400
Chem. Res. Toxicol. 2002, 15, 400-407
UVA Light-Induced DNA Cleavage by Isomeric Methylbenz[a]anthracenes Shiming Dong,† Peter P. Fu,‡ Rujendra N. Shirsat,† Huey-Min Hwang,§ Jerzy Leszczynski,† and Hongtao Yu*,† Department of Chemistry, Jackson State University, Jackson, Mississippi 39217, National Center for Toxicological Research, Jefferson, Arkansas 72079, and Department of Biology, Jackson State University, Jackson, Mississippi 39217 Received September 21, 2001
UVA light-induced DNA single strand cleavage by a set of 12 monomethyl substituted benz[a]anthracenes (MBAs) along with their parent compound, benz[a]anthracene (BA), and the potent carcinogen, 7,12-dimethylbenz[a]anthracene (DMBA), was studied. On the basis of the relative DNA single strand photocleavage efficiency of the fourteen compounds, they are divided into three groups: (1) strong DNA cleavers, 4-MBA, 5-MBA, 6-MBA, 8-MBA, 9-MBA, 10-MBA, and BA; (2) medium DNA cleavers, 1-MBA, 2-MBA, 3-MBA, and 11-MBA; and (3) weak DNA cleavers, 7-MBA, 12-MBA, and DMBA. The relative DNA photocleavage efficiency parallels very well with the energy gap between the highest-occupied-molecular-orbital (HOMO) and the lowest-unoccupied-molecular-orbital (LUMO) of each MBA, indicating that the DNA cleavage is related to their excited-state properties. The 7 and 12 positions of BA are two unique sites. Methyl substitution at either 7 or 12 (or both) positions lowers the HOMO-LUMO gap and greatly diminishes the DNA photocleavage efficiency. UVA light-induced photodegradation of selected MBAs reveals that methyl substitution at either 7 or 12 (or both) positions greatly enhances the degradation rate. Photodegradation of 7-MBA, 12-MBA, and DMBA yields products that are much less effective in mediating DNA cleavage. Photodegradation of other MBAs, exemplified by 5-MBA, yields a photooxidation product 5-MBA-7,12-quinone which is relatively stable under light and is a stronger DNA photocleaver than 5-MBA itself. The higher efficiency of DNA photocleavage for MBAs with methyl substitution at positions other than 7 or 12 is due, at least in part, to the formation of 7,12-quinone. Light-induced DNA single strand cleavage efficiency for several MBAs parallels the light-induced toxicity observed by other research groups, suggesting that light-induced DNA cleavage of MBAs are the source for phototoxicity. Since some PAHs such as coal tar are used commercially as creams, therapeutic agents, or ointments, or those roofers and asphalt workers that are subject to contamination with PAHs, the combination of PAHs and light (in the skin) may present a greater health risk to humans.
Introduction Polycyclic aromatic hydrocarbons (PAHs)1 are widespread environmental pollutants. Many of them are cytotoxic, mutagenic, and carcinogenic and can induce cancers in human (1-4). It is widely accepted that PAHs require metabolic activation in order to exert their biological activities, including carcinogenicity (1-5). After entering the cell, PAHs are either metabolized into diol epoxides, quinones, or free-radical intermediates, and all these species can react with cellular DNA to form PAHDNA covalent adducts and cause other forms of DNA * To whom correspondence should be addressed. Phone: (601) 9793727. Fax: (601) 979-3674. Email:
[email protected]. † Department of Chemistry. ‡ National Center for Toxicological Research. § Department of Biology. 1 Abbreviations: BA, benz[a]anthracene; MBA, monomethyl substituted benz[a]anthracene; DMBA, 7,12-dimethylbenz[a]anthracene; PAH, polycyclic aromatic hydrocarbon; DMF, dimethylformamide; DTT, dithiothreitol; SOD, superoxide dismutase; ROS, reactive oxygen species; 1O2, singlet oxygen; LUMO, lowest unoccupied molecular orbital; HOMO, highest occupied molecular orbital; UVA, ultraviolet light in the region of 320-400 nm; sc-DNA, supercoiled form I DNA; oc-DNA, open circular form II DNA.
damages (1-10). Some of the DNA adducts can produce mutations and are considered to be the source for carcinogenicity. It has been shown that light irradiation can activate PAHs to exert toxicity, as well. Previous studies (1114) have shown that some PAHs, if irradiated by light, are more toxic to microorganisms, plants, and other organisms than PAHs themselves without light irradiation. The light-induced toxicity, or phototoxicity, of some PAHs can be more than 100 times higher than their parent compounds (11, 12). Structure-activity relationship studies have found that the phototoxicity depends on PAH structures (15-19). DNA single strand cleavage (20), formation of DNA covalent adduct (21), and the formation of oxidative product 8-oxoguanine (22) have been observed when PAHs and DNA are irradiated together. It has been shown that DNA cleavage efficiency by individual PAHs depends on its structure and solvent used (20). Studies concerning how substituents affect the metabolism of PAHs have provided insight into the structure-activity relationships and consequently a better
10.1021/tx015567n CCC: $22.00 © 2002 American Chemical Society Published on Web 02/02/2002
Photoinduced DNA Damages and Phototoxicity of PAHs
Figure 1. Structure of benz[a]anthracene and its ring-numbering system.
understanding of the mechanisms by which these compounds exert their carcinogenic activities (1, 2, 23-28). Substitution strategy can also probe the geometric regions of PAHs that are involved in metabolic activation. In this regard, methyl and fluoro substituents have long been employed to determine the structural features of the parent PAHs that can correlate with tumorigenicity (1, 2, 23-27). The effects of the methyl and fluoro substituents include (1) inhibition or reduction of metabolism at the substituted aromatic double bond, (2) significant reduction of the enzymatic oxidation at the region ortho to the substituent, and (3) either complete or partial inhibition of metabolism peri to the substituent. Benz[a]anthracene (BA, Figure 1) and its 12 monomethyl substituted derivatives (MBAs) have been employed as model compounds for studying the structure-activity relationships of PAHs leading to tumorigenicity. Among the 12 MBAs, 6-, 7-, 8-, and 12-MBAs have been shown to be more tumorigenic than BA, their parent compound (23-25). In this study, relative UVA-induced DNA single strand cleavage efficiency for a set of 12 MBAs, their parent compound BA and the 7,12-dimethylated analogue DMBA was determined, and these results were compared with their HOMO-LUMO gaps and ground-state energies. The effects of the position of the methyl substitution on the photodegradation and DNA cleavage mechanisms for individual MBAs are discussed. The relative DNA cleavage efficiency of individual MBAs is compared with its relative phototoxicity, tumorigenicity, and carcinogenicity.
Materials and Methods Materials. 5-, 6-, 7-, and 12-MBAs were synthesized through total synthesis, as previously described (24, 29). 1-, 4-, 8-, and 11-MBAs were synthesized by methylation of 1- and 4-keto1,2,3,4-tetrahydro-BA, or 8- and 11-keto-8,9,10,11-tetrahydroBA, respectively, with CH3MgBr followed by p-toluenesulfonic acid catalyzed dehydration and aromatization by DDQ (24, 30). 2-, 3-, 9-, and 10-MBAs were purchased from the NCI Chemical Carcinogen Reference Standard Repositories at the Midwest Research Institute (Kansas City). BA, BA-7,12-quinone, and DMBA were purchased from Sigma-Aldrich. 5-MBA-7,12quinone was synthesized as previously described (29). All of the MBAs were analyzed by HPLC and found to be >99% pure. Other chemicals and solvents were purchased from either Sigma-Aldrich or Fisher Scientific. UVA Light-Induced DNA Cleavage by MBAs, BA, and DMBA. UVA light-induced DNA single strand cleavage by MBAs was carried out with protocols described in previous publications (20, 21). Supercoiled ΦX174 phage DNA (27 µM in base pairs, Sigma-Aldrich, St. Louis) was mixed with a substrate with or without the addition of a scavenger to make a total of 60 µL of solution. These solutions were placed in the wells of a 3 × 8 Titertek plate (ICN Biochemicals). The plate was placed on top of a Pyrex glass support and a UVA lamp (100 W type B from UVP Inc., Upland, CA) was used to irradiate the sample from below the glass support from a distance of 6.5 cm for 60
Chem. Res. Toxicol., Vol. 15, No. 3, 2002 401 min. The output light energy after the Pyrex filter was 170 J cm-2 h-1. A stream of cold air blowing on the bottom of the glass support was used to maintain the temperature. The Titertek plate was turned every 15 min to eliminate light heterogeneity. After irradiation, each sample was mixed with 8 µL of a dye solution (bromophenol blue and xylene cyanole in 50% glycerol) and 10 µL of the mixture was loaded onto a pre-prepared 1% agarose gel. The scavengers used for these experiments were dithiothreitol (DTT), NaN3, KI, mannitol, and superoxide dismutase (SOD). Due to single strand cleavage, the supercoiled form I DNA (sc-DNA) was converted into a relaxed open circular form II DNA (oc-DNA). The two DNA forms were separated by agarose gel electrophoresis and quantified to obtain the percent of DNA cleavage as previously described (20). A plot of the percent of DNA cleavage versus the substrate concentration was used to determine C25, the substrate concentration at which 25% of the sc-DNA is converted to the oc-DNA. Usually, a rough C25 value was determined using a larger concentration range of a substrate (1-100 µM); then a triplicate of experiments at a concentration range near the C25 value were carried out to obtain a more accurate C25. The solvent used for these experiments was 10% dimethylformamide (DMF) in 10 mM sodium phosphate buffer (pH 7.1) to achieve a better solubility for the substrates. The experiment under argon was carried out in a quartz cuvette containing 500 µL of solution. The cuvette was stoppered with a rubber septum. Argon gas (99.99%, Nordan Smith, Jackson) was bubbled for 30 min with an inlet needle through the septum. A second needle was used as an argon gas outlet. After bubbling, the outlet needle and then the inlet needle were removed and the cuvette was placed on top of the Pyrex glass support and subjected to UVA light irradiation from the bottom of the Pyrex glass as described above. The experiment in D2O was carried out in 10% DMF in a 10 mM sodium phosphate buffer prepared in D2O (pH 7.1). Since the original ΦX 174 DNA solution was not in D2O, the estimated deuterium content was >95%. Time course of the substrate-induced DNA photocleavage was carried out in a quartz cuvette containing 500 µL of solution. A sample of 40 µL was taken at a given time interval and immediately stored in the refrigerator with the exclusion of light. After all samples were collected, they were treated as above and subjected to gel electrophoresis and quantified. UVA Light-Induced Degradation of MBAs. A 500 µL solution of 60 µM of BA, 5-MBA, 7-MBA, 12-MBA, or DMBA in the 10% DMF buffer (pH 7.1) in a quartz cuvette was irradiated with UVA light as described above. Samples (40 µL) were taken at proper time intervals and stored in the dark before HPLC analysis (HP1100, Agilent Technologies). The mobile phase was 90% methanol (1 mL/min). The concentration of an MBA at each irradiation time interval, [MBA], was determined by comparing the peak area with that of the MBA at time zero ([MBA]o). Then the plot of ln [MBA]o/[MBA] versus irradiation time yielded a straight line with the slope being the degradation rate constant, k, assuming first-order degradation kinetics. The half-life was then calculated by t1/2 ) 0.693/k. Photoproducts of BA and 5-MBA were determined by comparing both the retention time and the UV-absorption spectrum obtained by the diode array detector with those for authentic samples: BA-7,12-quinone and 5-MBA-7,12-quinone, respectively. Quantum Chemical Calculations. Ab initio quantum chemical calculations of the 12 isomeric MBAs, BA, and DMBA were performed using the density functional theory (31) hybrid B3LYP (32) at the STO-3G and the 6-31G basis sets. The minimum energy structures have been obtained for all the molecules under consideration by performing geometry optimization using the GAUSSIAN 98 program (33). The calculated harmonic vibrational frequencies were used to determine the nature of stationary points found by geometry optimization. The minimum energy geometries obtained with the 6-31G basis sets were used further for single point level (34) calculations. Ground-state energies and energy gaps between the highest-
402
Chem. Res. Toxicol., Vol. 15, No. 3, 2002
Dong et al.
Figure 2. UVA light-induced DNA cleavage by 5-MBA. Solutions containing 27 µM ΦX 174 phage DNA and 4, 8, 12, 14, 16, 18, 20 µM of 5-MBA (lanes 3-9) in 10 mM sodium phosphate buffer with 10% DMF (pH 7.1) were irradiated with a UVA lamp (170 J cm-2 h-1) for 1 h. Control lanes: lane 1 had only DNA and was irradiated for 1 h and lane 2 had DNA and 20 µM 5-MBA and was not irradiated. Table 1. Relative UVA Light-Induced DNA Single Strand Cleavage Efficiency (C25), Ground-State Energy, and HOMO-LUMO Gapa
BA 1-MBA 2-MBA 3-MBA 4-MBA 5-MBA 6-MBA 7-MBA 8-MBA 9-MBA 10-MBA 11-MBA 12-MBA DMBA
C25 (µM)
HOMO/LUMO Gap (eV)
MP2 energy (au)b
18 ( 1 60 ( 4 74 ( 2 34 ( 2 12 ( 1 13 ( 1 20 ( 1 101 ( 5 18 ( 2 17 ( 1 12 ( 1 42 ( 5 93 ( 10 120 ( 8
8.7321 8.7267 8.6804 8.7049 8.6887 8.7386 8.7185 8.6015 8.6968 8.7593 8.7457 8.7049 8.5906 8.4519
-689.9710 -732.3215 -732.3327 -732.3328 -732.3306 -732.3316 -732.3317 -732.3256 -732.3318 -732.3331 -732.333 -732.3316 -732.3164 -768.1876
a C 25 is the concentration for a substrate at which 25% of the supercoiled DNA is converted into the relaxed form after irradiation with UVA light for 1 h. b The ground-state energy is given in au (1 au ) 627.51 kcal/mol).
occupied molecular orbital (HOMO) and the lowest-unoccupied molecular orbital (LUMO) are used to correlate with the experimental C25 values.
Results and Discussion DNA Single Strand Cleavage Induced by the Combination of Light and MBAs, BA, and DMBA. All of the 12 MBAs, plus the parent compound BA and the potent carcinogen DMBA are shown to be able to cause DNA single strand cleavage when irradiated by light. A typical gel is shown for 5-MBA (Figure 2). The original ΦX 174 phage DNA has about 20% oc-DNA and 80% sc-DNA (as in lane 1). In the control experiments, whether the sample was irradiated under light in the absence of 5-MBA (lane 1) or the mixture of DNA and the highest concentration of 5-MBA (20 µM) was left in the dark (lane 2), the amount of the oc-DNA remained about 20%. However, if the mixture was irradiated for 1 h under UVA light in the presence of 4, 8, 12, 14, 16, 18, and 20 µM of 5-MBA, a gradual increase of the amount of the oc-DNA was observed. After converting the amount of the oc-DNA into percent DNA cleavage (20), the plot of the percent of DNA cleavage versus MBA concentration was used to determine C25. The C25 values are listed in Table 1 and plotted in Figure 3 (panel A). The smaller the C25, the higher is the DNA photocleavage efficiency. The C25 values for these compounds range from 12 to more than 120 µM. The relative DNA single strand cleavage efficiency of the
Figure 3. Plot of relative light-induced DNA cleavage (C25, panel A), HOMO-LUMO gap (panel B in negative value of the gap), and relative ground-state energy (panel C) for MBAs. The ground-state energy was converted to kcal/mol from au in Table 1. The energy for 9-MBA was used as a zero energy reference point.
fourteen compounds can be divided into three groups: (1) strong DNA photocleavers, 4-MBA, 5-MBA, 6-MBA, 8-MBA, 9-MBA, 10-MBA, and BA; (2) medium DNA photocleavers, 1-MBA, 2-MBA, 3-MBA, and 11-MBA; and (3) weak DNA photocleavers, 7-MBA, 12-MBA, and DMBA. Comparing with the parent compound BA, methyl substitution at the 4, 5, 6, 8, 9, and 10 positions has no effect on the DNA photocleavage efficiency (similar C25 values), while methyl substitution at either the 7 or the 12 or both positions dramatically decreases the DNA photocleavage efficiency (higher C25 values). Substitution at the 1, 2, 3 and 11 positions moderately decreases the DNA photocleavage efficiency. Structure and DNA Photocleavage Efficiency Relationship Based on Molecular Orbital Calculations. All species reported here are fully optimized minimum energy structures obtained at the B3LYP/631G level. Zero imaginary frequencies are found for all the structures in the vibrational frequency calculations. As discussed earlier, these geometries are further used for MP2 calculations in order to account for electron correlation effects. The results of the ab initio calculations are presented in Table 1 along with C25. It has been suggested that HOMO-LUMO gaps for PAHs are strong indicators for phototoxicity (17-19). Therefore, C25 values (panel A), the calculated HOMOLUMO gaps (panel B), ground-state energies (panel C) for the 12 MBAs are plotted together versus the position of their methyl substitution in Figure 3. In this graph, the negative values of the HOMO-LUMO gaps are used in order to have a more visual comparison with the C25
Photoinduced DNA Damages and Phototoxicity of PAHs
Chem. Res. Toxicol., Vol. 15, No. 3, 2002 403
Table 2. Effect of Scavengers on Light-induced DNA Cleavage by 5- and 7-MBAsa 5-MBA
7-MBA
scavengers
ROS quenched
percent of DNA cleavage
percent of inhibition (-) or enhancement (+)
percent of DNA cleavage
percent of inhibition (-) or enhancement (+)
none KI (50 mM) argon DTT (50 mM) NaN3 (50 mM) mannitol (50 mM) SOD (200 unit/mL) D2O (> 95% D)
N/A singlet excited state O2 free radical 1O /free radical 2 free radical superoxide enhance 1O2 lifetime
35 ( 2 5.1 ( 2.5 18 ( 4 8.5 ( 3.1 13 ( 1 35 ( 2 33 ( 1 60 ( 4
-85 -47 -76 -62 b b +71
31 ( 2 5.9 ( 2.4 31 ( 6 5.8 ( 2.1 15 ( 2 24 ( 3 32 ( 2 73 ( 6
-81 b -81 -51 -23 b +135
a All data were in triplicate. A mixture of 27 µM ΦX-174 DNA, 20 µM 5-MBA or 120 µM 7-MBA, and a certain concentration of a scavenger was irradiated by a UVA lamp for 1 h. b These indicates that the DNA cleavage is not affected within error.
values. It clearly demonstrates that the C25 values correspond well with the HOMO-LUMO gaps and the ground-state energy for most of the MBAs (Figure 3 and Table 1). For example, 7- and 12-MBA are the two least efficient for DNA single strand photocleavage (have largest C25), and they also have the smallest HOMOLUMO gaps and highest energies. The most efficient derivatives for DNA photocleavage, 4-, 5-, 6-, 8-, 9-, and 10-MBAs (have smaller C25), also have larger HOMOLUMO gaps and lower energies. The exceptions are 1-MBA and 2-MBA, both have relatively high C25 values (64 and 72 µM, respectively), but their HOMO-LUMO gaps are about the same as those for MBAs with low C25 values. The data obtained here strongly suggest a direct relationship between electronic structure and lightinduced DNA cleavage efficiency of the isomeric MBAs. Effect of Scavengers on DNA Photocleavage by MBAs. Involvement of oxygen or free-radical intermediates during the light-induced DNA cleavage by 5-MBA and 7-MBA (a representative each for the MBAs with high or low DNA cleavage efficiency, respectively) was examined. Table 2 lists the relative DNA photocleavage efficiency in the presence of the excited-state quencher KI (35), the free-radical scavengers DTT, NaN3, and mannitol, and superoxide free-radical scavenger SOD (36, 37). NaN3 has also been regarded as a singlet molecular oxygen (1O2) scavenger (38). In addition, the experimental results obtained in D2O or under an argon atmosphere (degassing for 30 min) are also listed in Table 2. While the experiment in D2O tests the presence of 1O2 since the lifetime for 1O2 is 10 times longer in D2O than in H2O (39), the experiment carried out under argon gives a direct signal whether oxygen is involved during DNA cleavage. The presence of KI quenches the DNA photocleavage for both 5- and 7-MBA, indicating that the excited-state of the MBAs is involved in DNA photocleavage. This is in agreement with the correlation of the C25 values with the HOMO-LUMO gaps described earlier. The DNA photocleavage induced by 7-MBA is not affected by degassing with argon, while degassing with argon decreases 40% of the photocleavage by 5-MBA. This indicates that molecular oxygen does not play a role in the rate-determining step for 7-MBA-induced DNA photocleavage, whereas it does for 5-MBA induced DNA photocleavage. DNA photocleavage is decreased by a similar percentage in the presence of free-radical scavengers DTT or NaN3 for both 5-MBA and 7-MBA. The presence of a weaker free-radical scavenger, mannitol, decreases the 7-MBA induced DNA photocleavage by 23%, but it has no effect on the photocleavage induced
by 5-MBA. The diminished DNA cleavage in the presence of DTT, NaN3, and mannitol indicates that free-radical intermediates and, possibly, 1O2 are involved in generating DNA cleavage. It is likely that both free-radical intermediates and 1O2 are involved for 5-MBA-induced DNA photocleavage, but only free-radical intermediates are involved for 7-MBA induced DNA photocleavage since elimination of oxygen by argon degassing did not affect the DNA cleavage for the latter. The strong enhancement (+71%) for 5-MBA induced DNA photocleavage in D2O further supports the involvement of 1O2. Actually, as will be discussed later, photolysis of 5-MBA converts it into 5-MBA-7,12-quinone, a quinone. Quinones, upon light activation, can sensitize the production of 1O2 (40, 41). However, the even stronger enhancement of the 7-MBAinduced DNA photocleavage in D2O cannot be explained by the involvement of 1O2 since degassing with argon has no effect on its DNA photocleavage. This might be due to the involvement of water in DNA cleavage. It has been demonstrated that free-radical-induced DNA single strand cleavage can occur either via the involvement of oxygen or water (42). If water is involved, an isotope effect on the DNA cleavage may be observed for experiments conducted in D2O or H2O. The combination of light and MBA (or its photoproducts) can produce ROS and PAH centered free-radical intermediates as discussed above. Both of which, if generated in the cell, can lead to damages not only to DNA but also to other biological macromolecules such as proteins and lipids. It has been widely accepted that ROS is responsible for aging, inflammation, cardiovascular diseases, and cancer (43-45). Also the PAH-centered radicals generated by photoexcitation mimics the enzymatic activation of PAHs that generates PAH cation radicals through one electron oxidation (8, 10). This pathway is proposed to activate DMBA to insert genotoxicity (8). Light-Induced Degradation of BA, 5-MBA, 7-MBA, 12-MBA, and DMBA. Light-induced degradation of MBAs in buffer solutions containing 10% DMF was monitored by HPLC. From plotting ln [MBA]0/[MBA] versus irradiation time, the first-order degradation rate constant k and thus the degradation half-life (t1/2 ) 0.693/ k) were obtained. The degradation half-lives for BA, 5-MBA, 7-MBA, 12-MBA, and DMBA are 56, 11, 3.5, 0.63, and 0.28 min, respectively (Table 3). It is clear that the degradation of BA is slower than any of the MBAs and the degradation of 5-MBA is slower than 7-MBA, 12MBA, or DMBA. In other words, the photodegradation rate of BA is accelerated by methyl substitution. It is accelerated 5 times by 5-methyl substitution, and 17 or
404
Chem. Res. Toxicol., Vol. 15, No. 3, 2002
Dong et al.
Table 3. Degradation Rate Constants and Half-Lives for BA, 5-MBA, 7-MBA, 12-MBA, and DMBAa k (min-1) t1/2 (min)
BA
5-MBA
7-MBA
12-MBA
DMBA
0.012 56
0.062 11
0.20 3.5
1.1 0.63
2.5 0.28
a Individual substrates (60 µM) were dissolved in 10% DMF and 90% 10 mM sodium phosphate buffer (pH 7.1) and were irradiated by a UVA lamp (170 J cm-2 h-1). Samples were taken at various irradiation time intervals and analyzed with HPLC. Column, reversed phase C18; mobile phase, 90% methanol, 1 mL/min.
92 times by 7- or 12-methyl substitution, respectively. If both the 7 and 12 positions are methyl substituted, the photodegradation rate is accelerated more than 200 times. Two main photoproducts for 5-MBA photodegradation were detected by HPLC (Figure 4). 5-MBA has a retention time of 9.8 min. After 45 min of irradiation, two main peaks appeared at 8.3 and 3.3 min, respectively. The 8.3 min peak was identified to be 5-MBA-7,12-quinone by comparison with the authentic sample. The 3.3 min peak and other possible minor photoproducts eluting at 2-3 min were not identified. After 3 h of irradiation, all of the initial 5-MBA (60 µM) disappeared, and the resulting 5-MBA-7,12-quinone reached a concentration of 43 µM. In other words, 72% of the original 5-MBA was converted to 5-MBA-7,12-quinone. The 5-MBA-7,12-quinone is relatively stable under light irradiation since there is no significant degradation after it is irradiated for 1 h. The C25 value for 5-MBA-7,12-quinone was determined to be 11 ( 1 µM, slightly smaller than 13 ( 1 µM for 5-MBA. Therefore, DNA photocleavage induced by 5-MBA is contributed at least partially by its photoproduct, 5-MBA7,12-quinone, since 5-MBA was converted to 5-MBA-7,12-quinone during the 1 h irradiation used for DNA cleavage studies. In the same way, the photodegradation of BA under the same conditions produced BA-7,12quinone, identified by comparing with the authentic sample. Konig et al. had reported the same observation (46). The C25 for BA-7,12-quinone is 9.6 ( 0.4 µM, similar to the C25 value for 5-MBA-7,12-quinone. Therefore, it is logical to assume that, in addition to BA and 5-MBA, the photodegradation of 1-, 2-, 3-, 4-, 6-, 8-, 9-, 10-, and 11-MBAs, where both the 7 and 12 positions are not substituted, will also produce the respective 7,12-quinones. Both the MBAs themselves and the photoproduct 7,12-quinone contribute to DNA photocleavage. In contrast, if the methyl substituent is at either the 7 or the 12 or both positions, the degradation rate greatly increases. The photoproducts for the degradation of 7 or 12-MBA were not identified. However, BA7,12-quinone was not observed in the HPLC chromatogram (data not shown). This is different from the photodegradation of DMBA carried out by Wood and coworkers (47). They identified the methyl oxidation products, singlet oxygen addition product 7,12-epidioxyDMBA, and a minor amount of the BA-7,12-quinone. Nonetheless, the photoproducts for 7-MBA, 12-MBA, or DMBA do not appear to cause a significant amount of DNA photocleavage. Therefore, they are not as potent DNA photocleavers as the other MBAs since they degrade quickly. Time Course for MBA-Induced DNA Photocleavage. To demonstrate that both MBA and its photoproduct MBA-7,12-quinone participate in causing DNA photocleavage, time courses for DNA photocleavage induced
Figure 4. HPLC chromatogram for 5-MBA in 10% DMF after 45 min of irradiation. Mobile phase, 90% methanol; flow rate, 1 mL/min; column, reversed-phase C-18. Insert is the diode array detector-captured absorption spectrum for 5-MBA-7,12-quinone at 8.3 min.
Figure 5. Time course of UVA-induced DNA cleavage by 5-MBA, 7-MBA, and DMBA. A solution of 500 µL of 27 µM ΦX 174 phage DNA and an MBA was irradiated by UVA lamp (170 J cm-2 h-1) and samples were taken at various irradiation time intervals and analyzed by gel-electrophoresis to determine the percent of DNA cleavage. The error bars were calculated from the data of triplicate experiments. The concentrations for 5-MBA, 7-MBA, and DMBA were 20, 100, and 100 µM, respectively.
by 5-MBA, 7-MBA, and DMBA were examined and plotted in Figure 5. 5-MBA was used as an example for all the MBAs without a methyl group at either the 7 or the 12 positions. The percent of DNA photocleavage increases in a biphasic manner during the 60 min of irradiation for 5-MBA, 7-MBA, and DMBA. For 5-MBA, in the first phase (before 8 min), it is the 5-MBA that is mainly causing DNA cleavage and in the second phase (after 8 min), it is the 7,12-quinone that is mainly causing DNA cleavage. To fully understand this complex kinetics, detailed kinetic analyses of the photolysis reaction and structural identification of the intermediate eluting at 3.3 min on HPLC are necessary. Since both 5-MBA and 5-MBA-7,12-quinone can cause DNA cleavage, the actual C25 for pure 5-MBA may be larger than the value in Table
Photoinduced DNA Damages and Phototoxicity of PAHs
1 since 5-MBA-7,12-quinone contribute partially to DNA photocleavage. The same plot for DMBA and 7-MBA shows that the percent of DNA photocleavage increases quickly in the first 5 min for DMBA and 8 min for 7-MBA and then gradually flattens out. This indicates that DMBA and 7-MBA degrade quickly and their photoproduct(s) are less effective in causing DNA photocleavage. Comparison of Relative Carcinogenicity, TumorInitiation Activity, and Phototoxicity with C25. The relative carcinogenic activity is DMBA > 7-MBA > 8-MBA ∼ 6-MBA ∼ 12-MBA > 9-MBA ∼ 10-MBA ∼ 11MBA > 5-MBA ∼ 1-MBA ∼ 2-MBA ∼ 3-MBA ∼ 4-MBA ∼ BA (5). The relative tumor-initiating activity is DMBA > 7-MBA > 12-MBA > 8-MBA ∼ 6-MBA ∼ 9-MBA g 10MBA ∼ 11-MBA > 5-MBA ∼ 1-MBA ∼ 3-MBA > 2-MBA ∼ 4-MBA ∼ BA (10). The relative efficiency for UVAinduced DNA single strand cleavage of the 12 MBAs is 4-MBA ∼ 5-MBA ∼ 10-MBA > 6-MBA ∼ 8-MBA ∼ 9-MBA ∼ BA >> 3-MBA > 11-MBA > 1-MBA > 2-MBA > 12-MBA ∼ 7-MBA ∼ DMBA. This pattern does not parallel the relative carcinogenic activity or the tumorinitiation activity. For example, while 7-MBA and 12MBA are the two most potent tumorigens among the 12 isomeric MBAs, they exhibit the lowest activity in UVAinduced DNA single strand cleavage. This indicates that both carcinogenic and tumorigenic activities of the MBAs are not related to UVA light-induced DNA single strand cleavage. Phototoxicity for BA, BA-7,12-quinone, and DMBA on larvae parallels the C25 values of these compounds. BA and BA-7,12-quinone have the same toxicity and are more toxic than DMBA in the toxicity tests conducted by Fernandez and L’Haridon (14). BA and BA-7,12quinone, 5-MBA and 5-MBA-7,12-quinone have similar C25 values and are much stronger UVA light-induced DNA cleavers than DMBA. These results suggest that DNA single strand cleavage caused by the combination of light and MBA may be responsible for the light-induced toxicity enhancement for the MBAs. This assumption may also be true for other PAHs that are more toxic under light than if they are left in the dark (10, 11). The mechanism of light-induced toxicity and the mechanism of tumor induction in experimental rodents are different. As suggested, initiation of UVA-induced DNA single strand cleavage is through reactive oxygen species or free-radical intermediates (occurred at a specific carbon center). However, the formation of a vicinal bayregion dihydrodiol epoxide has been demonstrated to be the general in vitro and in vivo metabolic activation pathway for most carcinogenic PAHs. The formation of a DNA covalent adduct with a bay-region diol epoxide may lead to tumor initiation (2, 3, 23, 25-27). The formation of a vicinal bay-region diol epoxide involves the epoxidation of an aromatic double bond as an initial metabolism step, followed by catalytic hydration of the epoxy ring and epoxidation of the vicinal olefinic double bond. Thus, the effects of a methyl substitution of BA on UVA-induced DNA strand cleavage and animal tumor induction are different. For UVA-induced strand cleavage, the methyl group can enhance, reduce, or even block the most active carbon of BA so that the initial step takes place at another carbon position. For animal tumor initiation, the methyl group situated at the aromatic ring that involves diol epoxide formation can direct the formation of diol epoxide to the other regions of the MBA molecule and thus inhibit the formation of the tumori-
Chem. Res. Toxicol., Vol. 15, No. 3, 2002 405
genic bay-region diol epoxides. On the other hand, methyl substitution at other regions promotes the formation of bay-region diol epoxide and thus enhances tumorigenicity (2, 3, 23, 25-27).
Concluding Remarks In general, environmental organic chemicals, upon entering human body, are subject to degradation enzymatically, chemically, or photochemically (if light is accessible). Photodegradation of organic contaminants can be either incomplete to form relatively stable organic molecules such as quinones or it can be completely degraded to small molecular building blocks such as NH3, CO2, and H2O, thus become harmless. Usually, reactive intermediates such as ROS and free-radicals are formed during the degradation. The photodegradation of MBAs, including BA, produces primarily the 7,12-quinone, if the methyl group does not occupy any of the 7 or 12 positions. Methyl substitution at any positions of BA accelerates the photodegradation, especially if the methyl is at either or both of the 7 and 12 positions. It appears that both the MBA and the photoproduct quinone are able to induce the formation of reactive intermediates leading to DNA single strand cleavage. The photoproduct of MBAs, 7,12-quinone, may be more harmful since it is relatively stable under light radiation and can act as a photosensitizer to produce ROS that can cause various forms of DNA damages. PAHs are environmental contaminants and also present in food chains. Some commercial medicines contain PAHs as well. For example, coal tar, a complex mixture of PAHs, is widely used in pharmaceutical products such as creams, ointments, lotions, and shampoos and used for the treatment of psoriasis (48, 49). Topical application of coal tar on the skin followed by ultraviolet light radiation, known as the Goeckerman therapy for psoriasis, has an increased risk of developing cutaneous cancer (49). Later it was confirmed that combination of ultraviolet B radiation and coal tar has an additive effect on inducing metabolizing enzyme activities and DNA adduct formation in the mouse skin (50). Roofers and highway asphalt workers also have a high risk to be exposed to both PAHs and light at the same time (51). It is known that PAHs, including several MBAs, induce skin cancer (24, 48, 52, 53). Since human skin is exposed to light, it is of particular importance and significance to investigate human health risks posed by exposure to the combination of PAHs and light.
Acknowledgment. The authors wish to thank the National Institutes of Health for a generous grant NIHRCMI G122RR13459.
References (1) Dipple, A., Moschel, R. C., and Bigger, C. A. H. (1984) Polynuclear aromatic carcinogens. Chemical Carcinogens (Searle, C. E., Ed.) Vol. 1, pp 41-163, American Chemical Society Monograph 182, American Chemical Society, Washington, DC. (2) Yang, S. K. and Silverman, B. D., Eds. (1988) Polycyclic Aromatic Hydrocarbon Carcinogenesis: Structure-Activity Relationships, Vols. I and II, CRC Press, Boca Raton, FL. (3) Harvey, R. G. (1991) Polycyclic Aromatic Hydrocarbons: Chemistry and Carcinogenesis, Cambridge Monographs on Cancer Research, Cambridge University Press, New York. (4) Harvey, R. G. (1997) Polycyclic Aromatic Hydrocarbons, WileyVCH, New York.
406
Chem. Res. Toxicol., Vol. 15, No. 3, 2002
(5) Stevenson, J. L., and Von Haam, E. (1965) Carcinogenesis of benz[a]anthracene and benzo[c]phenanthrene derivatives. Am. Ind. Hyg. Assoc. J. 26, 475-478. (6) Conney, A. H. (1982) Induction of Microsomal Enzymes by Foreign Chemicals and Carcinogenesis by Polycyclic Aromatic Hydrocarbons, Cancer Res. 42, 4875-4917. (7) Lesko, S. A. (1984) Chemical Carcinogenesis: Benzopyrene System, Methods Enzymol. 105, 539-550. (8) RamaKrishna, N. V. S., Devanesan, P. D., Rogan, E. G., Cavalieri, E. L., Jeong, H., Jankowiak, R. and Small, G. J. (1992) Mechanism of Metabolic Activation of the Potent Carcinogen 7,12-Dimethylbenz[a]anthracene, Chem. Res. Toxicol. 5, 220-226. (9) Gelbroin, H. V. (1980) Benzo[a]pyrene Metabolism, Activation and Carcinogenesis: Role and Regulation of Mixed Function Oxidases and Related Enzymes, Physiol. Rev. 60, 1107-1166. (10) Penning, T. M., Burczynski, M. E., Hung, C. F., McCoull, K. D., Palackal, N. T. and Tsuruda, L. S. (1999) Dihydrodiol Dehydrogenases and Polycyclic Aromatic Hydrocarbon Activation: Generation of Reactive and Redox Active o-Quinones, Chem Res. Toxicol. 12, 1-18. (11) Pelletier, M. C., Burgess, R. M., Ho, K. T., Kuhn, A., McKinney, R. A., and Ryba, S. A. (1997) Phototoxicity of individual polycyclic aromatic hydrocarbons and petroleum to marine invertebrate larvae and juveniles. Environ. Toxicol. Chem. 16, 2190-2199. (12) Swartz, R. C., Ferraro, S. P., Lamberson, J. O., Cole, F. A., Ozretich, R. J., Boese, B. L., Schults, D. W., Behrenfeld, M., and Ankley, G. T. (1997) Photoactivation and toxicity of mixtures of polycyclic aromatic hydrocarbon compounds in marine sediment. Environ. Toxicol. Chem. 16, 2151-2157. (13) Kagan, J., Tuveson, R. W., and Gong, H.-H. (1989) The lightdependent cytotoxicity of benzo[a]pyrene: effect on human erythrocytes, Escherichia coli cells, and Haemophilus influenzae transforming DNA. Mutat. Res. 216, 231-242. (14) Fernandez, M., L’Haridon, J. (1992) Influence of lighting conditions on toxicity and genotoxicity of various PAH in the newt in vivo. Mutat. Res. 298, 31-41. (15) Mezey, P. G., Zimpel, Z., Warburton, P., Walker, P. D., Irvine, D. G., Huang, X.-D., Dixon, D. G., and Greenburg, B. M., (1998) Use of quantitative shape-activity relationship to model the photoinduced toxicity of polycyclic aromatic hydrocarbons: electron density shape features accurately predict toxicity. Environ. Toxicol. Chem. 17, 1207-1215. (16) Krylov, S. N., Huang, X.-D., Zeiler, L. F., Dixon, D. G., and Greenberg, B. M., (1997) Mechanistic quantitative structureactivity relationship model for photoinduced toxicity of polycyclic aromatic hydrocarbons: I. Physical model based on chemical kinetics in a two-compartment system. Environ. Toxicol. Chem. 16, 2283-2295. (17) Huang, X.-D., Krylov, S. N., Ren, L., McKonkey, B. J., Dixon, D. G., and Greenberg, B. M., (1997) Mechanistic quantitative structure-activity relationship model for photoinduced toxicity of polycyclic aromatic hydrocarbons: II. An empirical model for the toxicity of 16 polycyclic aromatic hydrocarbons to duckweed lemma gibba L. G-3. Environ. Toxicol. Chem. 16, 2296-2303. (18) Mekenyan, O. G., Ankley, G. T., Veith, G. D., and Call, D. J. (1994) QSARs for photoinduced toxicity: I. Acute lethality of polycyclic aromatic hydrocarbons to Draphnia magna. Chemosphere 28, 567-582. (19) Veith, G. D., Mekenyan, O. G., Ankley, G. T., and Call, D. J. (1995) A QSAR analysis of substituent effects on the photoinduced acute toxicity of PAHs. Chemosphere 30, 2129-2142. (20) Dong, S., Hwang, H.-M., Harrison, C., Holloway, L., Shi, X., Yu, H. (2000) UVA light-induced DNA single strand cleavage by selected polycyclic aromatic hydrocarbons. Bull. Environ. Contam. Toxicol. 64, 467-474. (21) Dong. S., Hwang, H.-M., Shi, X., Holloway, L., Yu, H. (2000) UVAinduced DNA single strand cleavage by 1-hydroxypyrene and formation of covalent adducts between DNA and 1-hydroxypyrene. Chem. Res. Toxicol. 13, 585-593. (22) Liu, Z., Lu, Y., Rosenstein, B., Lebwohl, M., and Wei, H., (1998) Benzo[a]pyrene enhances the formation of 8-hydroxy-2′-deoxyguanosine by ultraviolet A radiation in calf thymus DNA and human epidermoid carcinoma. Biochemistry 37, 10307-10312. (23) Yang, S. K., Chou, M. W., and Fu, P. P. (1981) Microsomal oxidation at methyl-substituted aromatic carbons of methyl benz[a]anthracenes. In Polycyclic Aromatic Hydrocarbons: Fifth International Symposium on Chemical Analysis and Biological Fate (Dennis, A. J., and Cooke, W. M., Eds.) pp 253-264, Battelle Press, Columbus, Ohio. (24) Wislocki, P. G., Fiorentini, K. M., Fu, P. P., Yang, S. K., and Lu, A. Y. H. (1982) Tumor-initiating ability of the twelve monomethylbenz[a]anthracenes. Carcinogenesis 3, 215-217.
Dong et al. (25) Hecht, S. S., Melikian, A. A., and Amin, S. (1986) Methylchrysenes as probes for the mechanism of metabolic activation of carcinogenic methylated polynuclear aromatic hydrocarbons. Acc. Chem. Res. 19, l74-l80. (26) Hecht, S. S., Melikian, A. A., and Amin, S., (1988) Methyl bay region diol epoxides: key intermediates in the metabolic activation of carcinogenic methylated polynuclear aromatic hydrocarbons. In Chemical Carcinogens, Activation Mechanisms, Structural and Electronic Factors, and Reactivity (Politzer, P., and Martin, F. J., Jr., Eds.) pp 291-311. Elsevier, Amsterdam. (27) Hecht, S. S., Melikian, A. A., and Amin, S. (1988) Effects of methyl substitution on the tumorigenicity and metabolic activation of polycyclic aromatic hydrocarbons. In Polycyclic Aromatic Hydrocarbon Carcinogenesis: Structure-Activity Relationships (Yang, S. K., and Silverman, B. D., Eds.) Vol. 1. pp 95-128, CRC Press, Boca Raton, FL. (28) Fu, P. P., Chou, M. W., and Beland, F. A. (1988) Effects of nitro substitution on the in vitro metabolic activation of polycyclic aromatic hydrocarbons. In Polycyclic Aromatic Hydrocarbon Carcinogenesis: Structure-Activity Relationships (Yang, S. K., and Silverman, B. D., Ed.) Vol. 2, pp 37-65, CRC Press, Boca Raton, FL. (29) Fu, P. P., Evans, F. E., Miller, D. W., Freeman, J. P., and Yang, S. K. (1982) A modified approach in the synthesis of 5- and 6-methylbenz[a]anthracenes. Org. Prep. Proc. Int. 14, 169-175. (30) Yang, S. K., Chou, M. W., Weems, H. B., and Fu, P. P. (1979) Enzymatic formation of an 8,9-diol from 8-methylbenz[a]anthracene. Biochem. Biophys. Res. Commun. 90, 1136-1141. (31) Parr, R. G., and Yang, W., (1989) Density Functional Theory of Atoms and Molecules, Oxford, New York. (32) Becke, A. D., (1993) Density-functional thermochemistry. III. The role of exact exchange. J. Chem. Phys. 98, 5648-5652. (33) Frisch, M. J., Trucks, G. W., Schlegel, H. B., Scuseria, G. E., Robb, M. A., Cheeseman, J. R., Zakrzewski, V. G., Montgomery, J. A. Jr., Stratmann, R. E., Burant, J. C., Dapprich, S., Millam, J. M., Daniels, A. D., Kudin, K. N., Strain, M. C., Farkas, O., Tomasi, J., Barone, V., Cossi, M., Cammi, R., Mennucci, B., Pomelli, C., Adamo, C., Clifford, S., Ochterski, J., Petersson, G. A., Ayala, P. Y., Cui, Q., Morokuma, K., Malick, D. K., Rabuck, A. D., Raghavachari, K., Foresman, J. B., Cioslowski, J., Ortiz, J. V., Baboul, A. G., Stefanov, B. B., Liu, G., Liashenko, A., Piskorz, P., Komaromi, I., Gomperts, R., Martin, R. L., Fox, D. J., Keith, T., Al-Laham, M. A., Peng, C. Y., Nanayakkara, A., Challacombe, M., Gill, P. M. W., Johnson, B., Chen, W., Wong, M. W., Andres, J. L., Gonzalez, C., Head-Gordon, M., Replogle, E. S., and Pople, J. A. (1998) Gaussian 98, Revision A9, Gaussian, Inc., Pittsburgh, PA. (34) Clementi, E., (1990) Modern Techniques in Computational Chemistry, MOTECC-90, ESCOM, The Netherlands. (35) McGlynn, S. P., Reynolds, M. J., Daigre, G. W., and Christodouleas, N. D. (1962) The external heavy-atom spin-orbital coupling effect. III. Phosphorescence spectra and lifetimes of externally perturbed naphthalenes. J. Phys. Chem. 66, 2499-2505. (36) Wiliams, R. M., Glinka, T., Flanagan, M. E., Gallegos, R., Coffman, H., and Pei, D. (1992) Cannizzaro-based O2-dependent cleavage of DNA by quinocarcin. J. Am. Chem. Soc. 114, 733740. (37) Ciulla, T. A., van Camp, J. R., Rosenfeld, E., and Kochevar, I. E. (1989) Photosensitization of single-strand breaks in pBR322 DNA by Rose Bengal. Photochem. Photobiol. 49, 293-298. (38) Sortino, S., Condorelli, G., de Guidi, G., and Giuffrida, S. (1998) Molecular mechanism of photosensitization XI. Membrane damage and DNA cleavage photoinduced by Enoxacin. Photochem. Photobiol. 68, 652-659. (39) Rodgers, M. A. J., and Snowden, P. T. (1982) Lifetime of O2 (1∆g) in liquid water as determined by time-resolved infrared luminescence measurements. J. Am. Chem. Soc. 104, 5541-5543. (40) Alegria, A. E., Ferrer, A., Sandiago, G., Sepulveda, E., and Flores, W. (1999) Photochemistry of water-soluble quinones. Production of the hydroxyl radical, singlet oxygen and the superoxide ion. J. Photochem. Photobiol. A. 127, 57-65. (41) Gutierrez, I., Bertolotti, S. G., Biasutti, M. A., Soltermann, A. T., Garcia, N. A. (1997) Quinones and hydroquinones as generators and quenchers of singlet molecular oxygen. Can. J. Chem. 75, 423-428. (42) Greenberg, M. M. (1998) Investigating nucleic acid damage processes via independent generation of reactive intermediates. Chem. Res. Toxicol. 11, 1235-1248. (43) Burkle, A. (2001) Mechansim of Aging. Eye June 15 (part 3), 371375. (44) Floyd, R. A., West, M., and Hensley, K. (2001) Oxidative biochemical markers: clues to understanding aging in long-lived species. Exp. Gerontol. 36, 619-640.
Photoinduced DNA Damages and Phototoxicity of PAHs (45) Loft, S., and Poulsen, H. E. (1996) Cancer risk and oxidative DNA damage in man, J. Mol. Med. 74(6), 297-312. (46) Konig, J., Balfanz, E., Funcke, W., and Romanowski, T. (1985) Structure-activity relationship for the photooxidation of anthracene and its anellated homologues, Polynuclear Aromatic Compounds: Mechansims, Methods and Metabolism (Cooke and Dennis Ed.), pp 739-748, Bartelle Press: lumbus. (47) Wood, J. L., Barker, C. L., and Grubbs, C. J. (1979) Photooxidation products of 7,12-dimethylbenz[a]anthracene. Chem.-Biol. Interact. 26, 339-347. (48) National Toxicology Program (1998) Soots, Tars, and Mineral Oils. Eighth Report on Carcinogens, pp 42-46 and 178-181. (49) Stern, R. S., Zierler, S., and Parrish, J. A. (1980) Skin carcinoma in patients with psoriasis treated with topical tar and artificial ultraviolet radiation. Lancet 2, 732-733. (50) Mukhtar, H., Del Tito, B. J., Jr., Matgouranis, P. M., Das, M., Asokan, P., and Bickers, D. R. Additive effects of ultraviolet B
Chem. Res. Toxicol., Vol. 15, No. 3, 2002 407 and crude coal tar on cutaneous carcinogen metabolism: Possible relevance to the tumorigenicity of the Goeckerman regimen. J. Invest. Dermatol. 87, 348-353. (51) Reed, L. D., and Liss, G. M. (1985) PAH exposure among pitch and asphalt roofing workers. In Polynuclear Aromatic Hydrocarbons: Mechanisms, Method, and Metabolism (Cooke and Dennis Ed.) pp 1089-1095, Batelle Press, Columbus, OH. (52) Mastrangelo, G., Fadda, E., and Marzia, V. (1996) Polycyclic aromatic hydrocarbons and cancers in man. Environ. Health Perspect. 104, 1166-1170. (53) Godschalk, R. W. L., Ostertag, J. U., Moonen, E. J. C., Neumann, H. A. M., Kleinjans, J. C. S., and van Schooten, F. J. (1998) Aromatic DNA adducts in human white blood cells and skin after dermal application of coal tar. Cancer Epidemiol. Biomark. Prev. 7, 767-773.
TX015567N
