Benzo[a]pyrene-DNA-adducts and monooxygenase activities in mice treated with benzo[a]pyrene, cigarette smoke or cigarette smoke condensate

Chem.-BioL Interactions, 70 (1989) 51-61 Elsevier Scientific Publishers Ireland Ltd.

51

BENZO[a]PYRENE-DNA-ADDUCTS AND MONOOXYGENASE ACTIVITIES IN MICE TREATED WITH BENZO[a]PYRENE, CIGARETTE SMOKE OR CIGARETTE SMOKE CONDENSATE*

NINA BJELOGRLIC, MUMTAZ ISCAN, HANNU RAUNIO, OLAVI PELKONEN and KIRSI VAH,~KANGAS Department of Pharmacology and Toxicology, University of Oulu~ SF-90~20 Oulu fFinland) (Received March 14th, 1988) (Revision received September 30th, 1988) (Accepted October 3rd, 1988)

SUMMARY S y n c h r o n o u s fluorescence s p e c t r o p h o t o m e t r y (SFS), developed to s t u d y benzo[a]pyrene-7,8-diol-9,10-epoxide(BPDE)-DNA, was used to m e a s u r e the in vivo formation of DNA-adducts in genetically r e s p o n s i v e C57BL/6 (B6) and non-responsive DBA/2 (D2) mice. T r e a t m e n t with c i g a r e t t e smoke by inhalation for 3 - - 1 6 days, or i.p. injection of c i g a r e t t e smoke c o n d e n s a t e or neutral fraction did not lead to d e t e c t a b l e levels of BPDE-DNA-adducts in e i t h e r lungs or liver, although aryl h y d r o c a r b o n h y d r o x y l a s e (AHH) activity, an indicator of benzo[a]pyrene (BP) metabolism, was clearly induced in lungs of B6 mouse. A dose-dependent a m o u n t of BPDE-DNA-adducts in lung and s o m e w h a t less in liver was found a f t e r i.p. injection with BP ( 2 0 - 8 0 mg/kg). Mice t r e a t e d with vehicle or 4 mg/kg of BP w e r e negative for adducts by SFS. In B6 mice A H H was induced both in lungs and livers while t h e r e was no A H H induction in D2 mice although the levels of BPDE-DNA-adducts w e r e s o m e w h a t h i g h e r t h a n in B6 mice. Thus, no clear correlation seems to exist b e t w e e n A H H activity and the formation of BPDE-DNA-adducts. Also, according to our r e s u l t s SFS can be used to q u a n t i t a t e adduct-formation in in vivo animal studies. K e y words: Benzo[a]pyrene-DNA-adducts -- S y n c h r o n o u s fluorescence spect r o p h o t o m e t r y -- A r y l h y d r o c a r b o n h y d r o x y l a s e -- M o n o o x y g e n a s e -C i g a r e t t e smoke -- Mouse

*A preliminary report of this study was presented at the International Symposium on Inhalation Toxicology, Hannover, F.R.G., March, 1987. Abbreviations: AHH, aryl hydrocarbon bydroxylase; BP, benzo[a]pyrene; BPDE, BP-7,8-diol-9,10epoxide; CS, cigarette smoke; CSC, cigarette smoke condensate; ECDE, 7-ethoxycoumarin O-deethylase; ERDE, 7-ethoxyresorufin O-deethylase; N.CSC, neutral fraction of CSC; PAHs, polycyclic aromatic hydrocarbons; SFS, synchronous fluorescence spectrophotometry. 0009-2797/89/$03.50 © 1989 Elsevier Scientific Publishers Ireland Ltd.

52 INTRODUCTION

Benzo[a]pyrene (BP) is a well-known animal carcinogen [1], the metabolism of which is traditionally monitored by measuring cytochrome P-450-catalyzed aryl hydrocarbon hydroxylase (AHH) activity [2]. While AHH measures the formation of hydroxylated metabolites, the activation of BP to putative ultimate carcinogen goes through BP-7,8-dihydrodiol to BP-7,8-diol-9,10-epoxide (BPDE) [3,4]. In animal studies, quantitatively the most important adduct and the one with the best correlation with carcinogenesis has been an (+ }antiisomer of BPDE covalently bound to the exocyclic nitrogen of guanine in DNA [3]. Cigarette smoking is the main source of BP in smokers who are not occupationally exposed to polycyclic aromatic hydrocarbons (PAHs) [5,6]. In some occupations the exposure to BP and other PAHs can be high, and putative BP-DNA-adducts have been detected in DNA from peripheral blood lymphocytes of such workers [7--10]. However, it is an open question whether and to what extent BP in cigarette smoke (CS) leads to the formation of BPDE-DNA-adducts, although BP-DNA-adducts found in placentas of some smokers indicate it is possible [11; K. Viih~ikangas, O. Pelkonen et al., unpublished results]. Methods currently used for DNA-adducts are immunoassay, 32p-postlabelling and synchronous fluorescence spectrophotometry (SFS). As new methods these need further validation before large-scale human studies. Here we used SFS to measure in vivo formed I}PDE-DNA-adducts in two inbred strains of mice, which are well-known for their differences in AHH inducibility [12]. This is the first study to show dose-response in an in vivo animal study for BPDE-DNA by SFS. MATERIALS AND METHODS

Chemicals BP, 7-hydroxycoumarin, coumarin and ribonuclease A (from bovine pancreas) were obtained from Sigma Chemical Co. (St. Louis, MO 63178 U.S.A.). 7-Ethoxyresorufin was obtained from Pierce Chemical Co. (Rockford, IL, U.S.A.) and resorufin from Eastman Kodak Co. (Rochester, NY 14650, U.S.A.). Phenol (GT, nucleic acid grade) was purchased from Orion Corporation Ltd. Fermion (Box 28, SF-02101 Espoo 10, Finland), proteinase (pronase E) and other chemicals (analytical grade) from Merck (E. Merck, Darmstadt, F.R.G.). The cigarettes used were commercial non-filter cigarettes containing 18 mg tar, 1.1 mg nicotine and 10 mg carbon monoxide per cigarette according to the manufacturer. Cigarette smoke condensate (CSC) and neutral fraction of CSC (N.CSC) as well as the chemically modified BPDE-DNA were generously provided by Dr. Curtis C. Harris, Laboratory of Human Carcinogenesis, National Cancer Institute, National Institutes of Health, U.S.A.

53

Mice The animals were 3--~month-old male inbred mice (25--30 g) of the strains C57BL/6 (B6, homozygous responsive, AhbAh b) and DBA/2 (D2, homozygous, non-responsive AhdAh d) originally obtained from the National Institutes of Health (Bethesda, MD, U.S.A.) through Dr. Daniel W. Nebert. Mice were caged in plastic cages (6 animals/cage) with softwood chips as bedding material, fed standard laboratory chow from Ewos-Alab (Box 618, 15127 SSdertiilje, Sweden) and given tap water ad libitum. The light/dark cycle was 12 h : 12 h (change at 06.30 h and 18.30 h). Room temperature was 22 _+ 1 °C and humidity 60--850/0.

Treatment of mice Minimum of three mice per dose were injected i.p. with BP (4--80 mg/kg, dissolved in DMSO and diluted with corn oil). Another set of B6 and D2 mice were exposed i.p. to CSC ( 1 2 - 6 0 mg/kg in DMSO/corn oil, 1 : 1) or N.CSC {10-90 mg/kg in DMSO/corn oil, 1 : 1). Controls were injected with the solvent. B6 mice were treated with CS in a way shown earlier to induce AHH activity, fetotoxicity and ovotoxicity [13,14]. Four to eight mice were placed into a chamber (14.2 l) into which the smoke from one cigarette at a time was blown by air stream: 30 puffs in 15 min from one cigarette, 15 min only fresh air (2 l/min) and cycle repeated for 1-h treatment. Groups of at least four mice were treated either for 3 days (0.5 h at a time, 7 times a day), or 5 or 16 days (1 h at a time, 3 times a day). Control mice were not treated. Mice were killed under ether anesthesia by cervical dislocation 24 h after the last treatment and tissues were removed. DNA isolation from lungs and all A H H assays were done fresh. Some liver tissue was stored at - 4 0 °C for DNA isolation.

DNA isolation DNA was isolated from lungs and livers by phenol extraction method as described earlier [9] with minor modifications: DNA was precipitated without LiCI, and the crude lung homogenate was not extracted with chloroform/ isoamyl alcohol before proteinase incubation.

Synchronous fluorescence spectrophotometry (SFS} DNA samples (100 ~g DNA/1 ml Tris-EDTA buffer) were hydrolysed in 0.1 M HC1 for 3 h at + 90°C, and measured without further purification by SFS [9]. Hydrolysis products of BPDE (BP-7,8,9,10-tetrols and BP-7,8,9-triols) give a peak in SFS at 340 nm of excitation when excitation and emission wavelengths are scanned simultaneously with a fixed wavelength difference of 34 nm (Fig. 1) [15]. With different BP-metabolites, eg. 3-OH-BP or 9-OH-BP, this peak appears only in DNA incubated with BP-7,8-diol, metabolized to BPDE (K. Viihiikangas, unpublished results). The sensitivity (0.3 fmol/~g DNA or 1 adduct in 10 7) and the standard curve with in vitro modified

54

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~ , , . . ~

Z

LUNG DNA(BP40MG/KGIP)

8 ._I Q

d 320

340

360

380

EXCI TAT ION WAVELENGIH (nm)

Fig. 1. BPDE-DNA-adducts measured by SFS using 34 nm wavelength difference between emission and excitation. 100 pg of lung DNA from control and BP-treated D2 mouse was hydrolyzed and measured as explained in Materials and Methods. A peak from DNA modified in vitro with BPDE is shown for comparison.

B P D E - D N A are practically identical by the machine used in this study (Hitachi F 3000) compared to Perkin-Elmer 650-40 used earlier [9].

Enzyme activities Enzyme activities were measured in tissue homogenates (in 0.1 M sodium/ potassium phosphate buffer, pH 7.4). A H H activity was measured by the fluorometric method of Nebert and Gelboin [2]. Frozen samples were thawed for ERDE (7-ethoxyresorufin O-deethylase) [16] and ECDE (7-ethoxycoumarin O-deethylase) [17] activities.

Statistical analysis The results are expressed as means _+ S.D. The significance of the differences between the groups was analysed with the Kruskal-Wallis test [18]. RESULTS

The only treatment leading to measurable amount of adducts was BP as such. With 20 mg/kg of BP, in lung DNA from two out of three B6 mice and one out of three D2 mice injected i.p. there were barely measurable bumps, while only one out of three D2 mouse livers and none of the B6 were positive. With higher doses all the lungs and livers were positive. There were more adducts in the lungs than livers with statistically significant (P < 0.05) difference in the highest dose-group of D2 mice (Fig. 2).

55

<

10

z O

0

m

05

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4

20

[1

,40

80 BP

(mg/kg)

Fig. 2. The amount of BPDE-DNA-adducts in lung and liver tissue after i.p. BP-treatment of B6 and D2 mice measured by SFS. Means ± S.D. of at least 3 mice for both strains and tissues is shown.

Although the level of BPDE-DNA-adducts was higher in the tissues of D2 than B6 mice, A H H activity in accordance with the literature [12] was induced only in B6 mice. Lung A H H was clearly induced already in lowest dose groups (Table I; Figs. 3 and 4) when no BPDE-DNA-adducts were detectable. The basal level of A H H in liver was 2 0 - 5 0 times higher compared to lung and was similar in B6 and D2 mice (Tables I and II). CS exposure for 3 days increased only slightly liver A H H activity in B6 mice, whereas there was about 3-fold induction after BP administration (Table I). However, liver A H H was not induced in either strains of mice treated with CSC or N.CSC (Fig. 4, data not shown for D2 mice). Because there have been reports of the induction of ERDE in the livers of smokers [19,20] we measured ERDE and ECDE activities in the livers of B6 mice exposed to CS. ERDE but not ECDE was clearly induced by CS exposure for 3 or 5 days (Table III). DISCUSSION

This is the first study where SFS for BPDE-DNA [9,15] has been applied to measure in vivo-formed adducts in animals. A dose-dependent formation of BPDE-DNA-adducts was found, and the results are comparable to the studies of Perera et al. using ELISA immunoassay [21]. CS-treatment of B6 mice

56 TABLE I A H H INDUCTION IN B6 MICE T R E A T E D WITH CS, BP, CSC OR N.CSC I Treatment

Lung A H H

Liver A H H

pmol/min/g tissue b Controls c Controls d CS (3 days) BP (40 mg/kg) CSC (60 mg/kg)" N.CSC (90 mg/kg)"

3.7 4.0 81.4 25.7 12.0 16.6

Fold induction

± 2.0 (13) ± 2.1 (7) ± 43.5*** (6) ± 17.5"** (7) ± 3.0** (3) ± 6.6*** (3)

22.0 6.4 3.0 4.2

pmol/min/g tissue b 296.1 207.1 376.7 702.5 209.4 166.5

Fold induction

± 102.7 ± 48.1 ± 30.8* ± 298.4*** ± 25.4 ± 52.2

(14) (8) (6) (8) (3) (3)

1.3 3.4 1.0 0.80

• Mice w e r e exposed to CS by inhalation and to BP, CSC or N.CSC by i.p. injection as described in Materials and Methods. b Mean ± S.D., *P < 0.05, **P < 0.01, ***P < 0.001 by Kruskal-Wallis test, compared to the A H H activity of controls (number of mice). c U n t r e a t e d controls, used for comparison with CS-treated mice. d DMSO/corn oil i.p. injected controls, used for comparison with BP-CSC- and N.CSC-treated groups. • Maximum dose used.

for several days did not lead to the formation of detectable amounts of BPDE-DNA-adducts in lungs or livers although lung A H H was clearly induced. Also, B6 and D2 mice injected i.p. with CSC or N.CSC, which contains all the PAHs of CS in a concentrated form [22], were negative for the adducts speaking for the specificity of the SFS-method used.

T A B L E II A H H INDUCTION IN D2 MICE T R E A T E D WITH BP, CSC OR N.CSC" Treatment

Lung AHH

Liver A H H

pmol/min/g tissue b Controls c BP (40 mg/kg) CSC (60 mg/kg) d N.CSC (90 mg/kg) d

6.0 5.5 15.0 10.9

± 5.0 ± 2.0 ± 2.5 ± 13.2

(7) (4) (3) (4)

Fold induction

pmol/min/g tissue b

0.92 2.5 1.8

127.5 108.3 266.8 202.0

± 57.9 ± 90.1 ± 70.5 ± 160.7

Fold induction (7) (5) (3) (4)

0.85 2.1 1.6

• BP, CSC or N.CSC w e r e a d m i n i s t e r e d by i.p. injection as described in Materials and Methods. No statistically significant differences w e r e found. b Mean ± S.D. (number of mice). c Solvent controls. d Maximum dose used.

57 TABLE III ERDE AND ECDE ACTIVITIES IN LIVERS OF B6 MICE EXPOSED TO CS OR BP" Treatment

ERDE

ECDE

nmol/g tissue/rain b Controls (untreated) CS 3 days 5 days 16 days BP (80 mg/kg)

Fold induction

26.9 ± 6.8

(7)

43.2 ± 1.9"** 50.2 ± 2.9*** 33.8 ± 0.9 82.5 ± 8.3***

(4) (3) (4) (4)

nmol/g tissue/rain b

Fold induction

10.6 ± 0.5 (4) 1.6 1.9 1.3 3.1

13.1 ± 3.7 (4) 10.8 ± 0.8 (3) 11.6 ± 1.0 (4) ND ~

1.2 1.0 1.1

" Mice were exposed to CS by inhalation and to BP by i.p. injection as described in Materials and Methods. b Mean ± S.D. ***P < 0.001 by Kruskal-Wallis test, compared to enzyme activities of controls (number of mice). c ND = not done.

These negative results for BPDE-DNA after smoke or smoke condensatetreatment are contrasted with the positive cases among human smokers [ 7 10,23; Viihiikangas et al., unpublished results]. However, human studies have been mainly from groups of workers occupationally exposed to PAHs, where the exposure is more extensive than from smoking [7-10]. Furthermore, ! 7.

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l,'

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LOtto -- -.................................... -0 D2 LIVER ' z.O

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BP (mg/kg) Fig. 3. AHH induction in lungs and livers of B6 and D2 mice injected i.p. with BP. AHH activities in control B6 and D2 mice were 4.0 ± 2.1 pmol/min/g tissue and 6.0 ± 5.0 pmol/min/g tissue in lungs and 207.1 ± 48.13 pmol/min/g tissue and 127.5 ± 57.9 pmol/min/g tissue in liver respectively. Each point is a mean of 3--5 animals.

58

Z C) (J

4

~

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.&J O

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CSC LIVER •• . . . . . . . . . . . •

30

60

NCSC- LIVER

90 CSC, NCSC (mg/kg)

Fig. 4. AHH induction in B6 mice after i.p. administration of different doses of CSC or N,CSC. AHH activities in control B6 mice were 4.0 ± 2.1 pmol/min/g tissue in lung and 207.1 _+ 48.1 pmol/min/g tissue in liver. Each point is a mean of 3 animals.

smoke from one cigarette contains 10--50 ng of BP [24] and the lowest dose of BP injected to mice in our study leading to detectable BPDE-DNA-adducts by SFS equals about 0.5 rag/mouse. Also, the period of smoke exposure used in this study was relatively short compared to human who may smoke for many years. According to this and earlier studies in rodents [25-27] and probably also in humans [28] lung A H H seems to be more sensitive to PAHs than liver AHH. Of the three monooxygenases studied in B6 mouse liver (AHH, ECDE and ERDE) CS induced only ERDE, which has been suggested to reflect very specifically the cytochrome P-450 form induced by PAHs or cigarette smoking [19,20]. An interesting observation here was that lung DNA seems to be also more vulnerable than liver DNA to the damage by BP. In certain instances A H H induction seems to provide a protection from the carcinogenic effect of P A H s [29] and the formation of BP-DNA-adducts [30], while in other cases a positive correlation between AHH-activity and the adduct formation has been shown [31,32]. It has to be kept in mind, however, that A H H only measures the formation of hydroxymetabolites of BP [2] and many other activation and detoxication pathways exist (for review of BP-metabolism see [3]). Furthermore, DNA-damage is repairable, and even already transformed cells can be eliminated by the immunological system [29]. The relationship between BP-DNA binding and A H H inducibility seems also to be different in vitro and in vivo. When tissue fractions are incubated

59

in vitro with BP and DNA, tissues from induced responsive mice catalyze a higher amount of covalent binding than tissues from induced non-responsive mice [33--36]. In this study, we found higher A H H induction but even lower amounts of BPDE-DNA-adducts formed in vivo in B6 than D2 mice. Similarly, Phillips et al. [37] have reported that there is no significant difference in the formation of BP-DNA-adducts in the skin of these same mouse strains. In conclusion, this study supports the applicability of SFS for BPDE-DNAadducts for in vivo studies. It also casts some doubt on BP-DNA-adducts as an indicator of DNA-damage by smoking although just one animal model is not enough to draw definitive conclusions. Our results indicate that A H H activity alone is not a good indicator for the in vivo formation of BPDEDNA-adducts in mice. ACKNOWLEDGEMENTS

The authors thank Ms Ritva Tauriainen and Ms P~iivi Kylli for the skillful technical assistance. The help of Ms Tuula Inkala in animal handling is also gratefully acknowledged. Financial support by the Finnish Cancer Society (to K.V.) and the Academy of Finland (to O.P.) is acknowledged. M. Iscan was supported by the grants from European Science Foundation Toxicology Program and from the European Medical Research Councils. REFERENCES 1 2

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61 31

32

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