Benzo[a]pyrene metabolism and induction of enzymealtered foci in regenerating rat liver

Chem.-BioL Interactions, 67 (1988) 243--253 Elsevier Scientific Publishers Ireland Ltd.

243

BENZO{a]PYRENE METABOLISM AND INDUCTION OF ENZYMEALTERED FOCI IN REGENERATING RAT LIVER

LENNART DOCK', GUNILLA SCHEU', BENGT JERNSTROM b, MARGARETA MARTINEZ b, ULLA-BRITTA TORNDAL c and LENNART ERIKSSON c •National Institute of Environmental Medicine, Box 60 208, S-10~ 01 Stockholm, bDepartment of Toxicology, Karolinska Institute~ Box 60 ~00, S-10~ 01 Stockholm and Department of Pathology, Huddinge University Hospita~ S-1~1 86 Huddinge (Swedenj (Received December 23rd, 1987) (Revision received February 15th, 1988) (Accepted April 19th, 1988)

SUMMARY

The metabolism of benzo[a]pyrene (BP) in regenerating rat liver and the induction of enzyme-altered loci (EAF) in the liver of partially hepatectomized rats, treated with BP and promoted with 2-acetylaminofluorene (2-AAF)/ CCI4 was investigated. The aim was to examine factors that might be of importance for the tumorigenicity of BP in the regenerating rat liver, such as cytochrome P-450 activity and glutathione levels. In regenerating rat liver, obtained 18 h after partial hepatectomy (PH), the amount of microsomal cytochrome P-450 was reduced by 200/0 whereas the level of glutathione was elevated by 15% and the cytosolic glutathione transferase activity towards chlorodinitrobenzene and (_+)-7~,8a~iihydroxy-9a,10a~poxy-7,8,9,10tetrahydro-BP (BPDE) was unaffected. Microsomes from these animals had a reduced capacity to activate (-)-trans-7,8-dihydroxy-7,8-dihydro-BP (BPD) to DNA-binding products but the pattern of BP metabolites was similar to that observed with control rat liver microsomes. Treatment of rats with 3-methylcholanthrene (MC, 50 mg/kg body wt.) increased cytochrome P-450 levels and glutathione transferase activity towards both substrates. Regenerating livers from these animals retained their cytochrome P-450 level and enzymatic activity towards BP and BPD. Regenerating rat liver microsomes from MCtreated animals were about 35 times more efficient in activating BPD than microsomes from uninduced, partially hepatectomized animals. Intraperitoheal administration of BP (50 mg/kg body wt.) 18 h after PH induced EAF in rats subsequently promoted with 2-AAF/CC14. Pretreatment of rats with MC Abbreviations: 2-AAF, 2-acetylaminofluorene; BNF, fl-naphtoflavone; BP, benzo[a]pyrene; BPD, ( - )-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene; BPDE, ( ± )-7~,Sa-dihydroxy-9a,10a-epoxy7,8,9,10-tetrahydrobenzo[a]pyrene; EAF, enzyme-altered loci; GSH, gldtathione; MC, 3-methyleholanthrene; PAH, polycyelie aromatic hydrocarbons; PH, partial hepatectomy. 0009-2797/88/$03.50 © 1988 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland

244 66 h before PH and 84 h before BP administration, increased the number of EAF. In accordance with results by Tsuda et al. (Cancer Res., 40 (1980) 1157 -1164), these studies demonstrate that BP is tumorigenic in regenerating rat liver, despite a reduced ability of the liver to activate this compound. Furthermore, MC, an inducer of certain cytochrome P-450 species ("aryl hydrocarbon hydroxylase"), potentiates the effect of BP.

Key words: Enzyme-altered foci -- Benzo[a]pyrene metabolism -- Regenerating liver INTRODUCTION

Polycyclic aromatic hydrocarbons (PAH) such as BP are mutagenic and carcinogenic in various experimental test systems and are also suspected to contribute to human cancer incidence [1-4]. PAH require metabolic activation to excert their toxic effects [2-5]. In the case of BP, formation of (+)7~,8adiihydroxy-9a,10a-epoxy- 7,8,9,10-tetrahydro-BP through oxidation of BPD and subsequent covalent binding of BPDE to DNA are considered essential events for tumor initiation [1-9]. Several metabolites of BP have been identified and the pattern of metabolites formed may be of importance for the biological effects of BP [1-3,10]. PAH are generally considered non-carcinogenic to the liver but may initiate the formation of hepatoceUular carcinomas in regenerating liver tissue [11]. An early sign of hepatic carcinogenesis is the occurrence of foci of altered hepatocytes. Induction of liver loci or nodules has been used as an indication of carcinogenicity using a multitude of different initiators, including BP and other PAH, and one of several promotion protocols [ 1 1 16]. Since regenerating rat liver is a target organ for initiation of BP-carcinogenesis whereas non-proliferating liver is not, we considered it to be of interest to compare the metabolism of BP under these conditions. The present paper is a summary of some of our findings. MATERIALS AND METHODS

Chemicals Unlabelled BP, calf thymus DNA, glutathione (GSH) chlorodinitrobenzene (CDNB) and dimethylsulfoxide (DMSO, grade I) were purchased from Sigma Chemical Co., St. Louis, MO; MC from Eastman Kodak Co., Rochester, NY; 2AAF from Fluka, Buchs, Switzerland; NADPH from Boehringer Mannheim GmbH, F.R.G. and InstaGel scintillation cocktail from United Technologies Packard, Bandhagen, Sweden. The 14C-labelled BP (25 mCi/mmol) was obtained from the Radiochemical Center, Amersham, Bucks., U.K. and diluted with unlabelled BP to 5.4 mCi/mmol. The BP was purified through chromatography on an A12Os-column eluted with toluene and further ana-

245

lyzed by HPLC. The purity was found to be greater than 98%. Racemic BPDE, unlabelled and tritium labelled BPD, as well as selected BP metabolites (used as HPLC reference standards) were obtained through the Cancer Research Program of the NCI, Division of Cancer Cause and Prevention, Bethesda, MD. The purity of BPD and BPDE (>95%) was determined by HPLC, the latter assayed as the ~-mercaptoethanol-conjugate [17]. BPD was stored dissolved in DMSO, BPDE in tetrahydrofuran/triethyl-amin (19 : 1). The specific activity of the BPD solution used in the experiments was 90 mCi/mmol. HPLC grade solvents were obtained from FSA Laboratory Supplies, Loughborough, U.K. All other chemicals and solvents were of analytical grade.

Treatment of animals Male Wistar rats (100-120 g) were purchased from M~bllegaards Breeding Centre, Ejby, Denmark and maintained on a basal diet (type R3, Ewos, SSdert~ilje, Sweden) for a week of acclimatization prior to the experiments. Liver regeneration was initiated by 2/3 PH [18] and microsomes and cytosols prepared 18, 48 and 96 h later [19]. Each group contained four animals and untreated animals served as controls. The cytosol preparations were treated with iodoacetamide and dialyzed to remove GSH [20]. Protein concentration was determined according to Lowry et al. [21]. Microsomes and cytosols were stored and used within three months of preparation. The formation of EAF was initiated with BP, injected intraperitoneally as a solution in corn oil (5 mg/ml, 80 mg/kg body wt.) 18 h after PH. Some animals were additionally treated with MC, dissolved in corn oil (5 mg/ml, 50 mg/kg body wt.), injected i.p. 66 h before PH. Following BP administration, the rats were maintained for 2 weeks on the basal diet, then for 2 weeks on this diet supplemented with 0.02% 2-AAF and a single i.g. dose of CC14 (2 ml/kg body wt.) was administered after 1 week of 2-AAFfeeding. The rats were killed after two additional weeks on basal diet. The experimental protocol used is concordant to that recommended by Solt and Farber [11] and modified by Tsuda et al. [12]. In all experiments the rats were starved 48 h before harvest of liver tissue. The three remaining liver lobes were cut into 2--3 mm thick slices. Two sections from each lobe were fixed in ice cold, fresh acetone, another two sections were fixed in neutral buffered 50/0 formaldehyde. After fixation the tissue was dehydrated in ethanol. The acetone-fixed material was embedded in low melting point ( 4 2 44°C) paraffin [22] and stained for y-glutamyl transferase [23]. The formalinfixed material was embedded in paraffin used for routine histopathology preparations (m.p., 5 6 ° - 5 8 ° C ) a n d stained with hematoxyline-eosine. ),-Glutamyl transferase-positive liver lesions were identified microscopically and quantitated using morphometric point counting [24] on photographic magnifications of the stained sections. Number of focal lesions/cm 8, focus volume as per cent of liver volume and mean focus diameter were calculated as suggested by Wiebel et al. [24].

246

Incubations and assays The hepatic concentrations of GSH and glutathione disulfide were determined by analyzing aliquots of the liver homogenates with a HPLC method modifed from Reed et al. [25]. With the use of a Waters Z-module equipped with a Radial-Pak NH2-column, the solvent flow rate could be increased to 5 ml/min and analysis time accordingly reduced. The solvents used were 800/0 (v/v) methanol in water (solvent A) and 200 ml of an ammonium acetate solution (154 g ammonium acetate, 122 ml H20 and 378 ml acetic acid) plus 800 ml of 800/0 methanol (solvent B). The gradient system was 1 0 200/0 B, linear gradient for 5 rain; 20-95°/0 B, linear gradient for 10 rain; 950/0 B, isocratic elution for 2 rain and finally 9 5 - 1 0 % B, linear gradient for 2 rain. The level of cytochrome P-450 was determined spectrophotometrically according to Omura and Sato [26] using freshly prepared microsomes. Metabolism of [14C]BP was determined by HPLC separation and liquid scintillation counting of ethyl acetate-extractable products as previously described [27]. Unlabelled derivatives of BP were co-injected as reference standards. The microsomal incubation system contained 1 mg microsomal protein, 100 ~mol Tris--HC1 buffer (pH 7.5), 2 ~mol NADPH and 160 nmol [14C]BP (added in 20 ~1 DMSO) in a final volume of 2 ml. The reaction was terminated after 10 rain of incubation at 37 °C by addition of 2 ml aceton, proteins were removed by centrifugation and the supernatants extracted twice with 4 ml ethyl acetate, containing butylated hydroxytoluene (0.12 mg/ml) to prevent oxidation of BP phenols. GSH-conjugation of BPDE, catalyzed by cytosolic GSH-transferases, was determined by the HPLC method of Robertson et al. [28]. The incubation system contained 50 ~g cytosolic protein, 12.5 ~g T r i s - H C l (pH 7.5), 0.625 ~mol KC1, 0.125/~mol EDTA, 250 pmol GSH and 20 ~mol BPDE (added in 10 /A DMSO) in a final volume of 250/A. After 1 rain of incubation at 37 °C the reaction was terminated by the addition of 250 }A aceton. The conjugates present in the aqueous phase following ethyl acetate extraction were analyzed by HPLC. The results were correlated for the spontaneous conjugation of BPDE with GSH (incubations without cytosol). GSH-transferase activity towards CDNB was measured as described by Habig and Jakoby [29]. The metabolic activation of BPD to DNA-binding products was measured by incubating 20 ~M [3H]BPD with calf thymus DNA (0.5 mg/ml) in the presence of microsomes (1 mg protein/ml) and NADPH (1 raM) for 30 rain at 37°C. Following purification of the DNA, the amount of metabolites bound were determined by scintillation counting and expressed as pmol bound/rag DNA [30]. Statistical evaluation of data Statistical significance between sets of data was determined by the T- or GT2-method, multiple comparison methods based on analysis of variance [31]. Differences were tested at the P = 0.05 level of significance against tabulated values.

247 TABLE I H E P A T I C L E V E L S O F GSH, C Y T O C H R O M E P-450 A N D C Y T O S O L I C T R A N S F E R A S E (GST) A C T I V I T Y T O W A R D S B P D E A N D C D N B

GLUTATHIONE

Treatment

Liver weight (% body wt.)

GSH (/~mol/g liver)

Cyt. P-450 (nmol/mg microsomal prot.)

GST, BPDE GST, CDNB (nmol/min (~ol/min ×mg ×mg cytosolic prot.) cytosolic protJ

Control PH" MC MC + PH

5.23 2,38 5.87 2,64

6.52 7.50 5.39 8.43

0.64 0.52 1.08 1.00

9.5 9.4 13.4 11.9

± ± ± ±

0.39 b 0.06 0.41 0.38

± ± ± ±

0.61 0.72 0.68 0.48

± ± ± ±

0.06 0.06 0.19 0.23

± ± ± -+

0.5 0.9 1.0 0.9

1.41 1.38 2.68 1.97

± _ ± ±

0.19 0.12 0.24 0.15

• Livers excised 18 h aRer PH. b All values represents means ± S.D. using livers or liver preparations from five different anirealsin each group. RESULTS

Characterization of livers The liver mass was reduced by 2/3 by removing the left and median lobes of the liver. After a recovery period of 18 h, the relative liver weight was about 2.5% of the body weight (Table I) and this increased to 3.6% after 96 h (Fig. 1). The relative liver weight in untreated animals was 5.20/0 of the body weight, slightly more in MC-treated animals. The hepatic concentration

150-

o

1,,,._ -l-a

100-

t.o o

50"

O,

I

I

I

0

48

96

Hours after PH Fig. 1. Relative liver weights (Q) and hepatic levels of GSH ( e ) and microsomal cytochrome P450 (O) in livers from partially hepatectomized rats. Control values were 5.23 ± 0.39% of body wt., 6.52 ± 0.61 i~mol/g liver and 0.64 ~- 0.06 nmol/mg microsomal protein, respectively (mean values ± S~D., n = 5).

248 of GSH was increased (although not significantly by our criteria) during liver regeneration (Table I) but came back towards control levels 96 h after PH (Fig. 1). The level of glutathione disulfide was consistently less than 5% of the GSH level (data not shown). The levels of cytochrome P-450 was reduced in the regenerating liver of control rats (Table I) and progressively decreased during the 96-h study period (Fig. 1). While an expected increase in cytochrome levels was observed in MCtreated rats, partial hepatectomy of these animals did not reduce the levels. This discrepancy between control and MC-treated rats after partial hepatectomy may be due to the differential reduction of various P-450 isoenzymes during regeneration as observed by Iversen et al. [32]. The cytosolic glutathione transferase activity towards both BPDE and CDNB was induced by MC-treatment (Table I). Partial hepatectomy of these animals led to a decreased activity of glutathione transferase with CDNB as the substrate. However, progressive liver regeneration significantly decreased this activity also in uninduced animals (Fig. 2). B P metabolism

The microsomal metabolism of BP to dihydrodiols was reduced by 50% in the regenerating liver (Table II) and this value remained fairly constant throughout the 96 h time period at study (data not shown). Consistent with the decreased rate of BP metabolism was the decrease in microsome-catalyzed activation of BPD to DNA-binding products (Table II). MC-treatment stimulated both dihydrodiol formation and BPD activation and the absence of cytochrome po450 reduction in microsomes from regenerating livers of MC-treated animals was consistent with the lack of effect both

150-

E

IO0-

oo

0

50-

O.

I

0

I

4'8

96

Hours after PH Fig. 2. Liver cytosolic GSH-transferase activity towards BPDE ( e ) and CDNB (O) in partially hepatectomized rats. Control values were 9.5 ± 0.5 nmol/min × mg protein and 1.41 ± 0.19 ~mollmin × mg protein, respectively (means ± S.D., n = 5).

249 T A B L E II BENZO(A)PYRENE DIHYDRODIOL FORMATION BY R A T LIVER M I C R O S O M E S

AND

BPD ACTIVATION CATALYZED

Treatment

Dihydrodiol f o r m a t i o n d (pmol/min x m g m i c r o s o m a l prot.)

BPD activation (pmol bound/rag DNA)

Control PH" MC MC + P H

170 78 322 270

60 36 1461 1355

± ± ± ±

38 b 12 70 78

± 4¢ ± 15 _+ 477 _+ 180

a L i v e r s excised 18 h a f t e r PH. b Values r e p r e s e n t m e a n s ± S.D u s i n g liver m i c r o s o m a l p r e p a r a t i o n s from t h r e e different anim a l s in each group. c M e a n s ± S.D. u s i n g liver m i c r o s o m a l p r e p a r a t i o n s from five d i f f e r e n t a n i m a l s in each group. d S u m of BP-7,8-, 4,5- a n d 9,10-dihydrodiols [2,3,27].

on dihydrodiol formation and BPD activation (Table II). No qualitative differences between control and regenerating rat liver in the formation of the various BP metabolites was observed within the treatment groups (uninduced versus MC, Table III). Microsomes from MC-treated animals metabolized BP preferentially to dihydrodiols and quinones while phenols were the main metabolites when microsomes from uninduced rats were used.

Induction of enzyme-altered foci Administration of BP by i.p. injection 18 h after PH, followed by 2-AAFfeeding and CCI 4 treatment was found to induce the formation of EAF (Table IV). Partial hepatectomy alone did not significantly initiate the formation of foci, nor did BP-treatment without partial hepatectomy. Since enzyme induction by MC increased the formation of dihydrodiols and the activation of BPD we investigated the effect of such treatment on the formation of altered foci. The administration of MC alone 66 h before PH did not significantly induce the formation of foci, although a slight increase in the mean values could be noted. MC-treatment in combination with PH and BP significantly increased the tumor-initiating activity of BP, the volume of the liver tissue occupied by E A F was however similar. T A B L E III BP METABOLITE TREATED RATS

PATTERN

USING

MICROSOMES

FROM

CONTROL,

PH-

OR MC-

Treatment

Dihydrodiols (% total m e t a b o l i t e s l

Quinones

Phenols

Control PH" MC MC + P H

16.6 14.7 45.4 33.4

18.1 18.9 30.5 38.6

61.0 63.0 18.7 20.6

± ± ± ±

2.5 b 3.2 6.0 3.8

± ± ± ±

1.6 3.4 6.4 9.1

__. ± _+ _+

4.1 0.4 2.9 10.2

• L i v e r s e x c i s e d 18 h a R e r P H . b M e a n s ± S.D. u s i n g liver m i c r o s o m a l p r e p a r a t i o n s from t h r e e d i f f e r e n t a n i m a l s in each group.

250 TABLE IV INDUCTION OF EAF IN REGENERATING RAT LIVER BY BP Treatment"

PH only BP only MC + PH PH + B P MC + PH + BP

No. of animals

Enzyme altered foci No./cm2

No./cm3

Mean focus area (mm2)

Volumedensity (%)

5 7 5 8 13

0.5 1.0 2.0 16.0 28.0

11 23 57 436 797

0.13 0.19 0.10 0.31 0.08

0.2 0.3 0.6 3.3 2.4

± ± ± ± ±

0.7~ 1 3 4 14

_+ 15 _+ 22 ± 69 ± 131 ± 392

± 0.07 ± 0.17 _+ 0.13 _ 0.28 ± 0.04

± 0.2 ± 0.4 ± 1.3 -+ 2.2 _+ 1.8

• See Materials and Methods for details. b Means ± S.D. using the indicated number of animals.

DISCUSSION Most chemical c a r c i n o g e n s show a c h a r a c t e r i s t i c t a r g e t o r g a n specificity. F o r instance, P A H are g e n e r a l l y not carcinogenic in r a t liver but m a y cause t u m o r in this o r g a n d u r i n g liver proliferation. This occurs in neonatal animals but can also be achieved in adult animals b y partial h e p a t e c t o m y [11-13]. Cell proliferation s e e m s to be one p r e r e q u i s i t e for cancer d e v e l o p m e n t b y chemicals, a n o t h e r one is induction of m u t a t i o n s t h r o u g h covalent binding of r e a c t i v e i n t e r m e d i a t e s to crucial t a r g e t g r o u p s in D N A [33]. BP is metabolized to a v a r i e t y of p r o d u c t s but the ones of particular i m p o r t a n c e for initiation of carcinogenesis are vicinal or bay-region diol epoxides [ 2 - 5 ] . In view of the susceptibility of r e g e n e r a t i n g r a t liver to BP-carcinogenesis we decided to i n v e s t i g a t e the m e t a b o l i s m of BP in this organ. A l t h o u g h we o b s e r v e d a 50% reduction in the m e t a b o l i s m of BP in r e g e n e r a t i n g r a t liver c o m p a r e d to non-proliferating tissue, the p a t t e r n of m e t a b o l i t e s r e m a i n e d the same. Thus, the susceptibility of r e g e n e r a t i n g r a t liver to BP-carcinogenesis, as opposed to normal r a t liver, is p r o b a b l y not related to qualitative differences in the activation of this carcinogen. Since the biological effects of toxic a g e n t s is d e t e r m i n e d t h r o u g h the balance b e t w e e n a c t i v a t i n g an d e t o x i f y i n g p a t h w a y s , we a t t e m p t e d to modulate the metabolism of BP in the r e g e n e r a t i n g liver in o r d e r to i n v e s t i g a t e w h a t results this would have on t h e induction of E A F . It is k n o w n t h a t the metabolism of P A H is increased following enzyme induction [4]. Consistent with

the increased activation of B P D to DNA-binding diol epoxides observed in the microsomal incubation system, pretreatment of rats with M C prior to P H and B P administration increased the formation of E A F in regenerating rat liver. However, this effect was only moderate compared to the 20-fold increase in microsomal activity towards BPD, which could be explained by the very efficientdetoxificationof B P D E by GSH-conjugation in the liver [3436], an activity that was also induced by MC-treatment. The lack of effect of partial hepatectomy on microsomal B P metabolism and B P D activation in

251 MC-pretreated rats is in agreement with a recent study by Raza and Levine [37] who used N,N-dimethyl-4-amino-azobenzene (DAB) as the substrate and /3-naphthoflavone (BNF) as the inducing agent (BNF has been shown to induce the same form(s) of cytochrome P-450 as MC [38]). In this study the metabolism of DAB was reduced in microsomes obtained from the regenerating liver of control animals but microsomes from BNF-pretreated and partially hepatectomized rats retained the increased metabolism of DAB observed by BNF-treatment alone. The authors conclude that the BNFinduced form of cytochrome P-450 is more stable during regeneration than the constitutive forms. Although normal liver produced the same reactive metabolites as regenerating liver did, and at a faster rate, BP did not initiate tumorigenesis in non-proliferating liver. The necessity of liver regeneration for initiation was also observed in a recent study by Kaufmann et al. [39] where racemic BPDE was used as the initiating agent. Cell division in association with BP administration thus seems to be a necessary prerequisite for the appearance of liver loci and nodules. In conclusion, our data show that there is a non-specific decrease in BP metabolism in regenerating rat liver as compared to non-proliferating tissue. The rate of BP-metabolism is thus probably of less importance for the susceptibility of the liver to tumor initiation by BP than the proliferative status of the tissue [40]. The higher incidence of E A F in the livers of MC-pretreated rats, highly efficient in activating BPD to BPDE but with only a moderate (=25°/o) increase in the capacity to detoxify BPDE through GSH-conjugation, indicate that the balance between activation and detoxification of BP is of importance for tumor formation. ACKNOWLEDGEMENTS

This investigation was supported by the Swedish Cancer Society and the Swedish Medical Research Council. The valuable advice given by Dr Lars W~rngard during the statistical evaluation of the data is gratefully acknowledged. REFERENCES 1

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