRUG METABOLISM AND DISPOSITION Vol. 29, No. 3, Part 2 Copyright © 2001 by The American Society for Pharmacology and Experimental Therapeutics 202/882806 DMD 29:213–216, 2001 Printed in U.S.A. 213 at A PE T Jornals on Jne 1, 2017 dm d.aspurnals.org D ow nladed from water extract. Starving rats were administered methanol or water extracts suspended in 0.1% carboxymethylcellulose or normal saline, respectively (50–500 mg/kg/day for 3 days). Isolation of Microsomal Fractions and Immunoblot Analysis. Hepatic microsomal fractions prepared by differential centrifugation were washed in pyrophosphate buffer and stored in 50 mM Tris-acetate buffer (pH 7.4) containing 1 mM EDTA and 20% glycerol (Kim et al., 1996). The subcellular preparations were stored at 270°C until use. SDS-polyacrylamide gel electrophoresis and immunoblot analyses were performed as described by Kim and Cho (1996). Microsomal proteins were separated by a 7.5% gel and electrophoretically transferred to nitrocellulose. The nitrocellulose was incubated with anti-CYP2E1 antibody (Kim et al., 1996), followed by incubation with alkaline phosphatase-conjugated secondary antibody and developed using 5-bromo-4-chloro-3-indolylphosphate and 4-nitroblue tetrazolium chloride. p-Nitrophenol (PNP) Hydroxylase Activity. The hydroxylation of PNP was determined spectrophotometrically (Koop et al., 1997). Reaction mixtures contained 100 mM PNP, 100 mM potassium phosphate buffer (pH 6.8), and 0.2 mg of microsomal proteins in a total volume of 1 ml. Reactions were initiated with 1 mM NADPH, carried out for 10 min, and terminated with 0.2 ml of 1.5 M perchloric acid. Northern Blot Hybridization. A specific cDNA probe for CYP2E1 gene was amplified by reverse transcriptase-polymerase chain reaction using selective primers (Kim and Novak, 1993) and cloned in the pGEM1T vector (Promega). Total RNA was isolated by thiocyanate-phenol-chloroform RNA extraction (Puissant and Houdebine, 1990). Northern blot analysis was carried out as described by Kim and Cho (1996). Hybridization was performed at 42°C for 18 h with a heat-denatured cDNA probe, which was random prime-labeled with [a-P]dCTP. Filters were washed twice in 23 SSC and 0.1% SDS for 10 min at 42°C, twice in 0.13 SSC and 0.1% SDS for 10 min at 42°C, and then in the solution containing 0.13 SSC and 0.1% SDS for 60 min at 55°C. After quantification of mRNA levels, the membranes were stripped and rehybridized with a labeled probe complementary to 18S rRNA to measure the amount of RNA loaded onto the membranes. Data Analysis. Scanning densitometry was performed with Image Scan & Analysis System (Alpha-Innotech Corporation, San Leandro, CA). One way analyses of variance were used to assess significant differences among treatment groups. For each significant treatment effect, the Newman-Keuls test was used for comparisons of multiple group means.