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RIGINAL ARTICLESetal contents of liver parenchyma afterercutaneous ethanol injection or radiofrequencyblation in patients with hepatocellular carcinomaefore and after trientine hydrochloride therapy

IROYUKI FUKUDA, MASAAKI EBARA, SHINICHIRO OKABE, MASAHARU YOSHIKAWA,OBUYUKI SUGIURA, HIROMITSU SAISHO, FUKUO KONDO, and MASAE YUKAWA

HIBA, JAPAN

We administered trientine hydrochloride, a drug used in the treatment of Wilson’sdisease, to patients with hepatocellular carcinoma after radical treatment withpercutaneous ethanol injection or radiofrequency ablation, and examined its effecton the reduction of liver-tissue copper content. We enrolled 24 patients with 3 orfewer primary lesions of Child class A or B hepatocellular carcinoma with diametersof 3 cm or less who had undergone radical treatment with percutaneous ethanolinjection or radiofrequency ablation. Trientine hydrochloride was orally adminis-tered in a single daily dose of 250 mg to 12 patients before a meal (at fasting, group1) or at a total daily dosage of 750 mg, divided into 3 doses, to 12 patients (group2). This study was a randomized between-groups comparative study of 12 weeks’duration. We used the particle-induced x-ray–emission method to determine liver-tissue mineral content. Urine copper and serum mineral levels were also measured,and transaminase levels were examined. Liver-tissue copper content decreasedsignificantly, to 160.1 �g/g dry weight, after treatment, compared with the pretreat-ment level of 306.8 �g/g dry weight (P < .05). We detected no significant differencein iron or zinc content before and after treatment. The copper content was signifi-cantly reduced after treatment in both groups (P < .05). The urine copper level wassignificantly increased after 1 week of treatment but decreased thereafter. Serumcopper levels were significantly reduced after treatment (P < .01). We detected nosignificant difference in transaminase level before and after treatment. Iron-defi-ciency anemia in 1 patient after 12 weeks’ treatment was the only adverse reaction,and it was improved by the administration of an iron product. We noted no otherovert adverse reactions. In patients with hepatocellular carcinoma, trientine hydro-chloride therapy may significantly reduce copper content in liver tissue. (J Lab ClinMed 2004;143:333-9)

Abbreviations: HCV � hepatitis C virus; HCC � hepatocellular carcinoma; LEC � Long-Evanscinnamon; MRI � magnetic resonance imaging; 8-OHdG � 8-hydroxydeoxyguanosine; PIXE �particle-induced x-ray emission

rom the Departments of Medicine and Clinical Oncology and Mo-ecular Pathology, Graduate School of Medicine, Chiba University;nd the National Institute of Radiological Sciences.

ubmitted for publication June 18, 2003; revision submitted February0, 2004; accepted February 23, 2004.

eprint requests: Dr. Hiroyuki Fukuda, Department of Medicine and

1-8-1 Inohana, Chuo-ku, Chiba-shi, Chiba 260-8670, Japan; e-mail:fukuhiro@ho.chiba-u.ac.jp.

0022-2143/$ – see front matter

© 2004 Elsevier Inc. All rights reserved.

linical Oncology, Graduate School of Medicine, Chiba University, doi:10.1016/j.lab.2004.02.010

333

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Walshe1 introduced the copper-chelating agent trien-ine hydrochloride for the oral treatment of Wilson’sisease because of its lower incidence of side effectshan that of D-penicillamine. Recent studies have re-ealed that copper is an important cofactor for severalngiogenic agents.2,3 It has been reported that trientineydrochloride suppresses tumor development and an-iogenesis of HCC in mice.4,5

In a previous study of patients with chronic hepatitis, we reported that liver-tissue copper content signifi-antly increased along with disease progression from aealthy state to chronic inactive hepatitis, chronic ac-ive hepatitis 2A, chronic active hepatitis 2B, and he-atic cirrhosis6 and that copper accumulation in fibroticiver caused by HCV may contribute to hepatocarcino-enesis.7 Furthermore, our studies involving the gel-ltration method showed that among HCC patientsith increased copper levels, copper existed as copperetallothionein in both cancerous and noncancerous

urrounding liver parenchyma, a finding similar tohose in LEC rats.8 Copper metallothionein in LEC ratsorms hydroxyl radicals by way of a Fenton-like reac-ion in the presence of hydrogen peroxide9 and maynduce a type of DNA damage associated with onco-enesis.10–13 The copper-chelating agents penicilla-ine14 and trientine15 are known to significantly reduce

he occurrence of HCC in LEC rats. In human subjects,opper-chelating agents may also decrease the occur-ence of HCC by reducing copper levels that are in-reased by liver cirrhosis. The study reported here is therst clinical investigation to examine the reduction of

iver-tissue copper by trientine hydrochloride in cir-hotic liver.

ETHODS

Patients. Between October 1999 and September 2000, wenrolled 24 patients with 3 or fewer primary lesions of Childlass A or B HCC with diameters of 3 cm or less who hadndergone radical treatment with percutaneous ethanol injec-ion (n � 9) or radiofrequency ablation (n � 15) in theepartment of Medicine and Clinical Oncology of Chibaniversity. The study group comprised 17 men and 7 women;

heir mean � standard deviation age was 65.8 � 6.2 years.he Child-Pugh score was 5 in 16 patients (66.7%), 6 in 6

25%), and 7 in 2 (8.3%). The origin of hepatic cirrhosis wass follows: HBsAg was present in 1 patient (4.2%) and HCVntibody was present in 22 (91.6%); both parameters wereegative in 1 patient (4.2%). HCV antibody was determinedith the use of a second- or third-generation enzyme-linked

mmunosorbent assay. This study was approved by the Eth-cal Committee of Chiba University and conducted in accor-ance with the Declaration of Helsinki. Informed consent wasbtained from all patients.

Study design. Trientine hydrochloride (Tsumura & Co,

okyo, Japan) was administered orally in a single daily dose a

f 250 mg to 12 patients before a meal (at fasting, group 1)nd in a daily dosage of 750 mg, divided in 3, to 12 patientsgroup 2). This study was a randomized between-group com-arative study lasting 12 weeks. The package insert specifieshat in patients with Wilson’s disease, 6 capsules of trientineydrochloride (1500 mg trientine hydrochloride) should beiven orally on a daily basis in 2 to 4 divided doses and thathe dose should be adjusted within a range of 4 to 10 capsules1000–2500 mg trientine hydrochloride) in accordance withuch factors as age, symptoms, and patient response. In thistudy, the daily dosage of trientine hydrochloride was spec-fied to be 1 or 3 capsules (250 or 750 mg trientine hydro-hloride) because the hepatic level of copper is 250 �g/g dryt or greater among patients with Wilson’s disease16,17 and0 and 169 �g/g dry wt in patients with cirrhosis with andithout cancer, respectively.7 Trientine hydrochloride was toe administered before meals because absorption of the drugay be reduced by food intake.Liver biopsy. We performed 2 liver biopsies in each pa-

ient, under imaging, with the use of an ultrasonic diagnosticpparatus (SSA-270A; Toshiba, Tokyo, Japan) to collect sur-ounding liver parenchyma before and after trientine hydro-hloride therapy. Biopsy specimens taken before trientineydrochloride therapy were likewise taken before percutane-us ethanol injection or radiofrequency ablation. Patientsere well informed of the nature of the procedure beforehand.21-gauge modified Menghini needle (Sonopsy; Hakko Co,

okyo, Japan) was used to determine trace-metal contents.Measurement of liver-tissue metal content by means of

IXE before and after trientine hydrochloride therapy. Sohat we might measure trace-mineral content in liver tissue,e air-dried biopsy samples, fixed them with carbon tape, and

nalyzed them with a PIXE analyzer unit.18–20 The PIXEethod simultaneously analyzes multiple elements in a sen-

itive, nondestructive fashion. In the PIXE analyzer unit, ahoton beam is accelerated by a Van de Graff accelerator andrradiated onto the samples, and the characteristic x-rays thatre generated are measured with a pulse-height analyzer. Inhis study we used a PIXE analyzer unit at the Nationalnstitute of Radiological Sciences, Science and Technologygency (Chiba, Japan). We determined the trace-mineral

ontent of liver tissue by regarding the tissue concentration ofotassium as constant and converting the measured contentso weight-base values (micrograms per gram dry wt), usingormal calf liver (National Institute of Standards and Tech-ology, Gaithersburg, Md) as standard sample.

Recurrence of HCC and follow-up monitoring of pa-ients. At the time of enrollment, we confirmed cure of HCCn each patient using a diagnostic imaging technique. Allatients were prospectively checked once a month, with de-ermination of �-fetoprotein and other biochemical laboratoryarameters (urine copper level, serum mineral level, transam-nase levels, tumor markers) every month and ultrasoundvery 3 months. All patients underwent computed tomogra-hy and MRI at least once a year. When HCC was suspected,definitive diagnosis was made on the basis of the findings ofomputed tomography, MRI, angiography, and fine-needle

spiration biopsy.

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Statistical analysis. All measurements are expressed asean � SEM. The Wilcoxon signed-rank test was used to

ssess liver-tissue mineral content before and after treatment.ne-way fractional analysis of variance and multiple-com-arisons tests were performed for data on urine copper level,erum mineral level, transaminase levels, and blood hemo-lobin. We used Abacus Concepts software Stat View (SASnstitute, Tokyo, Japan) to carry out these analyses. P valuesess than .05 were considered significant for all tests.

ESULTS

Toxicity. Trientine hydrochloride exerted no toxic ef-ect on the heart, lung, gastrointestinal tract, kidney, oriver. We found no evidence of hematologic, cutaneous,r neurologic toxicity, and no infection was induced.ild, reversible anemia occurred in 1 patient after 3onths of trientine hydrochloride therapy. The hemo-

lobin value was increased with the use of an ironreparation, with no need for transfusions. No addi-ional toxic effect was observed during the course ofhis patient’s treatment or follow-up.

Liver-tissue metal contents before and after trientineydrochloride therapy. Copper content was signifi-antly reduced from the pretreatment level of 306.9 �4.3 �g/g dry wt to 160.1 � 19.5 �g/g dry wt afterreatment (Fig 1). Iron content was 1837.7 � 359.3g/g dry wt at baseline and 1807.3 � 364.4 �g/g dryt after treatment (not statistically significant). Zinc

ontent was 367.3 � 41.5 �g/g dry wt at baseline and79.8 � 43.7 �g/g dry wt after treatment (not statisti-ally significant).

Urine copper level. The urine copper level was 2.4 �.3 �g/dL at baseline, 39.3 � 11.7 �g/dL after 1 weekf treatment, 29.0 � 6.7 �g/dL at 1 month, 24.1 � 5.0g/dL at 2 months, and 2.8 � 0.5 �g/dL at 3 months,

howing a significant increase at 1 week and reductionhereafter (Fig 2, A). In group 1, urine copper contentas 2.3 � 0.3 �g/dL at baseline and 29.7 � 7.1 �g/dL

t 1 week, 30.8 � 6.5 �g/dL at 1 month, 30.3 � 9.3g/dL at 2 months, and 3.4 � 1.0 �g/dL at 3 months,

ig 1. Hepatic copper content before and after trientine hydrochlorideherapy.

howing a significant increase at 1 week, 1 month, and f

months, and a reduction thereafter. In group 2, urineopper content was 2.5 � 0.6, 49.0 � 22.6, 27.1 �1.9, 17.9 � 3.5, and 2.2 � 0.4 �g/dL, respectively,lso showing a significant increase at 1 week and sub-equent reduction. We detected no significant differ-nce between the 2 groups (Fig 2, B).

Serum metal levels. The serum copper level was38.4 � 5.4 �g/dL at baseline, 134.8 � 5.0 �g/dL at 1eek, 125.2 � 4.8 �g/dL at 1 month, 121.5 � 4.5g/dL at 2 months, and 112.0 � 3.9 �g/dL at 3 months,

howing a significant decrease after the start of treat-ent (Fig 3, A). The urine copper level decreased

ignificantly in both groups, but we detected no signif-cant difference between the 2 groups (Fig 3, B).

Liver-function parameters. Aspartate aminotransfer-se and alanine aminotransferase showed no significantifferences before and after treatment. Likewise, weetected no significant differences before and afterreatment in either group or between the 2 groups.

Serum hemoglobin. The serum hemoglobin level was3.2 � 0.3 g/dL at baseline, 13.3 � 0.3 g/dL at 1 week,3.3 � 0.3 g/dL at 1 month, 13.5 � 0.3 g/dL at 2onths, and 12.7 � 0.4 g/dL at 3 months, showing a

ignificant decrease after treatment (Fig 4, A). The levelignificantly decreased in both groups. We detected noignificant difference between the 2 groups (Fig 4, B).

ISCUSSION

To our knowledge, there has been no report of mul-iple trace-metal contents in the liver parenchyma ofatients with Wilson’s disease after trientine hydro-hloride therapy; the amount of biopsy material is toocant for measurement with previously used methods.n this study, we exposed small biopsy samples to theIXE method,21 with which multiple trace metals cane simultaneously detected in samples on the order ofilligrams.In 1956, Walshe22 introduced the copper-chelating

gent D-penicillamine for the oral treatment of Wilson’sisease. It has since been reported that D-penicillaminehould be discontinued if a serious adverse reactionccurs.23 In 1969, Walshe1 introduced another oralopper-chelating agent, trientine hydrochloride, intohe clinical setting. Compared with D-penicillamine,dverse reactions occur much less frequently with tri-ntine. In this study, iron-deficiency anemia occurred inust 1 patient, so trientine hydrochloride was consideredafe for use in patients with hepatic cirrhosis.

Trientine hydrochloride forms chelate compoundsith copper contained in food in the intestine wheniven orally after a meal and therefore undergoes nontestinal absorption. It is therefore suitable to admin-ster this drug 3 times daily during between-meals

asting, ideally about 2 hours before the meal. Trientine

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ydrochloride has been administered at dosages of 40o 50 mg/kg/d in 3 divided doses.24 Such gastrointes-

Fig 2. Urine copper level (A) before and (B) afterSquares � group 1; triangles � group 2.

Fig 3. Serum copper levels (A) before and (B) afteSquares � group 1; triangles � group 2.

inal symptoms as gastric discomfort and nausea and s

omiting may occur in small numbers of patients earlyfter the start of trientine therapy, so the starting dosage

hydrochloride therapy.

e hydrochloride therapy.

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J Lab Clin MedVolume 143, Number 6 Fukuda et al 337

0 mg/kg/d over several days to 1 week. In this study,iver-tissue copper content decreased at daily dosagesf both 250 and 750 mg. These dosages were consid-red adequate for the cirrhosis patients with copperccumulation in liver tissue who were enrolled in thistudy. No gastrointestinal symptoms, including gastriciscomfort and nausea and vomiting, occurred at theow dosage level used in this study. Serum ceruloplas-in levels (21–37 mg/dL) were also measured through-

ut the treatment period. In 1 case, the serum cerulo-lasmin level was below normal (19 mg/dl) but did notecrease after the treatment. In another case, the serumeruloplasmin level was at the lower limit of normal21 mg/dL), decreased to 15 mg/dL with treatment, andhen recovered to a normal level after treatment. Nonef the other 22 patients demonstrated copper defi-iency. In this study, a reduction in the hepatic copperoncentration was seen after 12 weeks of therapy. Gen-rally, reduction in hepatic copper in patients withilson’s disease is not seen after just a few weeks of

herapy.1 The copper level of Wilson’s disease is higherhan that among HCC patients; this discrepancy isikely attributable to the difference in copper levels.16,17

It was shown in studies of rats and patients withilson’s disease that trientine induces a remarkable

ncrease in the excretion of urine copper compared withhat seen before administration. In this study as well,he urine copper level rapidly increased to 39.3 � 11.7g/dL after 1 week of treatment. Urine copper excre-

Fig 4. Serum hemoglobin levels (A) before and (BSquares � group 1; triangles � group 2.

ion was greater in group 1 than in group 2 at 1 week, c

lthough the difference was not significant. Of course,4-hour urine copper collection would have been morerecise than spot urine collection, but because ouratients were monitored on an outpatient basis, weere restricted to spot urine collection.Among patients with chronic hepatitis C, we re-

orted, liver-tissue copper content increased signifi-antly along with the extent of the disease,6 and weoted that the only factor significantly associated withhe coexistence of HCC was the copper level in theiver parenchyma.7 Ebara et al7 reported that the copperontent of liver parenchyma was significantly greater inatients with HCC (169 � 28.0 �g/g dry wt) than inatients without HCC (60.9 � 7.09 �g/g dry wt). Inhis study, the copper content of liver parenchyma was06.9 � 64.3 �g/g dry wt. The reason for the highopper level of this study could be ascribed to the facthat 3 cases had very high copper levels and the totalumber of cases was small.We also reported a relationship between the copper

ccumulation seen in small HCCs and the pattern onRI.25–27 Ito et al compared copper content in tumor

esions according to the degree of histologic differen-iation and reported that copper content was higher inetter-differentiated lesions.28 Higher copper content inell-differentiated small HCCs early after tumor onset

uggests that copper is associated with tumor develop-ent. Sakurai et al reported the results of a study

nvolving a gel-filtration technique and noted that most

entine hydrochloride therapy.

) after tri

opper accumulated in hepatocytes existed as copper

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etallothionein in LEC rats.29 Our previous studiesnvolving the gel-filtration method showed that amongCC patients with increased copper levels, copper ex-

sted as copper, zinc metallothionein, and copper me-allothionein in both cancerous and noncancerous sur-ounding liver parenchyma; this finding is similar tondings in LEC rats.8 Zinc metallothionein reportedlyas antioxidative activity,30 but copper metallothioneinn LEC rats forms hydroxyl radicals by way of a Fen-on-like reaction in the presence of hydrogen peroxide.9

ydroxyl radicals produce 8-OHdG, which indicatesNA damage.31 Shimada et al32 also examined tissue-OHdG levels in chronic hepatitis, liver cirrhosis, andCC; they reported that chronic inflammation in the

iver induces oxidative DNA damage. In the studyeported here, trientine hydrochloride therapy signifi-antly reduced liver-tissue copper content, so it is pos-ible that the occurrence of HCC could be reduced.

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4. Yoshii J, Yoshiji H, Kuriyama S, Ikenaka Y, Noguchi R, OkudaH, et al. The copper-chelating agent trientine suppresses tumordevelopment and angiogenesis in the murine hepatocellular car-cinoma cells. Int J Cancer 2001;94:768–73.

5. Moriguchi M, Nakajima T, Kimura H, Watanabe T, TakashimaH, Mitsumoto Y, et al. The copper chelator trientine has anantiangiogenic effect against hepatocellular carcinoma, possiblythrough inhibition of interleukin-8 production. Int J Cancer 2002;102:445–452.

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4. Yamaguchi Y. Triethylenetetramine therapy for D-penicilla-mine–intolerant patients with Wilson’s disease: preclinical andclinical studies on the safety and clinical efficacy of triethyl-enetetramine. J Med Soc Toho 1992;38:756–72.

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bases during DNA synthesis past the oxidation-damaged base8-oxodG. Nature 1991;349:431–4.

2. Shimoda R, Nagashima M, Sakamoto M, Yamaguchi N, Hiro-hashi S, Yokota J, et al. Increased formation of oxidative DNAdamage, 8-hydroxydeoxyguanosine, in human livers with

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