Free Radical Bmlogy & Medicine, Vol. 21, No. 6, pp. 755-761. 1996 Copyright 0 1996 Elsevier Science Inc.

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ELSEVIER PI1 SOSSl-5849(96)00222-5

-+ ’ Oriainal Contribution

THE EFFECTS OF a-PHENYL-TERT-BUTYL NITRONE (PBN) ON COPPER- INDUCED RAT FULMINANT HEPATITIS WITH JAUNDICE

TADASHI YAMASHITA,* HIDEKI OHSHIMA,+ TAKETOSHI ASANUMA,~ NAOKO INUKAI,’ ICHIRO MIYOSHI,* NORIYUKI KASAI,* YASUHIRO KoN,* TOMOMASA WATANABE,*

FUMIAKI SATO,+ and MIKINORI KUWABARA~ *Laboratory of Experimental Animal Science, Department of Animal Disease Control, ‘Laboratory of Radiation Biology,

Department of Environmental Veterinary Sciences, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo 060, Japan; and ‘Institute for Animal Experimentation, Tohoku University School of Medicine, Sendai 981, Japan

(Received 19 June 1995; Revised 27 December 1995; Accepted 1 April 1996)

Abstract-In the present study we demonstrated the protective effects of the spin-trapping agent a-phenyl-tert-butyl nitrone (PBN) against fulminant hepatitis with jaundice in LEC rats. In LEC rats an excess amount of copper is accumulated in the liver and causes hepatitis with severe jaundice. PBN was subcutaneously administered every 2 d at the concentration of 128 mg/kg, beginning with 13-week-old rats and continuing for 17 weeks. PBN prevented the loss of body weight, reduced death rate, and suppressed the increase in GTP and GOT values reflecting hepatic cell destruction. Ocular inspection also confirmed the suppressive effects of PBN on jaundice. In parallel with these phenomena, the amounts of thiobarbituric acid-reactive substances (TBARS) in livers of PBN-administered rats were found to be lower than those of non-PBN-administered rats. Little histological changes were observed in PBN- administered rats in comparison with non-PBN-administered rats. The protective effect of PBN on the formation of oxidative damage in liver DNA was observed but not so remarkable as that on lipid peroxidation. From these results, it was concluded that PBN had the liver-protective effects against fulminant hepatitis with jaundice. This suggested that free radicals play an important role in abnormally accumulated copper-induced liver injury and that PBN potentially has therapeutic value for the treatment of hepatitis.

Keywords--Phenyl-tert-butyl nitrone (PBN), LEC rat, Hepatitis, Copper, Lipid peroxides, Reactive oxygens, Free radicals

INTRODUCTION

LEC rat (Long-Evans rat having a cinnamon-colored coat) has been established as a new mutant strain with hereditary hepatitis,’ and is now used as an important animal model for studying the relationship between the chronic hepatitis and liver cancer. It was shown that if rats recover from hepatitis, they survive for more than 12 months during developing hepatoma.’ In relation to this phenomenon, abnormal accumulation of copper in the liver? and a marked decrease of ceruloplasmin and copper in serum4 are detected. From these results LEC rats are also considered to be a model of human Wil- son’s disease.4-7 Genetic analysis revealed that the hep- atitis in LEC rats is controlled by a single autosomal

Address correspondence to: M. Kuwabara, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo 060, Japan.

recessive gene, hts, and that the ceruloplasmin gene is not contained in the hts gene.*

The copper concentration in the liver reaches a high level soon after birth. The concentrations of 3- and 8- month-old rats are 40-50 times higher than those of age- and sex-matched normal LEA (Long-Evans Agouti) rats.4 Histochemical examination of livers re- vealed that copper was preferentially accumulated in the hepatocytes and was distributed throughout the cy- toplasm. The molecular mechanisms by which copper accumulation causes hepatotoxicity remain unknown. Copper toxicosis with hepatitis may occur when copper is not bound to metallothionein in the liver, and free copper may be a trigger for hepatocellular carcinoma.” Furthermore, the hepatitis reduces the induction of metallothionein.

Oxygen radicals are assumed to play important roles

155

756 T. YAMASHITA et al

in the course of multistep carcinogenesis.10-12 Recent studies have indicated that copper produces reactive oxygens and induces DNA damage in vitro in the pres- ence of hydrogen peroxide and reducing agents.13s14 In LEC rats the high concentration of copper in livers is thought to be responsible for the acute-phase hepatitis and hepatocarcinoma. 2,4 This might be explained by the fact that a metal chelator, D-penicillamine, effectively acts against hepatitis.4 Therefore, LEC rats having a high concentration of copper in the liver can be re- garded as a good model to examine the relationship between reactive oxygen toxicity and acute phase hep- atitis (hepatoma).

PBN is a typical spin trapping agent that is known to be able to trap not only oxygen radicals but also free radicals of CC4 in rats,” and to diminish the increase of oxidized proteins as well as the loss of glutamine synthetase activity accompanying ischemia/reperfusion injury in the brain.i6,” From a study concerning the tissue distribution, excretion, and metabolism of PBN, it was shown that this is rapidly absorbed, widely dis- tributed inside the body, and remains for a long period in many tissues when injected intraperitoneally into rats. l5 In the present study we attempted to demonstrate the protective effects of PBN against liver injury (ful- minant hepatitis) in LEC rats.

MATERIALS AND METHODS

Animals

The mutant inbred, Long-Evans Cinnamon (LEC) rat strain, was established from a closed colony of Long-Evans rats at a Center for Experimental Plants and Animals of Hokkaido University1,2 and maintained at the Institute for Animal Experimentation, School of Medicine, Hokkaido University. The animals were maintained under conventional conditions, and fed a regular diet and water ad Zibitum in an air-conditioned animal room at 22 +- 3°C with a relative humidity of 55 2 5%. Female rats from 13 to 30 months old were used in the present experiments.

Administration of PBN to rats

a-Phenyl-N-tert-butyl nitrone (PBN) (Sigma Chem- ical Co., St. Louis, MO) was dissolved in sesame oil (Nacalai Tesque Inc, Kyoto, Japan), and 0.1 ml of the oil was subcutaneously administered to rats every 2 days (corresponding to 128 mg/kg). This was started with 13-week-old rats and continued for 30 weeks. Subcutaneous administration of sesame oil only was regarded as a control experiment.

Measurements of glutamic pyruvic transaminase (GPT) and glutamic oxaloacetic transaminase (GOT) levels in sera

Serum GPT and GOT levels were measured by the UV method (Kokusai Shiyaku, Ltd., Japan) once a week from 13 to 30 weeks of age.

Measurement of lipid peroxidation in the liver

Liver tissues were obtained from four different groups of female LEC rats; non-PBN-administrated rats (30 weeks old), administrated rats (30 weeks old), rats before hepatitis (5 weeks old), and long-term sur- vivors (12 months old). After washing out blood, lipid peroxides in liver homogenates were measured as thio- barbituric acid-reactive substances (TBARS) according to the method of Uchiyama and Mihara.18

Detection of 7,8-dihydro&oxo-2’-deoxyguanosine (8-oxodG) by HPLC-ECD

Liver tissues were obtained from two groups of fe- male LEC rats; non-PBN-administered rats (30 weeks old), and PBN-administered rats (30 weeks old). High molecular genome DNA from liver tissues was di- gested with enzymes, alkaline phosphatase (Takara Shuzo Co., Ltd, Siga, Japan) and phosphodiesterase (Boehringer-Mannheim, GmbH, Mannheim, Ger- many). After digestion, HPLC-ECD was used to detect 8-oxodG. The HPLC-ECD system consisted of HPLC (CCP & 8000 system, Tosoh Company, Tokyo, Japan), a UV detector (W-8000, Tosoh), an EC detector (ECD-300, Eicom Company, Tokyo, Japan), and a re- verse phase column (TSK-Gel ODS-80TS, 4.6 mm i.d., 25 cm long, Tosoh). Elution was carried out with a mixed solution of 50 mM potassium phosphate buffer, pH6.0, and methanol at a ratio of 95:5 (v/v) with a flow rate of 1 rnl/min at room temperature. The effluent was monitored by UV detection at 260 nm and the EC de- tector with an electrode potential of +600 mV.” Quan- titative measurement of 8-oxodG was performed on the basis of the chemically synthesized sample according to the method of Lin et al.” The product finally ob- tained was identified as 8-oxodG by examining the UV absorption spectrum (Hitachi U-2000, Hitachi, Japan.), mass spectrum (JEOL, JMX-DX303, Akishima, Ja- pan), and NMR spectrum (JEOL, JMX-GX270, Aki- shima, Japan).20’21

Tissue processing

Tissue blocks from each animals anesthetized with ether and pentobarbital were removed immediately and

PBN inhibits copper-induced fulminant hepatitis 151

13 15 17 19 21 23 25 27 29

240, A

120’ . ’ ’ ’ ’ ’ ’ ’ I ’ ’ ’ 1 13 15 17 19 21 23 25 27 29

Age (week)

Fig. 1. Age-dependent changes of body weights in LEC rats. X-axis shows the ages of rats (weeks). Y-axis shows the body weights of rats (gram). Disappearance of lines means that rats died with jaun- dice. (A) non-PBN-administrated, and (B) PBN-administrated rats.

fixed with 4% paraformaldehyde solution in 0.1 M phosphate buffer (pH 7.3) overnight at 4°C. Paraffin sections (3 mm) prepared by routine procedure were stained with hematoxylin-eosin.

Statistical calculation

Data were expressed as mean -C SD, and the Wil- coxon rank sum test was used to determine the signif- icance of differences in the numbers of LEC rats and in the amounts of lipid peroxides.

RESULTS

Loss of body weight

The body weights of 13- to 30-week-old LEC rats were measured (Fig. 1). In non-PBN-administered rats

(n = 9) no increase in body weight in eight of nine rats was observed for 16-17 weeks, and they died within 18 weeks. For one rat, the body weight reached a minimum at 19 weeks and gradually increased up to 30 weeks. This rat survived for more than 30 weeks (Fig. 1A). In PBN-administered rats (128 mg/kg body weight), one of nine rats died at 17 weeks of age with no increase of body weight; two survived for 19 and 20 weeks, respectively without loss of body weight; one showed loss of body weight at 17 weeks but sur- vived for 22 weeks and one survived for 29 weeks with- out loss of body weight. A total of four rats survived for more than 30 weeks (Fig. 1B).

Survival times of non-PBN-administrated and PBN-administrated rats

LEC rats generally die of fulminant hepatitis within 1 week of the manifestation of jaundice if its symptoms are very severe, and they cannot recover from the hep- atitis.” Figure 2 shows the mean survival times of non- PBN-administrated and PBN-administrated rats. Of the non-PBN-administrated rats, eight of nine died within 17 weeks and only one survived after jaundice. The survival continued for more than 30 weeks (Fig. 1A). The mean survival time was 15.3 ? 1.5 weeks. Half of PBN-administrated rats (5 out of 9) survived for more than 29 weeks (Fig. 1B). The mean survival time was 21.4 t 4.6 weeks. Statistical analysis revealed that the mean survival time of PBN-administrated rats was sig- nificantly prolonged @ < .05).

Elevation of GPT and GOT

Figure 3 shows the GPT activities obtained from 13- to 30-week-old rats (n = 9). Generally, GPT activity is

p < 0.05

1

Control PBN

Fig. 2. Mean survival ages (weeks) of no PBN-administrated (control, n = 5) and PBN-administrated rats (n = 9). A significant difference between them was obtained at p < .05. Values represent means 2 SD.

758 T. YAMASHITA et al.

regarded as a reflection of hepatic cell destruction. All non-PBN-administered rats except one died before the GPT activity reached the maximum, but we could rec- ognize clinical signs of hepatitis with jaundice. The age at which GPT activity reached the maximum was be- tween 15 and 18 weeks (Fig. 3A). In PBN-treated rats, the maximal GPT activities were dispersed between 16 and 25 weeks, and prolongation of the maximal activ- ities was clearly recognized (Fig. 3B). No significant difference in the maximal activities of GPT between control and PBN-treated rats was observed. GOT ac- tivity is also regarded as a reflection of hepatic cell destruction. The time dependencies of GOT activities obtained from the two groups of rats were similar to those of GPT activities (data not shown).

Clinical signs of fulminant hepatitis

Severe jaundice is a symptom of fulminant hepatitis in LEC rats. The average week-ages at which the jaun- dice appeared in rats are presented in Fig. 4. The color changes in the coat, feet, ears, tail, and hair were re- garded as symptoms of jaundice. The period of jaun- dice is generally known to range from 16 to 18 weeks. In the present study, the mean period at which clinical signs of fulminant hepatitis appeared was 15.5 t 1.6 weeks in control rats, whereas this was observed to be 19.9 + 2.6 weeks in PBN-treated rats. The difference in these values between the two groups was statistically significant (p < .05).

Lipid peroxidation in the liver

The copper concentration is reported to increase with age in the LEC rat liver.4*5 Copper is thought to generate reactive oxygen species.13 This means that lipid peroxidation also occurs in the liver. To examine oxidative reactions in the liver, we measured TBARS of the liver tissues from four different groups. The re- sults are shown in Fig. 5. In the non-PBN-administrated group, only one rat was available as a 30-week survivor (Fig. 1A). This rat showed a TBARS level of about 250 nmol/g. In the PBN-treated group (30 weeks old, IZ = 5), the value of TBARS was 145 -+ 10 nmol/g. This was significantly lower than that (178 + 7 nmol/g) from the group before hepatitis (5 weeks old, n = 4) and that (250 f 13 nmol/g) from the group of l-year survivors (n = 6) @ < .Ol).

Formation of 8-oxodG in liver DNA

As another oxidative reaction in the liver, we mea- sured the formation of 8oxodG in liver DNA (Table 1). Though the results showed the lower amount of 8-

600

E 0 1200 )

1000

600

400

200

0” ’ ” t ’ ” ” “‘I 13 15 17 19 21 23 25 27 29

Age (week)

Fig. 3. Age-dependent changes of GPT activities in sera of LEC rats. X-axis shows the rat age (weeks). Y-axis shows the GPT activities (KU) of sera obtained from (A) non-PBN-administrated and (B) PBN-administrated rats. Disappearance of lines means that rats died with jaundice.

oxodG in PBN-administrated rats than in non-PBN-ad- ministrated rat, statistical calculation could not be car- ried out between two groups because only one rat was available as a 30-week survivor.

Histological changes of non-PBN-administrated and PBN-administrated rats

Microscopic changes were observed in non-PBN- administrated LEC rat in which the liver showed mas- sive and multifocal necrosis with filtration of a few inflammatory cells and giant hepatocytes. Proliferation of pseudobile ducts and increased numbers of kupper cells phagocyting were observed (Fig. 6A and B). In PBN-administrated LEC rats, only a minor morpholog- ical changes were observed in the liver (Fig. 6C and D).

PBN inhibits copper-induced fulminant hepatitis 159

25r

pco.05

Control PBN

Fig. 4. Mean ages of rats when they showed symptoms of hepatitis with jaundice. Control, non-PBN-administrated rats (n = 9); PBN, PBN-administrated rats (n = 9). A significant difference between two groups was observed @ < .05). Values represent means t SD.

DISCUSSION

This study was carried out to clarify whether abnor- mally accumulated copper induced lipid peroxidation in the liver through oxygen-radical reactions and whether the resulting lipid peroxidation was responsi- ble for hepatic cell injury with fulminant hepatitis in LEC rats. For this purpose, PBN was employed be- cause this agent was regarded to protect against lipid peroxidation. PBN not only effectively scavenges re- active oxygens but also suppresses the chain reactions leading to lipid peroxidation by trapping lipid radi- cals.23*24 As is shown in Fig. lA, no increase in body weight in eight of nine rats was observed for 16-17 weeks in the non-PBN-administrated rats. They died within 18 weeks. Acute-phase hepatitis with jaundice was inferred to cause a loss of body weight and to lead to death. However, when PBN was subcutaneously ad- ministrated to rats, this agent significantly suppressed from the loss of body weight in most rats and protected against death (Figs. 1 and 2). PBN also prolonged the period of the appearance of fulminant hepatitis (Fig. 3), and suppressed the elevation of GPT and GOT levels in sera (Fig. 4). Because high levels of GPT and GOP activities in sera are regarded as a reflection of hepatic cell destruction and are closely associated with hepa- titis, these observations indicated that PBN protected against hepatic cell death and thereby prolonged the appearance of hepatitis.

The measurements of lipid peroxides in livers from four groups of LEC rats indicated that (1) the amount of lipid peroxides increased with increasing age, and (2) administration of PBN certainly protected rats against accumulation of lipid peroxides in the liver (Fig. 5). These results proved the hypothesis that cop-

300 r

p < 0.01

p < 0.05 ’

Non-treated PBN-treated 5 weeks 1 year

Fig. 5. TBARS of liver homogenates obtained from four groups of rats: nontreated, 30-week survivor without PBN administration, n = 1; PBN-treated, 30-week survivors with PBN administration, n = 5; 5 weeks, rats before hepatitis, n = 4; 1 year, l-year survivors without PBN administration, n = 6. Significant differences between 30-week survivors with PBN administration and 5-week-old or l-year-old rats without PBN administration were observed (p < .Ol). A significant difference was observed between 5-week-old and l-year-old rats @ < .05). Values except for nontreated rats represent means 2 SD.

per accumulation caused the generation of reactive ox- ygens and the subsequent formation of lipid peroxides in the liver. To further confirm oxidative reaction in the liver due to copper accumulation, the formation of 8- oxodG in liver DNA was measured. PBN-administra- tion showed the tendency to lower the formation of 8- oxodG, but no statistical calculations could be carried out because of only one survivor at 30-weeks for con- trol (Table 1). Several experiments demonstrated that PBN inhibited cell injury related to lipid peroxidation in the brain’6*23-24 and liver.25-27 Mathews et al. sug- gested that lipid peroxides might be important as a mechanism damaging endothelial cells, a limited com- partment of the liver, which are close to Kupper cells and neutrophils, for example, the source cells of reac- tive oxygens.28 It is also reported that the pretreatment of gerbil rats with PBN significantly prevents free rad- ical-mediated protein oxidation and protects against the loss of glutamine synthetase activity in the brain after global cortical ischemia-reperfusion.‘“‘7,23

Table 1. 8-OxodC in Liver DNA Obtained from Two Rats

Animals

Nontreated PBN-treated

8-OxodG (pmol)

49.2 42.8 ? 2.6

Nontreated, 30-week survivor without PBN administration, n = 1; PBN-treated, 30-week survivor with PBN administration, n = 5.

760 T. YAMASHITA et al.

Fig. 6. Morphological changes of non-PBN-administrated and PBN-administrated rats: nontreated, 30-week survivor with non- PBN administration (A; x75, B; x300) and PBN-treated, 30-week survivor with PBN administration (C; ~75, D; x300).

In the present study we employed LEC rats that de- velop hepatic injury at 4-5 months of age followed by hepatocellular carcinoma. LEC rats exhibited abnormal accumulation of hepatic copper and a deficiency in se- rum ceruloplasmin activity. Therefore, it has been pro- posed that this strain is useful as an animal model for human Wilson’s disease.4-7 Low oxidase activity of ce- ruloplasmin in serum is one of the remarkable features of LEC rats.5 In contrast, copper-metallothionein (Cu- MT) was found to be induced in the LEC rat.29 Sakurai et al. reported that the Cu-MT of LEC rats generates hydroxyl radicals in the presence of hydrogen perox- ides.30 This suggests that the Cu-MT of LEC rats plays an important pathogenic role by generating hydroxyl radicals if hydrogen peroxides are produced in cells or tissues. Therefore, the LEC rat is a useful animal model for generating reactive oxygens in vivo. The present study demonstrated that PBN protected against hepatic cell injury due to fulminant hepatitis. Actually, differ- ence in the morphological changes between PBN-ad- ministrated LEC rat and non-PBN-administrated LEC

rat clearly showed that PBN protected against cell in- jury (Fig. 6). Therefore, this result can be explained by the fact that PBN effectively scavenges oxygen radicals that are induced by intracellular copper.

PBN was mixed with mineral oil and administrated subcutaneously (128 mg/kg body weights). This dose was higher than those used in other studies,‘“‘7*23-25 but this was an effective method to prolong the effects of PBN under conditions in which a high level of copper in the liver causes hepatitis.

In conclusion, the present study indicated that in vivo PBN administration caused a significant decrease in lipid peroxidation in the liver and prevented hepatic cell injury with fulminant hepatitis. This indicates that PBN has potential therapeutic value for hepatitis.

Acknowledgements - The authors thank Mr. Tsutomu Osanai of the Institute for Animal Experimentation, Hokkaido University School of Medicine, for technical suggestions and animal treatment. They also thank Rooibos Tea Japan Co., Ltd for financially supporting us. In conducting the research described in the report, the investigators

PBN inhibits copper-induced fulminant hepatitis 761

adhered to “The Guide for the Care and Use of Laboratory Animals, Hokkaido University School of Medicine.”

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