Female and male C57BL/6 mice were purchased from Daehan Biolink (Korea) and 7-8 weeks old at the beginning of experiments. The mice were housed in conventional animal house conditions with 12-hr light-dark cycle, temperature of 22±2℃, relative humidity of 50%, standard pelleted diet and tap water ad libitum. The animal care, handling, and experimental procedures were conducted in accordance with the guidelines approved by the Animal Care and Use Committee of the Korea Institute of Radiological & Medical Sciences (KIRAMS).

Whole-body irradiation and local whole-thorax irradiation of the mice were performed using ⁶⁰Co γ-rays from a Theratron 780 irradiation unit (Atomic Energy of Canada, Ltd., Canada). For whole-body irradiation, mice were placed into Plexiglas box and irradiated with a single dose of 10 Gy (0.5 Gy/min). For local whole-thorax irradiation, mice were anesthetized by i.p. injection of a mixture of ketamine (70 mg/kg) and xylazine (6 mg/kg), and fixed in a supine position on a Plexiglas tray. The thoracic regions (1.5 cm in length in cephalic-tail direction) of mice were placed in the field defined by collimators and irradiated with a single dose of 12.5 Gy (0.76 Gy/min). Other parts of the bodies were additionally shielded with 5-cm lead blocks. Before tissue sampling, irradiated and control mice were sacrificed by cervical dislocation in 1, 3, 5, and 7 days after whole-body irradiation and in 1, 2, 4, 12, 16, and 26 weeks after whole-thorax irradiation.

Immediately after sacrifice of mice by cervical dislocation, the cranial, peritoneal, and chest cavities of mice were opened, and the tissues (lung, liver, and brain) were quickly harvested and washed (except for brain) in cold 100 mM K-phosphate buffer (pH 7.4) containing 2 mM MgCl₂ (MgCl₂-phosphate buffer). The tissues were slightly dried by paper filter, minced with scissors, and homogenized in 4 volumes of MgCl₂-phosphate buffer on ice by a Tissue-Tearor with a 7-mm probe (BioSpec Products, Inc., WI, U.S.A.). The homogenates were centrifuged at 1,500×g for 10 min at 4℃, and the obtained supernatants were further centrifuged at 15,000×g for 15 min at 4℃. The 15,000×g supernatants were kept at 4℃ and used for determination of HO activity not later than 1-1.5 hr after preparation. Protein concentration of the 15,000×g supernatants was determined by the method of Schacterle and Pollack (17) with bovine serum albumin as a standard. The reaction mixture for determination of HO activity included MgCl₂-phosphate buffer, 412 mg of protein/mL of the 15,000×g supernatant, 1 mg of protein/mL of liver 105,000×g supernatant as a source of biliverdin reductase, 2 mM glucose 6-phosphate, 1 U glucose-6-phosphate dehydrogenase, 25 µM hemin, and 1 mM NADPH in a final volume of 500 µL in 1.7-mL Eppendorf microcentrifuge tubes. Reaction was initiated by the addition of NADPH, and the reaction mixtures were incubated in a water bath at 37℃ in the dark for 10 min (liver), 20 min (brain), or 40 min (lung). To terminate the reaction and extract produced bilirubin, 500 µL of chloroform were added to each sample, the samples were vortexed thoroughly for 30 sec, and centrifuged at 15,000×g for 10 min. The lower chloroform layer was transferred to a spectrophotometer microcuvette to measure the difference in optical density between 464 and 530 nm (ΔOD_464-530) using UV/Visible spectrophotometer Ultrospec 3100 pro (Biochrom, Ltd., England). The extinction coefficient (ε_464-530) of 40 mM/cm was used for calculation of bilirubin concentrations (18). HO activity was expressed in picomoles of bilirubin produced per milligram of protein per hour.

As a source of biliverdin reductase, 105,000×g supernatant of liver homogenate was used (18). To prepare the supernatant, livers of overnight-fasted non-irradiated mice were perfused in situ with cold (4℃) 1.15% KCl, harvested, slightly dried by paper filter, minced with scissors, and homogenized in 4 volumes of MgCl₂-phosphate buffer on ice. The homogenates were centrifuged at 1,500×g for 10 min at 4℃, and the pooled supernatant was further centrifuged at 15,000×g for 15 min at 4℃ and then at 105,000×g for 1.5 hr at 4℃. The 105,000×g supernatant was diluted to 5 mg of protein/mL with MgCl₂-phosphate buffer and stored at -20℃.

Semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR) was used for the mRNA expression level determination. After thawing liver samples frozen in liquid nitrogen, RNA was purified from the liver using RNAzol (Tel-Test, Friendswood, U.S.A.), and RNA concentration was spectrophotometrically determined by absorbance at 260 nm. One microgram of intact RNA was reverse-transcribed into cDNA in the reaction mixture containing 50 mM Tris-HCl (pH 8.3), 3 mM MgCl₂, 75 mM KCl, 2.5 µg/mL pd(N)₆ primer, 0.5 mM each of dNTP, and 10 U of AMV-RT (Amersham). Then produced cDNA was amplified by polymerase chain reaction (PCR) in the mixture containing 10 µL cDNA template from RT reaction, 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5mM MgCl₂, 0.5mM each of dNTP, 1.0 µM of each primer, and 0.5 U Tag DNA polymerase (Takara, Tokyo, Japan). The oligonucleotide primers used in these experiments are as followings; HO-1 PCR primer (5'-primer 5'-AACAAGCAGAACCCAGTC-3', 3'-primer: 5'-TGTCATCTCCAGAGT-GTTC-3'), β-actin primer (5'-primer: 5'-TGGAATCCTGTGGCATCCATGAAA-3', 3'-primer: 5'-TAAAACGCAGCTCAGTAACAGTCCG-3'). PCR was performed with a DNA thermal cycle (Hybaid, Ashford, U.K.) at 94℃ for 30 sec, at 56℃ for 30 sec, and at 72℃ for 30 sec per cycle. The PCR products were visualized by electrophoresis on 1% agarose gel in the presence of 0.5 µg/mL ethidium bromide. Density of bands was measured using an image analyzer (Flour-S Multimager, Bio-Rad, CA, U.S.A.).

NPSH content in animal tissues was determined with DTNB by the method of Sedlak and Lindsay (19). Briefly, immediately after sacrifice of mice, the tissues (50-90 mg of liver, 100-150 mg of brain and lung) were quickly harvested, placed on ice, slightly dried by paper filter (except for brain), weighed, minced with scissors, and homogenized on ice in a mixture of 2 mL of 10% TCA and 2 mL of 0.02 M EDTANa₂. The homogenates were centrifuged at 3,500×g for 20 min at 4℃. Then, 0.5 mL of the supernatant was mixed with 1 mL of 0.4 M Tris-HCl buffer (pH 8.9) containing 0.02 M EDTANa₂, and 25 µL of 0.01 M DTNB in methanol were added to each sample. OD was measured at 412 nm in 5 min. Blank sample contained all the above-mentioned components, except animal tissue. NPSH contents were calculated using an extinction coefficient (ε₄₁₂) of 13.1 mM/cm and expressed in micromoles per gram of wet tissue weight.

All experimental data were evaluated by GraphPad Prism software (San Diego, CA, U.S.A.). In the most of experiments, the significances of differences between irradiated and control groups were tested using Student's t test or Mann-Whitney when the data failed to pass a normality test. The effect of irradiation on HO-1 mRNA level was analyzed by Kruskal-Wallis test. Differences among the groups were considered as statistically significant at p<0.05.

Comparison of HO activity and NPSH level in lung, liver and brain of male and female control mice demonstrated that HO activity was the highest in brain and the lowest in lung; NPSH level was approximately same in lung and brain, and much higher in liver (Fig. 1). The significant difference between sexes was observed only in hepatic NPSH content: it was approximately 15% higher in males than in females.

In brain, HO activity in males was slightly decreased on the 5th day but no alteration of that in females (Fig. 2). Significant elevation of HO activity (approximately 25-30%) in lung was observed on the 5th day in females and on the 7th day in males after irradiation. The most impressed radiation-induced alterations of HO activity occurred in livers of both male and female mice. HO activity began to increase on the 3rd day after the irradiation in females and on the 5th day in males, and reached nearly 200% of the control level in females and 250% in males on the 7th day. To further confirm whether the elevation of HO activity is accompanied with the increase of gene expression of HO-1, we performed the RT-PCR for HO-1 mRNA in liver. As shown in Fig. 3, mRNA expression of HO-1 was significantly elevated on the 5th day and persisted until 7 days after irradiation (8.7 and 8.0-fold compared to the control group respectively).

NPSH level in the brain was not significantly changed in both male and female mice (Fig. 4). In case of the lung, NPSH level was significantly decreased on the 1st day after irradiation in males but increased in females. Like, irradiation induced different response of NPSH level in livers of males and females; it was increased in females on the 5-7th days in sharp contrast to decrease in males on the 3rd day. The alteration time of NPSH level in livers was shown later than that in lungs even the pattern of NPSH response was similar.

HO activity and NPSH level in the lung after whole-thorax irradiation at a dose of 12.5 Gy were evaluated during 26 weeks after exposure. As shown in Fig. 5, the whole-thorax irradiation resulted in different reaction of males and females. In females, there were no significant changes of both HO activity and NPSH level, although HO activity showed a tendency to increase on the 1st week and to decrease on the 12th week after irradiation. However, NPSH level was significantly decreased on the 2nd week and HO activity was increased on the 16th week (by 45%) after irradiation in male mice.