Vol. 1 INTERNATIONAL JOURNAL OF PHOTOENERGY 1999

Photochemical and photobiological propertiesof furocoumarins and homologues drugs

F. Bordin

Department of Pharmaceutical Sciences of Padova University, Padova, Italy

Abstract. Furocoumarins are natural photosensitizing drugs used in PUVA photochemotherapy and inphotopheresis. Their therapeutic effectiveness is connected to the lesions they induce to various cell compo-nents, membranes, ribosomes, mitochondria, and in particular to DNA, damaged by formation of monofunc-tional adducts and of inter-strand cross-links (ISC). ISC represent a severe damage, mainly correlated to themain side effects observed in photochemotherapy, skin phototoxicity and genotoxicity. Searching for newmonofunctional derivatives, two tetramethylfuroquinolinones, 1,4,6,8-tetramethyl-2H-furo[2,3-h]quinolin-2-one (FQ) and 4,6,8,9-tetramethyl-2H-furo[2,3-h]-quinolin-2-one (HFQ) were studied. Both compounds are veryactive; however while FQ produced many chromosomal aberrations and strong skin erythemas, HFQ practi-cally did not induce such side effects. FQ and HFQ formed high levels of monoadducts but no ISC in DNA,but both provoked many DNA-protein cross-links (DPC). FQ induced these lesions by a biphotonic reaction:at first a furan-side monoadduct is formed, which is then converted into a DPC; thus the FQ molecule seemedto form the bridge between DNA and proteins. HFQ formed DPC by a single step (DPC at zero length, likeUVC). For these features, HFQ appears to be the first molecule belonging to a new class of active but notphototoxic drugs for photomedicine.

1. INTRODUCTION

Furocoumarins are active photosensitizing drugswidely used in photomedicine [1], in research on thestructure of various biological macromolecules [2] andon DNA repair [3]. Some of them are natural compoundspresent in several plants, such as Psoralea corylifolia,Amni Majus and Angelica Archangelica (see Figure 1 forthe molecular structure of some natural derivatives).Furocoumarins are used in photomedicine

1′ 2′3′4′

5′5

67

8 1 23

4

Psoralen Angelicin(Isopsoralen)

5

8

5-Methoxypsoralen

(5-MOP, Bergapten)

8-Methoxypsoralen

(8-MOP, Xanthotoxin)

OCH3

O O OO

OO

O O OO

OCH3

OO

Figure 1. Molecular structure of some natural occurringfurocoumarins.

from centuries: in fact, ancient Egyptian and Indianphysicians used extracts of leaves, seeds or roots fromplants containing furocoumarins for the cure of vi-tiligo since thousands years BC [4]. These preparationswere applied on the skin or ingested and the patientwas then exposed to sunlight. In seventy years, pho-tochemotherapy PUVA (psoralen + UVA) has been intro-duced: 8-methoxypsoralen (8-MOP), the active principle

presented in Amni Majus, was administered orally; twohours later the skin area to be cured was exposed toUVA light (320–400 nm) [5]. PUVA is effective againstvarious skin diseases, such as vitiligo, psoriasis, alope-cia aerata, atopic dermatitis, mycosis fungoides, etc. [1].More recently the extracorporeal photochemotherapyor photopheresis has been introduced [6]. Accordingto this therapy 8-MOP is administered orally; after twohours about 0.5 litres of blood were taken out by thepatient, the lymphocytes were isolated and exposed toUVA light. Then, lymphocytes were returned back tothe patient. This treatment is practically an auto vac-cination against the same lymphocytes; because theyare specific for the disease, a selective immuno modu-lation is induced, without dangers of opportunistic in-fections. FDA approved photopheresis for the cure ofT-cell lymphoma but it is effective against various au-toimmune diseases and in the prevention of rejectionin organ transplantation [7].

By UVA irradiation furocoumarins induce various le-sions in a living cell: damage to unsatured fatty acidsand lecithins of membranes [8, 9] and to ribosomes[10,11]. However the main damage is provoked in DNA[12]. Linear furocoumarins (psoralens) have two reac-tive sites in their molecule, the double bonds at 3,4 po-sitions at pyron ring and at 4′,5′ at furan one. In thedark furocoumarins intercalate into base pairs of DNAwithout significant consequences; however, by light ac-tivation they react with pyrimidine bases via a C4-cycloaddition, forming covalent monoadducts (MA) en-gaging 3,4 (pyron side) or 4′,5′ (furan side) positions.Furan side monoadducts can absorb the UVA light andreact further with a second pyridine bases, thus form-ing a covalent bridge between the two DNA strands(inter-strand cross-links, ISC) [13]. The formation ofsuch bifunctional adducts represents a severe damage,

2 F. Bordin Vol. 1

which is regarded as mainly responsible for the sideeffects of PUVA therapy, the induction of skin erythe-mas [1] and for genotoxicity [14]. Thus, with the aimto obtain furocoumarins having a reduced toxicity, var-ious authors prepared and studied several monofunc-tional derivatives, incapable of forming ISC. A mono-functional furocoumarin can be obtained by severalways: blocking one of its two photoreactive sites by in-sertion of a suitable group, as for 3-carbethoxypsoralen[15], or by cumulating a fourth nucleus, e.g., the pyri-dopsoralens having a pyridine condensed at 3,4 posi-tions [16] and benzopsoralens in which the fourth nu-cleus is a benzenic one at 4′,5′ position [17]. We alsostudied several new angelicin derivatives, which, forthe geometric parameters of their angular molecularstructure, can hardly induce ISC [18]. Thus, we obtainedvarious active and interesting angelicins; the best oneis certainly 4,6,4′-trimethylangelicin [18]. Recently wealso studied some homologues drugs which can be con-sidered angelicin isosters; the most interesting com-pounds are two angular furoquinolinones, FQ (1,4,6,8-tetramethyl-2H-furo[2,3-h]quinolin-2-one, according toJUPAC) [19] and HFQ (4,6,8,9-tetramethyl-2H-furo[2,3-h]-quinolin-2-one) having a nitrogen atom replacing theoxygen at 1 position; the main difference between thesetwo derivatives is the presence or the absence of amethyl group at the nitrogen atom. Figure 2 shows theirmolecular structure.

O

H3C

H3C

CH3

H3C

H3C CH3

CH3

N

CH3

O N

H

OO

FQ HFQ

Figure 2. Molecular structure of the tetramethylfuroquino-linones.

2. MAIN PROPERTIES OF FUROQUINOLINONES

Both FQ and HFQ are characterized by a strong photo-sensitizing activity. In fact, upon UVA irradiation, bothcompounds induce a dramatic killing effect in CHOcells, even when they were employed in very mild exper-imental conditions. As we can see in Figure 3, there arenot significant differences between the survival curvesgenerated by FQ and HFQ. On the contrary, 8-MOP,tested at a double concentration, induced a very smalleffect. However, studying their capacity of inducing ery-themas on the skin, we obtained a surprising result:while FQ appeared to be as phototoxic as 8-MOP, HFQwas nearly inactive. The threshold doses for erythemainduction, that is the minimum amount of drug andof UVA light necessary to induce a barely visible ery-thema on albino guinea-pig skin, are shown in Table 1.FQ is only slightly more active than 8-MOP, while HFQis poorly effective.

0.0 0.2 0.4 0.6

UVA dose (kJ·m−2)

0.01

0.10

1.00

Surv

ivin

gfr

acti

on

FQ

HFQ

8-MOP

Figure 3. Clonal growth of CHO: cells were exposed to UVAlight in the presence of the drug (FQ and HFQ 2.3µM; 8-MOP4.6µM) and then the capacity of cells of forming clones wasassayed.

Table 1. Threshold doses for erythemas induction on guineapig skin.Compounds were applied on the skin as a 0.1% methanolicsolution and the skin was exposed to UVA light. The ani-mals were then kept in the dark and observed for 3 days.The dose unit is a parameter proportional to the activity,defined as follows: 1/((µM · cm−2)(kJ ·m−2)) in whichµM·cm−2 is the furocoumarin amount applied to a cm2

of the skin and kJ·m−2 is the UVA dose.

Compound µM·cm−2 kJ·m−2 Dose Relative

unit ×10−2 activity

8-MOP 4.6 5 4.3 1

FQ 3.3 5 6 1.39

HFQ 20 25 0.2 0.04

The skin phototoxicity is the furocoumarin photo-sensitising effect known from the longest time; in fact,in the past, a non-skin phototoxic compound was re-garded as totally inactive. At present we don’t knowwhat is the mechanism of erythema formation, evenif several hypothesis have been suggested: the gener-ation of active species of oxygen, such as singlet oxy-gen [20] or the ISC induction [1]. However until now, allexperimental data bore out neither of these hypothe-ses. Nevertheless, in 1990, Ortel et al., using the so-called double irradiation protocol, observed that theerythemas on human skin generated by 8-MOP are in-duced by a two steps reaction, as happen for ISC for-mation [21]. It is well known that furocoumarins in-duce ISC by the sequential absorption of two photons;the first one yields to the formation of a furan sidemonoadduct and the second photon converts it intoan inter-strand cross-link. The double-irradiation is animportant method which allows a selectively study ofthe consequence of MA and ISC [19]. After a smallUVA dose, which provokes mainly the formation of

Vol. 1 Photochemical and photobiological properties . . . 3

monoadducts, the unbound furocoumarin moleculesare removed by washing and the biological system isexposed again to the light. During the second irradia-tion step some monoadducts can be converted into ISCwithout an increase of the total number of the lesions.

3. FUROCOUMARINS INDUCE DNA-PROTEINCROSS-LINKS

On the bases of these data, we suggested that furo-coumarins could undergo to another kind of biphotonicreaction; because they can link covalently both to DNAand to proteins [22], we supposed they can form DNA-protein cross-links (DPC). Since it is impossible to de-tect such lesions in simplified systems, we studied themin whole mammalian cells, using alkaline elution [19].Thus, we observed that linear furocoumarins induceDPC, while angular ones, like angelicin derivatives, areless effective [23]. Therefore, the study of the mecha-nism and of the consequence of DPC appeared to bevery difficult, because linear furocoumarins form bothDPC and ISC.

The solution of this problem was found with FQ andHFQ, the two furoquinolinones above mentioned. Actu-ally, as reported in Table 2 and contrary to 8-MOP, bothcompounds are incapable of inducing ISC, while theycan form DPC to noticeable extents.

Table 2. Damage induced in DNA (8-MOP = 1). The dataare expressed as relative activity in comparison to 8-MOP.(a) detected in CHO cells by alkaline elution; (b) estimatedin vitro in the photoreaction with calf thymus DNA.

Compound ISC(a) DNA binding(b) DPC(a)

HFQ 0.01 5.70 2.40

FQ 0.01 9.56 8.50

8-MOP 1.0 1.0 1.0

4. MECHANISMS OF DPC FORMATION

Therefore, we studied the formation of DPC andsome their biological consequences using FQ and HFQ.At first, we studied DPC formation in CHO cells us-ing alkaline elution and the above mentioned double-irradiation protocol [19]. Figure 4 shows the resultsthus obtained. Furan-side monoadducts induced by FQcan produce a number of DPC, with a gradual increaseof their amount according to the UVA dose delivered inthe second step. On the contrary, with HFQ, after thesecond step the DPC number remained practically un-changed.

Therefore, it is evident that FQ induces a number ofDPC by a two step mechanism, while HFQ can form thislesion only by a one step reaction. Consequently, wecan suppose that FQ induces a new diadduct in whichits molecule forms the bridge between DNA and protein(DPC at length greater than zero); on the contrary, HFQinduces DPC probably by energy transfer (DPC at zerolength).

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

DPC

per

mil

lion

of

nu

cleo

tid

es

0.30 1.10 1.90

Total UVA dose ( kJ·m−2)

FQ HFQ

First step Second step

Figure 4. DPC formation in CHO cells by the double-irradiation method: CHO cells were exposed at a first ir-radiation step (0.06 KJ·m−2) in the presence of 2.3µM FQor HFQ. The cells were washed and then submitted to thesecond step, carried out with two different UVA dose. DPCwere detected by alkaline elution [25].

Single irradiation

DNA+Histones

Nucleoprotein

+ Drug

+ UVA

Extraction and

detection of DNA

UVA: 1◦ step

Dialysis

+ Histones(+ DNA)

UVA: 2◦ Step

(+ Histones)

Drug + DNADouble irradiation

Figure 5. Scheme of the methods used for DPC detection invitro. (1) Single irradiation: aqueous solutions of DNA, hi-stones and the drug (20µM) were mixed together, and themixture was exposed to UVA light (15 kJ·m−2); DNA wasextracted and determined by spectrophotometric determi-nation. (2) Double irradiation: an aqueous solution of calfthymus DNA was exposed to 10 kJ·m−2 in the presence ofthe drug (20µM) and then submitted to dialysis overnightagainst PBS; histones from calf thymus were added to thesolution which was then irradiated again (15 kJ·m−2); DNAwas extracted and its amount determined as above. Tocheck the adduct formation with proteins, histones weresubmitted to the first irradiation step: in this case after dial-ysis, DNA was added and the solution was irradiated for thesecond time and processed as above.

4 F. Bordin Vol. 1

To confirm these data we carried out some exper-iments in vitro; Figure 5 shows the protocols used,by single and double irradiation. In the single irradia-tion method, DNA and histones from calf thymus weremixed together thus obtaining a nucleoprotein com-plex, which was exposed, to UVA light in the pres-ence of furocoumarins. Then DNA was extracted andits amount determined. Because the formation of DPCreduced the amount of the extractable DNA, we canhave an evaluation of their amount. This protocolwas also modified according to the double-irradiationmethod: actually, we can submit to the first step DNAor histones. After the first irradiation the free furo-coumarin molecules were removed by dialysis, and thesecond macromolecule, histones or DNA respectively,was added to the mixture. Then it was submitted tothe second UVA exposure followed by DNA extraction.Figure 6 shows the results obtained with furoquinoli-nones and 8-MOP using these procedures. These dataare consistent with those obtained in vivo; actually, thedifferent mechanisms of DPC formation by FQ and HFQwere confirmed by these experiments. In fact, with FQ(and 8-MOP too) using the double-irradiation protocoland treating DNA in the first step we obtained DPCto an extent very close to that achieved by irradiat-ing the pre-formed nucleoprotein complex. On the con-trary, HFQ always yielded very poor amounts of DPC bythe two steps protocol. Moreover, using histones in thefirst step we obtained completely negative results. Thismeans that the biphotonic reaction involving FQ (or 8-MOP) requires the formation of MA during the first step.

0

5

10

15

20

25

Not

extr

acta

ble

DN

A(%

)

DNA Histones Mixture

HFQ

FQ

8-MOP

Figure 6. (1) DNA: double-irradiation procedure in whichDNA was submitted to the first irradiation step. (2) Histones:double-irradiation procedure in which histones were sub-mitted to the first irradiation step. (3) Mixture: accordingto single irradiation procedure DNA, histones and the drugwere submitted together to a single UVA light exposure.

The behaviour of HFQ can be explained by the low ab-sorption of its furan side monoadducts at 360 nm, thatis the maximum of emission of the lamp used in ourexperiments [24].

5. BIOLOGICAL CONSEQUENCES OF DPCFORMATION

As a first approach, we detected the formation ofdouble-strand breaks (DSB; DNA fragmentation) by neu-tral elution [25]; CHO cells were submitted to sensiti-zation with increasing UVA doses and then they wereincubated for 24 hours before testing DNA damage. Fig-ure 7 shows the results thus obtained. FQ induced avery large DNA fragmentation even at very low UVAdoses, much more pronounced than 8-MOP; on the con-trary, HFQ appeared to be ineffective. We must realizethat DNA fragmentation is not provoked by the photo-chemical reaction, because it can be observed only aftera suitable incubation time, performed after the treat-ment, at least 12 hours (data not shown). This means itis due to an enzymatic DNA processing that takes placeduring incubation, very likely by DNA repair.

0

20

40

60

80

100

DN

Afr

agm

enta

tion

(%of

tota

lD

NA

)

0.0 0.2 0.4 0.6

UVA dose ( kJ·m−2)

FQ

8-MOP

HFQ

Figure 7. DSB formation by sensitisation with FQ, HFQ(2.3µM) and 8-MOP (4.6µM). CHO cells were exposed to in-creasing UVA doses in the presence of the drug and thenincubated for 24 hours. DSB were detected by neutral elu-tion [25].

This behaviour is consistent with the data obtainedstudying the capacity of furoquinolinones of formingchromosomal aberrations (see Table 3). Actually FQ in-duced a lot of aberrations, while HFQ gave values veryclose to the controls.

To have direct information of the consequences in-duced by DPC at length greater than zero formed byFQ, we carried out some experiments with the double-irradiation protocol. Because both FQ and HFQ cannotform ISC at all, we can only detect the biological effect ofthe formation of DPC. Figure 8 shows the data obtainedwith this procedure in CHO cells testing the formationof chromosomal aberrations. We can see that their num-ber increases after the second irradiation step; becauseFQ with this mechanism induces only DPC, but not ISC,this result tells us that DPC are mainly responsible of FQgenotoxicity. On the contrary, HFQ was practically inac-tive. To establish this assumption, we plotted the num-ber of aberrations scored with FQ against DPC amounts

Vol. 1 Photochemical and photobiological properties . . . 5

Table 3. Chromosomal aberrations in CHO cells. After treat-ment, CHO cells were incubated for 24 hours at 37 ◦ ingrowth medium, and then the aberration frequency wasdetermined.

CompoundDrug

UVA doseTotal

concentration(kJ·m−2)

aberration

(µM) frequency (%)

None 0 2 0.8

(controls)

8-MOP 4.6 1.65 32.5

FQ 2.3 0.12 75.6

HFQ 2.3 0.12 2.65

0

20

40

60

80

100

Tota

lab

erra

tion

s(%

)

0.1 0.3 0.6

Total dose of UVA ( kJ· m−2)

HFQ FQ

First step Second step

Figure 8. Chromosomal aberrations induced by the double-irradiation protocol. The first step was carried out usinga 2.3µM concentration. After several washing, CHO cellswere irradiated further with different UVA doses. The aber-ration frequency was determined after 24 hours of incuba-tion.

formed in the same experimental conditions. A straightline was obtained, with a good linear relationship (datanot shown).

Using the same protocol, we also detected the lethal-ity of DPC greater than zero of FQ in CHO cells; asexpected, with HFQ the surviving fraction obtained af-ter the first irradiation was not further decreased sig-nificantly by the second step. As expected, during thesecond irradiation step DPC formed by FQ strongly re-duced the survival, according to the UVA dose, with anevident killing effect (see Figure 9).

6. DISCUSSION

FQ and HFQ induce the same kinds of damages intoDNA, roughly to the same order of magnitude (mainlymonofunctional adducts and DPC, but no ISC); in com-parison with 8-MOP both compounds are much more ef-fective. This behaviour is consistent with the very highantiproliferative activity tested on the clonal growth of

0.1

1.0

Surv

ivin

gfr

acti

on

0.5 1.0 1.5

UVA dose ( kJ·m−2)

FQ

HFQ

1◦ step

Figure 9. Clonal growth studied by the double-irradiationprotocol: CHO cells were exposed to the first irradiation step(0.06 KJ·m−2) in the presence of 2.3µM FQ or HFQ. The cellswere washed and submitted to the second irradiation step.Clonal growth was then determined.

CHO cells; in this test, 8-MOP, despite its capacity offorming ISC, appeared to be poorly active, even if as-sayed in more severe conditions. Using the double-irradiation method we observed that FQ could induce anumber of DPC by a two steps mechanism; on the con-trary, HFQ can form this lesion only by a single stepone. Because both drugs cannot form ISC, we studiedthe antiproliferative activity of DPC using the double-irradiation method; as expected, the second irradiationstep was ineffective with HFQ, but, on the contrary, FQinduced a strong reduction of the survival.

It is well known that the formation of ISC by furo-coumarins requires the sequential absorption of twophotons. We found that FQ induced DPC by a similarprocess. Conversely, DPC formation by HFQ occurs cer-tainly with the absorption of a single photon; thereforethey are certainly cross-links at zero length, like to thatformed by UVC. In conclusion, we could suppose DPCformed by the two drugs might be different. At presentthis is only a working hypothesis, but it seems to be con-sistent with the data we obtained studying the repairand the genotoxicity of such lesions. In fact, followingthe formation of DSB in DNA by incubating in growthmedium the sensitized CHO cells, we observed with FQa very high DNA fragmentation; with HFQ practically noDSB were formed. DSB are clearly not induced by a pho-tochemical reaction but by enzymatic processes occur-ring during cell incubation after the treatment. 8-MOP,used as a reference, formed significant amounts of DSBeven if to a much lower extent in comparison with FQ.We obtained the same picture studying the formationof chromosomal aberrations. FQ yielded a lot of aber-rations and their number increased with the second ir-radiation step: this probably means they are producedby DPC induction. Even in this test, HFQ appeared to be

6 F. Bordin Vol. 1

practically inactive.

Checking the classic effect induced by furo-coumarins, i.e., the formation of skin erythemas, wefound again the same situation: FQ is almost as activeas 8-MOP, while HFQ only provoked weak erythemas inmore severe experimental conditions.

Therefore, DPC formed by the furoquinolinones ap-pear to be very different, at least on the bases of theirdissimilar mechanism of formation and of their bio-logical consequences. As stated before, certainly DPCformed by HFQ are at zero length. DPC induction by FQrepresents a more complicated process. Actually, evenif these DPC are formed by a double-step mechanismlike ISC, thus suggesting they are cross-links at lengthgreater than zero, at present we have no direct evi-dences that the FQ molecule is really a physical part ofthe cross-links between the two macromolecules. How-ever this seems to be much more than an interestinghypothesis, because it is supported also by some dataalready published [25].

Finally, HFQ seems to be an interesting drug for pho-tomedicine. In fact, the data regarding its activity onmammalian cells together with its very low skin photo-toxicity and genotoxicity suggest that it could be a newpromising agent for photochemotherapy.

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[19] M. A. Pathak and C. Carraro, The Biological role ofReactive Oxygen species in skin, p. 75, University ofTokyo Press, Tokyo, 1987, O. Hayaishi, S. Imamuraand Y. Miyhachi (Eds.).

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