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New Zealand Veterinary Journal
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Phylloerythrin: Mechanisms for cellular uptake
and location, photosensitisation and spectroscopic
evaluation
E Scheie a , A Flåøyen a b , J Moan c & K Berg c
a National Veterinary Institute, Department of Food and Feed Hygiene, POB 8156 Dep.,
0033, Oslo, Norway E-mail:
b Department of Large Animal Clinical Sciences, Norwegian College of Veterinary
Medicine, POB 8146 Dep., 0033, Oslo, Norway
c Institute for Cancer Research, Department of Biophysics, The Norwegian Radium
Hospital, Oslo, Norway
Published online: 22 Feb 2011.
To cite this article: E Scheie , A Flåøyen , J Moan & K Berg (2002) Phylloerythrin: Mechanisms for cellular uptake
and location, photosensitisation and spectroscopic evaluation, New Zealand Veterinary Journal, 50:3, 104-110, DOI:
10.1080/00480169.2002.36291
To link to this article: http://dx.doi.org/10.1080/00480169.2002.36291
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104
New Zealand Veterinary Journal 50(3), 104-110, 2002
Scientific Article
Phylloerythrin: Mechanisms for cellular uptake and location,
photosensitisation and spectroscopic evaluation
*†
‡
‡
E Scheie*§, A Flåøyen , J Moan and K Berg
Abstract
Introduction
AIM: To elucidate the photobiological behaviour of phylloery-
thrin by studying the cellular uptake and intracellular
localisation pattern of phylloerythrin and its spectral properties
in Chinese hamster lung fibroblast cells (V79).
Hepatogenous photosensitisation occurs in ruminants when
a toxin, normally produced by a higher plant, a fungus or a
cyanobacterium (alga), causes liver injury or dysfunction, resulting in
retention of the photosensitising agent, phylloerythrin (Clare 1952;
Kellerman et al 1988; Flåøyen and Frøslie 1997). Phylloerythrin
(Figure 1) is a metabolic product of chlorophyll, produced in
ruminants by rumen microorganisms (Rimington and Quin
1933; Quin et al 1935). Normally, phylloerythrin is conjugated
by the liver and excreted into bile. However, liver injury or
dysfunction may lead to depression or cessation of hepatic elimin-
ation of phylloerythrin. In such cases phylloerythrin may enter
the blood stream and reach skin cells and accumulate.
METHODS: Phylloerythrin was diluted in dimethylsulfoxide
(DMSO). Fluorescence emission and excitation spectra were
measured using a luminescence spectrometer equipped with
a red-sensitive photomultiplier. V79 cells were cultured in
monolayers and labelled with 0.25 µg/ml phylloerythrin for
uptake, cell survival and intracellular localisation studies. For
cell survival and intracellular localisation studies, cells were
subsequently exposed to blue light at a fluence rate of 9.0
2
mW/cm .
An acute inflammatory response of the skin can be induced
when phylloerythrin is excited by sunlight. Excited phylloerythrin
RESULTS: The fluorescence excitation spectrum of
phylloerythrin in DMSO was characterised by a Soret band
exhibiting a maximum peak at 418 nm. The fluorescence
emission spectrum had peaks at 643 and 706 nm. The
corresponding spectra in cells were red-shifted to 422, 650 and
712 nm, respectively. The cellular uptake of phylloerythrin was
complete after about 10 h of incubation. The uptake together
with the activation energy and analysis of cells incubated with
phylloerythrin at 37°C and 0°C using fluorescence microscopy
indicated that the dye is taken up into cells via a diffusion-
mediated pathway. Measurements of subcellular marker enzymes
were performed immediately after light exposure of phylloerythrin-
treated cells. The mitochondrial marker enzyme, cytochrome-c
oxidase, and the marker enzyme for the Golgi apparatus, UDP
galactosyl transferase, but not those for lysosomes, β-N-acetyl-
D-glucosaminidase (β-AGA), and endoplasmic reticulum,
NADPH cytochrome-c reductase, were inactivated upon photo-
dynamic treatment.
1
molecules react with oxygen, resulting in singlet oxygen ( O ) and
2
1
possibly other reactive oxygen species being formed. O is believed
2
to be the main cytotoxic product formed during photochemical
treatment of cells (Weishaupt et al 1976; Moan and Sommer 1985).
1
O is short-lived in cells and has a diffusion length of 10–20 nm
2
(Moan and Berg 1991). Thus, the primary cytotoxic effect of a
1
photosensitiser after exposure to light occurs close to the site of O
formation.
2
The efficiency of a photosensitiser in sensitising cells to photo-
inactivation depends on its ability to be taken up by the cells as well
as its photochemical properties. Several factors may influence the
rate of uptake, such as the molecular structure of the photosensitiser
(Woodburn et al 1991; Boyle and Dolphin 1996), time after ad-
ministration (Peng et al 1990; Peng and Moan 1995), cell line,
and incubation conditions (Hanania and Malik 1992). Incubation
conditions, such as the temperature of the medium surrounding the
cells during light exposure, have a major affect on the rate of cellular
uptake of photosensitisers and the sensitivity to light (Moan et al
1981; Rud et al 2000).
CONCLUSION: These results indicate that phylloerythrin is
located mainly in the Golgi apparatus and mitochondria of V79
fibroblasts cells.
KEY WORDS: Golgi, mitochondria, fibroblast, phylloerythrin,
photosensitiser, hepatogenous photosensitisation.
A
Absorbance
β-AGA β-N-acetyl-D-glucosaminidase
CCD Cooled charge-coupled device
DMSO Dimethylsulfoxide
FCS
Foetal calf serum
MEM
Minimal essential medium
* National Veterinary Institute, Department of Food and Feed Hygiene, POB
8156 Dep., 0033, Oslo, Norway
† Department of Large Animal Clinical Sciences, Norwegian College of
Veterinary Medicine, POB 8146 Dep., 0033 Oslo, Norway
‡ Institute for Cancer Research, Department of Biophysics, The Norwegian
Radium Hospital, Oslo, Norway
NADPH Nicotinamide adenine dinucleotide phosphate
1
O
Singlet oxygen
Phosphate-buffered saline
Uridine diphospho
2
PBS
UDP
V79
Chinese hamster lung fibroblast cell line
§ Author for correspondence. Email: eldri.scheie@vetinst.no

Scheie et al
New Zealand Veterinary Journal 50(3), 2002
105
The rate of cellular uptake of a xenobioticum is also highly dependent
upon its lipophilic properties. In cells in vitro, lipophilic dyes generally
localise in membrane structures including plasma, mitochondrial,
endoplasmic reticulum, and nuclear membranes, while hydrophilic
dyes seem to accumulate in lysosomes (Moan et al 1989). Some
photosensitisers seem to be less phototoxic when located in the
plasma membrane than in other cellular compartments (Moan et al
1984). There is also some evidence for a lower quantum yield of cell
inactivation for lysosomally located dyes than for non-lysosomally
located ones (Moan et al 1992). Dyes that locate in mitochondria are
highly efficient at sensitising cells to photoinactivation (Woodburn et
al 1992; Morgan and Oseroff 2001).
equipped with an integrating sphere, suited for measurements of
scattering samples.
Uptake of phylloerythrin by V79 cells
5
2
V79 cells (5x10 ) were seeded out in 9.5 cm dishes and labelled
with 0.25 µg/ml phylloerythrin for up to 24 h. After incubation
the cells were washed three times with ice-cold 0.9% NaCl and
scraped off the substratum in 1 ml 0.9% NaCl. Part of the sample was
collected for protein assay and the rest was centrifuged at 1,100
rpm for 10 min in a Beckman microlitre centrifuge (Beckman
GS-15R, Beckman Instruments, Palo Alto CA, USA) and
resuspended in 1 ml of a solution containing 50% methanol and
1 M HClO . Phylloerythrin was measured spectrofluorometrically
4
To elucidate the photobiological behaviour of phylloerythrin in
skin cells we studied the spectral properties and the intracellular
localisation pattern of phylloerythrin in Chinese hamster fibroblasts.
using a Perkin-Elmer LS-50B and a 1 ml quartz cuvette (Hellma,
Baden, Germany). Fluorescence was measured using an excitation
wavelength of 422 nm and an emission wavelength of 650 nm.
A cut-off filter (545 nm) was used on the emission side. For
quantitative measurements, a standard of known concentration
of phylloerythrin in DMSO was added to the samples to increase
the fluorescence by 50–100%. The background, measured in
dishes without cells but otherwise treated the same way, was
subtracted. Protein was measured using the Bio-Rad assay as
described by Bradford (1976).
Materials and Methods
Cell line
V79 Chinese hamster lung fibroblasts were cultured in monolayers
at 37°C in an atmosphere of 100% humidity and 5% CO added
2
to air. Cells were grown in Eagle’s minimal essential medium
(MEM) with Hanks’ salts (Gibco, Paisley, UK) containing 10%
foetal calf serum (FCS), 100 U/ml penicillin and 100 µg/ml
streptomycin. The cells displayed a doubling time of 10 h and
were kept in exponential growth by subculturing twice weekly.
Labelling with photosensitiser and irradiation
2
V79 cells were seeded out in 9.5 cm dishes (Falcon NY, USA)
and kept in the incubator for about 5 h to allow attachment to
the substratum. The cells were exposed to 0.25, 0.5 and 1 µg/ml
phylloerythrin in MEM medium. If not otherwise described,
4
50x10 cells were incubated at 37°C with photosensitiser for
Chemicals
18 h, washed three times in MEM and incubated in the same
medium for 1 h before exposure to light. Cells were subsequently
exposed to blue light (400–500 nm) at a fluence rate of 9.0 mW/
cm from a bench of 4 fluorescent tubes (Osram 18W/67). After
irradiation, the cells were washed with medium and kept in the
incubator until fixation.
Phylloerythrin was provided by Porphyrin Products (Logan,
USA). The dye was dissolved in DMSO (Sigma, St Louis, USA)
to a concentration of 1 mg/ml as a stock solution and stored
at −80°C. Nocodazol, cytochrome c, the reduced form of β-
nicotinamide adenine dinucleotide phosphate (β-NADPH), and
p-nitrophenyl-N-acetyl-β-D-glucosaminide were purchased from
2
3
Sigma (St Louis, USA). Uridine diphospho-D-[6- H] galactose
Cell survival assay
was purchased from Amersham Pharmacia Biotech (Freiburg,
Germany).
The cytotoxic effect of phylloerythrin, with or without light, was
measured by incubating 400 cells with the photosensitiser for 18
h in 9.5 cm dishes and treated as described above. Cytotoxicity
2
Fluorescence and absorption of phylloerythrin in DMSO
Phylloerythrin wasdiluted in DMSO toaconcentration of1µg/ml
concentration. The solution was studied spectrofluorometrically
using a Perkin-Elmer LS-50B luminescence spectrometer with a
red-sensitive photomultiplier installed (Perkin-Elmer, Norwalk,
USA). Fluorescence was measured by exciting samples at 418
nm (Soret band) and detecting emission at 500–800 nm. A cut-
off filter (545 nm) was used on the emission side. Subsequently,
absorption was measured in a Perkin-Elmer Lambda-15
spectrophotometer equipped with an integrating sphere.
was determined by measuring the colony-forming ability of the
cells. After light exposure, cells were incubated for 5–6 days
at 37°C. Cells treated with phylloerythrin, but without light
exposure, were otherwise treated the same way. The colonies were
then fixed with 96% ethanol, stained with methylene blue, and
counted manually. Scored cell survival was calculated relative to
parallel samples of V79 cells that were neither incubated with
phylloerythrin nor irradiated. Cell death was defined as the
fraction of cells not surviving in the cell-survival assay.
Fluorescence microscopy
Fluorescence and absorption of cell-bound phylloerythrin
2
The intracellular distribution of phylloerythrin was studied after
Cells were seeded out in 20 cm dishes (Falcon NY, USA) and
4
incubation of 5x10 V79 cells with 0.5 µg/ml phylloerythrin.
treated with 1 µg/ml phylloerythrin for 18 h. After incubation
the cells were washed 3 times with ice cold Dulbecco’s phosphate-
buffered saline (PBS) and scraped off the dishes in 2 ml PBS.
The cell suspension was measured spectrofluorometrically using a
Perkin-Elmer LS-50B. The excitation wavelength was set to 422
nm, the maximum of the Soret band of phylloerythrin, and the
emission was scanned from 500–800 nm. A cut-off filter (545
nm) was used on the emission side. Subsequently, absorption
was measured in a Perkin-Elmer Lambda-15 spectrophotometer
Cells were washed three times in PBS at 37°C and a cover glass
was placed gently on the top of the PBS layer. Surplus PBS was
aspirated off. The cells were subsequently studied using a Zeiss
Axioplan fluorescence microscope equipped with a cooled charge-
coupled device (CCD) camera (TE 3/W, Astromed, Cambridge,
UK), operated by an image processing program. The microscope
was equipped with a 395 nm bandpass excitation filter, a 440
nm beam splitter and a 610 nm cut-off emission filter. In some

106
New Zealand Veterinary Journal 50(3), 2002
Scheie et al
experiments, cells were incubated with phylloerythrin at 0°C and
dishes subsequently put on ice in a room at 4°C.
706 nm. The absorption spectrum of phylloerythrin in DMSO
was similar to the excitation spectrum (Figure 2a), although the
Q-band (500–650 nm) was relatively weaker. The concentration
curve vs absorbance at the Soret band of phylloerythrin in
DMSO was linear for concentrations up to about 10 µg/ml. From
Enzyme analysis
β-N-acetyl-D-glucosaminidase (β-AGA)
Cells were isolated by scraping and pelleted immediately after
irradiation. They were then incubated for 3 h in a solution
containing 0.1% Brij 35, 100 mM acetate buffer (pH 4.6), 0.1%
Triton X-100 and 2 mM p-nitrophenyl-N-acetyl-β-glucosamine
according to the method of Beaufay et al (1974). The reaction was
5
this curve an extinction coefficient (ε) of 4.5 x10 was calculated
(Figure 3).
stopped by adding ice-cold 0.5 M Na CO . The β-AGA-activity
2
3
from the lysosomes was then measured by recording the formation
of p-nitrophenol (from the substrate p-nitrophenyl-N-acetyl-β-D-
glucosaminide), which can be registered spectrophotometrically
at 410 nm. The presence of phylloerythrin in the cells did not
influence the measurements.
NADPH cytochrome-c reductase
V79 cells were seeded out in 9.5 cm dishes and treated with
2
sensitiser as described above. The NADPH cytochrome-c reductase
activity, a marker enzyme for the endoplasmic reticulum, was
measured asdescribed byBeaufayet al(1974).Themethod isbased
on the formation of cytochrome c from cytochrome c, induced
red
by NADPH, which can be recorded spectrophotometrically at
550 nm.
Figure 1. Chemical structure of phylloerythrin.
UDP galactosyl transferase
V79 cells were seeded out in 9.5 cm dishes and treated with
2
sensitiser as described above. The UDP galactosyl transferase
activity, a marker enzyme for the Golgi apparatus, was measured
as described by Brandli et al (1988). The method is based on
3
binding of radioactively-labelled uridine diphospho-D-[6- H]
galactose to ovalbumin, which can be detected using an MR 300
automatic liquid scintillation system.
Cytochrome-c oxidase
6
2
V79 cells (1x10 ) were seeded out in 25 cm flasks and treated
with sensitiser as described above. Immediately after irradiation,
the cells were trypsinated and mitochondria were isolated
using electropermeabilisation. Repeated aspiration through
a needle followed by differential centrifugation ruptured the
permeabilised cells. The probes were first centrifuged at 2,500
rpm for 7 min, then the supernatant was centrifuged at 10,000
rpm for 20 min using a Beckman microlitre centrifuge. The
cytochrome-c oxidase activity in the mitochondria was then
measured as described by Gibson and Hilf (1983). The method
is based on spectrophotometric recording of the disappearance
of cytochrome c a reduced form of the substrate cytochrome c
red,
(Sigma) at 550 nm.
Statistical analysis
Results are presented as means and standard deviations (SD)
or standard errors (SE). Linear regression calculations were
conducted using the method of least squares in SimaPlot (SPSS
Inc, Chicago IL, USA).
Results
Fluorescence properties of phylloerythrin
Figure 2. Fluorescence excitation (1), emission (2), and absorption (3)
spectra of phylloerythrin (a) diluted in DMSO, and (b) in V79 fibroblasts.
The fluorescence excitation- and absorption spectra of phylloerythrin
in DMSO were normalised at 418 nm and the fluorescence-excitation
and absorption spectra in cells were normalised at 422 nm. The cells
were incubated for 18 h with 1 µg/ml phylloerythrin.
The fluorescence excitation spectrum of phylloerythrin in DMSO
(Figure 2a) was characterised by a Soret band with a maximum
at 418 nm, and a Q-band with vibrational structure (500–650
nm). The fluorescence emission spectrum had peaks at 643 and

Scheie et al
New Zealand Veterinary Journal 50(3), 2002
107
Figure 3. Absorbance-vs-concentration curve for phylloerythrin diluted
in DMSO ( ). The absorbance of phylloerythrin is a measurement of the
peak intensity at 418 nm. The straight line indicates linear regression
analysis based on results from the first five concentrations.
Figure 4. Time course and temperature dependency of cellular uptake
of phylloerythrin in V79 fibroblasts. The cells were incubated with 0.25
µg/ml phylloerythrin in MEM at 0°C ( ) and 37°C ( ) and cell-bound
phylloerythrin analysed. Bars=SD from three independent experiments.
a
c
b
d
Figure 5. Fluorescence micrograph of V79 fibroblast
cells incubated with phylloerythrin for (a) 1 and (b)
4 h at 0°C, or (c) 1, (d) 4 and (e) 18 h at 37°C. The
micrographs were monitored using a CCD camera.
Arrows indicates artefacts from the background.
e

108
New Zealand Veterinary Journal 50(3), 2002
Scheie et al
Figure 6. Concentration dependency of cellular survival after
incubation for 18 h with phylloerythrin. V79 fibroblasts were incubated
with different concentrations of phylloerythrin in the dark. Bars=SD
from three independent experiments.
Figure 8. Relative activities of different intracellular enzymes in V79
fibroblast cells immediately after treatment with phylloerythrin and
light. Inhibition of the activity of (a) β-N-acetyl-D-glucosaminidase
located mainly in lysosomes, (b) NADPH cytochrome-c reductase in
the endoplasmic reticulum, (c) UDP galactosyl transferase in the Golgi
apparatus, and (d) cytochrome-c oxidase in the mitochondria. The cells
were incubated with 0.25 µg/ml phylloerythrin for 18 h and incubated
in sensitiser-free medium 1 h before exposed to various light doses.
Bars=SE from three independent experiments.
for the first hour of incubation at 0°C. The uptake after 1 h at
0°C was about 60% of the uptake at 37°C and about 90% of
the total uptake at this temperature (Figure 4). Linear regression
analysis indicated an initial rate of phylloerythrin uptake of
0.60 µg/mg protein during the first hour of incubation at 0°C
2
(R =0.94) compared with 0.96 µg/mg protein during the first
hour of incubation at 37°C (R =0.80). The activation energy was
estimated from these results to be about 10 kJ/mol, assuming a
linear Arrhenius plot within this temperature range.
Figure 7. Survival curves for V79 fibroblast cells incubated for 18 h with
0.25 µg/ml ( ) and 0.5 µg/ml ( ) phylloerythrin and exposed to blue
light. The cells were washed three times and incubated in sensitiser
free medium for 1 h before light exposure. Survival curve of V79 cells
incubated with 0.5 µg/ml phylloerythrin, but incubated directly in
sensitiser free medium 1 h before light exposure ( ). Bars=SD from
three independent experiments.
2
Fluorescence microscopy of cells exposed to phylloerythrin for 1
h at 0°C clearly showed the fluorescence originated from intracellular
regions (Figure 5). When the treatment was extended to 4 h, the
pattern of fluorescence was similar. A similar pattern was observed
in cells treated with phylloerythrin at 37°C, although the fluorescence
intensity was higher. After 1 h of incubation, the fluorescence
pattern from phylloerythrin seemed to be almost independent
of time of incubation at both 0°C and 37°C. The intracellular
localisation of phylloerythrin was mainly extranuclear, was not
homogeneous, and covered most of the cytoplasm. However, the
fluorescence pattern was more granular after incubation at 37°C
than after 0°C.
The Soret band of phylloerythrin had its maximum at 422 nm
when bound to cells, i.e. slightly red-shifted compared with the
spectrum in DMSO. When the dye in cells was excited by 422
nm light, two fluorescence emission peaks were evident, at 650
and 712 nm (Figure 2b). The absorption and the corrected
fluorescence excitation spectra for phylloerythrin bound to V79
cells were almost similar, and showed a main peak at about 422
nm (Figure 2b).
Cellular uptake of phylloerythrin
Cytotoxic effects in the absence and presence of light
Cellular uptake of phylloerythrin was measured up to 24 h, and
for the first hour of incubation uptake increased almost linearly
with time (Figure 4). Uptake occurred quickly during the first
few hours after addition of phylloerythrin, then reached a plateau
within about 10 h; 55% of uptake over 24 h occurred within the
first hour of incubation.
Cytotoxic effects of phylloerythrin, measured both in the absence
of light and after light exposure, are presented in Figures 6 and 7,
respectively. Samples incubated with different concentrations of
phylloerythrin in the dark for 18 h showed that the clonogenicity
of the V79 cells was not influenced by treatment with up to 8
µg/ml phylloerythrin (Figure 6). However, cells exposed to 0.25
µg/ml phylloerythrin for 18 h followed by 1 h in sensitiser-free
medium were highly sensitive to light exposure (Figure 7). About
Temperature dependency of cellular uptake
The cellular uptake of phylloerythrin was almost linear over time

Scheie et al
New Zealand Veterinary Journal 50(3), 2002
109
90% of the cells were inactivated after 60 sec of light exposure,
corresponding to 0.54 J/cm . After incubation with 0.5 µg/ml
phylloerythrin only 40 secs of light exposure was required to
inactivate 90% of cells. Incubation in sensitiser-free medium
for 1 h before exposure to light reduced the sensitivity of cells to
photoinactivation by only 15% (Figure 7).
transport mechanisms are more temperature dependent and
have activation energies of more than 50 kJ/mol (Macy 1979).
Active transport is normally completely inhibited at 0°C. These
results demonstrate that phylloerythrin clearly penetrated the
plasma membrane at 0°C. It is also evident from the fluorescence
micrographs that phylloerythrin penetrated the plasma membrane
and localised in intracellular compartments, even when the
cells were incubated at 0°C. This would not be expected for
compounds taken up by an active transport mechanism.
2
Inactivation of intracellular enzymes
The enzymatic activity of UDP galactosyl transferase associated
with the Golgi apparatus was reduced by approximately 55% after
treatment with phylloerythrin and 1 min exposure to light (Figure
8c), while the activity of the mitochondrial cytochrome-c oxidase
was reduced by approximately 70% after 2 min exposure to
light (Figure 8d). Lysosomal β-AGA and NADPH cytochrome-c
reductase located in endoplasmic reticulum showed no significant
loss in enzymatic activities after treatment with phylloerythrin
and 2 min light (Figure 8ab).
Photoinactivation of cells and tissues containing photosensitisers
is strongly dependent on the localisation of the dye (Moan
and Christensen 1981; Benstead and Moore 1988; Peng et
al 1991). In the present study, localisation of phylloerythrin
was investigated using fluorescence microscopy as well as
measurements of photochemically induced damage to enzymes
known to be markers for subcellular organelles. The activities of
UDP galactosyl transferase, which localises in the Golgi apparatus,
and cytochrome-c oxidase, a marker enzyme for mitochondria,
were found to decrease in cells treated with phylloerythrin and
light. This is in accordance with the fluorescence microscopic
analysis, which showed a high concentration of fluorescence in
the perinuclear area, i.e. where the Golgi apparatus is located,
and a widespread extranuclear granular fluorescence that might
resemble the localisation of mitochondria. Thus, our results
are consistent with localisation of phylloerythrin in the Golgi
apparatus and mitochondria in V79 cells and that these organelles
are targets in phylloerythrin-sensitised photodynamic action. The
lack of inactivation of the lysosomal marker enzyme, β-AGA, by
treatment with phylloerythrin in combination with light indicates
low or no lysosomal localisation of phylloerythrin in V79 cells.
This is in agreement with the uptake studies, which indicated that
endocytosis was not involved in the uptake of phylloerythrin in
this cell line, as well as the intracellular localisation pattern evident
using fluorescence microscopy (Figure 7). The marker enzyme for
endoplasmic reticulum, NADPH cytochrome-c reductase, was
not inactivated by phylloerythrin and light, indicating minor
localisation of phylloerythrin in the endoplasmic reticulum.
Discussion
The spectral characteristics of phylloerythrin were similar
to those found for other porphyrins, which have their main
absorption at about 400 nm, with a distinct Soret band near
this wavelength (Blum 1941; Rimington 1960). Perrin (1958)
reported that phylloerythrin in ether had a Soret band at 414
nm, whereas Rimington (1960) reported the spectral properties
of phylloerythrin in acid solution with a Soret band at 421 nm
5
and an extinction coefficient (ε) to 2.78x10 . He also found that
porphyrins obey Beer’s law up to an absorbance (A) of about
1.0. The present work with phylloerythrin dissolved in DMSO
showed linearity of absorption vs concentration up to about
A=2.5. The extinction coefficient in this solution was found to
5
be 4.5x10 , i.e. slightly larger than that found by Rimington in
1960. The spectra in cells were slightly red-shifted compared with
the spectra in DMSO, which may indicate membrane binding of
the dye in cells.
In the case of porphyrins, it is well known that aggregates have a
low fluorescence quantum yield compared to monomers (Moan
and Sommer 1983) and absorption spectra that are different from
those of monomeric dyes (Moan and Christensen 1981; Berg et
al 1989). In the present study the absorption and the fluorescence
excitation spectra of phylloerythrin in V79 cells were found
to be similar in shape. This indicates little or no aggregation
of phylloerythrin in V79 cells. This is to be expected for an
efficient photosensitiser, since monomeric species are much more
photoactive than aggregates (Moan 1984; Berg et al 1989). In
DMSO, however, the Q-bands of the absorption and excitation
spectra were different, indicating that the dye was not only in a
monomeric form.
Phylloerythrin is a lipophilic compound (Figure 1). Our results
demonstrating that phylloerythrin located in both the Golgi
apparatus and mitochondria support previous findings that lipophilic
dyes in cells in vitro generally localise in membrane structures
(including plasma, mitochondrial, endoplasmic reticulum and
nuclear membranes), while hydrophilic dyes appear to accumulate in
lysosomes (Moan et al 1989; Boyle and Dolphin 1996). Our results
are not in accordance with results from a previous study that reported
that phylloerythrin and light caused inactivation of the lysosomal
enzyme, acid phosphatase, in frozen sections of rat tail skin (Slater
and Riley 1966). However, in that study, phylloerythrin and light
had only minor effects on release of acid phosphatase from isolated
lysosomes from liver homogenate, and no comparisons with other
enzymes or organelles were reported to indicate that lysosomes were a
preferred target. Results from studies using frozen sections are difficult
to equate with those such as ours that used live cells in culture.
Photosensitisers may enter cells either by penetrating the
plasma membrane by passive diffusion (Bohmer and Morstyn
1985; Dellinger et al 1986) or by endocytosis (Berg and Moan
1994). Plasma membrane transporters have not been reported
to be involved in the cellular uptake of porphyrin-based photo-
sensitisers. The present results indicate that the cellular uptake of
phylloerythrin is due to a diffusion-controlled process. Although
the uptake of phylloerythrin was slightly temperature dependent,
the activation energy for the process was only about 10 kJ/
mol, which is within the range of diffusion-controlled uptake
mechanisms (Le Cam and Freychet 1977). In contrast, active
The localisation of other porphyrins in mitochondria and
photochemical damage to these organelles is well documented,
and it has been suggested by several authors that the most
efficient sensitisers of cells to photoinactivation localise in the
mitochondria (Morgan and Oseroff 2001). This may explain the
severe lesions to light seen in the skin of ruminants suffering from
phylloerythrin-induced photosensitisation.

110
New Zealand Veterinary Journal 50(3), 2002
Scheie et al
Moan J, Sommer S. Uptake of the components of hematoporphyrin derivative by
cells and tumours. Cancer Letters 21, 167−74, 1983
Acknowledgements
Thank you to Lena Moen for assistance with the intracellular
localisation of the dye.
Moan J, Sommer S. Oxygen dependence of the photosensitizing effect of
hematoporphyrin derivative in NHIK 3025 cells. Cancer Research 45,
1608−10, 1985
Moan J, Steen HB, Feren K, Christensen T. Uptake of hematoporphyrin
derivative and sensitized photoinactivation of C3H cells with different
oncogenic potential. Cancer Letters 14, 291−6, 1981
Moan J, Christensen T, Jacobsen PB. Photodynamic effects on cells in vitro
labelled with hematoporphyrin derivative. Photochemistry and Photobiology 7,
349−58, 1984
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