Formation and persistence of benzo(a)pyrene metabolite-DNA adducts.

Benzo(a)pyrene (BP) and other polycyclic aromatic hydrocarbons (PAH) are ubiquitous environmental pollutants and are suspected to be carcinogenic in man. The in vivo formation of BP metabolite-DNA adducts has been characterized in a variety of target and nontarget tissues of mice and rabbits. Tissues included were lung, liver, forestomach, colon, kidney, muscle, and brain. The major adduct identified in each tissue was the (+)-7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydro-BP (BPDEI)-deoxyguanosine adduct. A 7 beta, 8 alpha-dihydroxy-9 beta,10 beta-epoxy-7,8,9,10-tetrahydro-BP (BPDEII)-deoxyguanosine adduct, a (-)-BPDEI-deoxyguanosine adduct, and an unidentified adduct were also observed. The adduct levels are unexpectedly similar in all the tissues examined from the same BP-treated animal. For example, the BPDEI-DNA adduct levels in muscle and brain of mice were approximately 50% of those in lung and liver at each oral BP dose used. We have also examined adduct levels formed in vivo in several cell types of lung and liver. Macrophages, type II cells, and Clara cells from lung and hepatocytes and nonpparenchymal cells from liver were isolated from BP-treated rabbits. BPDEI-deoxyguanosine adduct was observed in each cell type and, moreover, the levels were similar in various cell types. These and previous results strongly suggest that DNA in many human tissues is continuously damaged from known exposure of humans to BP and other PAH. Moreover, DNA adducts formed from BP are persistent in lung and brain.(ABSTRACT TRUNCATED AT 250 WORDS)


Introduction
Benzo(a)pyrene (BP) and other polycyclic aromatic hydrocarbons (PAH) are ubiquitous environmental pollutants produced mainly by industrial and transportational sources (1,2). Since these compounds are carcinogenic in laboratory animals and since human exposure to these compounds in food, air and water has been increasing, these chemicals pose a threat as potential human carcinogens.
Laboratory studies as well as epidemiological studies support this idea (3)(4)(5)(6)(7)(8)(9)(10)(11)(12). PAH induce tumors in various tissues of animal species, regardless of route of administration. One model of BP-induced neoplasia is the intratracheal administration to hamsters of BP adsorbed to particulate matter such as Fe2O3 (13). This causes respiratory tract tumors in the hamster and is relevant to human exposure, since atmosphere BP is also adsorbed to particulates. Epidemiological studies on health effects of PAH, though difficult to obtain because of widespread but low or varied exposure, have shown that *National Institute of Environmental Health Sciences, P.O. Box 12233, Research Triangle Park, NC 27709. gas production workers and coal tar pitch workers do have higher incidences of lung cancer in addition to skin and bladder cancers (14)(15)(16)(17)(18). Living in areas with high pollution increases lung cancer incidence (19)(20)(21). Studies have also shown that cigarette smoking is the major cause of lung cancer (20,22). All of these conditions result in exposure to PAH as well as other classes of carcinogens.
The mechanism(s) by which BP and other PAH induce neoplasia are quite complicated. In the body, these lipophilic compounds are oxidatively metabolized to epoxides, phenols, etc. (Fig. 1), by the cytochrome P-450dependent monooxygenase and epoxide hydrase. These metabolites are then conjugated to more hydrophillic metabolites by various conjugating enzyme systems, presumably for excretion. Most of the metabolites are excreted but sometimes the enzyme system will convert the parent compound to a more reactive form that can bind extensively and covalently to cellular macromolecules (23)(24)(25)(26). This covalent binding of reactive metabolites of PAH to DNA appears to be an essential first step in PAH-induced neoplasia (23)(24)(25)(26)(27)(28). If the cell cannot repair the damaged DNA before synthesis occurs, then replication on the damaged template can result in a mutation.
In Vivo Formation of PAH Metabolite-DNA Adducts Several PAH have been examined for their formation of DNA adducts in vivo. In this section, the in vivo DNA binding of four PAH: BP, 3-MC, DMBA, and 15, 16-dihydro-11-methylcyclopenta[a]phenanthrene-17one (11-methyl ketone) will be discussed. BP, the most extensively studied PAH, will be the topic of most of this discussion. Anderson (30) have shown that BPDEI and BPDEII bind to lung, liver, colon, kidney, muscle, brain, and forestomach of the A/HeJ mouse and to the lung, liver, colon, muscle, brain, and blood of the New Zealand White rabbit (Tables 1 and 2). Eastman et al. (32) observed the formation of BP metabolite-DNA adducts in lung, liver, and kidney of mice. Dunn et al. (31) showed that BP metabolites bind to DNA in liver, stomach, colon, and intestine of Swiss mice following oral doses of BP. The adduct levels in liver and intestines were similar and these levels were 2 to 4 times higher than those in stomach and colon. Several investigators have shown that BP metabolites also bind to DNA in the skin of several mouse strains (29,(69)(70)(71)(72)(73)(74)(75). In all cases, the major metabolite is the (+)-BPDEI bound to the N-2 of guanine residues as shown in Figure 4. As shown in the chromatogram, other adducts are also consistently observed in vivo: (-)-BPDEI-dGuo, BPDEII-dGuo and an unidentified peak. BPDEI and BPDEII may also bind to adenine residues except in smaller amounts. It is important to note that these adducts are formed in both tissues susceptible to PAH-induced neoplasia (target tissues) and tissues resistant to PAH-induced neoplasia. Similar adduct patterns are seen in each examined tissue in mice and rabbits regardless of dose, route of administration, or time of sacrifice after dosing (30). In addition to similar adduct patterns in different tissues and species, it is surprising to find that the levels of the BP metabolite-DNA adducts are similar in tissues of the same BP-treated animal and that this similarity in adduct levels is independent of the oral dose level. As seen in Table 1, Stowers and Anderson (30) observed similar (+)-BPDEI-dGuo adduct levels in several tissues of the A/HeJ mouse. At both dose levels of BP, there was no more than a 2-fold difference in the specific activities of the BPDEI-dGuo adduct in the seven tissues examined. Adriaenssens et al. (76) showed similar results in the lung, liver, and forestomach of mice over a wider dose range of 2 to 1351 ,umole/kg. These studies suggest no first pass effect in the liver for adduct formation after oral administration of BP to mice.
Similar BP metabolite-DNA adduct levels may also be seen after IV administration of BP. Table 2 shows that (+ )-BPDEI-dGuo levels in several tissues of the rabbit after an IV dose of 4 ,mole BP/kg. The liver, brain, and colon had the same adduct levels while the adduct levels in the muscle and blood were slightly higher. The levels of (+)-BPDEI-dGuo adduct in the lung were even higher, almost three to four times those in the other tissues. Eastman et al. (32) saw a similar pattern of binding levels in the A/J mouse after IV administration of BP. The levels of (+ )-BPDEI-dGuo in the lung were three to five times those in the liver or kidney. Another study by Eastman and Bresnick (77) with a different PAH, 3-MC, shows the similar pattern of relative binding of adducts in various tissues after an IV dose in several mouse strains. In the four mouse strains examined, there is a 2-to 4-fold higher amount   Table 2. The arrows (l) denote positions of the internal standards (MB, methyl-p-hydroxybenzoate; PB, propylp-hydroxybenzoate; and 9,10-diol, BP-9,10-diol).
of 3-MC metabolites bound to DNA in the lung than in the liver. These reports indicate that there is a firstpass effect in the lung for adduct formation after IV adminstration of BP as well as other PAH causing the higher relative binding of PAH metabolites to DNA of the lung. In contrast to mice and rabbits, no generalization can be made at present on the binding of BP metabolites to DNA of rat tissue. In the study by Boroujerdi et al. (78), the BPDEI-dGuo adduct was not the major adduct observed in lung and liver 1 hr after an IV dose of BP. isolated from each pooled tissue (lung, liver, forestomach, brain, colon, kidney, and muscle) and enzymatically digested to deoxyribonucleosides. The deoxyribonucleosides were chromatographed on HPLC. bThese numbers represent the specific activity (pmole/mg DNA) of the (+ )-BPDEI-dGuo (peak III in the chromatogram in Fig. 2).
The major adduct formed probably results from the interaction of 9-hydroxy-BP-4,5-oxide with DNA. However, Baer-Dubowska and Alexandrov (79) observed the same proffle in rat skin as seen in mouse after topical application of BP. It is also interesting that studies in cultured rat hepatocytes yield confficting data: Ashurst and Cohen (80) observed that the BPDEI-dGuo as the major adduct whereas Jernstrom et al. (81) observed the same major adduct as seen by Boroujerdi et al. (78). Obviously, more data are needed to resolve these differences observed in BP metabolite-DNA adduct proifies in rat tissue. In any case, as in rabbit and mouse, adducts were observed in each tissue examined and in the study by Boroujerdi et al. (78), the adduct binding levels were similar in lung and liver.
DMBA binding to DNA has been studied in the skin of several mouse strains (69). In all mouse strains studied, Sephadex LH20 chromatography of the DMBA metabolite-deoxyribonucleoside adducts in the skin were similar, with three peaks being observed (69). The major DMBA-DNA adducts appear to arise through the reaction of the bay region diol epoxide of DMBA with deoxyguanosine and deoxyadenine residues in DNA.
Abbott and Crew have examined the in vivo binding of 11-methyl ketone metabolites to DNA in lung, liver and skin of TO mice (82,83). The major adduct was formed from the interaction of the anti-3,4-dihydro-3,4, trans-dihydroxy-1,2-dihydro-1,2-epoxide metabolite with deoxyguanosine (84). This diol epoxide bound to deoxyguanosine was observed regardless of tissue susceptibility to PAH-induced neoplasia or route of administration (intramuscular, topical, or intraperitoneal). Similar total carcinogen-DNA binding levels were also seen in the three tissues after intravenous adminstration.
There are several possible explanations for the binding of PAH metabolites to the DNA of all tissue of the mouse and rabbit that have been studied. It is possible that oxidative metabolism of PAH in each tissue is sufficient to account for the observed DNA binding since monooxygenase activity has been detected in most tissues (85,86). However, with BP, there is obviously no correlation between the cytochrome P-450-dependent monooxygenase activity and the DNA binding. Because there is a 400-fold difference between mouse brain microsomes and liver microsomes in the metabolism of BP (87), it is surprising to see only a 2-fold difference in the BPDEI-dGuo adduct between the two tissues. Another incongruity is that the muscle, a tissue that has no detectable activity, still shows an appreciable amount of binding in the mouse and the rabbit. Another possible cause of similar adduct levels in various tissues could be compartmentalization of some cytochrome P-450 activity in the nucleus; although no data has been obtained to support this theory.
Other studies suggest that transport via a carrier protein can be responsible for the presence of BP metabolites in cells unable to metabolize this carcinogen. Hanson-Painton et al. (88) have shown that cytosolic proteins that transport BP from microsomes do exist. b Specific activity of a peak not quantitated unless the counts in the peak were at least 100 dpm above background.
Others have shown a similar cytosolic protein to bind to and transport 3-MC. These proteins probably bind and transport BP and other PAH to their site of metabolism in microsomes (89)(90)(91) ,umole/mouse. In lung and liver, the dose-response curve were sigmoidal whereas the forestomach dose-response curve was more linear. In each ofthese studies the doseresponse relationships for PAH metabolite-DNA adducts approached linearity at low doses and, thus, there does not appear to be a threshold dose below which the binding of PAH metabolites to DNA does not occur. In summary, PAH metabolites have been found to bind to DNA in vivo in every tissue that has been examined. This occurs regardless of species, dose, and route of administration. It is also quite surprising to find that similar levels of adducts are formned in vivo in the various tissues of the same PAH-treated animal whether the tissue is a target for PAH-induced neopla-sia or not. Small differences in the relative binding levels are most likely due to route of administration and probable first-pass effects in particular tissues. The major PAH metabolite-DNA adduct observed has been a bay region diol epoxide metabolite bound to a deoxyguanosine residue. Previous studies have shown that this bay region diol epoxide-deoxyguanosine adduct is also the major adduct formed in human cell culture incubated with BP. These findings have strong toxicological implications because of the low but continuous exposure of humans to BP and other PAH in the environment.

Persistence and Repair of Adducts
Several in vivo studies have attempted to determine if persistence of PAH metabolite-DNA adducts in a particular tissue is causally related to its susceptibility to PAH-induced neoplasia. The data of Kulkarni et al. on persistence of BPDE adducts offer no explanation for the strain difference in susceptibility to BP-induced pulmonary adenoma (94). The data suggest that adducts may be more persistent in lungs of the resistant C57BL/ 6J strain than in the susceptible A/HeJ strain (Fig. 5). Several other studies are in agreement with this conclusion. Phillips et al. (69) showed that there was no correlation between persistence of DMBA metabolite-DNA adducts in mouse skin and susceptibility ofvarious mice to PAH-induced neoplasia. Similar conclusions were reached with BP and 3-MC, although adduct levels were examined at only two time points (32). Pelkonen et al. (95) examined the disappearance of BP metabolite-DNA adducts in skin and subcutaneous tissues of C3H and C57BL/6 mice. The rates of disappearance of the adducts do not differentiate between the C57BL/6 resistance and the C3H susceptibility to BP-initiated subcutaneous fibrosarcomas. In contrast, Eastman and Bresnick (77) reported that the persistence of 3-MC metabolite-DNA adducts in mouse lung correlated with susceptibility of the various mouse strains to 3-MC-induced pulmonary adenomas (Fig. 5). The reasons for the discrepancy between the results of Eastman and Bresnick (77)  strains of mice, respectively (94); (U,*, 0, x) disappearance of 3-MC metabolite-DNA adducts in livers of A/J, C3H/HeJ, DBA/ 2J, C57BL/6J strains of mice, respectively (77). not explain tissue susceptibility to PAH-induced neoplasia in various mice strains. However, it should be emphasized that the specific activities of the PAH metabolite-DNA adducts reported in the above-mentioned studies are calculated on the basis of the total DNA in the organ. It is possible that the amounts of adducts formed as well as their repair rates in different cell types of the target organ may vary considerably. Examination of the formation and persistence of PAH metabolite-DNA adducts in individual cell types of the target tissue might allow differentiation of tissues with respect to susceptibility and resistance to PAH-induced neoplasia. A summary of the in vivo disappearance of PAH metabolite-DNA adducts in lung and liver of various mice strains is illustrated in Figures 5 and 6. The adduct level present in the tissue at a given time point is expressed as a percent of the initial value measured after administration of the PAH. The variations in disappearance rates are much greater in liver than in lung. In fact, the similarity in disappearance rates in lung is surprising.
Measurements of in vivo disappearance rates may reflect enzymatic excision repair; however, cell turnover can also account for adduct disappearance. In a recent study, Kulkarni and Anderson (96) examined BPinduced unscheduled DNA synthesis (UDS) in lung and liver of A/HeJ mice. UDS is a direct measurement of excision repair. BP induced UDS in liver but not in lung at the doses and time points examined. The procedure was able to detect UDS in lung, since 4NQO-induced UDS was observed (96). Thus the observed disappearance of BPDE adducts in liver of A/HeJ mice (Fig. 6) is due, at least in part, to excision repair whereas the disappearance in lung (Fig. 5) results from cell turnover (96). Abbott and Crew (83) also showed that normal DNA turnover rates could account for the disappearance of DNA adducts formed from metabolites of 11- z methyl ketone in lungs and skin of mice whereas excision repair was probably involved in removal of adducts from liver. Thus, both excision repair and cell turnover must be considered in assessing the mechanisms of in vivo removal of carcinogen metabolite-DNA adducts.
The presence of excision repair in the liver and the low rate of DNA synthesis by this tissue may provide an explanation for the relative resistance of this tissue to carcinogenesis by BP, since, under altered conditions of DNA replication following hepatectomy, tumors can be induced by PAH treatment (12,97,98). In contrast to the liver, the lack of excision repair in vivo of BP metabolites and the relatively high rate of DNA turnover in lung may be favorable conditions for the fixation of promutagenic lesions. The same arguments would hold for 11-methyl ketone, since lung and skin are target tissues whereas the liver is resistant to carcinogenesis by 11-methyl ketone under normal conditions. Thus, the balance between DNA repair and DNA replication is an important consideration in the study of mutagenesis and carcinogenesis.
In summary, DNA adducts formed from BP and other PAH are relatively persistent in tissues such as lung, skin, and brain. Persistence of PAH metabolite-DNA adducts in these tissues could be the result of particular cell types lacking excision repair. If these cell types also have slow turnover rates, accumulation of significant levels of PAH metabolite-DNA adducts could occur, especially if there is continuous long-term exposure of even low levels of PAH. The result of the persistence of bulky adducts on the DNA template could be inhibition of replication or transcription (99). Even if environmental exposure to PAH is too low to be tumorigenic, the persistence of DNA adducts may produce aberrations in transcripts of genetic information in various organs and lead to other toxic effects.