Toxicology, 60 (1990) 127--135 Elsevier Scientific Publishers Ireland Ltd.
Concentration- and time-dependent formation of D N A adducts in lungs of rats exposed to diesel exhaust James A. Bond**, Joe L. Mauderly and Ronald K. Wolff* Inhalation Toxicology Research Institute, Lovelace Biomedical and Environmental Research Institute, P.O. Box 5890, Albuquerque, N M 87185 (U.S.A.) (Received June 13th, 1989; accepted September 2nd, 1989)
Summary Diesel exhaust (DE) is a pulmonary carcinogen in rodents. Previous studies have demonstrated that following exposure of rats to high concentrations (10 mg soot/m 3) of DE, DNA adducts can be measured in lung tissue. The purpose of the present study was to characterize the kinetics of formation and persistence of lung DNA adducts after a 12-week exposure to DE and to determine the effect of exposure concentration on adduct formation. Rats were exposed for 7 h/day, 5 days/week, for up to 12 weeks, to filtered air (controls) or to diluted DE (0.35--10 mg soot/m3). DNA from lungs of rats was analyzed for the presence of DNA adducts by the 32P-postlabeling method. Levels of DNA adducts in lungs of rats exposed to the different exhaust concentrations were similar, and were about twice control values (,,o 14 vs. 7 adducts/109 bases). DNA adduct levels in lungs of control rats remained relatively constant throughout the 12-week exposure period. In contrast, lung DNA adduct levels in rats exposed to DE soot accumulated slowly during the 12-week exposure, and were highest at the end of exposure. DNA adduct levels declined rapidly after the termination of exposure. Because adduct levels in lungs were similar at all concentrations examined and the finding that adducts were increased in rats at an exposure level that does not significantly increase tumor incidence (0.35 mg soot/m3), it is likely that factors in addition to lung DNA adduct formation must be involved in DE-induced carcinogenicity. We concluded that the formation of lung DNA adducts by metabolites of soot-associated organic compounds may be only one step in the initiation of DEinduced pulmonary carcinogenesis. Key words: DNA adducts; Diesel exhaust; Lung; F344 rats; Carcinogenesis.
Introduction The
health
effects of diesel exhaust
(DE)
have
been
a topic of intensive
research d u r i n g the last d e c a d e . D E consists o f a m i x t u r e o f gases, v a p o r s , a n d *Present address: Lilly Research Laboratory, Greenfield, IN 46140, U.S.A. Address all correspondence to: Dr. James A. Bond, Lovelace Inhalation Toxicology, Research Institute, P.O. Box 5890, Albuquerque, NM 87185, U.S.A. **Present address: Chemical Industry, Institute of Toxicology, P.O. Box 12137, Research Triangle Park, NC 27709, U.S.A. 030o-483x/90/$03.50 © 1990 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
127
soot particles of respirable size [1]. Recent results from several laboratories [2--5] indicated that DE, inhaled chronically at high concentrations, is a pulmonary carcinogen in laboratory rats. The carcinogenicity of inhaled DE was shown to be both concentration- and time-dependent [2]. Organic extracts of DE particles are genotoxic in bacterial and mammalian test systems [6,7]. Genetic mechanisms may be responsible for the carcinogenic action of inhaled DE. Several studies suggest a potential role of the organic chemicals associated with DE soot as causative factors in the carcinogenic response of rodents (reviewed in [8]). One hypothesis regarding a potential genetic mechanism for the tumorigenic response is that the organic chemicals, desorbed from DE particles, must interact with lung DNA to initiate carcinogenesis. DNA adducts have been identified in the rat lung after both short-term (i.e., 12 weeks; [9]) and long-term exposures (i.e., 30 months; [10]) to DE. After 12 weeks of exposure to DE, higher levels of total DNA adducts and exhaust-induced adducts (i.e., numbers of adducts in exposed minus numbers of adducts in unexposed) were present in tissues in which tumors developed after chronic exposure to DE [9]. There have been no studies characterizing the concentration- and timedependency of lung DNA adduct formation following exposure to DE. The purpose of the present work was to characterize the kinetics of formation and persistence of lung DNA adducts during and after a 12-week exposure to DE and to determine the effect of exposure concentration on adduct formation. The 32p_ post-labeling assay was used to quantitate lung DNA adducts. The results indicate that DNA adduct formation was independent of exposure concentration over the range of concentrations tested (0.35--10 mg/m3). In addition, DNA adducts increased with increasing exposure duration up to 12 weeks of exposure and rapidly decreased to control values by 4 weeks after the termination of exposure. Materials and methods
Chemicals Calf thymus DNA, ethylenediamine tetraacetic acid, micrococcal endonuclease (from Staphylococcus aureus; Grade VI, 100 U/rag), spermidine, sodium pyruvate (Type II), adenosine diphosphate, RNase A (Type Ilia, 84 U/mg), N, N-Ms(2-hydroxyethyl)glycine (bicine), proteinase K (from Tritirachium album; Type XI), nuclease P1 (from penicillum citrinum), 2'-deoxyadenosine-3'-phosphate (dAp), lithium chloride, phenol, chloroform, and isoamyl alcohol were purchased from Sigma Chemical Co. (St. Louis, MO). Calf spleen endonuclease (phosphodiesterase) (2 U/mg), glycerol-3-phosphate dehydrogenase (rabbit muscle; 60 U / mg), triose phosphate isomerase (rabbit muscle; 5000 U/rag), 3-phosphogylcerate kinease (yeast; 450 U/mg), glyceraldehyde-3-phosphate dehydrogenase (rabbit muscle; 80 U/mg), lactate dehydrogenase (rabbit muscle; 550 U/mg), and NAD (Grade I, 100%) were purchased from Boehringer Mannheim (Indianapolis, IN). Ribonuclease (RNase) T1 (Aspergillus oryzae," 5000 U/ml) was purchased from CalBiochem (San Diego, CA). Ammonium formate, sodium dodecylsulfate (SDS), ammonium sulfate, and formic acid were purchased from Fisher Scientific
128
(Houston, TX). Urea and dithiothreitol were purchased from Bio-Rad (Richmond, CA). MN 300 cellulose powder was purchased from Brinkman Instruments (Westbury, NY). Corcut P600 XE polyethylenimine (PEI) was purchased from Virginia Chemicals, Inc. (Portsmouth, VA). [32p]H3PO 4 (carrier free) was purchased from ICN Biochemicals (Irvine, CA). Plastic vinyl sheets were custom made by Plastic Supply and Fabrication (Albuquerque, NM). PEI-cellulose thin-layer plastic-backed sheets for D N A adduct chromatography are best made in the laboratory to ensure high-quality sheets (K. Randerath, pers. comm.). Thus, D N A adduct analyses performed for this study were performed using PEI-cellulose sheets made at this Institute. All sheets were predeveloped with distilled water before use and stored in the dark at 4°C.
Animals Male F344/N rats, 14--15 weeks of age at the start of expost:re, were used in these studies. The rats were born and raised in the Institute's barrier-maintained colony and were housed two per polycarbonate cage supplied with sterilized hardwood-chip bedding and filter tops. Animal rooms were maintained at 2 0 - 22°C with a relative humidity of 2 0 - - 5 0 % and a 12-h light/dark cycle starting at 0600 h. Feed (Lab Blox, Allied Mills, Chicago, IL) and water were provided ad libitum. Before exposure to DE, rats were transferred from the animal housing quarters to inhalation exposure chambers (H2000, Hazelton Systems, Aberdeen, MD). All rats were allowed to acclimate to the exposure chambers for 3 weeks before initiation of DE exposures. Water from an automated watering system was supplied ad libitum during the acclimation period. None of the rats used in these studies had clinical signs of respiratory disease prior to exposure. Exposure system Details of the DE exposure system have been reported [11]. Briefly, exhaust was generated by 1980 Model, 5.7 liter, Oldsmobile V-8 engines mounted on test stands and connected via automatic transmissions to dynamometers and flywheels to simulate a mid-sized passenger car operated on continuously repeating, U.S. Federal Test Procedure urban cycles. Fuel meeting U S E P A certification standards was used (D-2 diesel control fuel, Phillips Chemical Co., Borger, TX) [1]. The exhaust was routed through a standard automotive muffler and tailpipe, diluted 10:1 with filtered air, and then diluted to the final concentration. Exposure chamber atmospheres were measured for particles by daily filter samples and for carbon monoxide, carbon dioxide, hydrocarbon vapors, nitrogen oxides, and a m m o n i a by weekly bag samples. The particle size distribution was measured using a Lovelace multijet cascade impactor and a parallel flow diffusion battery operated in series [1]. Rats were exposed for 7 h / d a y , 5 days/week, for up to 12 weeks, to filtered air (controls) or to diluted DE (0.35--10 mg/m3). For the concentration-response studies, rats were exposed to air or to 0.35, 3.5, 7.0, and 10 mg s o o t / m 3 for 12 weeks, and then were killed for D N A adduct analysis. For the time course studies, rats were exposed to 7 mg s o o t / m 3 for up to 12 weeks, and were killed at 2, 4, 8, 12, 14, 16, or 20 weeks after the start of exposure.
129
DNA isolation Rats were killed by CO 2 asphyxiation and the lungs were excised. DNA was isolated from lung tissue by a modification [12] of the Marmur procedure [13]. DNA concentration was estimated spectrophotometrically (1 A26o = 50 tag D N A / ml).
[y-32p]ATP synthesis [y-aEp]ATP was synthesized by the method of Johnson and Walseth [14], as described by Reddy and Randerath [15], except that the reaction cocktail was made fresh for each assay. The final concentration of the reaction mixture was 100 taCi/tal. The specific activity of [y-32p]ATP was determined using a known amount of 2'-deoxyadenosine-3'-phosphate, as described by Reddy and Randerath [15].
32P-labeling o f DNA adducts The nuclease P~ procedure, as described in detail by Reddy and Randerath [15], was used to separate DNA adducts. Reddy and Randerath [15] have shown that incubation of the micrococcal endonuclease/spleen exonuclease digest of DNA with nuclease P~ leads to removal of normal nucleotides prior to labeling. We have previously used this procedure with DNA from respiratory tract tissues of rats exposed to DE [9]. Furthermore, we have previously demonstrated (unpublished observations) that the nuclease P~ and butanol extraction enrichment procedures of the postlabeling assay yield similar patterns of adducts derived from DE exposures. A reference DNA adduct was also run along with the samples described above. This adduct, the nucleoside adduct from the reaction of benzo[a]pyrene-7,8-dihydrodiol-9,10-oxide with calf thymus DNA, was synthesized as previously described [16]. The adduct obtained using this method is predominantly N 2(7,8,9-trihydroxy-7,8,9,10-tetrahydrobenzo [a] pyren- 10-yl)deoxyguanosine. Separation o f DNA adducts by TLC To separate DNA adducts chromatographically, the 32p-postlabeled solutions were applied to a PEI-cellulose layer. The sheets were developed according to published methods [15,17,18], using the following solvent conditions: D1, 1 M sodium phosphate buffer (pH 6.8); D3, 4.2 M lithium formate, 7.5 M urea (pH 3.5); D4, 0.8 M lithium chloride, 6.0 M urea, 0.5 M Tris--HC1 (pH 8.0); D5, 1 M sodium phosphate buffer (pH 6.0). Areas of radioactivity on the chromatograms were located by autoradiography [17--19], after exposing the film for 65 h at - 8 0 ° C . Count rates for adducts were determined by liquid scintillation spectrometry (counting efficiency > 99%) after subtracting count rates from adjacent blank areas. Because adducts were derived from 8.2 tag of DNA (or 2.66 x 104 pmol dNp, assuming 1 tag = 3240 pmol dNp) and the specific activity of [y32p]ATP ranged from 2.68 x 106 cpm/pmol to 6.00 × 106 cpm/pmol, DNA adduct levels were calculated according to Reddy and Randerath [15] as follows: cpm in adduct nuelcotides Relative adduct labeling (RAL) = 2.66 x 104 pmol × specific activity 130
Results and discussion
The exposures did not cause overt signs of toxicity and there were no significant exposure-related differences in body weight (data not shown). Gross observations of the lungs of rats exposed to DE showed darkly pigmented lungs, while the lungs f r o m sham-exposed rats were unpigmented. Autoradiograms of D N A adducts in lungs from control and diesel exhaustexposed (7 mg s o o t / m 3) rats showed m a n y more adducts in the D N A from rats exposed to DE than in D N A f r o m control rats, and m a n y of these adducts were unique to DE-exposed rats (Fig. 1). It is possible, however, that the difference in the autoradiographic patterns o f adducts m a y be due to alterations in fragmentation patterns or incomplete digestion by nuclease P~. The identities of the chemicals in DE that are responsible for the observed D N A adducts have not been determined. Radioactivity was measured in the same region as the B P D E - D N A adduct, however, there was no significant difference in the levels of this adduct in D N A f r o m either control or f r o m DE-exposed rats. Levels of D N A adducts in lungs of rats exposed to the different exhaust concentrations for 12 weeks were similar (Fig. 2). The overall mean level of 14 adducts per 109 bases in exhaust-exposed rats was significantly (P < 0.05; Student's t-test) higher than the level of adducts in sham-exposed rats. These data demonstrated that, while D N A adduct formation in lung tissue from DE-exposed rats was significantly elevated compared to adduct formation in lung tissue from control rats, adduct formation was independent of exhaust exposure concentration at the exhaust exposure levels tested. Bond and Mauderly [20] demonstrated that the capacity of isolated, perfused rat lungs to metabolize chemicals associated with DE could be saturated at high concentrations. One explanation for the results reported here is that, at the exhaust concentrations tested, the lung enzymes responsible for formation of metabolites that bind to D N A were saturated. Furthermore, the lack of an exposure-response relationship for D N A adduct formation as reported here, and the earlier finding that D N A adducts were increased at an exposure level that did not significantly increase lung t u m o r incidence (0.35 mg/m3; [2]), suggest that additional factors are also important in exhaust-induced carcinogenicity. The presence of adducts in D N A from rats sham-exposed to air has been reported previously [9,10]. These adducts, termed I-spots, are derived from D N A derivatives termed I-compounds [21,22] and have been reported to increase with age [22]. I-spots may be related to exogenous factors (e.g., dietary) or to small amounts of reactive, electrophilic by-products of normal metabolic activities. Experiments were designed to determine whether the number of adducts increased linearly with time during the 12-week exposure, and whether steadystate levels of adducts could be predicted after a chronic exposure to DE. Lung D N A adduct levels in rats exposed to 7 m g / m ~ of DE soot were less than controls by 4 weeks of exposure, after which levels of adducts accumulated slowly during the remainder o f the 12-week exposure (Fig. 3). By the end of exposure, adduct levels in exposed rat lungs were about 160% of controls. D N A adduct levels declined rapidly after the termination of exposure (Fig. 3); by 4
131
t~
CONTROL
DIESEL EXHAUST
?ig. 1. Autoradiograms of chromatograms from lungs of control and diesel exhaust-exposed rats (7 mg s o o t / m s) after 12 weeks of exposure. The origin (©) ind the BPDE-DNA adduct (arrow) are indicated on each chromatogram.
D
16
12 o) A Ec4 uJ~
Q Q
0
1
0.35
3.5
7.0
10
EXPOSURE CONCENTRATIONOF DIESEL SOOT (mg/m3 )
Fig. 2. E f f e c t o f diesel e x h a u s t c o n c e n t r a t i o n o n D N A a d d u c t levels in r a t l u n g a f t e r 12 w e e k s o f e x p o s u r e . V a l u e s r e p r e s e n t m e a n _+ S . E . ; n = 6.
weeks after the end of exposure, adduct levels were not significantly different from those of controls. The observation that repair of adducts was faster than adduct formation suggests that steady-state levels of D N A adducts would be reached during long-term exposure to DE. Wong et al. [10] reported adduct levels of 20_+8 adducts/109 bases (,~ _+ S.D.; n = 6) in lungs of rats exposed for 30 months to 7 mg s o o t / m 3. This level of adducts is not significantly different from 220 200 .J O OC IZ O (J " O IZ UJ O OC UJ O.
160
120
8O
40 EXPOSURE DURATION
O0
L
I
I
I
i
i
i
I
I
4 8 12 16 20, T I M E S I N C E T H E S T A R T OF E X P O S U R E ( w e e k s )
Fig. 3. T i m e c o u r s e f o r t h e f o r m a t i o n a n d p e r s i s t e n c e o f D N A a d d u c t s in l u n g s o f r a t s e x p o s e d f o r u p to 12 w e e k s t o a i r o r to 7 m g s o o t / m 3.
133
the 12 _+ 4 adducts/109 bases present at 12 weeks o f e x p o s u r e to 7 m g / m 3 in the present study. This suggests that a d d u c t levels were at, or near, steady-state by 12 weeks o f exposure. In s u m m a r y , the results o f these studies suggest that the f o r m a t i o n o f lung D N A adducts by m e t a b o l i t e s o f s o o t - a s s o c i a t e d o r g a n i c c o m p o u n d s is only o n e step in the initiation o f D E - i n d u c e d , p u l m o n a r y carcinogenesis in the rat. Because a d d u c t levels in lungs were similar at all 4 exhaust c o n c e n t r a t i o n s e x a m i n e d , and were increased in rats at an e x p o s u r e level that did n o t increase lung t u m o r incidence (0.35 m g / m 3 ; [2]), we c o n c l u d e d that a d d i t i o n a l factors m u s t also be i n v o l v e d in D E - i n d u c e d carcinogenicity.
Acknowledgements T h e a u t h o r s gratefully a c k n o w l e d g e the technical assistance o f Mr. E. Barr an d Ms. L. Blair d u r i n g the c o n d u c t o f the e x p o s u r e and Ms. M. M a r q u e z f o r her w o r k o n the 32p-post-labeling assay. T h e a u t h o r s also a c k n o w l e d g e the critical review an d discussions with a n u m b e r o f o u r colleagues. This research was supp o r t e d by the O f f i c e o f H e a l t h a n d E n v i r o n m e n t a l Research, U . S . D e p a r t m e n t o f Energy, u n d e r C o n t r a c t N o . D E - A C 0 4 - 7 6 E V 0 1 0 1 3 , in facilities fully accredited by the A m e r i c a n A s s o c i a t i o n f o r A c c r e d i t a t i o n o f L a b o r a t o r y A n i m a l Care.
References 1 2
3
4 5
6
7
8 9
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Y.S. Cheng, H.C. Yeh, J.L. Mauderly, and B.V. Mokler, Characterization of diesel exhaust in a chronic inhalation study. Am. Ind. Hyg. Assoc. J., 45 (1984) 547. J.L. Mauderly, R.K. Jones, W.C. Griffith, R.F. Henderson and R.O. McClellan, Diesel exhaust is a pulmonary carcinogen in rats exposed chronically by inhalation. Fundam. Appl. Toxicol., 9 (1987) 208. W. St6ber, Experimental induction of tumors in hamsters, mice and rats after long-term inhalation of filtered and unfiltered diesel engine exhaust, in N. Ishinishi, A. Koizumi, R. McClellan and W. St6ber (Eds.), Carcinogenicity and Mutagenicity of Diesel Engine Exhaust, Elsevier, Amsterdam, 1986, p. 421. N. Ishinishi, A. Koizumi, R.O. McClellan and W. St6ber (Eds.), Carcinogenicity and Mutagenicity of Diesel Engine Exhaust, Elsevier, Amsterdam, 1986. J. Brightwell, X. Fouillet, A.L. Cassano-Zopi, R. Gatz and F. Duchosal, Neoplastic and functional changes in rodents after chronic inhalation of engine exhaust emissions, in N. Ishinishi, A. Koizumi, R. McClellan and W. St6ber (Eds.), Carcinogenicity and Mutagenicity of Diesel Engine Exhaust, Elsevier, Amsterdam, 1986, p. 471. A.L. Brooks, A.P. Li, J.S. Dutcher, C.R. Clark, S.J. Rothenberg, R. Kiyoura, W.E. Bechtold and R.O. McClellan, A comparison of genotoxicity of automotive exhaust particles from laboratory and environmental sources. Environ. Mutagen., 6 (1984) 651. J.M. Lockard, C.J. Viau, C. Lee-Stevens, J.C. Caldwell, J.P. Wojciechowski, H.G. Enoch and P.S. Sabharwal, Induction of sister chromatid exchanges and bacterial revertants by organic extracts of airborne particles. Environ. Mutagen., 3 (1981) 671. R.O. McClellan, Health effects of exposure to diesel exhaust particles. Annu. Rev. Pharmacol. Toxicol., 27 (1987) 279. J.A. Bond, R.K. Wolff, J.R. Harkema, J.L. Mauderly, R. F. Henderson, W.C. Griffith and R.O. McClellan, Distribution of DNA adducts in the respiratory tract of rats exposed to diesel exhaust. Toxicol. Appl. Pharmacol., 96 (1988) 336.
I0
11
12 13 14
15 16
17 18
19 20 21
22
D. Wong, C.E. Mitchell, R.K. Wolff, J.L. Mauderly and A. M. Jeffrey, Short communication: Identification of D N A damage as a result of exposure of rats to diesel exhaust. Carcinogenesis, 7 (1986) 1595. B.V. Mokler, F.A. Archibeque, R.L. Beethe, C.P.J. Kelly, J.A. Lopez, J.L. Mauderly and D.S. Stafford, Diesel exhaust exposure system for animal studies. Fundam. Appl. Toxicol., 4 (1984) 270. R.C. Gupta, Nonrandom binding of the carcinogen N-hydroxy-2-acetylaminofluorene to repetitive sequences of rat liver DNA in vivo. Proc. Natl. Acad. Sci. USA, 81 (1984) 6943. J.A. Marmur, Procedure for the isolation of deoxyribonucleic acid from micro-organisms. J. Mol. Biol., 3 (1961) 208. R.A. Johnson and T.F. Walseth, The enzymatic preparation of [a-32P]ATP, [a-32p]GTP, [~32p]cAMP, [aJ2p]cGMP, and their use in assay of adenylate and guanylate cyclases, and cyclic nucleotide phosphodiesterases. Adv. Cyclic Nucleotide Res., l0 (1979) 135. M.V. Reddy and K. Randerath, Nuclease Pj-mediated enhancement of sensitivity of ~2p-postlabeling test for structurally diverse DNA adducts. Carcinogenesis, 7 (1986) 1543. K.W. Jennette, A.M. Jeffrey, S.H. Blobstein, F.A. Beland, R.G. Harvey and I.B. Weinstein, Nucleoside adducts from the in vitro reaction of benzo[a]pyrene-7,8-dihydrodiol 9,10-oxide or benzo[a]pyrene 4,5-oxide with nucleic acids. Biochemistry, 16 (1977) 932. R.C. Gupta, M.V. Reddy and K. Randerath, 32p-postlabeling analysis of nonradioactive aromatic carcinogen-DNA adducts. Carcinogenesis, 3 (1983) 1081. M.V. Reddy, R.C. Gupta, E. Randerath and K. Randerath, 32p-postlabeling test for covalent DNA binding of chemicals in vivo: Application to a variety of aromatic carcinogens and methylating agents. Carcinogenesis, 5 (1984)231. K. Randerath, M.V. Reddy and R.C. Gupta, 32p-postlabeling test for DNA damage. Proc. Natl. Acad. Sci. USA, 78 (1981) 6126. J.A. Bond and J.L. Mauderly, Metabolism and macromolecular covalent binding of ~4C-l-nitropyrene in isolated perfused/ventilated rat lungs. Cancer Res., 44 (1984) 3924. K. Randerath, M. Reddy and R. Disher, Age- and tissue-related DNA modifications in untreated rats: Detection by 32p-postlabeling assay and possible significance for spontaneous tumor induction and aging. Carcinogenesis, 7 (1986) 1615. K. Randerath, L-J.W. Lu and D. Li, A comparison between different types of covalent DNA modifications (I-compounds, persistent carcinogen adducts and 5-methylcytosine) in regenerating rat liver. Carcinogenesis, 9 (1988) 1843.
135
Concentration- and time-dependent formation of D N A adducts in lungs of rats exposed to diesel exhaust James A. Bond**, Joe L. Mauderly and Ronald K. Wolff* Inhalation Toxicology Research Institute, Lovelace Biomedical and Environmental Research Institute, P.O. Box 5890, Albuquerque, N M 87185 (U.S.A.) (Received June 13th, 1989; accepted September 2nd, 1989)
Summary Diesel exhaust (DE) is a pulmonary carcinogen in rodents. Previous studies have demonstrated that following exposure of rats to high concentrations (10 mg soot/m 3) of DE, DNA adducts can be measured in lung tissue. The purpose of the present study was to characterize the kinetics of formation and persistence of lung DNA adducts after a 12-week exposure to DE and to determine the effect of exposure concentration on adduct formation. Rats were exposed for 7 h/day, 5 days/week, for up to 12 weeks, to filtered air (controls) or to diluted DE (0.35--10 mg soot/m3). DNA from lungs of rats was analyzed for the presence of DNA adducts by the 32P-postlabeling method. Levels of DNA adducts in lungs of rats exposed to the different exhaust concentrations were similar, and were about twice control values (,,o 14 vs. 7 adducts/109 bases). DNA adduct levels in lungs of control rats remained relatively constant throughout the 12-week exposure period. In contrast, lung DNA adduct levels in rats exposed to DE soot accumulated slowly during the 12-week exposure, and were highest at the end of exposure. DNA adduct levels declined rapidly after the termination of exposure. Because adduct levels in lungs were similar at all concentrations examined and the finding that adducts were increased in rats at an exposure level that does not significantly increase tumor incidence (0.35 mg soot/m3), it is likely that factors in addition to lung DNA adduct formation must be involved in DE-induced carcinogenicity. We concluded that the formation of lung DNA adducts by metabolites of soot-associated organic compounds may be only one step in the initiation of DEinduced pulmonary carcinogenesis. Key words: DNA adducts; Diesel exhaust; Lung; F344 rats; Carcinogenesis.
Introduction The
health
effects of diesel exhaust
(DE)
have
been
a topic of intensive
research d u r i n g the last d e c a d e . D E consists o f a m i x t u r e o f gases, v a p o r s , a n d *Present address: Lilly Research Laboratory, Greenfield, IN 46140, U.S.A. Address all correspondence to: Dr. James A. Bond, Lovelace Inhalation Toxicology, Research Institute, P.O. Box 5890, Albuquerque, NM 87185, U.S.A. **Present address: Chemical Industry, Institute of Toxicology, P.O. Box 12137, Research Triangle Park, NC 27709, U.S.A. 030o-483x/90/$03.50 © 1990 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
127
soot particles of respirable size [1]. Recent results from several laboratories [2--5] indicated that DE, inhaled chronically at high concentrations, is a pulmonary carcinogen in laboratory rats. The carcinogenicity of inhaled DE was shown to be both concentration- and time-dependent [2]. Organic extracts of DE particles are genotoxic in bacterial and mammalian test systems [6,7]. Genetic mechanisms may be responsible for the carcinogenic action of inhaled DE. Several studies suggest a potential role of the organic chemicals associated with DE soot as causative factors in the carcinogenic response of rodents (reviewed in [8]). One hypothesis regarding a potential genetic mechanism for the tumorigenic response is that the organic chemicals, desorbed from DE particles, must interact with lung DNA to initiate carcinogenesis. DNA adducts have been identified in the rat lung after both short-term (i.e., 12 weeks; [9]) and long-term exposures (i.e., 30 months; [10]) to DE. After 12 weeks of exposure to DE, higher levels of total DNA adducts and exhaust-induced adducts (i.e., numbers of adducts in exposed minus numbers of adducts in unexposed) were present in tissues in which tumors developed after chronic exposure to DE [9]. There have been no studies characterizing the concentration- and timedependency of lung DNA adduct formation following exposure to DE. The purpose of the present work was to characterize the kinetics of formation and persistence of lung DNA adducts during and after a 12-week exposure to DE and to determine the effect of exposure concentration on adduct formation. The 32p_ post-labeling assay was used to quantitate lung DNA adducts. The results indicate that DNA adduct formation was independent of exposure concentration over the range of concentrations tested (0.35--10 mg/m3). In addition, DNA adducts increased with increasing exposure duration up to 12 weeks of exposure and rapidly decreased to control values by 4 weeks after the termination of exposure. Materials and methods
Chemicals Calf thymus DNA, ethylenediamine tetraacetic acid, micrococcal endonuclease (from Staphylococcus aureus; Grade VI, 100 U/rag), spermidine, sodium pyruvate (Type II), adenosine diphosphate, RNase A (Type Ilia, 84 U/mg), N, N-Ms(2-hydroxyethyl)glycine (bicine), proteinase K (from Tritirachium album; Type XI), nuclease P1 (from penicillum citrinum), 2'-deoxyadenosine-3'-phosphate (dAp), lithium chloride, phenol, chloroform, and isoamyl alcohol were purchased from Sigma Chemical Co. (St. Louis, MO). Calf spleen endonuclease (phosphodiesterase) (2 U/mg), glycerol-3-phosphate dehydrogenase (rabbit muscle; 60 U / mg), triose phosphate isomerase (rabbit muscle; 5000 U/rag), 3-phosphogylcerate kinease (yeast; 450 U/mg), glyceraldehyde-3-phosphate dehydrogenase (rabbit muscle; 80 U/mg), lactate dehydrogenase (rabbit muscle; 550 U/mg), and NAD (Grade I, 100%) were purchased from Boehringer Mannheim (Indianapolis, IN). Ribonuclease (RNase) T1 (Aspergillus oryzae," 5000 U/ml) was purchased from CalBiochem (San Diego, CA). Ammonium formate, sodium dodecylsulfate (SDS), ammonium sulfate, and formic acid were purchased from Fisher Scientific
128
(Houston, TX). Urea and dithiothreitol were purchased from Bio-Rad (Richmond, CA). MN 300 cellulose powder was purchased from Brinkman Instruments (Westbury, NY). Corcut P600 XE polyethylenimine (PEI) was purchased from Virginia Chemicals, Inc. (Portsmouth, VA). [32p]H3PO 4 (carrier free) was purchased from ICN Biochemicals (Irvine, CA). Plastic vinyl sheets were custom made by Plastic Supply and Fabrication (Albuquerque, NM). PEI-cellulose thin-layer plastic-backed sheets for D N A adduct chromatography are best made in the laboratory to ensure high-quality sheets (K. Randerath, pers. comm.). Thus, D N A adduct analyses performed for this study were performed using PEI-cellulose sheets made at this Institute. All sheets were predeveloped with distilled water before use and stored in the dark at 4°C.
Animals Male F344/N rats, 14--15 weeks of age at the start of expost:re, were used in these studies. The rats were born and raised in the Institute's barrier-maintained colony and were housed two per polycarbonate cage supplied with sterilized hardwood-chip bedding and filter tops. Animal rooms were maintained at 2 0 - 22°C with a relative humidity of 2 0 - - 5 0 % and a 12-h light/dark cycle starting at 0600 h. Feed (Lab Blox, Allied Mills, Chicago, IL) and water were provided ad libitum. Before exposure to DE, rats were transferred from the animal housing quarters to inhalation exposure chambers (H2000, Hazelton Systems, Aberdeen, MD). All rats were allowed to acclimate to the exposure chambers for 3 weeks before initiation of DE exposures. Water from an automated watering system was supplied ad libitum during the acclimation period. None of the rats used in these studies had clinical signs of respiratory disease prior to exposure. Exposure system Details of the DE exposure system have been reported [11]. Briefly, exhaust was generated by 1980 Model, 5.7 liter, Oldsmobile V-8 engines mounted on test stands and connected via automatic transmissions to dynamometers and flywheels to simulate a mid-sized passenger car operated on continuously repeating, U.S. Federal Test Procedure urban cycles. Fuel meeting U S E P A certification standards was used (D-2 diesel control fuel, Phillips Chemical Co., Borger, TX) [1]. The exhaust was routed through a standard automotive muffler and tailpipe, diluted 10:1 with filtered air, and then diluted to the final concentration. Exposure chamber atmospheres were measured for particles by daily filter samples and for carbon monoxide, carbon dioxide, hydrocarbon vapors, nitrogen oxides, and a m m o n i a by weekly bag samples. The particle size distribution was measured using a Lovelace multijet cascade impactor and a parallel flow diffusion battery operated in series [1]. Rats were exposed for 7 h / d a y , 5 days/week, for up to 12 weeks, to filtered air (controls) or to diluted DE (0.35--10 mg/m3). For the concentration-response studies, rats were exposed to air or to 0.35, 3.5, 7.0, and 10 mg s o o t / m 3 for 12 weeks, and then were killed for D N A adduct analysis. For the time course studies, rats were exposed to 7 mg s o o t / m 3 for up to 12 weeks, and were killed at 2, 4, 8, 12, 14, 16, or 20 weeks after the start of exposure.
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DNA isolation Rats were killed by CO 2 asphyxiation and the lungs were excised. DNA was isolated from lung tissue by a modification [12] of the Marmur procedure [13]. DNA concentration was estimated spectrophotometrically (1 A26o = 50 tag D N A / ml).
[y-32p]ATP synthesis [y-aEp]ATP was synthesized by the method of Johnson and Walseth [14], as described by Reddy and Randerath [15], except that the reaction cocktail was made fresh for each assay. The final concentration of the reaction mixture was 100 taCi/tal. The specific activity of [y-32p]ATP was determined using a known amount of 2'-deoxyadenosine-3'-phosphate, as described by Reddy and Randerath [15].
32P-labeling o f DNA adducts The nuclease P~ procedure, as described in detail by Reddy and Randerath [15], was used to separate DNA adducts. Reddy and Randerath [15] have shown that incubation of the micrococcal endonuclease/spleen exonuclease digest of DNA with nuclease P~ leads to removal of normal nucleotides prior to labeling. We have previously used this procedure with DNA from respiratory tract tissues of rats exposed to DE [9]. Furthermore, we have previously demonstrated (unpublished observations) that the nuclease P~ and butanol extraction enrichment procedures of the postlabeling assay yield similar patterns of adducts derived from DE exposures. A reference DNA adduct was also run along with the samples described above. This adduct, the nucleoside adduct from the reaction of benzo[a]pyrene-7,8-dihydrodiol-9,10-oxide with calf thymus DNA, was synthesized as previously described [16]. The adduct obtained using this method is predominantly N 2(7,8,9-trihydroxy-7,8,9,10-tetrahydrobenzo [a] pyren- 10-yl)deoxyguanosine. Separation o f DNA adducts by TLC To separate DNA adducts chromatographically, the 32p-postlabeled solutions were applied to a PEI-cellulose layer. The sheets were developed according to published methods [15,17,18], using the following solvent conditions: D1, 1 M sodium phosphate buffer (pH 6.8); D3, 4.2 M lithium formate, 7.5 M urea (pH 3.5); D4, 0.8 M lithium chloride, 6.0 M urea, 0.5 M Tris--HC1 (pH 8.0); D5, 1 M sodium phosphate buffer (pH 6.0). Areas of radioactivity on the chromatograms were located by autoradiography [17--19], after exposing the film for 65 h at - 8 0 ° C . Count rates for adducts were determined by liquid scintillation spectrometry (counting efficiency > 99%) after subtracting count rates from adjacent blank areas. Because adducts were derived from 8.2 tag of DNA (or 2.66 x 104 pmol dNp, assuming 1 tag = 3240 pmol dNp) and the specific activity of [y32p]ATP ranged from 2.68 x 106 cpm/pmol to 6.00 × 106 cpm/pmol, DNA adduct levels were calculated according to Reddy and Randerath [15] as follows: cpm in adduct nuelcotides Relative adduct labeling (RAL) = 2.66 x 104 pmol × specific activity 130
Results and discussion
The exposures did not cause overt signs of toxicity and there were no significant exposure-related differences in body weight (data not shown). Gross observations of the lungs of rats exposed to DE showed darkly pigmented lungs, while the lungs f r o m sham-exposed rats were unpigmented. Autoradiograms of D N A adducts in lungs from control and diesel exhaustexposed (7 mg s o o t / m 3) rats showed m a n y more adducts in the D N A from rats exposed to DE than in D N A f r o m control rats, and m a n y of these adducts were unique to DE-exposed rats (Fig. 1). It is possible, however, that the difference in the autoradiographic patterns o f adducts m a y be due to alterations in fragmentation patterns or incomplete digestion by nuclease P~. The identities of the chemicals in DE that are responsible for the observed D N A adducts have not been determined. Radioactivity was measured in the same region as the B P D E - D N A adduct, however, there was no significant difference in the levels of this adduct in D N A f r o m either control or f r o m DE-exposed rats. Levels of D N A adducts in lungs of rats exposed to the different exhaust concentrations for 12 weeks were similar (Fig. 2). The overall mean level of 14 adducts per 109 bases in exhaust-exposed rats was significantly (P < 0.05; Student's t-test) higher than the level of adducts in sham-exposed rats. These data demonstrated that, while D N A adduct formation in lung tissue from DE-exposed rats was significantly elevated compared to adduct formation in lung tissue from control rats, adduct formation was independent of exhaust exposure concentration at the exhaust exposure levels tested. Bond and Mauderly [20] demonstrated that the capacity of isolated, perfused rat lungs to metabolize chemicals associated with DE could be saturated at high concentrations. One explanation for the results reported here is that, at the exhaust concentrations tested, the lung enzymes responsible for formation of metabolites that bind to D N A were saturated. Furthermore, the lack of an exposure-response relationship for D N A adduct formation as reported here, and the earlier finding that D N A adducts were increased at an exposure level that did not significantly increase lung t u m o r incidence (0.35 mg/m3; [2]), suggest that additional factors are also important in exhaust-induced carcinogenicity. The presence of adducts in D N A from rats sham-exposed to air has been reported previously [9,10]. These adducts, termed I-spots, are derived from D N A derivatives termed I-compounds [21,22] and have been reported to increase with age [22]. I-spots may be related to exogenous factors (e.g., dietary) or to small amounts of reactive, electrophilic by-products of normal metabolic activities. Experiments were designed to determine whether the number of adducts increased linearly with time during the 12-week exposure, and whether steadystate levels of adducts could be predicted after a chronic exposure to DE. Lung D N A adduct levels in rats exposed to 7 m g / m ~ of DE soot were less than controls by 4 weeks of exposure, after which levels of adducts accumulated slowly during the remainder o f the 12-week exposure (Fig. 3). By the end of exposure, adduct levels in exposed rat lungs were about 160% of controls. D N A adduct levels declined rapidly after the termination of exposure (Fig. 3); by 4
131
t~
CONTROL
DIESEL EXHAUST
?ig. 1. Autoradiograms of chromatograms from lungs of control and diesel exhaust-exposed rats (7 mg s o o t / m s) after 12 weeks of exposure. The origin (©) ind the BPDE-DNA adduct (arrow) are indicated on each chromatogram.
D
16
12 o) A Ec4 uJ~
Q Q
0
1
0.35
3.5
7.0
10
EXPOSURE CONCENTRATIONOF DIESEL SOOT (mg/m3 )
Fig. 2. E f f e c t o f diesel e x h a u s t c o n c e n t r a t i o n o n D N A a d d u c t levels in r a t l u n g a f t e r 12 w e e k s o f e x p o s u r e . V a l u e s r e p r e s e n t m e a n _+ S . E . ; n = 6.
weeks after the end of exposure, adduct levels were not significantly different from those of controls. The observation that repair of adducts was faster than adduct formation suggests that steady-state levels of D N A adducts would be reached during long-term exposure to DE. Wong et al. [10] reported adduct levels of 20_+8 adducts/109 bases (,~ _+ S.D.; n = 6) in lungs of rats exposed for 30 months to 7 mg s o o t / m 3. This level of adducts is not significantly different from 220 200 .J O OC IZ O (J " O IZ UJ O OC UJ O.
160
120
8O
40 EXPOSURE DURATION
O0
L
I
I
I
i
i
i
I
I
4 8 12 16 20, T I M E S I N C E T H E S T A R T OF E X P O S U R E ( w e e k s )
Fig. 3. T i m e c o u r s e f o r t h e f o r m a t i o n a n d p e r s i s t e n c e o f D N A a d d u c t s in l u n g s o f r a t s e x p o s e d f o r u p to 12 w e e k s t o a i r o r to 7 m g s o o t / m 3.
133
the 12 _+ 4 adducts/109 bases present at 12 weeks o f e x p o s u r e to 7 m g / m 3 in the present study. This suggests that a d d u c t levels were at, or near, steady-state by 12 weeks o f exposure. In s u m m a r y , the results o f these studies suggest that the f o r m a t i o n o f lung D N A adducts by m e t a b o l i t e s o f s o o t - a s s o c i a t e d o r g a n i c c o m p o u n d s is only o n e step in the initiation o f D E - i n d u c e d , p u l m o n a r y carcinogenesis in the rat. Because a d d u c t levels in lungs were similar at all 4 exhaust c o n c e n t r a t i o n s e x a m i n e d , and were increased in rats at an e x p o s u r e level that did n o t increase lung t u m o r incidence (0.35 m g / m 3 ; [2]), we c o n c l u d e d that a d d i t i o n a l factors m u s t also be i n v o l v e d in D E - i n d u c e d carcinogenicity.
Acknowledgements T h e a u t h o r s gratefully a c k n o w l e d g e the technical assistance o f Mr. E. Barr an d Ms. L. Blair d u r i n g the c o n d u c t o f the e x p o s u r e and Ms. M. M a r q u e z f o r her w o r k o n the 32p-post-labeling assay. T h e a u t h o r s also a c k n o w l e d g e the critical review an d discussions with a n u m b e r o f o u r colleagues. This research was supp o r t e d by the O f f i c e o f H e a l t h a n d E n v i r o n m e n t a l Research, U . S . D e p a r t m e n t o f Energy, u n d e r C o n t r a c t N o . D E - A C 0 4 - 7 6 E V 0 1 0 1 3 , in facilities fully accredited by the A m e r i c a n A s s o c i a t i o n f o r A c c r e d i t a t i o n o f L a b o r a t o r y A n i m a l Care.
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