DNA adduct formation and persistence in rat tissues following exposure to the mammary carcinogen dibenzo[a,l]pyrene

Jamal M. Arif1, Wendy A. Smith2 and Ramesh C. Gupta1,2,3

1 Department of Preventive Medicine and Environmental Health and
2 Graduate Center for Toxicology, University of Kentucky Medical Center, Lexington, KY 40536, USA


    Abstract
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 Abstract
 Introduction
 References
 
Dibenzo[a,l]pyrene (DBP), an environmentally significant polycyclic aromatic hydrocarbon (PAH), is one of the most potent carcinogens with greater carcinogenicity in rodent mammary glands and skin than 7,12-dimethylbenz[a]anthracene or benzo[a]pyrene, respectively. In this study, we have examined the formation and persistence of stable DNA adducts in rats administered a carcinogenic intramammillary (i.m.) dose of DBP (0.25 µmol/gland). 32P-post-labeling analysis of mammary epithelial DNA 6 h, and 2, 5 and 14 days post-treatment produced one major (~30%) and at least six minor adducts. Non-target tissue DNA (lung, heart, bladder and pancreas) also showed essentially the same adduct pattern as did mammary DNA, except liver which resulted in four additional adduct spots. The mammary DNA was most adducted (2640 ± 532 adducts/109 nucleotides) on day 5 while the other tissue DNAs had 10- to 65-fold lower adduct levels (lung > liver > heart > bladder > pancreas). Adduct levels continued to increase at all time points examined for all tissues, except mammary tissue which showed a decline (~40%) on day 14. Chromatographic comparison with adducts formed in vitro by reaction of syn- and anti-DBP-11,12-diol-13,14-epoxides (DBPDEs) with DNA and individual nucleotides indicated that the in vivo adducts were deoxyadenosine- and deoxyguanosine-derived, formed by interaction with both the anti- and syn-isomers; the adenine-derived adducts comprised 60–75% of the total adducts. However, the liver-specific DNA adducts (nos 8–11) were not derived from any of the DBPDE isomers. Our data show: (i) significantly higher DBP–DNA adduction in mammary tissue as compared with non-target tissues, which is consistent with its mammary carcinogenicity; (ii) adenine is highly reactive towards DBP metabolites as has been observed for many other PAHs; and (iii) the peak binding of DBP with DNA was shifted beyond 14 days for the non-target tissues by i.m. route of exposure.

Abbreviations: B[a]P, benzo[a]pyrene; CYP, cytochrome P450; DBP, dibenzo[a,l]pyrene; DBPDE, DBP-11,12-diol-13,14-epoxide; DMBA, 7,12-dimethylbenz[a]anthracene; DMSO, dimethyl sulfoxide; i.g., intragastric; i.m., intramammillary; i.p., intraperitoneal; PAH, polycyclic aromatic hydrocarbon; PEI, polyethyleneimine; RAL, relative adduct labeling.


    Introduction
 Top
 Abstract
 Introduction
 References
 
Dibenzo[a,l]pyrene (DBP), an environmentally significant polycyclic aromatic hydrocarbon (PAH) (1), has been reported to be one of the most potent animal carcinogens (24) and mutagens (5). It is a hexacyclic aromatic hydrocarbon, having both a bay region and a hindered bay region (Fjord region). Bioactivation of DBP to DNA-reactive species involves metabolic activation to either diolepoxides through cytochrome P450 (CYP)-dependent monooxygenases and epoxide hydrolases or to reactive metabolites by the one-electron oxidation pathway (69). 3-Methylcholanthrene-, ß-naphthoflavone- or Aroclor 1254-induced rat liver microsomes were able to produce enhanced levels of syn- and anti-DBP-11,12-diol-13,14-epoxides (DBPDEs) when incubated with DBP (8,9). Furthermore, recombinant human CYP1A1 also demonstrated the ability to oxidize DBP to various DBP dihydrodiols (7). In addition, human primary mammary epithelial cells and human mammary carcinoma MCF-7 cells, have been shown to activate DBP efficiently to several DNA binding species (6,10,11).

In rat mammary and mouse skin models, DBP was found to be several fold more carcinogenic than either 7,12-dimethylbenz[a]anthracene (DMBA) or benzo[a]pyrene (B[a]P), respectively (2,3). This observation is consistent with the DNA adduct levels found in rat mammary epithelial DNA following intramammillary exposure to DBP, DMBA and B[a]P (12). Our previous studies with microsomal bioactivation showed that >60% of the total DNA adducts are adenine-derived and both syn- and anti-DBPDE are involved in DBP–DNA adduction (8).

In this study, we have examined the formation and persistence of stable DNA adducts in target and non-target tissues of rats treated with a carcinogenic dose of DBP by an intramammillary (i.m.) route.

Female Sprague–Dawley rats (60-days-old) were injected with DBP (0.25 µmol/gland/50 µl DMSO) by i.m. injection under the nipple region of third, fourth and fifth mammary glands on both the sides (12). This model was chosen since it has been documented to produce mammary tumors in rats (2). In a separate experiment, animals were also administered DBP (1.5 µmol/300 µl vehicle) by either intragastric (i.g.) or intraperitoneal (i.p.) routes. Control animals received either DMSO (i.m. and i.p.) or corn oil (i.g.). The animals were maintained on standard rodent chow diet and water ad libitum and were killed 6 h, and 2, 5 and 14 days post-treatment, respectively. Crude tissue nuclei and mammary epithelial cells, obtained by collagenase treatment (13), were sequentially digested with RNases T1 and A and proteinase K, and solvent extracted, followed by precipitation with ethanol to obtain pure DNA (14). The DNA preparations were virtually free of RNA and protein. DNAs were digested with micrococcal nuclease and spleen phosphodiesterase (enzyme:DNA, 1:5; 5 h, 37°C), followed by adduct enrichment with nuclease P1, as described (14). Enriched adducts were then labeled with molar excess of [{gamma}-32P]ATP (100 µCi, <2 µM; <3000 Ci/mmol) and separated by multi-directional PEI–cellulose TLC using the following solvents: D1, 1.7 M sodium phosphate (pH 6.0); D3, 4 M lithium formate/7 M urea (pH 3.5); D4, isopropanol:4 M ammonia (1.2:1); D5, 1.0 M sodium phosphate (pH 6.0) (8,12). DNA adducts were visualized and quantified by a Packard InstantImager, and adduct levels were determined by relative adduct labeling (14).

Intramammillary injection of DBP produced one predominant and at least six other adducts in the mammary epithelial DNA at all time points tested (Figure 1BGo). Various non-target tissues (lung, heart, pancreas and bladder) had a qualitatively identical adduct pattern (Figure 1C, GoE, F and G), which is chromatographically comparable with that found following metabolism of DBP by rat liver microsomes (8) and MCF-7 cells (10). Interestingly, the liver showed at least four additional adduct spots (Figure 1DGo, nos 8–11) which comprise 30% of the total adduct level. Adduct 6 was the major adduct in all tissues. Intensities of adduct spots 4 and 5, presumably derived from syn-DBPDE, in the liver were significantly lower than those in other tissues such as the heart and lung. This suggests that the ratio of metabolically formed syn- and anti-DBPDE is different or the syn-DBPDE adducts are more efficiently repaired in the liver as compared with the other tissues examined.



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Fig. 1. Representative 32P-adduct maps of mammary (B), lung (C), liver (D), heart (E), pancreas (F) and bladder (G) DNA isolated from rats treated with a single i.m. injection of DBP (0.25 µmol/gland) or vehicle (DMSO)-treated lung DNA (A). Five micrograms of labeled DNA digest were loaded onto PEI–cellulose sheets and the adducts were resolved by multidirectional TLC. The DNA adducts in various tissues were visualized by screen enhanced autoradiography at –80°C for 24 h, using Dupont Cronex-4 films, except in mammary tissue where the maps were exposed for 5 h at –80°C.

 
Measurement of adduct radioactivity revealed that the mammary DNA was most adducted (2640 ± 532 adducts/109 nucleotides) after a single i.m. injection of DBP, while the non-target tissue DNAs were 10- to 65-fold less adducted (Figure 2Go). Adduct levels were the highest on day 14 in all tissues, except mammary tissue which showed a decline of ~40% as compared with the peak binding on day 5. The higher DNA binding in mammary tissue may be due to the direct injection of carcinogen at the target site, resulting in its trapping in the adipose tissue, which may eventually make it biologically more readily available to the mammary tissues (12,15). In the mammary gland, the cell turnover is generally reported to be higher than the other tissues, for example lung and liver (16). Thus, the decrease in the DBP–DNA adducts in mammary tissue on day 14 could be in part attributable to the increased cell turnover rate, in addition to DNA repair. On the contrary, the increase in the DBP–DNA adduction over a period of time in the non-target tissues could be due to gradual and continuous release of the trapped compound to the distant organs through systemic circulation (15). Recently, it has been reported that the majority of DBP–DNA adducts in mouse skin were found to adopt an intercalated conformation which render them less efficiently repaired than the external adducts (17). Furthermore, as a result of less efficient repair, DBP–DNA adducts can accumulate over a period of time which may eventually shift the peak binding time in the non-target tissues. Peak DNA bindings for several PAHs such as B[a]P, DMBA, dibenzo[a,e]pyrene, dibenzo[a,h]pyrene, and DBP in mouse skin and lung, have been reported to be between 1 and 4 days (18,19).



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Fig. 2. Comparative DBP–DNA adduct levels in mammary, lung, liver, heart, pancreas and bladder. All tissues, except lung and mammary were pooled from three to four animals and data represent averages of three analytical replicates ± SD (the liver data are from a single analysis).

 
Administration of DBP by i.p. and i.g. routes resulted in essentially the same adduct patterns in the mammary and lung tissues examined (maps not shown), as formed by the i.m. route (Figure 1Go). However, quantitatively, the i.m. route resulted in significantly higher DNA adduction; the i.g. route was least effective (Table IGo).


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Table I. DBP–DNA adduct levels in mammary and lung tissues of rats treated by different routesa
 
Multiple isoforms of CYP have been suggested to be involved in the metabolism of a number of PAHs, including DBP in vitro (7,8,12,20). Our previous results showed that rat liver microsomal activation of DBP was almost completely abolished in the presence of {alpha}-naphthoflavone, a known inhibitor of CYP1A family (8). Furthermore, it was supported by another report where the recombinant human CYP1A1 was found most active in metabolizing DBP (7). However, in human tissues and MCF-7 cells, CYP1A1 has been reported to be virtually inactive in the absence of inducers (21). Recently, CYP1B1, a PAH-inducible isoform found in several extrahepatic human tissues, has been suggested to play an important role in the metabolic activation of various PAHs, including DBP (2225). Studies with MCF-7 cells also revealed that CYP1A1 was not inducible with low doses of DBP and, thus, the DBPDE–DNA adducts resulted exclusively through the constitutive expression of CYP1B1 (25). Furthermore, the presence of highly polar DNA adducts in V79 cells expressing human CYP1A1, in contrast to the MCF-7 cells, suggested that CYP1B1 could be responsible for the metabolic activation of low doses of DBP in MCF-7 cells (24,26). It appears from several reports that CYP1A1 may be playing a major role in DBP metabolism by rat liver microsomes in vitro; however, in MCF-7 cells and in vivo systems, it could be preferentially metabolized by CYP1B1 (7,8,24).

Furthermore, CYP1A1 can metabolize DBP to at least three intermediate metabolites, namely 8,9-, 11,12- and 13,14-dihydrodiols (7). Recently, Nesnow et al. have shown that the 8,9-diol of DBP can be further metabolized to at least three ultimate metabolites (27) which can bind to DNA. In addition, several highly polar adducts have been observed (24,27). The present results along with our published data (12) revealed the presence of several liver-specific DBP–DNA adducts, which were chromatographically distinct from syn- and anti-DBPDE–DNA adducts. It is possible that these liver specific adducts are derived from further metabolism of the 8,9-dihydrodiol; however, it is unlikely that these adducts are highly polar since they did not migrate in D1 (1.7 M sodium phosphate).

Chromatographic comparison of DNA and individual nucleotide adducts formed by reaction with syn- and anti-DBPDEs showed that the in vivo adducts were both deoxyadenosine- and deoxyguanosine-derived, formed by interaction with both syn- and anti-DBPDEs, as published earlier (12). However, the adenine-derived adducts were found to be ~60–75% of the total adducts. Our results indicate that adenine in DNA is the major site of adduction with DBP in rat tissues and is in agreement with the published reports (8,10,12,17,26). The extensive binding of DBP to adenine residues in DNA, as reported earlier for other PAHs such as DMBA, benzo[c]phenanthrene and benzo[g]chrysene (2831), may actually be responsible for its high carcinogenicity.

Our results suggest that DBP exposure can produce several lipophilic DNA adducts in target as well as non-target tissues of rats which can persist for a long period of time. Furthermore, abundance of adenine-derived adducts of DBP in the tissue DNA may eventually contribute to its exceptionally known high carcinogenicity.


    Acknowledgments
 
This work was supported in part by a grant from the National Cancer Institute (CA77114). W.A.S. was supported in part from the NIEHS training grant ES07266.


    Notes
 
3 To whom correspondence should be addressed Email: rcgupta{at}pop.uky.edu Back


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Received December 11, 1998; revised February 1, 1999; accepted February 5, 1999.