Mitochondrial dysfunction by γ-irradiation accompanies the induction of cytochrome P450 2E1 (CYP2E1) in rat liver

Mitochondrial dysfunction by γ-irradiation accompanies the induction of cytochrome P450 2E1 (CYP2E1) in rat liver

Toxicology 161 (2001) 79 – 91 www.elsevier.com/locate/toxicol Mitochondrial dysfunction by g-irradiation accompanies the induction of cytochrome P450...

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Toxicology 161 (2001) 79 – 91 www.elsevier.com/locate/toxicol

Mitochondrial dysfunction by g-irradiation accompanies the induction of cytochrome P450 2E1 (CYP2E1) in rat liver Hye Chin Chung a, So Hee Kim a, Myung Gull Lee a, Chul Koo Cho b, Tae Hwan Kim b, Dong Han Lee b, Sang Geon Kim a,* a

College of Pharmacy and Research Institute of Pharmaceutical Sciences, Seoul National Uni6ersity, Sillim-dong, Kwanak-gu, Seoul 151 -742, South Korea b Laboratory of Radiation Effect, Korea Cancer Center Hospital, Korean Atomic Energy Research Institute, Seoul, South Korea Received 8 September 2000; received in revised form 25 September 2000; accepted 14 December 2000

Abstract Multiple biological effects are induced by ionizing radiation through dysfunction of cellular organelles, direct interaction with nucleic acids and production of free radical species. The expression of cytochrome P450s was assessed in the livers of 60Co g-irradiated rats. Three gray (G) of g-irradiation caused CYP2E1 induction with a 3.6-fold increase in the mRNA at 24 h, whereas the expression of CYP1A2 and CYP3A was not changed. Pharmacokinetics of chlorzoxazone, a specific substrate of CYP2E1, was studied in 3 G-irradiated rats. The area under the plasma concentration–time curve from time zero to infinity of 6-hydroxychlorzoxazone and the amount of 6-hydroxychlorzoxazone excreted in 8 h urine were both significantly greater than those in control rats. Hepatic CYP2E1 was not induced in rats exposed to 0.5–1 G of g-rays. Rats irradiated at 6 – 9 G accumulated doses of g-rays exhibited smaller increases in the mRNA due to liver injury than those irradiated at a single dose of 3 G g-rays. The plasma glucose and insulin levels were not altered in rats with 3 G of g-irradiation. As the exposure level of g-irradiation increased, the activity of hepatic aconitase, a key enzyme in energy metabolism in mitochondria, was 30 – 90% decreased. The amount of mitochondrial DNA per gram of wet liver was 50% decreased in rats exposed to 3 G of g-rays. These results demonstrated that g-ray irradiation at the exposure level inducing organelle dysfunction induced CYP2E1 in the liver, which might be associated with mitochondrial damage, but not with alterations in glucose or insulin levels. © 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: CYP2E1; Ionizing radiation; Mitochondrial injury; Aconitase; Chlorzoxazone

Abbre6iations: ALT, alanine aminotransferase; CYP2El, cytochrome P450 2E1; G, gray; ROS, reactive oxygen species; SDS, sodium dodecylsulfate; SSC, standard saline citrate; CZX, chlorzoxazone; OH-CZX, 6-hydroxychlorzoxazone; iv, intravenous; HPLC, high-performance liquid chromatography; AUC, total area under the plasma concentration– time curve from time zero to infinity; AUMC, first moment of AUC; MRT, mean residence time; CL, time-averaged total body clearance; Vss, apparent volume of distribution at steady state; Ae0 “ 8 h, total amount excreted unchanged in urine between 0 and 8 h. * Corresponding author. Tel.: + 82-2-8807840; fax: + 82-2-8721795. E-mail address: [email protected] (S.G. Kim). 0300-483X/01/$ - see front matter © 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 0 0 - 4 8 3 X ( 0 1 ) 0 0 3 3 2 - 8

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1. Introduction

2. Materials and methods

Cytochrome P450 2E1 (CYP2E1) is inducible by small organic molecules and pathophysiological states (Hong et al., 1987; Kim and Novak, 1993; Lieber, 1997). The mechanisms governing regulation of CYP2E1 expression are complex and involve transcriptional, post-transcriptional, translational and post-translational events (Kim et al., 1990; Woodcroft and Novak, 1997). The expression of CYP2E1 is also affected by insulin, thyroid hormones and growth hormone in hepatocytes (Waxman et al., 1989; Peng and Coon, 1998; Son et al., 2000). CYP2E1 expression appeared to be affected by the plasma glucose level (Son et al., 2000) in conjunction with the aforementioned hormones. It seems that glucose utilization is an important determinant for CYP2E1 expression. Ionizing radiation induces multiple biological effects through direct interaction with DNA or production of activated free radical species from water (Tubiana et al., 1990). Previous studies from our laboratories have shown that g-irradiation induces hepatic glutathione S-transferases and microsomal epoxide hydrolase in the liver with concomitant increases in the mRNAs (Kim et al., 1997; Nam et al., 1997). Induction of the phase II detoxifying enzymes by g-irradiation may represent adaptive responses against oxidative stress and protect cells against subsequent exposure to radiation. In spite of the studies on the expression of phase II detoxifying enzymes, no studies have been conducted on the expression of cytochrome P450s in response to g-irradiation. Many studies have shown that the expression of cytochrome P450 is highly associated with oxidative stress (Gergel et al., 1997; Schlezinger et al., 1999; Morel et al., 2000). In view of the close relationship of oxidative stress with the expression of cytochrome P450, the present study was designed to determine the expression of major cytochrome P450 forms in the liver after g-irradiation. g-Irradiation damages cellular organelles and induces subsequent cellular changes. Because CYP2E1 induction is associated with change in energy metabolism, mitochondrial dysfunction after g-irradiation was correlated with CYP2E1 expression in the present study.

2.1. Materials Alkaline-phosphatase conjugated goat anti-rabbit IgG was supplied from Life Technologies (Gaithersburg, MD, USA). [a-32P]dCTP (3000 mCi/mmol) was purchased from New England Nuclear (Arlington Heights, IL, USA). Random prime-labeling kit was obtained from Promega (Madison, WI, USA). The assay kit for determination of plasma insulin was purchased from Amersham Pharmacia Biotech (Arlington Heights). Chlorzoxazone (CZX), b-glucuronidase (Type H-1, from Helix pomatia) and reagentgrade ammonium acetate were purchased from Sigma Chemical (St. Louis, MO, USA). 6-Hydroxychlorzoxazone (OH-CZX) and 3aminophenyl sulfone (an internal standard of HPLC assay for CZX and OH-CZX) were obtained from Research Biomedical International (Natick, MA, USA) and Aldrich Chemical (Milwaukee, WI, USA), respectively. Most of the reagents in the molecular studies were supplied from Sigma Chemical.

2.2. Animal treatments Five-week-old male Sprague –Dawley rats (1509 20 g) were supplied from the Korean Food and Drug Administration (Seoul, South Korea). Animals were maintained in a clean room at the Animal Center for Pharmaceutical Research, College of Pharmacy, Seoul National University (Seoul, South Korea), at a temperature between 20 and 23°C with 12 h light and dark cycles and a relative humidity of 50%. Animals were caged under the supply of filtered pathogen-free air and water ad libitum. Food intake and body weight were recorded everyday during experiments. Rats were subjected to total body irradiation of 0.5, 1, 3 or 9 gray (G) at a dosage rate of 12.5 cG/min from a 60Co radiation source. Groups of animals (four animals per group) were irradiated with a dose of 3 G g-rays per day for 1–3 day(s) and sacrificed 24 h after the last irradiation. The body weight was not significantly changed after irradiation. Food intake of 3 G-irradiated rats

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was  30% reduced. Data points represent the results from at least four animals. Assays were performed with the samples prepared from individual animals. Blood was collected from the heart and analyzed for glucose level and alanine aminotransferase (ALT) activity. In addition, rats were sacrificed at 1, 3, 5, 7 and 14 day(s) after a single dose of 3 G irradiation to determine changes in plasma insulin levels.

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Hepatic microsomal fractions were prepared by differential centrifugations at 10 000 g for 30 min and then at 100 000 g for 90 min. Microsomes were washed in pyrophosphate buffer and stored in 50 mM Tris –acetate buffer (pH 7.4) containing 1 mM ethylenediamine tetracetate (EDTA) and 20% glycerol, as described previously (Kim et al., 1996). Protein contents were assayed (Lowry et al., 1951) and the subcellular preparations were stored at −7°C until use.

phenol –chloroform RNA extraction according to the method of Puissant and Houdebine (1990). Northern blot analysis was carried out according to the procedure, as described previously (Kim and Cho, 1996). Briefly, total RNA isolated from the liver was resolved by electrophoresis in a 1% agarose gel containing 2.2 M of formaldehyde, and transferred to nitrocellulose paper. The nitrocellulose paper was baked in a vacuum oven at 80°C for 2 h. The blot was hybridized, as described previously (Kim and Cho, 1996). Filters were washed in 2× standard saline citrate and 0.1% SDS for 10 min at room temperature three times and in 0.1× standard saline citrate and 0.1% SDS for 10 min at 42°C twice. Filters were finally washed in the solution containing 0.1× standard saline citrate 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 quantify the amount of RNA loaded onto the membranes.

2.4. Immunoblot analysis

2.6. Alanine aminotransferase (ALT) acti6ity

Sodium dodecylsulfate (SDS) – polyacrylamide gel electrophoresis and immunoblot analyses were performed according to the previously published procedures (Kim et al., 1996). Microsomal proteins were separated by a 7.5% gel, and electrophoretically transferred to nitrocellulose paper. The nitrocellulose paper was incubated with antiCYP2E1 antibody (Kim et al., 1996), followed by incubation with alkaline phosphatase-conjugated secondary antibody and developed using 5bromo-4-chloro-3-indolylphosphate and 4-nitroblue tetrazolium chloride.

The activity of plasma ALT was assayed using a commercial assay kit (Yeongdong Pharmaceutical, Seoul, South Korea). It was used as a parameter for liver damage.

2.3. Preparation of microsomal proteins

2.5. Northern blot hybridization Specific cDNA probes for the CYP2E1, CYP1A2 and CYP3A genes were amplified by reverse transcription – polymerase chain reaction using the selective primers (Kim and Novak, 1993), cloned in the pGEM+ T vector (Promega, Madison, WI, USA), as described previously (Cho et al., 1999). Total RNA was isolated using the improved single-step method of thiocyanate –

2.7. Intra6enous pharmacokinetic study of CZX The carotid artery and the jugular vein of each rat (Charles River, Atsugi, Japan) were cannulated with polyethylene tubing (Clay Adams, Parsippany, NJ, USA) under light ether anesthesia. Each cannula was exteriorized to the dorsal side of the neck, where each cannula terminated with long Silastic tubing (Dow Corning, Midland, MI, USA). The Silastic tubings were inserted in a wire coil to allow free movement of the rat. The exposed areas were surgically sutured. Each rat was housed individually in a rat metabolic cage (Daejong Scientific Company, Seoul, South Korea) and allowed to recover from anesthesia for 20 h before the study began. They were not restrained at any time during the study. Heparinized 0.9% NaCl-injectable solution (20 units/ml), 0.3 ml, was

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used to flush each cannula to prevent blood clotting. CZX (25 mg/kg), dissolved in a minimum amount of 2 N NaOH solution and filtered through 0.45 mm filter, was administered by iv infusion in 1 min via the jugular vein (total injection volume was approximately 0.6 ml) to control (n= 6) and irradiated (n = 7) rats 24 h after 3 G irradiation. Approximately 0.12 ml aliquot of blood sample was collected via the carotid artery at 0 (to serve as a control), 1 (at the end of the infusion), 5, 15, 30, 45, 60, 90, 120, 180,240, 300, 360 and 480 min after administration of CZX. After centrifugation, a 0.05 ml aliquots of plasma samples were kept in −70°C freezer (Revco ULT 1490 D-N-S; Western Mednics, Asheville, NC, USA) until HPLC analysis of CZX and OH-CZX (Frye and Stiff, 1996). Approximately 0.3 ml aliquot of heparinized 0.9% NaCl-injectable solution (20 units/ml) was used to flush the cannula immediately after each blood sampling. At the end of 8 h, each metabolic cage was rinsed with 20 ml of distilled water and the rinses were combined with the 8 h urine. After measuring the exact volume of the combined urine, two 0.05 ml aliquots of combined urine samples were kept in −70°C freezer until HPLC analysis of CZX and OH-CZX (Frye and Stiff, 1996).

2.8. HPLC analysis The concentrations of CZX and OH-CZX in the above biological samples were analyzed by the reported HPLC method (Frye and Stiff, 1996). To 0.05 ml of plasma or urine sample were added 0.1 ml of 0.2 M sodium acetate buffer (pH 4.75) and 200 units of b-glucuronidase dissolved in 0.1 ml of isotonic phosphate buffer (pH 7.4). Samples were manually mixed and incubated in a water-bath shaker kept at 37°C and at a rate of 50 oscillations per min for 2 h. After incubation, 0.05 ml methanol containing 10 mg/ml of 3-aminophenyl sulfone was added, vortex-mixed, and 1 ml of diethyl ether was added. The mixture was shaken for 10 min and centrifuged at 2000 g for 10 min. The upper organic layer was transferred to a clean tube and evaporated at 37°C under a stream of nitrogen. The residues were reconstituted in 100 ml

of the mobile phase and a 50 ml aliquot was injected directly onto the HPLC column. The mobile phase, 0.1 M ammonium acetate:acetonitrile:tetrahydrofuran (72:22:5.5, v/v/v) was run at a flow rate of 1.5 ml/min. An UV detector set at 283 nm monitored the column effluent. The retention times for OH-CZX, 3aminophenyl sulfone and CZX were approximately 6, 10 and 18 min, respectively. The detection limits for CZX and OH-CZX in plasma were both 0.1 mg/ml and the corresponding values in urine were 4 mg/ml. The coefficients of variation of the assay (within- and between-day) were generally low (below 8.2%).

2.9. Pharmacokinetic analysis The area under the plasma concentration –time curve from time zero to infinity (AUC) was calculated by the trapezoidal rule-extrapolation method; this method employed the logarithmic trapezoidal rule for the calculation of the area during the declining plasma level phase (Chiou, 1978) and the linear trapezoidal rule for the rising plasma level phase. The area from the last data point to time infinity was estimated by dividing the last measured plasma concentration by the terminal rate constant. Standard methods (Gibaldi and Perrier, 1982) were used to calculate the time-averaged total body clearance (CL), first moment of AUC (AUMC), mean residence time (MRT), and apparent volume of distribution at steady state (Vss) (Kim et al., 1993). CL =

Dose , AUC

AUMC =

&

(1)

t·Cp dt,

(2)

0

MRT =

AUMC , AUC

Vss = CL × MRT,

(3) (4)

where Cp is the plasma concentration of CZX at time t.

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The harmonic mean method was used to calculate the mean values of Vss (Chiou, 1979), terminal half-life (Eatman et al., 1977), and CL (Chiou, 1980). A P-value of less than 0.05 was considered to be statistically significant using t-test between two means for the unpaired data (Statistical Research Institute, College of Natural Sciences, Seoul National University, Seoul, South Korea). All data were expressed as mean9SD.

2.10. Plasma glucose and insulin le6els Glucose was colorimetrically assayed using a commercial kit (Glucose-E, Yeongdong Pharmaceutical). The plasma insulin content was measured by the enzyme-linked immunosorbent assay according to the manufacturer’s protocol (Amersham Pharmacia Biotech).

2.11. Preparation of mitochondrial fraction The livers were homogenized in nine volumes of 2 mM Hepes buffer (pH 7.4) containing 0.3 M mannitol, as described by Raijman and Bartulis (1979). The homogenate was diluted with the buffer, followed by centrifugations at 600 and 15 000 g for 5 min each, and the post-nuclear supernatant was carefully decanted. Then, the mitochondrial fraction was washed with the Hepes buffer, and resuspended. This method yielded a mitochondrial preparation minimally contaminated with lysosomes and endoplasmic reticulum, as described previously (Katz et al., 1983; Cohen and Cheung, 1984).

2.12. Aconitase acti6ity Aconitase activity was assessed in the post-nuclear fraction by measuring the conversion of citrate to isocitrate, and subsequent production of a-ketoglutarate was spectrophotometrically determined at the wavelength of 340 nm. The reaction mixture contained the 50 mM Tris – Cl buffer (pH 7.4) containing 30 mM citrate, 0.2 mM NADP+, 0.6 mM MnCl2, 1–2 unit(s) of isocitrate dehydrogenase and 0.1 mg of post-nuclear fraction in a total volume of 1 ml. The reaction was carried out at 25°C for 1 h. One unit of aconitase activity is

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equivalent to the production of 1 mmole of isocitrate per min.

2.13. Isolation of mtDNA Mitochondrial DNA (mtDNA) was prepared using a commercially available Trizol® Reagent kit according to the manufacturer’s instruction (GIBCOBRL, NY, USA). The DNA content was spectrophotometrically quantified by measuring the absorbance ratio at 260 –280 nm (Richter et al., 1988).

2.14. Data analysis Scanning densitometry of Northern and Western blots was performed with the Image Scan and Analysis System (Alpha-Innotech, San Leandro, CA, USA) to assess the expression of CYP2E1. Data were analyzed using the SigmaStat® program (SPSS Inc, San Rafael, CA, USA) and expressed as means9 SD. One way analysis of variance procedures were used to assess significant differences among treatment groups. For each significant effect of treatment, the Newman – Keuls test was used for comparisons of multiple group means.

3. Results

3.1. Effects of k-irradiation on cytochrome P450 expression The expression of cytochrome P450 was monitored in the livers of rats 24 h after 3 G of g-irradiation. Whereas the expression of hepatic CYP1A2 and CYP3A was not affected by g-irradiation, CYP2E1 protein was 2.5-fold induced, as compared to control (Fig. 1). Northern blot analysis confirmed that the CYP2E1 mRNA level was 3.6-fold increased in 3 G-irradiated rats. In contrast, the mRNA levels of CYP1A2 and CYP3A were not changed. To monitor the level of CYP2E1 in rats exposed to varying doses of g-rays, rats were irradiated at the doses from 0.5 to 9 G of g-rays. g-Rays at 0.5 or 1 G, however, failed to increase

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the CYP2E1 mRNA in the liver. g-Irradiation at the doses from 3 to 9 G caused significant increases in the mRNA (Fig. 2A). Nonetheless, the increase in CYP2E1 mRNA by 9 G was less than that by 3 G presumably due to liver toxicity. Studies were extended to assess the extent of CYP2E1 induction following multiple doses of daily 3 G g-irradiation (Fig. 2B). The CYP2E1 mRNA level failed to be further increased following 2–3 repeated exposures. Rats irradiated at 6 – 9 G accumulated g-rays exhibited smaller increases in the mRNA than that caused by a single dose of 3 G g-rays. This would result from diminished gene expression due to radiation-induced liver injury.

3.2. ALT acti6ity To assess the liver injury by g-rays, ALT activity was monitored in the plasma of g-irradiated rats. Whereas rats exposed to a single dose of 3 G

Fig. 1. Expression of hepatic cytochrome P450s in g-irradiated rats. Rats were exposed to a single dose of 3 G g-rays. The mRNA and protein levels for CYP2E1, CYP1A2 and CYP3A were assessed 24 h after irradiation. Northern blot analyses were carried out with total RNA fractions (20 mg of each) prepared from the livers of rats 24 h after g-irradiation. The amount of RNA loaded in each lane was confirmed by rehybridization of the stripped membrane with a 32P-labeled probe complementary to 18S rRNA. Western blot analyses for the cytochrome P450s were performed with 15, 10 and 5 mg of hepatic microsomal proteins per lane, respectively. For details, see Section 2. Con, Control.

g-rays or less showed minimal changes in the plasma ALT activity, a single dose of 9 G or multiple doses of 3 G irradiation increased the plasma ALT activity (Fig. 3A and Fig. 3B). These results confirmed the possibility that the diminished CYP2E1 induction by 6–9 G g-rays resulted from the tissue injury.

3.3. Pharmacokinetics of chlorzoxazone Studies were extended to determine whether the induction of CYP2E1 in the liver by 3 G of g-irradiation indeed changed CYP2E1-mediated drug metabolism in vivo. Pharmacokinetic studies were performed with CZX, which is known to be primarily catalyzed to OH-CZX by CYP2E1 (Peter et al., 1990). The mean arterial plasma concentration –time profiles of CZX and OH-CZX after iv administration of CZX, 25 mg/kg, to control and 3 G-irradiated rats are shown in Fig. 4, and some relevant pharmacokinetic parameters are listed in Table 1. After iv administration of CZX, the plasma levels of CZX declined in a polyexponential fashion for both groups of rats (Fig. 4A) with mean terminal half-lives of 44.5 and 33.4 min for control and irradiated rats, respectively; they were significantly different (Table 1). The MRT of CZX was also significantly shorter (26% decrease) in irradiated rats (Table 1). Although the AUCs of CZX were not significantly different between both groups of rats, the amount of CZX excreted in 8 h urine as unchanged drug (Ae0 “ 8 h) in irradiated rats was significantly smaller (84% decrease, expressed as the percentage of iv dose of CZX) than that in control rats (Table 1). The CL and Vss of CZX were not significantly different between two groups of rats (Table 1). Formation of OH-CZX after iv administration of CZX seemed to be rapid; for both groups of rats, the plasma concentration of OH-CZX detected from the first blood sampling time (5 min), reached its peak at approximately 45 min and declined thereafter in a monoexponential fashion (Fig. 4B). The plasma concentrations of OH-CZX in irradiated rats were higher than those in control rats (Fig. 4B). This resulted in a significantly greater AUC of OH-CZX (72% increase). In irradiated rats, the Ae0 “ 8 h of OH-CZX was signifi-

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Fig. 2. Hepatic CYP2E1 mRNA and protein levels in rats irradiated with g-rays. CYP2E1 expression was assessed in rats exposed to a single dose from 0.5 to 9 G of g-rays (A) or in rats daily exposed to a dose of 3 G g-rays for 2 – 3 days (B). The mRNA and protein levels were measured 24 h after the last treatment, as described in Fig. 1. Upper panels show representative Northern blots. Data represent means 9 SD from 4 animals. One way analysis of variance was used for comparisons of multiple group means, followed by Newman– Keuls test (significant as compared to control, **PB 0.0l). Con, Control.

cantly greater (24% increase, expressed as the percentage of iv dose of CZX) and terminal halflife was significantly shorter (25% decrease) than those in control rats (Table 1). These results demonstrated that 3 G of g-irradiation altered CYP2E1-mediated drug metabolism in vivo.

3.4. Plasma glucose and insulin le6els Previous studies from our laboratories have shown that the blood glucose level and glucose utilization affected the expression of CYP2E1 in hypophysectomized and dehydrated animals (Kim et al., 2000; Son et al., 2000). Therefore, it was assessed whether the plasma glucose level was altered in rats exposed to g-rays. The plasma glucose content was not changed in rats g-irradiated at the doses from 0.5 to 9 G (Table 2).

Multiple exposures of rats to g-irradiation failed to change the glucose level (Table 2). The plasma insulin level was also monitored. The plasma insulin level was not changed in rats at 1 through 14 day(s) after a single dose of 3 G g-irradiation (i.e. 16 IU/ml of plasma). These results raised the notion that hepatic CYP2E1 induction by 3 G of g-rays was not related with alterations in the plasma glucose or insulin levels.

3.5. Aconitase acti6ity and mtDNA contents Given no changes in the plasma glucose and insulin levels after g-irradiation, we were interested in whether the cellular glucose utilization would be affected by irradiation. Mitochondria are the organelles of eukaryotic cells for carbohydrate metabolism, and mitochondrial oxidative

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phosphorylation is the major energy transduction pathway. Aconitase activity, which represents mitochondrial function, was measured by the turnover of citrate to isocitrate as an index of cellular energy utilization. The aconitase activity decreased in the post-nuclear fraction of the liver, as the exposure level of g-irradiation increased (Fig. 5A). Hepatic aconitase activities in rats irradiated at the doses of 3 G and 9 G were 70 and 10% of control, respectively. The aconitase activity in the livers of rats g-irradiated with 3 G per day for 2–3 consecutive days was 40 – 70% reduced (Fig. 5A). Reactive oxygen intermediates modify and reduce mtDNA because the mtDNA is localized in the proximity of the inner membrane and has limited repair capacity (Croteau et al., 1997). The mitochondrial DNA content, which represents mitochondrial function, was measured in the livers of rats exposed to g-rays. The amounts of mtDNA per gram wet liver were 33, 50 and 80% decreased in rats exposed to a single dose of 1, 3 and 9 G g-rays, respectively, as compared to control (Fig. 5B). Exposure of daily 3 G of g-rays for 2 –3 days also reduced the content of mtDNA by 63–70% (Fig. 5B). It is likely that mitochondrial dysfunction by g-irradiation and subsequent change in energy production may be associated

with the induction of CYP2E1.

4. Discussion Previous studies provided evidence that many of the damaging effects of ionizing irradiation are due to the generation of reactive oxygen species (ROS), in particular hydroxyl radicals. Ionizing radiation includes g-rays and X-rays. The effects of 60Co g-rays and X-rays on biological systems seem to be comparable. The relative biological effectiveness of X-rays radiation for the production of acute lethality in mice was 1.3 –1.4 of 60Co g-rays (Storer et al., 1957). Other studies showed that there were only minor differences between gand X-ray beam dose distributions for sphericalshape targets for radiosurgery (Luxton et al., 1993) and that the energy deposited to cells per decay from g-rays was similar to that from X-rays (Bingham et al., 2000). In the present study, we chose g-rays as a source of representative ionizing radiation to assess the effects of ionizing irradiation on cytochrome P450 expression. The present study revealed that relatively strong irradiation induced CYP2E1 in the liver with concomitant increase in its mRNA. Multiple radiation exposures, however, failed to further

Fig. 3. The plasma alanine aminotransferase (ALT) activity in rats exposed to g-irradiation. The plasma ALT activity was measured in rats exposed to a single dose from 0.5 to 9 G (A) or in rats daily exposed to a dose of 3 G of g-irradiation for 2 – 3 days (B). Data represent means 9SD from 4 animals. One way analysis of variance was used for comparisons of multiple group means, followed by Newman– Keuls test (significant as compared to control, **PB 0.0l). Con, Control.

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Fig. 4. Mean arterial plasma concentration-time profiles of CZX (A) and OH-CZX (B) after 1 min iv infusion of CZX, 25 mg/kg, to control (O, n =7) and 3 G g-irradiated ( , n=6) rats. Bars represent SD.

increase the extent of CYP2E1 induction. CYP2E1 induction by a single dose of 9 G g-irradiation was 30% less than that by 3 G g-irradiation. The smaller induction resulted from severe hepatocyte injury. In the present study, we used CZX as a non-invasive probe to assess CYP2E1 induction in vivo. CZX primarily undergoes hydroxylation to form OH-CZX which is known to be catalyzed mainly by CYP2E1 (Conney and Burns, 1960; Peter et al., 1990). OH-CZX is then rapidly glucuronidated and excreted in the urine (Conney and Burns, 1960; Desiraju et al., 1983). Recently, however, CZX has been suggested for use as a chemical probe to assess the activity of CYP2E1 in vitro and in vivo (Peter et al., 1990; Rockich and Blouin, 1999). We examined the pharmacokinetics of CZX and its main metabolite, OH-CZX, after iv administration of CZX to control and irradiated rats. Three gray of g-irradiation increased formation of OH-CZX; in irradiated rats, the AUC and Ae0 “ 8 h of OH-CZX were significantly greater (Table 1). This could also be supported by significant decrease in Ae0 “ 8 h of CZX in irradiated rats. Moreover, the AUCOH-CZX/AUCCZX ratio also increased 73% in irradiated rats compared with control rats (Table 1). These results indicate that formation of OHCZX from CZX was increased by the induction of

CYP2E1. The pharmacokinetic data was consistent with increases in the hepatic CYP2E1 protein and mRNA expression (Figs. 1 and 2). Table 1 Mean ( 9 SD) pharmacokinetic parameters of CZX and OHCZX after 1-min iv infusion of CZX, 25 mg/kg, to control and 3 G g-irradiated rats Parameter

CZX Terminal half-life (min) AUC (mg min/ml) MRT (min) CL (ml/min/kg) Vss (ml/kg) Ae0 “ 8 h (% of iv dose) OH-CZX Terminal half-life (min) AUC (mg min/ml) Ae0 “ 8 h (% of iv dose)a a

Control rats (n = 7)

g-Irradiated rats (n =6)

44.5 92.92

33.4 94.75c

3010 9936 57.4 9 5.71 8.30 9 2.87 473 9 169 1.58 9 0.321

3058 9 481 42.4 9 9.10b 8.17 91.62 335 9 99.8 0.249 9 0.116d

44.9 912.0

33.9 92.37b

309 962.5 41.8 9 6.44

530 9 99.4c 51.8 9 6.35b

Expressed in terms of CZX. Significant as compared to control: PB0.05. c PB0.01. d PB0.001. b

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Table 2 The plasma glucose level in g-irradiated ratsa Treatments [×day(s)]

Plasma glucose level (mg/dl)

Untreated 0.5 G×1 1 G×1 3 G×1 9 G×1 3 G×2 3 G×3

147 916 14094 1499 4 16599 143915 1649 5 14895

a The plasma glucose level was measured in rats exposed to a single dose of 0.5–9 G or to the daily dose of 3 G g-irradiation for 2–3 days. Glucose levels were determined 24 h after the last dose of g-irradiation. Data represent means 9 SD from 4 animals. g-Irradiation resulted in no significant change in the plasma glucose content, as compared to untreated animals (control).

Previous studies have shown that the metabolic activity of CYP2E1 is highly associated with production of reactive oxygens due to the high rate of uncoupling of electron transfer and oxygen reduction by CYP2E1 (Bell and Guengerich, 1997; Lieber, 1997). Taking the relationship between g-irradiation and oxidative stress into consideration, g-irradiation may induce changes in the expression of certain cytochrome P450(s). In addition, other mediators, which arise at specific sites of irradiated cells, such as cellular organelles and plasma membrane may contribute to the effects evoked by irradiation. Aconitase, a citric acid cycle enzyme, is a superoxide anion-sensitive hydratase and an important site of the toxicity by ROS. Aconitase, estimated to be  15% of total mitochondrial matrix proteins in a certain species, catalyzes interconversion between citrate and isocitrate in the citric acid cycle (Yan et al., 1997). The activity is extremely sensitive to superoxide anion. The loss of aconitase activity limits citric acid cycle activity and mitochondrial respiratory capacity in vivo. Studies have shown that the activity of aconitase is decreased by lethal oxygen exposure of mammalian cells (Gardner et al., 1994) or aging (Yan et al., 1997). g-Irradiation induces physical and chemical damage to tissues, leading to organelle injuries, cell death or neoplastic transformation. Because aconitase activity is essential to normal metabolic function, the reac-

tive oxygens produced from g-irradiation would inactivate aconitase in mitochondria and lead to the loss of oxidative phosphorylation. Hence, the impairment of aconitase activity alters energy metabolism. The present study demonstrated that the decrease in aconitase activity by g-irradiation correlated with CYP2E1 induction in the liver prior to severe cell damage. This was consistent with the previous observation that impaired glucose utilization caused induction of hepatic CYP2E1 (Son et al., 2000), supporting the hypothesis that CYP2E1 induction by g-irradiation might result from the change in energy metabolism involving the citric acid cycle. Mitochondria are the major cellular sources of ROS. The mitochondrial genome (mtDNA) is a circular double-stranded DNA that is replicated within mitochondria. The mtDNA located in the matrix, near the inner mitochondrial membrane, is exposed to the constant generation of semiquinone radicals and reactive oxygens produced by aerobic respiration. The mtDNA is particularly vulnerable to the damaging effects mediated by ROS (Yakes and Houten, 1996; Croteau et al., 1997). The mitochondrial genome lacks protective histone-like proteins and is replicated by DNA polymerase-g without proofreading. These features contribute to the high susceptibility of this genome to oxidative damage. In the present study, decrease in mtDNA following g-irradiation was in parallel with that in aconitase activity. Mitochondrial dysfunction such as reduction in aconitase activity and decrease in mtDNA by g-rays accompanied CYP2E1 induction. Hence, CYP2E1 might be induced by injuries of energy-producing organelles prior to cell death. CYP2E1 expression is affected by a variety of pathophysiological situations such as diabetes, starvation and dehydration (Hong et al., 1987; Yamazoe et al., 1989; Chen et al., 1999; Kim et al., 2000; Chung et al., 2001). It is highly likely that the induction of CYP2E1 by the aforementioned pathophysiological situations resulted from the altered plasma glucose level and consequent change in glucose utilization. Lack of growth hormone induced CYP2E1 in the livers of hypophysectomized rats, which accompanied decreases

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in the plasma glucose content and in the utilization of glucose by hepatocytes (Son et al., 2000). Insulin level may be an important determinant for CYP2E1 expression (Woodcroft and Novak, 1997; Peng and Coon, 1998). In the present study, the plasma glucose and insulin levels were not altered in g-irradiated rats. In conclusion, the current study demonstrated that g-irradiation increased CYP2E1 protein and

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mRNA levels in the rat liver, which might result from mitochondrial dysfunction but not from alterations in the plasma glucose and insulin levels.

Acknowledgements This work was financially supported by research funds from Korea Cancer Center Hospital,

Fig. 5. Mitochondrial aconitase activity and DNA content after g-irradiation. (A) Aconitase activity in the post-nuclear fraction prepared from the livers of rats irradiated with g-rays. The aconitase activity was measured in rats exposed to a single dose from 0.5 to 9 G g-irradiation or in rats daily exposed to a dose of 3 G g-irradiation for 2 – 3 days. One unit of aconitase activity per mg of post-nuclear proteins was defined as the conversion of 1 mmole of citrate to isocitrate per min. (B) The mtDNA content in rat liver. The mtDNA level was measured in the irradiated rats, as described above. Data represent the amount of mtDNA (mg) per mg of mitochondrial proteins. Data shows the means 9 SD from 4 animals. One way analysis of variance was used for comparisons of multiple group means, followed by Newman–Keuls test (significant as compared to control, **PB 0.01) Con, Control.

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Korea Atomic Energy Research Institute (SGK) and BK21 project for Medicine, Dentistry and Pharmacy.

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