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Hepatology Research 36 (2006) 78–85

Zonal differences in gallium-67 uptakes between perivenous versusperiportal regions of rat liver following carbon tetrachloride treatment

Shinya Abe a,∗, Hajime Nakao a, Kana Hatakeyama a, Yosuke Ohtake a,Soichi Kojima b, Manabu Fukumoto c, Yasuhito Ohkubo a

a Department of Radiopharmacy, Tohoku Pharmaceutical University, 4-4-1 Komatsushima, Aoba-ku, Sendai, Miyagi 981-8558, Japanb Molecular Cellular Pathology Research Unit, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan

c Department of Pathology, Institute of Development, Aging and Cancer, Tohoku University, 4-1 Seiryo-machi, Aoba-ku, Sendai, Miyagi 980-8575, Japan

Received 17 February 2006; received in revised form 24 May 2006; accepted 6 July 2006Available online 14 August 2006

bstract

Gallium-67 (67Ga) has been used as a tumor or inflammation-imaging agent in nuclear medicine, although underlying mechanism hasot been fully elucidated. To gain some insights into the mechanism of 67Ga uptake by injured liver, we analyzed the difference betweenerivenous and periportal regions of rat liver in terms of 67Ga uptake by hepatocytes at the site of inflammation caused by carbon tetrachlorideCCl4)-treatment. Distribution of 67Ga in rat liver sections was monitored with a BAS5000 system following hepatic injury by CCl4-treatment.eriportal hepatocytes (PPH) and perivenous hepatocytes (PVH) were prepared by modified digitonin–collagenase perfusion technique.ptakes of 67Ga in PVH region and PPH region reached to a maximum 2 days after CCl4-treatment, and the amount of maximum uptake of

7Ga in PVH was twice as much as that in PPH. Liver damage as measured by lipid peroxidation and 45Ca uptake occurred in PVH region

ithin 1 day after CCl4-treatment. Incorporation of bromodeoxyuridine into hepatocytes reached to a maximum 2 days after CCl4-treatment,

nd peaked amount of DNA synthesis in PVH was twice as much as that in PPH. These results indicated that the uptake of 67Ga by the PVHegion was carried out during hepatic regeneration phase rather than hepatic damage period by CCl4-treatment.

2006 Elsevier Ireland Ltd. All rights reserved.

67 tes; Ca

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eywords: Gallium-67 ( Ga); Periportal hepatocytes; Perivenous hepatocy

. Introduction

Gallium-67 (67Ga) has been used for detecting variousumors [1–4] as well as acute and chronic inflammations5,6] since the first observation of its accumulation in thoseesions was reported [7–9]. However, underlying molecular

echanisms of 67Ga uptakes by these tissues has not beenully elucidated. We recently reported that free 67Ga wasaken up into hepatocytes of CCl4-injured liver [10] and theptake of 67Ga by the hepatocytes was carried out during hep-

tic regeneration phase rather than hepatic damage period byCl4-treatment [11].

∗ Corresponding author. Tel.: +81 22 727 0121; fax: +81 22 275 2013.E-mail address: shin-abe@tohoku-pharm.ac.jp (S. Abe).

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386-6346/$ – see front matter © 2006 Elsevier Ireland Ltd. All rights reserved.oi:10.1016/j.hepres.2006.07.006

rbon tetrachloride; Lipid peroxidation; Liver regeneration

Hepatotoxic damages by CCl4 are mostly attributed to itsoxic metabolite, trichloromethyl radical (CCl3*), formed byn action of cytochrome P450, especially by P450 2E1 [12].his radical reacts with oxygen to generate a highly reactivepecies, trichloromethylperoxy radical (CCl3OO*) [13] thatnitiates chain reactions of lipid peroxidation [14,15], andhereby subsequent cellular damages occur [16–18].

Parenchymal cells nearest the sinusoidal inflow of theiver acinus are termed periportal hepatocytes (PPH), whereashose nearest the sinusoidal outflow are termed perivenousepatocytes (PVH). PPH and PVH differ in their cellular con-tituents, as a result of differences in enzyme content, carry

ut different metabolic functions. For example, gluconeogen-sis, ureogenesis, and amino acid uptake and degradation arereferentially undertaken by PPH, whereas glycolysis andlutamine synthesis are preferentially undertaken by PVH

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19]. Because many cytochrome P450 isozymes have a higherxpression in the PVH region [20–23], CCl4 preferentiallyaused damage in PVH [24–26]. PVH region must regener-te to gain lost number of hepatocytes by CCl4-treatment.owever, there has been no report on detailed studies for the

egeneration of PVH region. We have recently reported thatrowth potential of PPH was higher than PVH after partialepatectomy [27]. In order to investigate whether or not PVHfter CCl4-treatment acquires the same growth potential asPH, we examined the uptakes of 67Ga by the PVH and PPHegions after that. Distribution of 67Ga among the liver sec-ion of CCl4-treated rat was investigated by use of a BAS5000ystem, and PPH and PVH were prepared by modified digi-onin/collagenase perfusion technique to carry out detailedxamination.

. Materials and methods

.1. Animals

Male Wistar rats weighing 180–200 g (SLC, Hamamatsu,apan) were kept under conditions of a 12 h light–dark cycle,3 ± 1 ◦C and 55 ± 5% humidity, and had free access to foodnd water. Animals were cared following the standard pro-edures indicated in the “Guide for the Care and Use ofaboratory Animals” published by the Tohoku Pharmaceu-

ical University.

.2. Chemicals

Gallium-67 citrate was purchased from Daiichi Radioiso-ope Laboratory, Ltd. (Tokyo, Japan). Calcium-45 was pur-hased from Perkin-Elmer Life Sciences, Inc. (Boston, MA).anks’ balanced salt solution (HBSS) was purchased fromigma (St. Louis, MO). Collagenase was purchased fromitta Gelatin (Osaka, Japan). All other reagents were pur-

hased from Nakarai Tesque (Tokyo, Japan).

.3. Administration of CCl4

Rats were given a 20% suspension of CCl4 in olive oil5 ml/kg) orally, after being fasted for 16 h. Control rats werereated similarly with equivalent amounts of olive oil alone.ats were sacrificed at 1, 2, and 3 days after starting treatment.

.4. Detection of 67Ga radioactivity on frozen sectionsf the liver

Gallium-67 citrate was diluted with isotonic physiologicalaline. A group of 4–6 rats ware injected intravenously with.2 mL of a solution containing 111 kBq of 67Ga 1, 2, and 3

ays after CCl4-administration under urethane anesthetiza-ion (1.5 g/kg, i.p.).

Four hours after 67Ga injection, CCl4-treated rats weremmediately perfused with 0.9% NaCl solution. The whole

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arch 36 (2006) 78–85 79

iver was then removed. After removal of the liver, 3-o 6-mm-thick slices were taken, and then tissue samplesere embedded in OCT compounds, immersed for dried

ce/isopentane. Frozen sections were cut at 20 �m thick andere exposed in a cassette with imaging plate (BAS-SR type,uji Photo Film, Japan) for 18 h at room temperature. Then

he imaging plate was analyzed with an imaging analyzerBAS5000, Fuji Film, Japan) to quantify the radioactivity andetermine its distribution in the sections. After exposure, sec-ions were counterstained with hematoxylin and eosin (HE)or observation of periportal and perivenous regions.

.5. The uptake of 67 Ga and 45Ca by PPH and PVH

Gallium-67 citrate and 45Ca were diluted with isotonichysiological saline. A group of 4–6 rats ware injected intra-enously with 0.2 mL of a solution containing 37 kBq of7Ga and 148 kBq of 45Ca 1, 2, and 3 days after CCl4-dministration under urethane anesthetization (1.5 g/kg, i.p.).our hours after 67Ga or 45Ca injection, CCl4-treated ratsere sacrificed.

.6. Isolaton of PPH and PVH from a single rat liver

PPH and PVH were isolated from a single rat liver andharacterized as described previously [28]. Briefly, the por-al vein and caudal vena cava were cathererized, and a salineuffer (solution A, containing 115 mmol/L NaCl, 5.6 mmol/LCl, 1.2 mmol/L MgCl2, 1.2 mmol/L NaH2PO4, 25 mmol/LaHCO3, 4.4 mmol/L CaCl2, and 5.56 mmol/L glucose) atH 7.4, was infused via the portal vein at a rate of 20 mL/minor 5 min at room temperature, to remove the blood from theiver. The median lobe vessels at the hilum were clamped,nd the digitonin solution (4 mg/mL) was infused via the por-al vein at a rate of 10 mL/min at room temperature until aegularly scattered PPH discoloration pattern was observed.his took 10–30 s. Digitonin was then washed out by perfu-ion with solution A in the retrograde direction (through theaudal vena cava) at a rate of 20 mL/min for 2 min. It is impor-ant to note that the clamp was not removed during the first

inute of washing to allow the digitonin to leave the circuitithout contaminating the median lobe. After removal of the

lamp, retrograde washing was continued for another 1 min.clamp was then placed on the left lateral lobe vessels at the

ilum, and digitonin was infused via the caudal vena cava atrate of 10 mL/min until a regularly scattered PVH discol-ration pattern was observed. This took 45–60 s. Digitoninas then washed out by perfusion with a Ca2+-free solution

solution A without CaCl2, but containing 2 mmol/L ethylenelycol-bis (�-aminoethyl ether)-N,N,N′,N′-tetraacetic acid atH 7.4), in the antegrade direction via portal vein at a rate of0 mL/min for 10 min, at 37 ◦C. Again, the clamp was main-

ained on the left lateral lobe vessels for the first minute ofashing to ensure that this lobe was not contaminated withportal load of digitonin; then, both lobes were perfusedith the Ca2+-free solution. The Ca2+-free solution was per-

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ormed to prepare the subsequent infusion of collagenase.he isolation procedure was then undertaken as describedy Abe et al. [29], except that the median and left laterialobes were separated after collagenase perfusion and pro-essed independently. PPH-and PVH-enriched populationsere simultaneously obtained from the same liver.The hepatocytes were counted with a well-type NaI-

cintillation counter (ARC-370M, Aloka, Japan) (67Ga) andiquid scintillation counter (LS7800, Beckman) (45Ca). Theesults were expressed as cpm/mg protein. Hepatocytes wereissolved by NP-40 solution. Protein concentration wasetermined according to the method by Bradford [30].

.7. Enzyme assay

Alanine aminotransferase (l-alanine-oxoglutarate amino-ransferase, EC 2.6.1.2) (ALT) activity was determined byhe method of Reitman and Frankel [31], and expressed asarman units (KU). Glutamine synthetase (GS) activity wasetermined as previously described [27].

.8. Determination of the amount of hepatic lipideroxidation

The extent of lipid peroxidation in hepatocyteomogenates was estimated by determining malondi-ldehyde (MDA) formation using the thiobarbituric acidethod [32]. The MDA concentration from the sam-

les was calculated using an extinction coefficient of.56 × 105 M−1 cm−1. The results were expressed as theoncentration of MDA/mg protein.

.9. Flow cytometric analysis of DNA synthesis

Cell cycle was analyzed using a bromodeoxyuridineBrdU) Flow Kit (Becton Dickinson, San Diego, CA). BrdUas dissolved in phosphate buffered saline and was given

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ig. 1. Distribution of 67Ga in liver sections. Pseudocolor images of autoradiograpnd 2 days (C) after administration of carbon tetrachloride (CCl4). Blue color, least

arch 36 (2006) 78–85

ntravenously (10 mg/kg) 2 h before killing. Incorporation ofrdU into hepatocytes was detected by FACScan (Bectonickinson).

.10. Statistical analysis

The statistical significance of difference was analyzedsing Student’s t-test.

. Results

.1. Distribution of 67Ga in liver sections ofCl4-treated rat

To see distribution of 67Ga in the liver, we visualizedadioactivity within liver by a BAS5000 system. As seenn Fig. 1A–C, 67Ga signals increased time-dependentlyfter injection of CCl4, and heterogeneous distributionsere observed. Identification of hepatic region in the het-

rogeneous distribution was confirmed by using HE stainFigs. 2–4). These results indicated that 67Ga signal intensityn perivenous region was higher than that in periportal region.

.2. Characterization of cell populations isolated from aingle liver by digitonin–collagenase perfusion

To investigate in detail zonal differences in 67Ga uptake,e isolated PPH and PVH from damaged liver following

dministration of 67Ga. PPH and PVH, isolated by modifiedigitonin/collagenase perfusion technique, were identified byeasuring activities of each of specific marker enzymes (ALT

nd GS) (Fig. 5). ALT activity was about two-fold highern PPH than in PVH in both normal and CCl4-treated rats

Fig. 5A), whereas GS activity was 6.3-fold higher in PVHhan in PPH only in normal rats (Fig. 5B). No differenceetween PVH and PPH in GS activity was observed uponCl4-treatment.

hy showing the radioactivity of 67Ga in the liver at control (A), 1 day (B),intense; red color, most intense.

S. Abe et al. / Hepatology Research 36 (2006) 78–85 81

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ig. 2. Comparison of autoradiography (A) with hematoxylin–eosin stainiV: portal vein; CV: central vein. (B) Magnified image of a part (rectangle)

.3. Zonal difference in uptakes of 67Ga by PPH andVH after CCl4-treatment

To verify uptakes of 67Ga by PPH and PVH, we isolatedPH and PVH using an improved digitonin/collagenase per-usion technique and measured their radioactivities. Uptakesf 67Ga by PPH and PVH reached to a maximum 2 daysfter CCl4-treatment, and then declined at day 3 (Fig. 6).

he peaked uptake of 67Ga in PVH was twice as much as

hat in PPH. On the other hand, 67Ga was not taken up intoonparenchymal cells (data not shown).

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ig. 3. Comparison of radioluminography (A) with hematoxylin–eosin staining (etrachloride (CCl4). Hematoxylin–eosin stained section; 40×. PV: portal vein; CV:ectangles in (A).

n liver sections in a control rat. Hematoxylin–eosin stained section; 40×.

.4. Zonal difference in an index of degenerationetween PPH and PVH after CCl4-treatment

To quantify degree of degeneration in PPH and PVH, weetermined the lipid peroxidation by thiobarbituric acid assayFig. 7). The lipid peroxidation of PVH was higher than thatf PPH, and increased within 1 day after CCl4-treatment. Theptake of 45Ca by PVH was also higher than that by PPH,

nd also increased within 1 day of treatment (Fig. 8). SerumLT levels increased within 1 day after CCl4-treatment andeclined (Fig. 9).

B), (C) in liver sections in a rat of 1 day after administration of carboncentral vein. (B) and (C) are magnified image of respective parts shown by

82 S. Abe et al. / Hepatology Research 36 (2006) 78–85

Fig. 4. Comparison of radioluminography (A) with hematoxylin–eosin staining (B), (C) in liver sections in a rat of 2 days after admnistration of carbontetrachloride (CCl4). Hematoxylin–eosin stained section; 40×. PV: portal vein; CV: central vein. (B) and (C) are magnified image of respective parts shown byrectangles in (A).

Fig. 5. Time course of alanine aminotransferase (ALT) (A) and glutaminesynthetase (GS) (B) activities in cytosolic fractions of PPH and PVH aftertreatment of carbon tetrachloride (CCl4). PPH and PVH were isolatedby modified digitonin/collagenase perfusion technique. Experimental tech-nique was performed as described in the text. Each point represents themean ± S.E. of four to six rats.

Fig. 6. Time course of 67Ga uptake by PPH and PVH after treatmentof carbon tetrachloride (CCl4). PPH and PVH were isolated by modifieddigitonin/collagenase perfusion technique. Experimental technique was per-formed as described in the text. Each point represents the mean ± S.E. of fourto six rats. Each point represents the mean ± S.E. of four to six rats. The datawere analyzed by Student’s t-test. *p < 0.01 compared to PPH with PVH.#p < 0.01 compared to normal rats (PPH) with treatment of CCl4 (PPH).§p < 0.01 compared to normal rats (PVH) with treatment of CCl4 (PVH).

S. Abe et al. / Hepatology Research 36 (2006) 78–85 83

Fig. 7. Time course of thiobarbituric acid-reactive substances (TBARS)production in whole homogenates of PPH and PVH after treatment ofcarbon tetrachloride (CCl4). PPH and PVH were isolated by modifieddigitonin/collagenase perfusion technique. Experimental technique was per-formed as described in the text. Each point represents the mean ± S.E. of fourto six rats. The data were analyzed by Student’s t-test. *p < 0.01 comparedto PPH with PVH. ##p < 0.05 compared to normal rats (PPH) with treatmentof CCl4 (PPH). §p < 0.01 compared to normal rats (PVH) with treatment ofCCl4 (PVH).

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Fig. 8. Time course of 45Ca uptake by PPH and PVH after treatmentof carbon tetrachloride (CCl4). PPH and PVH were isolated by modifieddigitonin/collagenase perfusion technique. Experimental technique was per-formed as described in the text. Each point represents the mean ± S.E. of fourto six rats. The data were analyzed by Student’s t-test. *p < 0.01 comparedto PPH with PVH. #p < 0.01 compared to normal rats (PPH) with treatmentof CCl4 (PPH). §p < 0.01 compared to normal rats (PVH) with treatment ofCCl4 (PVH).

Fig. 9. Time course of serum alanine aminotransferase (ALT) levelsam#

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.5. Zonal difference in DNA synthesis between PPHnd PVH after CCl4-treatment

To monitor DNA synthesis of hepatocytes during liveregeneration after CCl4-induced liver damage, we measuredhe incorporation of BrdU into PPH and PVH (Fig. 10). Incor-oration reached to a maximum 2 days after CCl4-treatment,nd the peaked amount of DNA synthesis in PVH was twices much as that in PPH.

. Discussion

In the present study, we confirmed zonal differences of7Ga uptake by hepatocytes of CCl4-treated rats. We isolatedepatocyte populations enriched in either PPH or PVH usingn improved digitonin–collagenase perfusion technique [28].tandard enzymatic markers were assayed to assess the zonalurity of each cell population. ALT is distributed as a lineareclining gradient from the PPH to PVH area [33], whereasS activity is known to be confined to two or three rings ofepatocytes surrounding the centrilobular vein [34]. Our data

f PVH/PPH ratios in the ALT activity were in good agree-ent with published values [28]. It was reported that CCl4

ffected preferentially the perivenous region of the acinusnd led to disappearance of cells containing GS [35]. In the

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fter treatment of carbon tetrachloride (CCl4). Each point represents theean ± S.E. of four to six rats. The data were analyzed by Student’s t-test.

p < 0.01 compared to normal rats with treatment of CCl4.

resent study, the GS activity of PVH markedly decreased

fter CCl4-treatment and that of PVH was the same as PPH.herefore, GS activity could not be used as enzymatic markerf PVH after CCl4-treatment. On the other hand, ALT activ-ty insured successful isolation of PPH and PVH region.

84 S. Abe et al. / Hepatology Rese

Fig. 10. Time course of incorporation of bromodeoxyuridine (BrdU) intoPPH and PVH after the treatment of carbon tetrachloride (CCl4). PPH andPVH were isolated by modified digitonin/collagenase perfusion technique.Experimental technique was performed as described in the text. Each pointrepresents the mean ± S.E. of four to six rats. The data were analyzed by Stu-dent’s t-test. *p < 0.01 and **p < 0.05 compared to PPH with PVH. #p < 0.01and ##p < 0.05 compared to normal rats (PPH) with treatment of CCl4 (PPH).§C

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p < 0.01 and §§p < 0.05 compared to normal rats (PVH) with treatment ofCl4 (PVH).

he lipid peroxidation of PVH after CCl4-treatment greatlyncreased within 1 day after the treatment and two-fold largerhan PPH. This result was in agreement with the reports thatCl4 preferentially caused damage in PVH region [24–26].

t had been reported that a critical step in cellular dam-ge by CCl4-treatment consisted with an increase of Ca2+

nflux into cells [36]. In the present study, uptake of 45Ca byVH increased within 1 day after the treatment and higher

han that of PPH. Serum ALT also increased within 1 dayfter the treatment. These results indicated that preferentialestruction of PVH occurred by 1 day after CCl4-treatment.owever, uptakes of 67Ga by PVH and PPH regions reached

o a maximum 2 days after CCl4-treatment, and the valuef 67Ga uptake by PVH was twice as much as that by PPH.hese results were consistent with the data of a BAS5000nd HE stain. These results suggested that maximum uptakef 67Ga by PVH region occurred during liver regenerationhase rather than liver damage period. We suspected that7Ga uptake by PVH region might correlate with hepatocyteroliferation of still alive PVH after the damage caused byCl4-treatment.

To ascertain the relationship between 67Ga uptake and

ell proliferation, we measured DNA synthesis of regenerat-ng hepatocytes after CCl4-treatment. Incorporation of BrdUnto still alive PVH reached to a maximum 2 days after CCl4-

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arch 36 (2006) 78–85

reatment, and that into still alive PVH was twice as much ashat into PPH. This fluctuation of hepatic DNA synthesis wasonsistent with the pattern of 67Ga uptake. This result showedhat still alive PVH after CCl4-treatment acquired higherrowth potential than PPH. This is supported by the reporthat the protein kinase C in the PVH region was detectedt 2 days after CCl4-treatment [37]. In this study, it was sug-ested that still alive PVH after CCl4-treatment acquired highrowth potential to gain lost number of hepatocytes in perive-ous region.

Our data indicated that the uptake of 67Ga by the PVHegion was carried out during hepatic regeneration phaseather than hepatic damage period by CCl4-treatment.

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