Alternative Mechanisms of Psoralen Phototoxicity - American

Chapter 15

Alternative Mechanisms of Psoralen Phototoxicity 1

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Karen A. Marley , Richard A. Larson , and Richard Davenport 1

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Downloaded by COLUMBIA UNIV on September 9, 2012 | http://pubs.acs.org Publication Date: May 5, 1995 | doi: 10.1021/bk-1995-0616.ch015

Institute for Environmental Studies and School of Life Sciences, University of Illinois, Urbana, IL 61801

Traditional studies of the mechanism of phototoxicity of psoralens (furocoumarins) have emphasized their photoaddition reactions with DNA. Photooxidation products of furocumarins, although they have been observed in some studies, have been given little attention. By the use of improved extraction and separation techniques, we recently identified a group of salicylaldehyde derivatives from psoralen photooxidation in vitro. The occurrence of these phenolic aldehydes could possibly account for some of the observed photobiological effects now attributed to the parent compounds. Psoralen and 8methoxypsoralen are quite susceptible to photolysis in polar solvents such as water, giving many cleavage products including aldehydes, carboxylic acids, and hydrogen peroxide. Studies with photolyzed psoralens and isolated photoproducts such as aromatic aldehydes reveal them to be toxic, even in the absence of light, to Xenopus embryos.

Naturally occurring substances having a variety of structures have been found to display insecticidal effects in sunlight. Many other phototoxic effects have also been attributed to these compounds. In particular, furocoumarins (also known as psoralens) induce blistering, "accelerated" sunburn and hyperpigmentation when humans or animals come into contact with furocoumarincontaining plants (7). Structure-activity studies have established that psoralen, the parent compound, is the most phototoxic furocoumarin and that ultraviolet light in the region of 320-360 nm is the most effective region of Psoralen irradiation (2). The concept of phototoxicity implies some biochemical target within the cell (or at the cell surface) that interacts in some fashion with the excited state of the toxic agent, leading to a deleterious change. However, in no case of phototoxicity

0097-6156/95/0616-0179$12.00/0 © 1995 American Chemical Society In Light-Activated Pest Control; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1995.

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Table I. Products identified from the aqueous photolysis of psoralen

Status

Identification Method

5-formyl-6-hydroxybenzofuran (FHBF)

New product (75)

from SPE; HPLC-PDAD, gc-ms, IR, N M R

6-formyl-7-hydroxycoumarin (FHC)

Previously identified

from SPE; H P L C - P D A D , gc-ms

furocoumaric acid (FCA)

Previously identified


Compound

Structure

(20)

\

COOH

(33)

from polar residue (gc-ms); methylated deriv by gc-ms

2,5-dihydroxy-l,4benzenedicarboxaldehyde

New photoproduct; Acid previously identified as an alkaline oxidation product (27)

from SPE (gc-ms); also found corresponding acid as methylated deriv by gc-ms

2,4-dihydroxybenzaldehyde

New product

From polar residue (gc-ms); also found corresponding acid as methylated deriv by gc-ms

New product

From polar residue (gc-ms); also found corresponding acid as methylated deriv by gc-ms

salicylaldehyde

XT

cc

Source: Reprinted with permission from The Spectrum (in press). Copyright 1995 Center for Photochemical Sciences, Bowling Green State University, Bowling Green, Ohio.

In Light-Activated Pest Control; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1995.

181 Alternative Mechanisms of Psoralen Phototoxicity

15. MARLEY ET AL.

has one particular target or group of targets been definitively identified. Experiments in vivo with furocoumarins, for example, have demonstrated that they react with DNA, proteins, and lipids to a significant degree (3,4). Various mechanisms have been proposed for the action of furocoumarin phototoxicity. In summary, these are: 1) intercalation of the furocoumarin molecule into the DNA helix and photoiniated [2+2] cyclobutane adduct formation leading to crosslinked DNA; 2) [2+2] cycloadducts with lipids; 3) formation of reactive oxygen, either 0 and/or Η 0 · ; 4) formation of reactive intermediates or "transients", such as free radicals, peroxides, epoxides or dioxetanes. (For review articles, see 4-6). The majority of in vitro experiments conducted by photobiologists have highlighted psoralen reactions with DNA. However, DNA adducts are usually formed only in a dry or solid state with a quantum yield of < 1% (7). Quantum yields of the reactive oxygen species are also quite low (8). Although the importance of light induction has been well-established, the subsequent reactivity of furocoumarins without light has also been noted. Several studies have demonstrated that pre-illuminated furocoumarins (sometimes called "photooxided psoralens") can hemolyze red blood cells (9) and oxidize lipids (10). Other laboratory investigations of the inflammatory response initiated by psoralens note that these effects are not immediate — usually there is a delay (or induction period) of 6-12 hours with maximum effect noted at 24-48 hours. In contrast, rose bengal, a well-known 0 producer, produces erythemal effects within three hours (77). Additional notes in various experimental protocols also indicate that enhanced toxicity can be observed if the reaction mixtures are held for a short period of time following illumination (e.g., see 72). Among the structural classes of furocoumarin products that may be formed, synthetic furocoumarin-dioxetanes can form adducts with DNA (73) and synthetic furocoumarin-peroxides can cleave DNA (14). However, there is no good evidence for these exact structural types in mixtures of photooxidized psoralens. Recently we demonstrated that 5-formyl-6-hydroxybenzofuran (FHBF) (Table I) is a major early photodecomposition product of psoralen in aqueous solution (75). This aldehyde is structurally related to phototoxic keto benzofuran derivatives that occur in the plant genus Encelia (16). Preliminary testing of this compound suggests that it also may have phototoxic properties. In the aqueous photolysis of psoralen, additional aldehydes are formed, including short-chain aldehydes, such as acetaldehyde, as well as phenolic aldehydes; minor products include the corresponding acid derivatives; H 0 is also formed. None of the previous papers on furocoumarin photooxidation has identified either FHBF or these aldehydes (and acids) as reaction products. !


2

2

l

2

2

2

Results and Discussion Psoralens are somewhat hydrophobic compounds and might be expected to partition into lipid-rich regions of the cell. However, much metabolic activity (such as transport through membranes and vitamin C-vitamin Ε interactions (77)) occurs at lipid-water interfaces, and aqueous processes can be expected to play important roles. Most solution photochemistry by previous investigators on furocoumarins has been conducted with 8-methoxypsoralen (8-MOP), at millimolar concentrations in



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methylene chloride, of which it was noted that photolysis was slow and "intractable" (18-19). However, in our laboratory, using simulated sunlight, the half-life of a 20 μΜ air-saturated aqueous solution of 8-MOP was found to be approximately 6 hours; for 20 μΜ psoralen the half-life was 20 minutes. Because the product mixtures were less complex and the yields much higher with psoralen than with 8MOP, most of the following work describes psoralen photoproducts, although some of the 8-MOP analogues have also been detected. Reverse phase HPLC analysis of the reaction mixtures immediately following illumination showed that one major comparatively non-polar product (eluting after psoralen) was formed. However this product was not present if the analysis was delayed following irradiation (e.g., if analyzed after an overnight delay) or during various isolation procedures. Finally, a combination of solid phase extraction and semi-prep HPLC purification allowed the identification of 5formyl-6-hydroxybenzofuran (FHBF), a lactone ring-opened product (75). In addition, a relatively minor polar product was identified as 6-formyl-7hydroxycoumarin (FHC) as previously described (20). Further investigation of the polar residue showed that some phenolic aldehydes and the corresponding acids, all of which are structurally related to FHBF or FHC, were also formed (Table I). Of these compounds, 2,5-dihydroxy-l,4benzenedicarboxylic acid has been identified as a product of alkaline permanganateinduced oxidative degradation of psoralen (27). None of the remaining compounds have been described as a photolytic or oxidative product of psoralen. Short-chain aldehydes were also detected in the reaction mixtures; they are most likely the result of ring-cleavage reactions. During the photolysis of psoralen, the formation of H 0 was also observed. Small amounts of H 0 were produced in aqueous solutions; however, when better Η-donor solvents were used such as ethanol or isopropanol, the yields of H 0 increased by factors of 10 and 100, respectively. Previous work has shown that these solvents participate in H 0 production by a mechanism of hydrogen atom abstraction by the excited state of a carbonyl group [e.g., for the phototoxic aldehyde, citral (22)]. In fact, high yields of H 0 continued long after psoralen was photodegraded (Figure 1) thus indicating that aldehyde products could also be precursors of H 0 . Preliminary work on the model compounds salicylaldehyde and isophthalaldehyde also confirm high yields of H 0 in Η-donor solvents (manuscript in prepration). Independently, a group of para phenolic aldehydes, such as vanillin, have also been shown to yield H 0 under aqueous photolysis conditions (Anastasio, personal communication). The participation of carbonyl compounds in furocoumarin phototoxicity may occur by at least three pathways: 1) reaction of the carbonyl group with electron-rich cellular constituents, such as sulfhydryl and amino groups of proteins and amino groups of guanine, through Schiff base formation: R.-CHO + H N-R.' — RCH=NR' 2

2

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protein crosslinking or DNA adduct formation may result. 2) reactions requiring the presence of H 0 , as in the addition of the HOOH to a carbonyl group to form a hydroxyhydroperoxide (23): 2

2



80 0-0·

o

2

Figure 1. Loss of psoralen and formation of hydrogen peroxide in a Hidonating solvent (ethanol). (Reproduced with permission from The Spectrum (in press). Copyright 1995 Center for Photochemical Sciences, Bowling Green State University, Bowling Green, Ohio.)

Time (min)

40

C

OH

HOO

Alternative Mechanisms of Psoralen Phototoxicity - American

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