Synthesis and photoirradiation of isomeric ethylchrysenes by UVA light leading to lipid peroxidation.

Polycyclic aromatic hydrocarbons (PAHs) are widespread genotoxic environmental pollutants. We have recently demonstrated that photoirradiation of PAHs leads to cytotoxicity, DNA damage, and induction of lipid peroxidation. In this paper we report the synthesis of all the six isomeric ethylchrysenes and the study of light-induced lipid peroxidation by these ethylchrysenes. 5-Ethylchrysene was synthesized by reaction of 5-keto-5,6,6a,7,8,9,10,10a-octahydrochrysene with CH3CH2MgBr followed by dehydration catalyzed by p-toluenesulfonic acid and dehydrogenation with DDQ in benzene. 1- and 4-Ethylchrysenes were similarly prepared by reaction of 1-keto-1,2,3,4,5,6-hexahydrochrysene and 4-keto-1,2,3,4-tetrahydrochrysenes, respectively with CH3CH2MgBr followed by dehydration and dehydrogenation. Direct acetylation of chrysene followed by Wolff-Kishner or Clemmensen reduction resulted in the formation of 2-, 3-, and 6-ethylchrysenes in 4%, 16%, and 43% yields, respectively. Photoirradiation of these compounds with 7 and 21 J/cm2 UVA light in the presence of methyl linoleate all resulted in lipid peroxidation. For comparison, photoirradiation of 4-methylchrysene and 5-methylchrysene was similarly conducted. For irradiation at a UVA light dose of 21 J/cm2, the level of induced lipid peroxidation is in the order 4-methylchrysene = 5-methylchrysene = 5-ethylchrysene = 4-ethylchrysene = chrysene > 1-ethylchrysene = 2-ethylchrysene > 3-ethylchrysene > 6-ethylchrysene. Compared with chrysene, these results indicate that the ethyl group at C4 or C5 position either slightly enhances or has no effect on the light-induced lipid peroxidation, while at C1-, C2-, C3-, or C6 position reduces light-induced lipid peroxidation.

It has also been reported that PAHs can be activated by light irradiation to produce phototoxicity [10,11]. Since skin is the largest body organ in humans and is unavoidably 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. However, this activation pathway has received relatively much less attention [12]. We previously reported the photoirradiation of a series of parent and methyl-substituted PAHs with UVA light in the presence of methyl linoleate. It was found that 20 out of the 21 tested PAHs induce lipid peroxidation, forming lipid peroxides (methyl linoleate hydroperoxides). We also determined that the photoinduced lipid peroxidation by PAH is mediated by reactive oxygen species (ROS), specifically singlet oxygen and superoxide, which are generated during the photoirradiation of PAHs [13]. The study of the twelve isomeric methylbenz [a]anthracenes determined that the level of lipid peroxidation induced by methylbenz[a]anthracenes does not correlate with energy gap between the highest occupied molecular orbital (HOMO) and the lowest unoccupied molecular orbital (LUMO).
These results indicate that lipid peroxidation induced by the twelve methylbenz[a]anthracenes is not directly related to their excited-state properties [13]. Also, the relative levels of UVA-induced lipid peroxidation by the twelve methylbenz[a]anthracenes do not correlate with the relative carcinogenic activity or the tumor-initiation activity [13].
As a continuation of the study on a structure-activity basis, it is interesting to study whether or not ethylated PAHs concomitantly exposed to UVA light would also induce lipid peroxidation. We report in this paper the synthesis of all the six isomeric ethylchrysenes and photoirradiation of these compounds by UVA light in the presence of methyl linoleate.
For comparison, photoirradiation of 4-methylchrysene and 5-methylchrysene was also studied. The results indicate that under experimental conditions, all the tested PAHs induce lipid peroxidation and the potency of lipid peroxidation depends on the position of the ethyl substitution.

Materials and General Procedures
Chrysene, 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ), sodium thiosulfate, acetic anhydride, and ethyl magnesium bromide were purchased from Aldrich Chemical Co. (Milwaukee, WI). 4-Methylchrysene and 5-methylchrysene were obtained from the National Cancer Institute's Chemical Repository. Xanthine oxidase (XOD) was purchased from Roche Applied Science (Indianapolis, IN). All other reagents were obtained through commercial sources and were the highest quality available. All solvents used were HPLC grade. Tetrahydrofuran (THF) was distilled from lithium aluminum hydride prior to use.

Instrumentation
A Waters Alliance HPLC system, consisting of a 2695 separation module and a 2996 photodiode array detector, was used for the separation and analysis of the lipid hydroperoxide products. The mass spectra were determined on JEOL-DX 300 mass spectrometer. The NMR spectra were obtained on Varian VXR-300 spectrometer with tetramethylene silane as an internal standard in CDCl 3 .

Light Source
The UVA light box was custom made using 4 UVA lamps (National Biologics, Twinsburg, OH) [13]. The irradiance of the light box was determined using an Optronics OL754 Spectroradiometer (Optronics Laboratories, Orlando, FL), and the light dose was routinely measured using a Solar Light PMA-2110 UVA detector (Solar Light Inc., Philadelphia, PA). The UVA intensity is 5.1 mW/cm 2 at the distance of 13 cm. The maximum emission of the UVA light box was 350-352 nm with the following spectral distribution: UVA (315-400 nm), 98.93%; UVB (280-315 nm), 1.07%; UVC (250-280 nm), <0.0001%.

Peroxidation of Methyl Linoleate Initiated by Photoirradiation of Chrysene, Ethylchrysenes, and Methylchrysenes with UVA Light
Experiments were conducted with a solution of 100 mM methyl linoleate and 0.2 mM substrate in methanol. Samples were placed in a UV-transparent cuvette and irradiated with 7 and 21 J/cm 2 of UVA light, respectively. After irradiation, the methyl linoleate hydroperoxide products were separated by HPLC using a Prodigy 5µm ODS column (4.6 x 250 mm, Phenomenex, Torrance, CA) eluted isocratically with 10% water in methanol (v/v) at 1 mL/min. The levels of lipid peroxidation were quantified by monitoring the HPLC peak areas at 235 nm [21,22] followed by convertion to the concentration based on the molar extinction coefficient (at 235 nm) of the hydroperoxide as reported by Gibian and Vandenberg [23].

Effect of NaN 3 on Peroxidation of Methyl Lineolate Initiated by Photoirradiation of 5-Ethylchrysene and 5-Methylchrysene
NaN 3 is a single oxygen and free radical scavenger [13]. The effect of NaN 3 on peroxidation of methyl lineolate was determined. 4-Ethylchrysene and 5-ethylchrysene were selected for the study. The experiments were similarly conducted as described above, with the exception that 50 mM NaN 3 was present in the irradiation.

Synthesis of Isomeric Ethylchrysenes
To study the induction of lipid peroxidation by PAHs on structure-activity bases, the six isomeric ethylchrysenes were synthesized. In our study, acetylation of chrysene generated 2-, 3-, and 6-acetylchrysenes in 4%, 16%, and 43% yields, respectively (Figure 1). The 6-carbon position is the most active site of chrysene in chemical reactions. Thus, acetylation of chrysene providing 6-acetylchrysene as the highest yield product is consistent with theoretical consideration. In our study, both Wolff-Kishner reduction and Clemmensen reduction were employed to reduce acetylchrysenes to the corresponding 6-, 3-, and 2-ethylchrysenes in good yields. Thus, direct acetylation of chrysene followed by Wolff-Kishner reduction or Clemmensen reduction is a convenient method to prepare 6-, 3-, and 2-acetylchrysenes at the same time.

Photoirradiation of Ethylchrysenes and Methylchrysenes
Photoirradiation of the six isomeric ethylchrysenes with UVA in the presence of a lipid, methyl linoleate, was studied. To compare the effect of the ethyl and methyl groups on lipid peroxidation, photoirradiation of 4-methylchrysene and 5-methylchrysene was similarly conducted. Each compound received two light doses, 7 and 21 J/cm 2 , respectively ( Table 1). The results summarized in Table 1 and Figures 5 and 6 indicated that lipid peroxidation exhibited a dose and structural dependence. Higher light dose generate more peroxides as expected. The level of induced lipid peroxidation due to concomitant exposure to UVA irradiation (21 J/cm 2 ) is in the order 4-methylchrysene = 5-methylchrysene = 5-ethylchrysene = 4-ethylchrysene = chrysene > 1-ethylchrysene = 2-ethylchrysene > 3-ethylchrysene > 6-ethylchrysene (Statistically significant differences were identified when p < 0.05). Thus, a methyl or an ethyl substitution at the same poison does not affect the ability of the chrysene molecule to generate UVA light-induced lipid peroxidation.

Discussion
We previously determined that when irradiated with UVA light in the presence of methyl linoleate, 20 out of 21 PAHs initiated lipid peroxidation, forming the methyl linoleate hydroperoxide [13]. We also determined that the photoinduced lipid peroxidation is mediated by reactive oxygen species (ROS), specifically singlet oxygen and superoxide, which are generated during the photoirradiation of PAHs [13]. The results also indicate that there is no correlation between the level of lipid peroxidation induced and the tumorigenic potency of parent PAHs and the twelve isomeric methylbenz[a]anthracenes.
In this paper, we report the induction of lipid peroxidation by photoirradiation of the six isomeric ethylchrysenes with UVA. We found that the addition of an ethyl or a methyl group to the C4-or C5-position of chrysene either slightly enhances the level of induction or there is no apparent effect (Table 1) on the UVA-induced lipid peroxidation. In contrast, the addition of an ethyl group at the other positions (C1-, C2-, C3-, or C6-position) lowers the extent of lipid peroxidation. These results suggest that under our experimental conditions, an ethyl group located at the bay-region (the 4-and 5-positions) of chrysene can enhance lipid peroxidition or exerts no effect, while an ethyl group located at a non-bay region position exhibits an inhibitive effect.
5-Methylchrysene is a potent carcinogen while 4-methylsene is not [2]. The similar level of UVA-induced lipid peroxidation by these two compounds indicates that there is no correlation between the level of lipid peroxidation and the tumorigenic potency of the compounds studied. This is consistent with the previous finding from the twelve methylbenz[a]anthracenes [13]. The lack of correlation is understandable since the mechanism leading to lipid peroxidation and the mechanism involving enzymatic metabolic activation leading to tumorigenicity are different. Thus, our overall results indicate that photoirradiation of PAHs by UVA light generates reactive oxygen species (ROS), which induce lipid peroxidation. Lipid peroxidation can cause tumor induction in experimental animals [24,25]. It has been well established that ROS can damage nucleic acids and proteins leading to aging, inflammation, cardiovascular diseases, cancer and other age-related diseases [26,27]. Human body is unavoidably exposed to both sunlight and PAHs present in the environment or contaminated in the commercial products. As such, it warrants further investigation to determine the possible human health risks posed by exposure to the combination of PAHs and light, especially since coal tar, which contains PAHs, has been classified as a human carcinogen [28].