Pentachlorophenol-induced oxidative DNA damage in mouse liver and protective effect of antioxidants

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Fd Chem. Toxic. Vol. 33, No. 10, pp. 877-882, 1995 Copyright © 1995ElsevierScienceLtd Printed in Great Britain.All rights reserved 0278-6915/95$9.50+ 0.00

BRIEF COMMUNICATIONS

Pentachlorophenol-induced Oxidative DNA Damage in Mouse Liver and Protective Effect of Antioxidants K. S A I - K A T O * , T. U M E M U R A , A. T A K A G I , R. H A S E G A W A , A. T A N I M U R A t a n d Y. K U R O K A W A Division of Toxicology, National Institute of Health Sciences, I-18-I Kamiyoga, Setagaya-ku, Tokyo 158 and tLaboratory of Life Science, Faculty of Living Sciences, Showa Women's University, 1-7 Taishido, Setagaya-ku, Tokyo 154, Japan (Accepted 11 April 1995)

Abstraet--8-Hydroxydeoxyguanosine (8-OH-dG) was determined as a marker of oxidative DNA damage in male B6C3F~ mice treated with the hepatocarcinogen pentachlorophenol (PCP). A single oral administration of PCP (0-80 mg/kg) significantly and dose-dependently increased the 8-OH-dG level specifically in the liver at 6 hr. Repeated doses (0-80 mg/kg) over 5 days caused a further increase. Elevation of the 8-OH-dG levelcaused by a single dose of PCP (60 mg/kg) was not affected by ip injection of buthionine sulfoximine (2 mmol/kg), an inhibitor of GSH synthesis, or aminotriazole (1 g/kg), an inhibitor of catalase, showing no clear evidence for enhancement by the oxidative stress due to reduction of antioxidative factors under these experimental conditions. However, examination of the effects of natural antioxidants on repeated PCP treatment (60 mg/kg/day, for 5 days) revealed that oral administration of vitamin E and diallyl sulfide 3 hr before each PCP challenge significantly protected against elevation of hepatic 8-OH-dG levels, fl-Carotene did not have any effect. Ellagic acid, epigallocatechin gallate and vitamin C demonstrated partial protection. These findings indicate that PCP causes oxidative DNA damage in the target organ liver which can be blocked by a number of antioxidant agents.

Introduction

therefore thought to contribute to PCP-induced carcinogenesis. Pentachlorophenol (PCP) has been used in large In recent years, we have analysed levels of 8-hyquantities, mainly as a wood preservative (WHO, droxydeoxyguanosine (8-OH-dG), an oxidative DNA 1987) and also in herbicide or insecticide preparation product having mutagenic potential (Shibutani et al., (Crosby et al., 1981). Because biodegradation of PCP 1991), after administration of chemical carcinogens, is relatively slow and it has been detected in foods showing a close relationship between 8-OH-dG for(Agriculture Canada, 1989) and human urine mation and carcinogenesis (Sai et al., 1991, 1992 and (Gomez-Catalan e t a / . , 1987), its contamination of 1994; Takagi et al., 1993; Umemura et al., 1990). In the environment has l:ecome a serious problem. PCP the study reported here, we examined whether PCP has been reported to be negative for mutagenicity in could induce 8-OH-dG formation in the liver of mice, most Ames tests (Seiler, 1991). However, in a 2-yr in order to consider the involvement of oxidative carcinogenicity study~ PCP (600ppm in diet) was DNA damage in its carcinogenesis. found to induce hepatocellular adenomas and adenoThe effective role of naturally occurring antioxicarcinomas in mice [National Toxicology Program dants, such as ellagic acid (Tanaka et al., 1988), (NTP) 1989]. Meanwhile, tetrachlorohydroquinone, E-carotene (Suda et al., 1986), vitamin E (Shklar, a metabolite of PCP (Ahlborg et al., 1974), was 1982), vitamin C (Kallistratos and Fasske, 1980), revealed to have genotoxic potential (Naito et al., diallyl sulfide (Wargovich et al., 1988) and epigallo1994; Witte et al., 1985) and, in this genotoxic catechin gailate (Wei and Frenkel, 1993; Xu et al., mechanism, involvement of superoxide anions pro1992), in protection against tumorigenesis has been a duced in further oxidation to tetrachlorobenzo- recent focus of attention. This finding prompted us to quinone is presumed. Oxidative DNA damage by investigate further the influence of PCP-induced active oxygen species generated in metabolism is 8-OH-dG formation by reducing the presence of antioxidative factor with buthionine sulfoximine or aminotriazole, and the protective effects on the oxi*Author for correspondence. Abbreviations: 8-OH-dG = 8-hydroxydeoxyguanosine; dative DNA damage by the application of six antioxidants. PCP = pentachlorophenol. 877

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Materials and Methods

Animals and housing conditions 5-wk-old male B6C3F~ mice (specific pathogen free, SLC, Shizuoka, Japan) were housed at 23 + I°C under 55 + 5% relative humidity, and given F2 pellet basal diet (Funabashi Farm Co., Chiba, Japan) and tap water freely. Animals were used after I wk of acclimatization. Chemicals PCP (purity 98.6%), aminotriazole, epigallocatechin gallate, ellagic acid, vitamin E, vitamin C and fl-carotene were purchased from Wako Pure Chemical Industries, Ltd (Osaka, Japan), and diallyl sulfide was from Aldrich Chemical Co., Inc. (Milwaukee, WI, USA). Buthionine sulfoximine and alkaline phosphatase were obtained from Sigma Chemical Co. (St Louis, MO, USA) and nuclease Pi was from Yamasa Shoyu Co., Ltd (Chiba, Japan). All other chemicals used were of specific analytical or HPLC grade.

phosphatase. Digested nucleoside samples were analysed within 12hr after the digestion. Deoxyguanosine (dG) was quantified by analysing absorbance at 290 nm and 8-OH-dG was analysed by HPLC (HPLC pump 420, Kontron AG. Instruments, Zurich, Switzerland) with an electrochemical detection system (Coulochem 5100A, esa, Bedford, MA, USA) as described previously (Sai et al., 1991). Other measurements For the analysis of GSH levels, 10% liver homogenates in 6% trichloroacetic acid were prepared and non-protein thiol (nmol/mg protein) was estimated spectrophotometrically at 412nm by the method of Eliman (1959). Catalase activity was determined spectrophotometrically at 240nm by the method of Beers and Sizer (1952). Briefly, 10% liver homogenates were prepared in 50 mM K-Na phosphate buffer (pH 7.0) and the activity at room temperature was expressed as decomposed hydrogen peroxide (#mol/min/mg protein).

Treatment of animals

Statistical analysis

Mice (five mice per group) were given a single oral administration of PCP in olive oil by gavage at doses of 0, 30, 60 and 80 mg/kg. Livers were excised 6 hr after treatment and used for 8-OH-dG analysis. To study organ specificity, mice were given a single oral administration of 60 mg PCP/kg and liver, kidney and spleen were excised at the same 6-hr time point. For investigation of repeated oral administration of PCP, mice were treated with 30, 60 or 80 mg PCP kg/day for 5 days and livers were excised 6 hr after the last treatment. For the study of buthionine suifoximine or aminotriazole treatment, mice were injected ip with 2 mmol buthionine sulfoximine/kg in saline or 1 g aminotriazole/kg in saline 30 min before a single administration of PCP at doses of 30 and 60 mg/kg, and livers were excised 6 hr after PCP treatment. In the antioxidant-treatment study, 100mg/kg/day of either vitamin E, ellagic acid, epigallocatechin gallate, fl-carotene or diallyl sulfide, or 300 mg/kg/day vitamin C were daily administered to mice orally 3 hr before PCP challenge (60 mg/kg/day) for 5 days. Livers were excised 6 hr after the last PCP administration. Control animals were treated with vehicle (olive oil or saline) alone.

All data are expressed as mean + SD of values for five mice/group and were anlaysed for intergroup significance using Student's t-test.

Analysis of 8-OH-dG level in nuclear DNA Extraction of DNA took place shielded from light and air. Organs were homogenized in 5 msi phosphate buffered saline (pH7.4) containing 20msl EDTA using a teflon homogenizer. After centrifugation (1000g, 10 min at 4°C), the nuclear fraction was suspended in the same buffer and mixed with 62.5 U proteinase K. DNA from the nuclear fraction was extracted with DNA purification system (Model 341, Applied Biosystems, Foster City, CA, USA) and digested by treatment with nuclease Pl and alkaline

Results

8-OH-dG level after administration of PCP First we measured 8-OH-dG levels in the liver 24 hr after a single oral administration of PCP (0, 40 and 60 mg/kg), because, in our previous investigation of 8-OH-dG level after a single administration of a renal carcinogen to rats, maximum kidney adducts were seen more than 24 hr after the treatment. Regarding PCP treatment, however, 8-OH-dG levels in the liver DNA were not changed at that time point. The effects of PCP at earlier time points were then tested with doses up to 80 mg/kg. As a result, 8-OH-dG level at 6 hr was found to be significantly increased with doses of 60 mg/kg and above. The levels were elevated 1.4-fold by 60mg/kg (1.07+0.23 8-OHdG/105 dG) and 1.7-fold by 80 mg/kg (1.34 + 0.38 8-OH-dG/105dG) compared with control (0.77-10.15 8-OH-dG/10 SdG) (Fig. 1). In order to clarify the organ specificity of PCP-induced 8-OH-dG formation, the 8-OH-dG levels in liver and non-target organs (kidney and spleen) were compared after a single administration of PCP. Significant elevation of 8-OH-dG level in the liver was again seen, whereas no obvious changes were observed in the levels in the kidney and spleen (Table 1). With repeated administration of PCP, significant elevation of 8-OH-dG levels was found from 30 mg/kg. The levels were raised to 1.5-fold at 30 mg/kg/day (1.17 _+0.31 8-OHdG/105dG) and 1.9-fold at 60 or 80mg/kg/day (1.44 + 0.41 and 1.43 + 0.31 8-OH-dG/10 s dG) compared with control (0.76 + 0.10 8-OH-dG/105 dG),

Oxidative D N A damage by pentachlorophenol 2 --

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Fig. 1.8-OH-dG levels in the mouse liver 6 hr after a single dose o f PCP. PCP (0, 30, 60 and 80 mg/kg) was administered by gavage to mice and 8-OH-dG levels in the liver were analysed 6 hr later. Values are the means + SD of data for five mice. Asterisks indicate significant differences from control (*P < 0.05; Student's t-test).

Fig. 2. 8-OH-dG levels in the mouse liver after daily administration o f PCP for 5 days. PCP (0, 30, 60 and 80 mg/kg/day) was administered by gavage to mice for 5 days and 8-OH-dG levels in the liver were analysed 6 hr after the last administration. Values are the means + SD of data for five mice. Asterisks indicate significant differences from control (*P < 0.05; **P < 0.01; Student's t-test).

respectively (Fig. 2), b e i n g h i g h e r t h a n with the single administration.

levels, a d o s e - d e p e n d e n t increase in the levels was seen with b o t h P C P a l o n e a n d c o t r e a t m e n t with buthionine sulfoximine and PCP. However, the magn i t u d e o f the increase with c o t r e a t m e n t w a s a l m o s t t h e s a m e as t h a t w i t h P C P t r e a t m e n t alone, respectively b e i n g 1.1- a n d 1.2-fold at 3 0 m g / k g P C P a n d 1.4- a n d 1.4-fold at 60 m g / k g , c o m p a r e d with t h e c o n t r o l (Table 2). T o investigate the c o n t r i b u t i o n o f h y d r o g e n peroxide g e n e r a t i o n to P C P - i n d u c e d 8 - O H - d G form a t i o n , effects o f the catalase inhibitor, a m i n o triazole, were e x a m i n e d . T r e a t m e n t with a m i n o t r i a zole c a u s e d a 7 5 % i n h i b i t i o n in catalase activity in all t r e a t e d g r o u p s (Table 3). W i t h r e s p e c t to 8 - O H - d G levels, n o effect o f a m i n o t r i a z o l e o n 8 - O H - d G level was revealed, w h e r e a s a d o s e - d e p e n d e n t increase in 8 - O H - d G level was o b s e r v e d with b o t h P C P a l o n e a n d c o t r e a t m e n t with P C P a n d a m i n o t r i a z o l e , the values being, respectively, 1.2- a n d 1.2-fold

Treatment with buthionine sulfoximine or aminotriazole In o r d e r to e x a m i n e w h e t h e r intracellular G S H levels m i g h t influence g e n e r a t i o n o f 8 - O H - d G by P C P , b u t h i o n i n e sui[foximine, a n i n h i b i t o r o f G S H synthesis, w a s a d m i n i s t e r e d b e f o r e P C P . G S H levels in t h e liver were r e d u c e d to 6 0 % o f the c o n t r o l level after p r e t r e a t m e n t with b u t h i o n i n e s u l f o x i m i n e a l o n e o r c o t r e a t m e n t with b u t h i o n i n e s u l f o x i m i n e a n d P C P (30 m g / k g ) . A l t h o u g h n o c h a n g e w a s o b s e r v e d in liver G S H levels in aninaals t r e a t e d with P C P alone, cotreatment with buthionine sulfoximine and PCP (60 m g / k g ) c a u s e d far m o r e decrease t h a n b u t h i o n i n e s u l f o x i m i n e t r e a t m e n t alone. A l t h o u g h b u t h i o n i n e s u l f o x i m i n e t r e a t m e n t a l o n e did n o t affect 8 - O H - d G

Table 1. 8-Hydroxydeoxyguanosine (8-OH-dG) levels in the mouse liver, kidney and spleen after a single dose of pcntachlorophenol (PCP) 8-OH-dG(/10 s dG) Organ Control PCP Liver 0.97 + 0.26 1.32 + 0.19" Kidney 0.93 + 0.15 1.06 + 0.26 Spleen 0.87 + 0.26 0.94 __0.14 8-OH-dG = 8-hydroxydeoxyguanosine PCP = pentachlorophenol PCP (60 mg/kg) was administered by savage to mice and 8-OH-dG levels in the liver, kidney and spleen were analysed 6 hr thereafter. Values are means + SD of data for five mice. Asterisk indicales significant difference from control (*P < 0.05; Student's t-test). FCT 33/10--F

Table 2. Effects of buthionine sulfoximine (BSO) on glutathione (GSH) and 8-OH-dG levels in the mouse liver after a single dose of PCP Group

GSH (gmol/g wet tissue) 8-OH-dG(/105dG)

Control 5.77 + 0.66 0.83 + 0.18 PCP (30 mg/kg) 6.51 + 1.26 1.01 + 0.51 PCP (60 mg/kg) 6.06 + 0.58 1.14 + 0.20* BSO 3.35 _+0.50* 0.84 + 0.25 BSO + PCP (30 mg/kg) 3.46+0.83* 0.91 +0.15 BSO + PCP (60 m g / k g ) 1.56 + 1.02*t I. 15 + 0.14" BSO (2 mmol/kg) was given ip 30 rain before PCP (0, 30 or 60 mg/kg) oral administration to mice. GSH and 8-OH-dG levels in the liver were analysed 6 hr after PCP treatment. Values are means + SD of data for five mice. Asterisks indicate significant differences from control (*P < 0.05; Student's t-test). Dagger indicates significant difference from BSO-treated group (~P < 0.05; Student's t-test).

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K. Sai-Kato et al. Table 3. Effects of aminotriazole (AT) on catalase activity and 8-OH-dG levels in the mouse liver after a single dose of PCP Catalase activity Group (#mol H202/min/mg protein) 8-OH-dG(/10~dG) Control 76.8 _+7.4 0.91 + 0.18 PCP (30 mg/kg) 85.0 + 7.3 1.06 ~ 0.11 PCP (60 mg/kg) 85.8 + 5.6 1.39 + 0.06** AT 19.6 + 1.6"* 0.97 +_0.20 AT + PCP (30 mg/kg) 21.4 +_3.6** 1.11 + 0.14 AT + PCP (60 mg/kg) 17.3 + 1.5"*I" 1.49 _ 0.13"'5"t AT (1 g/kg) was given ip 30 min before PCP (0, 30 and 60 mg/kg) oral administration to mice. Catalase activity and 8-OH-dG levelsin the liver were analysed 6 hr after PCP treatment. Values are means + SD of data for five mice. Asterisks indicate significantdifferences from control (**P < 0.01; Student's t-test). Daggers indicate significant differences from AT-treated group (~'P <0.05; t~'P < 0.01; Student's t-test).

for 3 0 m g / k g a n d 1.5- a n d 1.6-fold for 6 0 m g / k g (Table 3). Treatment with antioxidants

The effects of daily t r e a t m e n t with vitamin E, ellagic acid or epigallocatechin gallate o n PCPinduced oxidative D N A d a m a g e were first examined after repeated a d m i n i s t r a t i o n of P C P (Fig. 3). T r e a t m e n t with vitamin E resulted in significant protection against 8 - O H - d G production, n o t completely to the extent of control (0.43 +__0.13 8-OH-dG/105 d G ) but p r o d u c t i o n being 75 % inhibited (0.63 + 0.01 8 - O H - d G / 1 0 5 d G ) c o m p a r e d with the effect of P C P alone (1.21 ___0.44 8-OH-dG/105 dG). T r e a t m e n t with ellagic acid or epigallocatechin gailate showed more t h a n 50% inhibition (1.49 ___0.20

a n d 1.23 + 0.17 8-OH-dG/105 d G , respectively), a l t h o u g h this was n o t statistically significant. Effects o f daily a d m i n i s t r a t i o n o f fl-carotene, vitam i n C or diallyl sulfide are s h o w n in Fig. 4. flC a r o t e n e - t r e a t m e n t did n o t exert any protective effect (1.63 + 0.41 8 - O H - d G / 1 0 5 d G ) on PCPinduced 8 - O H - d G f o r m a t i o n ( 1 . 4 9 + 0 . 2 0 8-OHdG/105 dG), being more t h a n two-fold higher t h a n the control (0.72 + 0.26 8-OH-dG/105 dG). With vitam i n C treatment, increase o f 8 - O H - d G levels was partially inhibited by a b o u t 30% (1.23 + 0.17 8-OHdG/105 dG), but this was not statistically significant. T r e a t m e n t with diallyl sulfide d e m o n s t r a t e d a significant inhibitory effect on PCP-induced 8 - O H - d G f o r m a t i o n o f more t h a n 60% (1.01 + 0.20 8-OH-dG/105 dG).

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Fig. 3. Effects of vitamin E (VE), ellagic acid (EA) and epigallocatechin gallate (EGCG) on 8-OH-dG levels in the mouse liver after daily oral administration of PCP for 5 days. VE, EA or EGCG (100 mg/kg/day) were daily given orally 3 hr before PCP (60 mg/kg/day) administration to mice and 8-OH-dG levels in the liver were analysed 6 hr after the last PCP treatment. Values are means ± SD of data for five mice. Asterisks indicate significant differences from control (*P < 0.05; **P < 0.01; Student's t-test). Dagger indicates significant difference from PCP-treated group ( t P < 0.05; Student's t-test).

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Fig. 4. Effects of /~-carotene (tiC), vitamin C (VC) and diallyl sulfide (DAS) on 8-OH-dG levels in the mouse liver after daily oral administration of PCP for 5 days. /~C or DAS (100 mg/kg/day) or VC (300 mg/kg/day) were daily given orally 3 hr before PCP (60 mg/kg/day) administration to mice and 8-OH-dG levels in the liver were analysed 6 hr after the last PCP treatment. Values are means _ SD of data for five mice. Asterisks indicate significant differences from control (**P < 0.01; Student's t-test). Daggers indicate significant difference from PCP-treated group (~'?P <0.01; Student's t-test).

Oxidative DNA damage by pentachlorophenol Discussion

This study demonstrated that PCP induces oxidative D N A damage in mouse target organ D N A after a single or repeated oral administration. Moreover, 8-OH-dG formation was specifically detected in the liver and not in the kidney or spleen. Since the presence of a repair enzyme for 8-OH-dG in mammalian cells has been demonstrated (Yamamoto et al., 1992), the finding that elevation of 8-OH-dG level was not observed at 24 hr after a single dose administration suggests that repair of this oxidative DNA damage had occurred by that time point. However, repeated administration of PCP caused higher levels of 8-OH-dG than single doses, more obviously at low doses (30 or 60 mg/kg/day). This implies that long-term exposure of PCP may induce gradual accumulation of oxidative D N A damage in the liver by overwhelming the repair potential, and that this cumulative oxidative D N A damage could cause critical mutations leading to carcinogenesis. There is evidence of generation of active oxygen species (Witte et al., 1985) or a semiquinone radical from tetrachlorohydroquinone (Naito et al., 1994), and 8-OH-dG formation caused by tetrachlorohydroquinone was demonstrated in studies in vitro (Naito et al., 1994) and in vivo (Dahlhaus et al., 1994). These findings suggest that active oxygen species generated by metabolic processes may cause PCP-induced oxidative D N A damage. Although enhancement of hepatotoxicity by buthionine sulfoximine treatment has been reported (Mizutani et al., 1994), we did not observe any apparent effects of this inhibitor in the present study. This result suggests that oxidative D N A damage by PCP is not influenced by the cytoplasmic GSH pool, which is supported by the finding of no change in liver GSH levels vAth PCP treatment (Table 2). Another possibility is that a compensating response of other antioxidative factors, such as elevation of ascorbic acid content (Martensson and Meister, 1992), could act against buthionine sulfoximinetreatment. Regarding the negative effects of aminotriazole in our study, it can be concluded that hydrogen peroxide production from superoxide anions produced during redox cycling of PCP metabolites, at least in the cytoplasm, is not of direct relevance. However, a possible role for hydrogen peroxide inside nuclei in PCP-induced oxidative damage could not be excluded in the present study. The fact that several antioxidants, such as vitamin E and diallyl sulfide, effectively reduced 8-OH-dG formation by PCP clearly indicates some involvement of active oxygen sp,~cies. This finding also suggests that such antioxidants may inhibit PCP-induced carcinogenesis, in line with the report of inhibitory effects of epigallocal:echin gallate on both 8-OH-dG formation and tumorigenesis (Xu et al., 1992). Finding of no effect of fl-carotene implies that this

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antioxidant could not interact with PCP-induced active oxygen species and/or its derived products in this experimental condition. It seems that application of this antioxidant alone could not be effective in some cases such as 2-nitropropane-induced oxidative D N A damage (Takagi et aL, 1995). However, since synergistic effects of mixtures of antioxidants has been reported (Shklar et aL, 1993), combination with the other antioxidants might be very effective at blocking PCP-induced oxidative D N A damage. Recent epidemiological data indicate chemopreventive effects on cancer of supplementation with vitamins (Blot et al., 1993). This suggests that supplementation of the diet with several antioxidants might be helpful for prevention of carcinogenesis induced by environmental chemicals such as PCP. In conclusion, this study has shown that PCP causes oxidative D N A damage in the target organ liver, which can be blocked by antioxidants. This result suggests that oxidative D N A damage may be involved in PCP-induced carcinogenic mechanisms and also that naturally occurring antioxidants may have a protective effect against such carcinogenesis. Further research is in progress to investigate the relationship between levels of PCP-induced oxidative DNA damage and generation of tumours as well as the effects of antioxidants on both phenomena in a long-term feeding study. Acknowledgement--This work was supported by a Grant-

in-Aid for cancer research from the Ministry of Health and Welfare.

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