6-Aminoquinolones: photostability, cellular distribution and phototoxicity

Toxicology in Vitro 18 (2004) 581–592

www.elsevier.com/locate/toxinvit

6-Aminoquinolones: photostability, cellular distributionand phototoxicity

G. Viola a,*, L. Facciolo a, S. Dall�Acqua a, F. Di Lisa b, M. Canton b, D. Vedaldi a,A. Fravolini c, O. Tabarrini c, V. Cecchetti c

a Department of Pharmaceutical Sciences, University of Padova, via Marzolo 5, 35131 Padova, Italyb Department of Biological Chemistry, University of Padova, via G. Colombo 3, 35121 Padova, Italy

c Department of Chemistry and Technology of Drug, University of Perugia via del Liceo, 06123 Perugia, Italy

Received 28 July 2003; accepted 16 January 2004

Abstract

Three selected aminoquinolones endowed with a potent antibacterial (compounds 1 and 2) and antiviral activity (compound 3)

have been evaluated for their phototoxic properties in vitro. Photostability studies of these compounds indicate that compound 3 is

photostable whereas compound 1 and in particular, compound 2 are rapidly photodegraded upon UVA irradiation, yielding a toxic

photoproduct. Intracellular localization of these compounds has been evaluated by means of fluorescence microscopy using te-

tramethylrhodamine methyl ester and acridine orange, which are specific fluorescent probes for mitochondria and lysosomes,

respectively. No co-staining was observed with lysosomal stain for all the test compounds. On the contrary compound 3 was found

to be specifically incorporated in mitochondria. The compounds exhibited remarkable phototoxicity in two cell culture lines: human

promyelocytic leukaemia (HL-60) and human fibrosarcoma (HT-1080). The quinolone-induced photodamage was also evaluated

measuring the photosensitizing cross-linking in erythrocyte ghost membranes, the strand breaks activity and oxidative damage on

plasmid DNA. The results show that these derivatives are able to photoinduce crosslink of erythrocytes spectrin, whereas do not

significantly photocleavage DNA directly, but single strand breaks were observed after treatment of photosensitized DNA with two

base excision repair enzymes, Fpg and Endo III respectively.

� 2004 Elsevier Ltd. All rights reserved.

Keywords: Phototoxicity; Photodegradation; Quinolones; Protein photo-crosslink; DNA-photocleavage

1. Introduction

Quinolones are potent chemotherapeutic agents of

first choice for the treatment of a broad range of bac-

terial infections. Toxicity of quinolones is low and

comparable to that of other commonly used antimi-

crobial agents; so they can be considered relatively well-

Abbreviations: AO, acridine orange; BER, base excision repair

enzymes; BPB, bromophenol blue; BSA, bovine serum albumin;

DMSO, dimethyl sulfoxide; Endo III Endonuclease III; Fpg, for-

mammido pyrimidin glycosilase; His, Histidine; MTT, 3-(4,5-dimeth-

ylthiazol-2-yl)-2,5 diphenyl tetrazolium) bromide; ROS, reactive

oxygen species; ssb, single strand breaks; TMRM, tetramethyl

rhodamine methyl ester; Trp, tryptophan; Tyr, Tyrosine*Corresponding author. Tel.: +39-498275363; fax: +39-498275366.

E-mail address: [email protected] (G. Viola).

0887-2333/$ - see front matter � 2004 Elsevier Ltd. All rights reserved.

doi:10.1016/j.tiv.2004.01.008

tolerated. However, this class of antibiotics exhibits

moderate to severe phototoxicity manifested by a dra-matic sunburn in patients exposed to direct sunlight

upon treatment with some members of this class of

drugs (Andriole, 1999).

The phototoxic mechanism(s) underlines abnormal

photosensitivity but its relationship with chemical

structure remains in many cases unclear. The action of

these photosensitizers depends on the production of

extremely reactive molecules (reactive oxygen species,free radicals, photoproducts), able to modify the cell

components, including lipids, proteins and nucleic acids,

when combinated with UVA (Martinez and Chignell,

1998; Wada et al., 1994; Sortino et al., 1998). It has been

suggested that the most phototoxic quinolones induce

the formation of reactive oxygen species (ROS) able to

cause severe tissue damage (Wagai and Tawara, 1992a;

Wagai and Tawara, 1992b; Umezawa et al., 1997),

mail to: [email protected]

582 G. Viola et al. / Toxicology in Vitro 18 (2004) 581–592

although other mechanisms, i.e. generation of highly

reactive photodegradation products, seem to be in-

volved (Martinez et al., 1998).

Many studies on the phototoxic properties of thesedrugs have been reported in the last decades and struc-

ture-side effects relationship of newly developed fluo-

roquinolones has been evaluated (Domagala, 1994;

Traynor et al., 2000; Miolo et al., 2002).

Photoreactivity is mostly influenced by the sub-

stituent at the C-8 position; in fact, halogen atoms,

such as fluorine and chlorine, have relatively high inci-

dence (Domagala, 1994), while the introduction of amethoxy group greatly reduces the phototoxicity

(Von Keutz and Schulter, 1999). The current challenge

in this field is therefore to develop less phototoxic

quinolones.

Previously, some of us have described a new series of

quinolones in which the usual fluorine atom at C-6 po-

sition was replaced with a hydrogen atom or an amino

group. These compounds showed strong activity againstGram-positive bacteria (Cecchetti et al., 1995; Cecchetti

et al., 1996a; Cecchetti et al., 1996b). Moreover an

interesting result is that 6-aminoquinolones, if appro-

priately functionalised, may be useful as antiviral agents.

In particular, the introduction of a 4-aryl-piperazinyl

moiety at C-7 position coupled with a small substituent

at N-1 position (the best being the methyl group), gave

compounds with anti-HIV-1 and anti-HIV-2 activity inboth acutely and chronically infected cells (Cecchetti

et al., 2000; Tabarrini et al., 2002). The antiviral

mechanism of 6-aminoquinolones is clearly distinct

from that of the other known inhibitors of HIV, such as

reverse transcriptase and protease inhibitors. In fact

they interfere with the trans-activation step of the

transcription, thus becoming promising candidates for

N

ONH2

NCH3

COOH

N

ONH2

NCH3

COOH

N

ONH2

NCH3

COOH

NN

1 2

3

Fig. 1. Chemical structure of the examined compounds.

the combination therapy of the AIDS (Parolin et al.,

2003).

Therefore, this paper aims at analyzing the photo-

stability and the phototoxic properties of one of themost active and selective anti-HIV 6-aminoquinolone 3,

as well as of two potent antibacterials 1 and 2 (Fig. 1).

In particular, observations on the in vitro effects on

two cell lines are reported. We have also extended our

studies on the photochemical damages induced by these

new derivatives on biological molecules, such as proteins

and DNA, to better characterize the cellular targets

involved in their phototoxic reactions.

2. Materials and methods

Chemicals. Aminoquinolones 1–3 were synthesized as

previously described (Cecchetti et al., 1996a; Cecchetti

et al., 2000). and their chemical structure is depicted in

Fig. 1. Bovine serum albumin (BSA), Ribonuclease A

from bovine pancreas, 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyl tetrazolium bromide (MTT), Acridine Orange

(AO) and agarose were purchased from Sigma Chemical

Company (St. Louis, MO USA). pBR322 DNA was

purchased from InVitrogen (Milano, Italy). Coomassie

Brilliant Blue G-250 was purchased from Bio Rad

Laboratories (Segrate, Milano, Italy). Acrilamide and

N ;N 0-methylenebisacrylamide, ammonium persulphate,

TEMED (N ;N ;N 0;N 0-tetramethylethylethylenediaminewere purchased from Pharmacia Biotech.

Tetramethylrhodamine methyl ester (TMRM) was

purchased by Molecular Probes (Eugene, OR, USA).

The two base excision repair enzymes (BER) forma-

mydo-pyrimidine glycosilase (FPG) and Endonuclease

III were a generous gift from Dr. S. Boiteux (CEA,

Fontenay aux Roses FRANCE).

Irradiation procedure. HPW 125 Philips lamps,mainly emitting at 365 nm, were used for irradiation

experiments. The spectral irradiance of the source was

4.0 mWcm�2 as measured, at the sample level, by a

Cole-Parmer Instrument Company radiometer (Niles,

IL), equipped with a 365-CX sensor.

2.1. HPLC separation and photoproducts characteriza-

tion

HPLC was performed using a Perkin–Elmer Series

200 equipped with a diode array detector. A Vydac C18

column was used (5 lm, 250 · 4.3 ID) for chromato-graphic separations. The mobile phase consisted of:

Eluent A, water: formic acid (99:1); Eluent B, aceto-

nitrile: formic acid (99:1). Gradient: 5% B (0 min), 95%

B (15 min); flow rate 1 ml/min. ESI/MS measurements

were performed with an API-TOF Mariner. 1H-NMR

analysis were performed by a 200 MHz Varian Gemini

spectrometer.

G. Viola et al. / Toxicology in Vitro 18 (2004) 581–592 583

2.2. Cellular phototoxicity

Human promyelocytic leukaemia cells (HL-60) were

grown in RPMI-1640 medium, human fibrosarcomacell (HT-1080) were grown in DMEM medium both

supplemented with 115 units/mL of penicillin G (Invit-

rogen, Milano, Italy), 115 lg/ml streptomycin (Invit-

rogen, Milano, Italy) and 10% fetal calf serum

(Invitrogen, Milano, Italy). Individual wells of a 96-well

tissue culture microtiter plate (IWAKI, Japan) were

inoculated with 100 ll of complete medium containing

8 · 103 HL-60 cells or 5 · 103 HT-1080 cells. The plateswere incubated at 37 C� in a humidified 5% incubator

for 18 h prior the experiments. After medium removal,

100 ll of the drug solution, dissolved in DMSO and

diluted with Hank�s balanced salt solution (HBSS

pH¼ 7.2), was added to each well. After irradiation, the

solution was replaced with the medium, and the plates

were incubated for 72 h. Cell viability was assayed by

the MTT [(3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl tet-razolium bromide)] test, as described previously (Elisei

et al., 2002; Miolo et al., 2002).

2.3. Fluorescence microscopy

HT-1080 fibrosarcoma cells were grown in a sterile

microscope slides and treated with the three compounds

at the concentration of 50 lm for 30 min, then washed

with HBSS. Cellular fluorescence images were acquired

with an Olympus IMT-2 inverted microscope, as previ-ously described (Petronilli et al., 1999). For subcellular

co-localization studies Tetramethyl Rhodamine methyl

ester (TMRM) and Acridine Orange (AO), were used as

fluorescent probes which stain mitochondria and lyso-

somes, respectively. Quinolones excitation was per-

formed at 350 nm and emission was read at 440 nm.

Longpass emission filter settings were used to separate

the emission of the probes from that of the test com-pounds. Data were acquired and analysed using the

Metamorph software (Universal Imaging).

2.4. Erithrocyte ghost preparation

Ghosts were prepared from heparinized human blood

following the gradual osmotic lysis method of Steck and

Kant (1974). White membranes (ghosts) were resus-

pended in PBS buffer. Membrane protein contents weredetermined as described (Peterson, 1977), using bovine

serum albumin (BSA) as standard.

2.5. Protein photo cross-link

The test compounds were added to the membrane

suspension (1.0 mg/ml protein concentration) and irra-

diated. The membrane samples were reduced and

denatured by addition of b-mercaptoethanol and SDS at

90 �C for 3 min, and bromophenol blue (BPB) was ad-

ded before polyacrylamide gel electrophoresis analysis

(5% running gel, 3% stacking gel). The quantitation of

the bands, stained with Coomassie Brilliant Blue R-250,was achieved by image analyzer software Quantity

One (BIO RAD, Milano, Italy).

2.6. Studies on isolated proteins

Solutions of Bovine serum albumin (BSA), (0.5 mg/

ml) in phosphate buffer 10 mM were irradiated in the

presence of the test compounds for various time using a

quartz cuvette. At each time the Tryptophan (Trp)

content was followed by monitoring the characteristicTrp fluorescence as described (Balasubramanian et al.,

1990). Solutions of Ribonuclease A (RNAseA), 0.5 mg/

ml in phosphate buffer 10 mM were irradiated in the

presence of the test compounds for various time periods

in a quartz cuvette. Analogous experiments were per-

formed by bubbling nitrogen before and during the

irradiation.

2.7. Studies on isolated amino acids

Irradiation system and buffer in these experiments

were the same used for the irradiation of BSA. Tryp-

tophan solutions (100 lM) were irradiated in the pres-

ence of the test compounds and analysed as described

for BSA. Histidine solutions were irradiated for various

time periods and the resulting samples were analysed for

His content as described (Figueiredo et al., 1993).Tyrosine solutions (1 mM) were irradiated in PBS for

various times and the resulting samples were analysed

for Tyr content as described (Peterson, 1977) Analogous

experiments were performed by bubbling nitrogen prior

and during the irradiation and using phosphate buffer

prepared with deuterium oxide instead of water.

2.8. pBR322 DNA strand breaks

Each pBR322 DNA sample (100 ng) dissolved in TEbuffer (10 mM Tris-HCl, 1 mM EDTA, pH 7.5) was

irradiated with increasing UVA doses in the presence of

the compounds under examination. After irradiation

two aliquots of the sample were incubated for 30 min at

37 �C with Fpg (formamydo pyrimidin glycosilase) and

Endo III (Endonuclease III) as described by Pflaum

et al. (1994). The samples were loaded on 1% agarose

gel, and the run was carried out in TAE buffer (0.04 MTris-acetate, 1 mM EDTA) at 50 V for 3 h, using the

GNA-100 electrophoretic apparatus (Amersham Bio-

sciences). After staining in ethidium bromide solution,

the gel was washed with water and the DNA bands were

detected under with a UV transilluminator. Photo-

graphs were taken with a digital photocamera Kodak

DC265 and the quantitation of the bands was achieved

Table 1

Mass spectral data of photodegradation products of aminoquinolones

Photoproduct Retention time (min) m/z

P1 8.6 359 (+17 amu)a


by image analyzer software Quantity One (BIO RAD,

Milano, Italy). The fractions of supercoiled (Form I)

and open circular DNA (Form II) were calculated

as described (Ciulla et al., 1989).
P2 10.3 357 (+15 amu)
P3 9.1 315 ()75 amu)

P4 11.9 386 ()4 amu)a In parenthesis is indicated the variation of mass in comparison to

the parent compound.
3. Results
3.1. Photostability

Previous studies (Miolo et al., 2002), showed that

compounds 1 and 2 underwent photodegradation, as

shown by the changes in their absorption spectra upon

UVA irradiation in buffered aqueous solution. Com-

pound 3, in comparison to the other two derivatives,

does not show any variation of the absorption spectrum

even after a long time of irradiation (data not shown).

To obtain precise informations of the photodegra-dation kinetics, at appropriate time intervals the irra-

diated solutions were analysed by HPLC (Fig. 2).

Irradiation of 1 and 2 induces for both compounds the

formation of two photoproducts, while compound 3

practically remains unmodified even at high doses of

UVA (23 J cm�2). Kinetic analysis of the photodegra-

dation process reveals an apparent first order kinetics

for the three derivatives. The rate constants (k) wereobtained from the slopes of the plots by linear regression

analysis. Degradation kinetics followed a first order

reaction with photolysis constants of 2.3 · 10�3,

9.8 · 10�3 and 1.6 · 10�4 min�1, for compounds 1, 2 and

3 respectively.

The characterization of the photoproducts was per-

formed by ESI-MS and the mass spectral data are re-

ported in Table 1. In the case of compound 2, thestructure of the main product, P4, that is produced in

good yield, was elucidated by NMR spectroscopy. Fig. 3

illustrates the 1H-NMR spectra of the photoproduct

(panel A) and of the compound 2 (panel B).

Abso

rban

ce u

nits

0.00

0.02

0.04

RetentionTime (min)0 5 10 15 20 25

Abso

rban

ceun

its

0.00

0.02

0.04

0.06

0.00

0.02

0.04

Retention0 5 10

0.00

0.02

(A) (B)

(D) (E)

P1P2P3

Fig. 2. HPLC profile of aminoquinolones upon UVA irradiation in phosph

panel D¼ 15 J cm�2); compound 2 (panel B¼ 0 J cm�2, panel E¼ 15 J cm�2

The 1H-NMR spectra of P4 and compound 2 showed

similar patterns: two singlets in the aromatic region at

8.9 and 8.4 ppm, respectively; a multiplet between 7.22

and 7.23 ppm which has been assigned to the aromatic

protons of the tetrahydroisoquinolinic ring; a multiplet

at 4.3 ppm; the methyl group in position 8 has been

assigned to the signal at 2.6 ppm; two multiplets be-

tween 1.3 and 0.80 ppm which correspond to the cy-clopropyl group. It is also possible to see a signal at 4.95

ppm which has been assigned to the protons of the

amino group in position 6. The substantial difference

between the two spectra lies in the region from 7 to 9

ppm. In fact, while the spectrum of compound 2 only

displays the presence of the singlet belonging to the

carboxyl group (7.35 ppm), the spectrum of P4 shows a

signal of a doublet at 8.3 ppm which counterparts is amultiplet at 7.20 ppm.

These data, also supported by the mass spectrometry

analysis, lead to postulate a photoinduced aromatisa-

tion of the tetrahydroisoquinolinic nucleus in position 7

of compound 2. The proposed structure is showed in

Fig. 3.

3.2. Intracellular localization

It has recently been demonstrated by microspectro-

fluorimetric techniques on fibroblast cells that some

fluoroquinolones localize in subcellular structures. For

example, lysosomes are a preferential site for lomeflox-

0.00

0.04

0.08

0 8 120.00

0.04

0.08

Time (min)15 20 25

Retention Time (min)

P4

(C)

(F)

4

ate buffer under aerobic conditions. Compound 1 (panel A¼ 0 J cm�2,

); compound 3 (panel C¼ 0 J cm�2, panel F¼ 23 J cm�2).

Fig. 3. 1H-NMR spectra of photoproduct P4 (panel A) with the proposed chemical structure. In the inset is also shown a magnification of the region

between 7 and 9 ppm. Panel B show for the sake of comparison the 1H-NMR spectra of compound 2.

Fig. 4. Fluorescence microphotographs showing the intracellular

localization of the aminoquinolones in HT-1080 fibrosarcoma cells in

the presence of TMRM. The cells were treated as described in Section 2.

Upper panels, compound 1 middle panels compound 2; lower panels

compound 2. Column A, fluorescence image of the aminoquinolones;

Column B, fluorescence image of TMRM; Column C, Overlay image of

aminoquinolones localization generated by transferring the blue color


acin and ciprofloxacin whereas for norfloxacin the cel-

lular localization is less clear (Ouedraogo et al., 1999).

Other subcellular structures could be the target of these

compounds. To investigate the intracellular localizationof 6-aminoquinolones we used two fluorescent probes:

TMRM, a lipophilic cation commonly used for the

assessment of the mitochondrial potential (Bernardi

et al., 1999; Petronilli et al., 1999), and acridine orange,

which stains lysosomes (Kessel et al., 2000). These

markers fluoresce in the visible region (about 550 nm)

while the test compounds emit in the blue region. Both

fluorescences can be easily separated using suitablebandpass optical filters. After an incubation in HT-1080

fibrosarcoma cells, all the compounds were found to

incorporate and associate with subcellular structures.

It can be observed that, despite the studied quino-

lones have similar structure, they localize differently in-

side cells. Compound 1 diffuses largely in the cytoplasm

but does not co-localize with the two fluorescence

probes. Compound 2 does not accumulate in lysosomesbut only partially co-localizes with TMRM. A more

evident accumulation in mitochondria can be observed

for compound 3, suggesting that mitochondria may be a

site of photosensitization (Figs. 4 and 5).

of the compounds seen in column A onto the corresponding fluores-

cence image of column B. Pink is used as a false color to indicate the

localization of aminoquinolones. (For interpretation with reference to

the color picture the reader is referred to the web version of this article.)
3.3. Cellular phototoxicity
Cellular phototoxicity was investigated using two cell

lines: HT-1080 fibrosarcoma and HL-60 leukemiarespectively. In Fig. 6 the viability curves after irradia-

tion with various UVA-doses and different concentra-

tion of aminoquinolones are depicted. The most potent

compound in both cell lines is compound 3 which

exhibits high levels of toxicity even at very low con-

centrations and low UVA doses. Compound 2 has also a

remarkable toxicity, whereas compound 1 is less pho-

totoxic in comparison to the other two studied amino-

quinolones.

HL-60 cells incubated in the presence of a pre-irra-

diated solution of compound 2 showed a dose depen-dent decrease of viability (data not shown) suggesting

the production of a toxic photoproduct. Further experi-

ments performed without UVA irradiation demon-

strated that the isolated photoproduct P4 induces a

Fig. 5. Fluorescence microphotographs showing the intracellular

localization of the aminoquinolones in HT-1080 fibrosarcoma cells in

the presence of AO. The cells were treated as described in Section 2.

Column A, fluorescence image of the aminoquinolones; Column B,

fluorescence image of AO; Column C, Overlay image of aminoqui-

nolones localization generated by transferring the blue color of the

compounds seen in column A onto the corresponding fluorescence

image of column B. Clear blue is used as a false color to indicate the

localization of aminoquinolones. (For interpretation with reference to

the color picture the reader is referred to the web version of this

article.)


remarkable decrease of cellular viability with an esti-

mated IC50 of about 4 lm (Fig. 7, panel A), whereas the

(A)

0 1 2 3 4

% o

f via

ble

cells

0

20

40

60

80

100

(B)

0 1 2

0

20

40

60

80

100

(E)

UVA do

0 1 20

20

40

60

80

100

(D)

0 1 2 3 40

20

40

60

80

100

% o

f via

ble

cells

UVA dose (Jcm )-2

Fig. 6. Effect of the quinolones derivatives photosensitization upon HL-60 c

and F). The cells were treated with the compounds (1, panels A and D

(r¼ irradiated controls,�¼ 2 lM,j¼ 5 lM,N¼ 10 lM,.¼ 20 lM panels

N¼ 2.55 lM, .¼ 5 lM panel C); (r¼ irradiated controls, �¼ 1.25 lM, j¼as indicated in the figure. The viability was measured with the MTT method a

independent experiments performed in quadruplicate.

value for the parent compound is higher than 20 lm.

Moreover, the isolated photoproduct has also been

evaluated for its phototoxicity (Fig. 7, panel B), upon

irradiation showed a moderate induction of cytotoxicityas compared with parent compound 2.

3.4. Protein photodamage

The photosensitization ability of test compounds to-

wards other components of cellular membranes, such as

proteins, was estimated by measuring the photoinduced

cross-linking in erythrocyte ghost proteins (Merville

et al., 1983). Light-induced cross-linking of spectrin, a

protein associated with the cytoplasmic side of the RBC

membrane, in the presence of these compounds wasdetected by the partial or total disappearance of the two

spectrin bands (220.000 and 245.000 Daltons) on SDS-

PAGE while cross-linked aggregates can not run inside

the gel and remain at the top.

Fig. 8 (panel A) shows the pictures of the SDS-PAGE

gels of ghosts irradiated in the presence of the three

aminoquinolones, at the concentration of 100 lm and

at UVA dose ranging from 3.6 to 14.4 J cm�2. Underaerobic conditions compound 2 causes the almost total

disappearance of the two spectrin subunit bands,

whereas in the presence of nitrogen this effect is lower

indicating the involvement of reactive oxygen species.

The other two compounds exhibit a lower efficacy, al-

though in both cases the effect is partially reduced in

anaerobic conditions. The gel scans of erythrocytes

3 4

se (Jcm )-2

3 4

(C)

0 1 2 3 4

0

20

40

60

80

100

(F)

0 1 2 3 40

20

40

60

80

100

UVA dose (Jcm )-2

ells (panels A, B and C) and HT-1080 fibrosarcoma cells (panels D, E,

; 2, panels B and E; 3, panel C and F) at different concentrations

A, B, D and E); (r¼ irradiated controls,�¼ 0.625 lM,j¼ 1.25 lM,

2.5 lM, N¼ 5 lM, .¼ 10 lM panel G), and various doses of UVA,

s described in Materials and methods. Points are mean±SEM for three

Concentration (µm)0 5 10 15 20

% o

f via

ble

cells

0

20

40

60

80

100

UVA Dose (Jcm-2)0 1 2 3 4

% o

f via

ble

cells

0

20

40

60

80

100

(A)

(B)

Fig. 7. Effect of photoproduct P4 on HT-1080 cells. Panel A: Dark

cytotoxicity evaluated after 72 h of incubation in the presence of P4

(j) and for the sake of comparison compound 2 (�), at different

concentrations. Panel B: Phototoxicity of P4 at different concentra-

tions (r¼ irradiated controls, �¼ 1.25 lM, j¼ 2.5 lM, N¼ 5 lM,

.¼ 10 lM) and UVA doses.


spectrin for all the three drugs investigated are shown in

Fig. 8 (panel B).

To further investigate the photosensitizing properties

of the three compounds towards proteins, solutions

containing bovine serum albumin, as a model system

(Miranda et al., 1998), and the drugs in phosphate buffer

were irradiate for different time periods. Trp contentwas directly analysed by monitoring the characteristics

fluorescence of Trp residues (Balasubramanian et al.,

1990). No effects were observed with the three com-

pounds indicating that Trp is not involved in the protein

photodamage. Another protein model used in this study

was RNAseA, which is devoid of Trp but has Tyr resi-

dues in its sequence. Its emission band centered at about

350 nm does not change after irradiation with com-pound 1 and 3. Interestingly, compound 2 rapidly de-

creases the emission of the protein by about 80% after

irradiation for 15 min (Fig. 9, panel A). This effect is

markedly reduced in anaerobic conditions. Deuterated

phosphate buffer which increases singlet oxygen lifetime,

did not induce any variation of the degradation profile.

Furthermore, gel electrophoresis of RNAseA showed

protein cross link after irradiation in the presence ofcompound 2 (data not shown).

Subsequent analysis were performed to confirm the

results on pure amino acids. Data obtained with Tyr are

in excellent agreement with the observation on the pure

RNAseA. Under aerobic conditions 30% of Tyr was

degradated (Fig. 9, panel B), whereas in anaerobic

conditions no effect was observed. In this case too, Tyr

degradation was not potentiated in deuterated buffer,suggesting that singlet oxygen should not be involved in

the reaction.

Histidine reacts which singlet oxygen faster than

other amino acids, such as Trp and Tyr, so it is useful to

test its degradation if we expect a type II mechanism. In

the case of compounds 1 and 2 we observed a rapid

decrease of His content (Fig. 9, panels C and D). This

effect was abolished in N2-purged solution and poten-tiated in deuterated phosphate buffer, suggesting the

involvement of singlet oxygen. Compound 3 did not

show any effect on this amino acid.

3.5. DNA photodamage

pBR322 Plasmid DNA was used as a model system to

evaluate DNA breaking activity. None of the test

compounds was able to induce DNA damage without

UVA irradiation.

Supercoiled circular DNA allows the detection of

structural alterations such as strand breaks or damagedbases. DNA strand breaks can be induced either directly

(frank strand breaks) or indirectly. In the latter case we

used DNA repair enzymes in order to determine the

characteristics of DNA modifications: Fpg protein rec-

ognizes 8-hydroxyguanine, purines whose imidazole ring

is open (Fapy residues) and sites of base loss (apurinic

sites) and Endonuclease III recognizes apurinic sites and

5,6-dihydropirimidine derivatives. Release of damagedbase is followed by a b–d reaction and a b elimination

step respectively, resulting in DNA breakage (Burrows

and Muller, 1998).

In addition to endonuclease sensitive modifications,

the number of single strand breaks generated by the

excited photosensitizers were quantified. Fig. 10 shows

the results obtained for the three aminoquinolones tes-

ted at a [drug]/[DNA] ratio of 1. For all compounds theformation of single strand breaks expressed as the per-

centage of form II is low and becomes significantly

different from the irradiated control, only at the highest

UVA dose employed (22.5 J cm�2). On the contrary,

high levels of single strand breaks can be detected after

enzyme digestion for 2 and 3 whereas only a slight in-

crease was observed for 1.

Fig. 8. Electrophoretic pattern (A) of the photoinduced cross-link of spectrin in RBC ghosts irradiated in aerobic and anaerobic conditions, for the

indicated times, corresponding to UVA doses of 3.6 J cm�2 (15 min) to 14.4 J cm�2 (60 min), in the presence of aminoquinolones at the concentration

of 50 lm in PBS buffer pH¼ 7.2. (B) Quantitation of the relative abundance of spectrum by gel densitometry; (�¼Air,j¼N2). The spectrin band is

indicated by an arrow.


4. Discussion

In this study we have evaluated the phototoxic po-tential of three aminoquinolones, using various in vitro

methods. Particular attention has been given towards

the investigation of the mechanism involved in their

phototoxicity, extending our studies on the photo-

chemical damage on biological molecules, such as pro-

teins and DNA, to better characterize the cellular targets

involved in their phototoxic reactions.

To have a complete phototoxicological profile westudied the photoreactivity of the test molecules

including degradation reactions. The three aminoqui-

nolone derivatives showed different patterns of degra-

dation depending on their molecular structures. The

UVA lamp emits light with maximum intensity at 365nm and the kmax of the test compounds has similar molar

extinction coefficients at this wavelength, thus differ-

ences underlying in their photodegradation profiles may

not be related to a different absorption of light.

By a photochemical point of view the strength of a

photosensitiser is determined by the nature of the ex-

cited states or by its ability to form reactive species

while, in a phototoxicological context, other factorssuch as subcellular localization, are important parame-

ters. Thus, it is critically important to evaluate the dis-

% o

f und

ecom

pos

ed H

is

0

20

40

60

80

100

UVA Dose (J cm-2)

% o

f und

e co

mpo

sed

His

0

20

40

60

80

100

UVA Dose (J cm )

UVA Dose (J cm )

-2 UVA Dose (J cm )-2

-2

0 4 8 12 16

0 4 8 12 16 0 4 8 12 16

0 4 8 12 16

I 0/I f

0

20

40

60

80

100

% o

fu n

deco

mpo

sed

Tyr

0

20

40

60

80

100

(A) (B)

(C) (D)

Fig. 9. Photosensitising effects of compound 2 on RNAseA (panel A) and Tyr (panel B) after irradiation at the dose of 50 lm in different conditions

(�¼Air, j¼N2 purged solution, N¼Deuterated phosphate buffer purged solution). Photosensitising effects of compounds 1 (panel C) and 2

(panel D) on His after irradiation at the dose of 100 lm in different conditions (� ¼ Air,j¼N2 purged solution,N¼Deuterated phosphate buffer).


tribution of photosensitisers inside cells. Subcellular

localization of a photosensitiser is due to its physico-

chemical properties such as hydrophobicity, charge, pKa

etc. Previous studies have shown that fluoroquinolones

principally accumulate in lysosomes (Ouedraogo et al.,

1999), while the present compounds (aminoquinolones),

interestingly do not shown the same pattern. The

physico-chemical properties of the aminoquinolones

amply differ from that of fluoroquinolones used in

therapy. They are hydrophobic, as demonstrated by

their high values of partition coefficients (Miolo et al.,2002) and do not possess a pKa close to neutrality, while

many fluoroquinolones are present in solution as zwit-

terionic ions. Consistently with these characteristics,

none of the aminoquinolones investigated shows a

lysosome incorporation. On the contrary, preferential

accumulation in mitochondria has been observed in

particular for compound 3. On HL-60 and HT-1080

cells, compound 3 was highly phototoxic, even at thelowest concentrations (0.625 and 1.25 lm respectively)

and UVA dose applied (1 J cm�2) followed by com-

pound 2 and compound 1. Numerous reports show that

photosensitizers which localize in mitochondria are

more efficient in killing cells than those that localize in

other cellular sites (Oleinick et al., 2002 and references

therein).

This study clearly demonstrates that compound 2 has

a phototoxic potential which may be partially mediated

by the induction of a toxic photoproduct. The cytotoxicactivity and the concentration of the photoproduct

are not related and this could be due to the low solu-

bility. Furthermore the presence of a positive charge on

the molecule could be an hindrance for its diffusion into

the cells. It is possible that the cytotoxic effect of this

photoproduct may be caused by a small amount pro-

duced inside the cell from the parent compound. The

target for the P4-induced cell damage is not clear at themoment, although preliminary results obtained with

fluorescence microscopy (data not shown) suggest spe-

cific accumulation of this photoproduct in mitochon-

dria.

An important damage photoinduced by aminoqui-

nolones is the cross-link in membrane proteins. A good

model is represented by erythrocyte membranes because

they contain spectrin, an excellent target of the cross-link reactions (Dubbelman et al., 1978; Merville et al.,

1983). Although the three derivatives promote spectrin

cross-link, their, efficiency differs from each other.

Compound 2 is the most efficient whereas the other two

compounds seem to be less active. The spectrin cross-

link is reduced in anaerobic conditions indicating the

involvement of molecular oxygen in the mechanism.

Fig. 10. DNA strand breaks photoinduced by the aminoquinolones.

pBR322 supercoiled circular DNA was irradiated at the indicated

doses of UVA and then treated as described in materials and methods.

Data are expressed as percentage of form II obtained after densito-

metric analysis of the agarose gel.


The amino acids that are more easily photooxidized inproteins are tryptophan, tyrosine, histidine, cysteine and

methionine (Davies and Truscott, 2001). Thus, in order

to elucidate which amino acids are involved in the

protein photosensitization by the test compounds, we

studied the photoreaction with BSA and RNAseA as

protein models. The experimental results, obtained with

BSA indicated that Trp does not constitute a target for

the photoreaction with test compounds. On the contraryTyr residues in RNAseA are promptly photoxidized

upon irradiation in the presence of compound 2. How-

ever, it should be note thatin a native protein the reac-

tivity of the above amino acids might be affected by the

location of the given amino acid residue or to the par-

ticular binding site of the photosensitiser, thus the

photosensitised reactions of proteins turn out to be very

complex.The behaviour of the three compounds largely differs

in respect of pure amino acids. Compound 1 neither

photooxidizes Trp nor Tyr, but it is able to photoxidize

His, although with a low efficiency. In contrast, com-

pound 2 is able to efficiently photooxidize Tyr and His.

From a mechanistic point of view, the photoreactions

with these two amino acids are strongly reduced in

anaerobic conditions but only in the case of His thephotodegradation is remarkable potentiated in deuter-

ated phosphate buffer, suggesting that this effects may

be mediated by singlet oxygen.

Most of the fluoroquinolones present in therapy have

demonstrated a high extent of DNA photodamage and

this can explain their capability of enhancing UVA-

induced phototumorigenesis (Reavy et al., 1997; Cheta-

lat et al., 1996; Martinez and Chignell, 1998; Belvedereet al., 2002; Cuquerella et al., 2003).

In order to determine whether test compounds were

able to photosensitize DNA, strand break activity was

evaluated using supercoiled circular DNA, a very sen-

sitive tool for damage detection (Marrot et al., 2001). In

addition to frank strand breaks we have also evaluated if

purine and/or pyrimidine bases were involved in the

oxidative damage using base excision repair enzymesFpg and Endo III, respectively. Specific repair enzymes,

such as Fpg or Endo III, induce DNA single strand

breaks at the sites of damaged purine bases such as 8-

oxoguanine or thymine glycol. The aminoquinolones

showed different responses in terms of oxidative dam-

age. While they do not induce a significant direct DNA

breakage, they are able to photooxidize nucleobases, as

assessed after enzymatic digestion with Fpg. No effectswere seen with Endo III. This damage profile for com-

pounds 2 and 3 is an indication that singlet oxygen could

be an intermediate in the DNA damage and is in good

agreement with the data obtained on His photosensiti-

zation.

It should be noted, however, that at the present stage

a definite mechanism for the photoinduced cellular

damage in the presence of aminoquinolones cannot bepresented. Further studies along these lines are neces-

sary and are presently underway to clarify the mecha-

nism.

In conclusion, the three aminoquinolones demon-

strated a potential phototoxicity, in particular com-

pounds 2 and 3. An increase of the phototoxic activity

can be observed in all the utilized tests when the pipe-

ridinyl group is replaced by the more hydrophobic1,2,3,4-tetrahydroisoquinolinyl group. The activity of

compound 3 remains unclear: further experiments are in

progress aimed at defining the targets at cellular level

and the mechanism of phototoxicity.

Acknowledgements

This project was funded by MIUR (Ministero

dell�Istruzione, dell�Universit�a e della Ricerca), pro-

gram: ‘‘Photoprocesses of interest for application’’.


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6-Aminoquinolones: photostability, cellular distribution and phototoxicity

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