Effect of dietary chlorophyll derivatives on mutagenesis and tumor cell growth

Article abstract of DOI:10.1002/(SICI)1520-6866(1999)19:5<313::AID-TCM1>3.0.CO;2-G

Much attention in recent years has been given to the antigenotoxicity of chlorophyll. Chlorophyll, however, is known to be converted into pheophytin, pyropheophytin, and pheophorbide in processed vegetable food and following ingestion by humans. Studies were conducted on the antimutagenic and tumoricidal potencies of these compounds. All the chlorophyll derivatives tested exhibit identical antimutagenic effect towards 3-methylcholanthrene (3-MC), suggesting that the porphyrin nucleus may complex directly with the mutagen. It does not exclude, however, another mechanism of activity involving inactivation the enzymatic transformation of 3-MC. In contrast, the action of N'-nitro-N'-nitrosoguanidine (MNNG) depends upon structural differences between the chlorophyll derivatives. It is significantly lower when the phytol-containing pheophytin and pyropheophytin are tested as to that of the phytol-lacking pheophorbide. The higher concentrations of the chlorophyll derivatives were required to reduce the mutagenicity of MNNG than needed for 3-MC. The cytotoxicity of chlorophyll derivatives against tumor cells also was evaluated. The cellular uptake and inhibition of myeloma cell multiplicity were found to be greater for pheophorbide than for pheophytin. Calculated on the amount of cell associated chlorophyll derivative, however, pheophytin was more cytostatic/cytotoxic than pheophorbide. The results presented in this report indicate that food sources that yield chlorophyll derivatives may play a significant role in cancer prevention.

Full text of DOI:10.1002/(SICI)1520-6866(1999)19:5<313::AID-TCM1>3.0.CO;2-G

Teratogenesis, Carcinogenesis, and Mutagenesis 19:313–322 (1999)
Effect of Dietary Chlorophyll Derivatives
on Mutagenesis and Tumor Cell Growth
1
2
1
*
Simon Chernomorsky, Alvin Segelman, and Ronald D. Poretz
1Department of Biochemistry and Microbiology, Rutgers University, New
Brunswick, New Jersey
2Department of Chemical Biology and Pharmacology, Rutgers University,
New Brunswick, New Jersey
Much attention in recent years has been given to the antigenotoxicity of chlorophyll.
Chlorophyll, however, is known to be converted into pheophytin, pyropheophytin,
and pheophorbide in processed vegetable food and following ingestion by humans.
Studies were conducted on the antimutagenic and tumoricidal potencies of these com-
pounds. All the chlorophyll derivatives tested exhibit identical antimutagenic effect
towards 3-methylcholanthrene (3-MC), suggesting that the porphyrin nucleus may
complex directly with the mutagen. It does not exclude, however, another mecha-
nism of activity involving inactivation the enzymatic transformation of 3-MC. In
contrast, the action of N′-nitro-N′-nitrosoguanidine (MNNG) depends upon struc-
tural differences between the chlorophyll derivatives. It is significantly lower when
the phytol-containing pheophytin and pyropheophytin are tested as to that of the
phytol-lacking pheophorbide. The higher concentrations of the chlorophyll deriva-
tives were required to reduce the mutagenicity of MNNG than needed for 3-MC. The
cytotoxicity of chlorophyll derivatives against tumor cells also was evaluated. The
cellular uptake and inhibition of myeloma cell multiplicity were found to be greater
for pheophorbide than for pheophytin. Calculated on the amount of cell associated
chlorophyll derivative, however, pheophytin was more cytostatic/cytotoxic than
pheophorbide. The results presented in this report indicate that food sources that
yield chlorophyll derivatives may play a significant role in cancer prevention. Ter-
atogenesis Carcinog. Mutagen. 19:313–322, 1999.
© 1999 Wiley-Liss, Inc.
Key words: chlorophyll derivatives; pheophytin; pyropheophytin; pheophorbide; antimutagens;
cytotoxicity
*Correspondence to: Simon Chernomorsky, Department of Biochemistry and Microbiology, Rutgers
University, 76 Lipman Drive, New Brunswick, NJ 08901-8525.
Alvin Segelman is now at Nature’s Sunshine Products, Inc., 75 East 1700 South, Provo, UT 84605.
© 1999 Wiley-Liss, Inc.

314
INTRODUCTION
Along with other dietary constituents of vegetables tested for their effect on
Chernomorsky et al.
chemically induced mutagenesis/carcinogenesis, much attention in recent years has
been given to chlorophyll and its water-soluble commercial version, chlorophyllin
copper complex (CHL) [1–4]. A close positive relationship was established between
the chlorophyll content of various vegetable extracts and their ability to inhibit mu-
tations in the Ames Salmonella system [5–7]. Extracts from parsley and lettuce, which
have high levels of chlorophyll, were more effective in modulating mutagenicity
caused by 3-methylcholanthrene (3-MC) and benzo[a]pyrene (BP) compared to other
common vegetables in the diet of the Western population [6]. Such a correlation with
chlorophyll content was also found among the vegetables favored by the Oriental
(Korean) population. Extracts of mustard leaves and Korean radish leaves exhibited
the strongest antimutagenic activity against N′-nitro-N′-nitrosoguanidine (MNNG)
[7]. Chlorophyll was also tested individually towards cigarette-smoke condensate
and BP [8]; and 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2) and 3-hydro-
xiamino-1-methyl-5H-pyrido[4,3-b]indole [1] and 4-nitro-o-phenylenediamine [9]. For
all mutagens used, chlorophyll demonstrated an inhibitory effect, though various con-
ditions were required to fully reveal its potency.
The antimutagenic effect of chlorophyll and its derivatives has been well docu-
mented. Recently, limited data appeared on the tumoricidal activity of these com-
pounds in a tissue culture model. For example, CHL was shown to exhibit considerable
cytotoxicity against myeloma cells [10,11].
CHL, the most common among chlorophyll derivatives used in cancer related
studies, is a semi-synthetic preparation. Its parent compound, chlorophyll, however,
is a naturally occurring porphyrin. It is noteworthy that chlorophyll is known to be
converted into pheophytin, pyropheophytin, and pheophorbide (Fig. 1) in stored plant
tissues, processed vegetable food, and following ingestion by humans [12–14]. In
this report, we present data on the antimutagenic and tumoricidal potencies of these
compounds that may contribute to the practical application of chlorophyll and its
derivatives as dietary supplements in cancer prevention.
MATERIALS AND METHODS
Chemicals
Chlorophyll a, isolated from Anacystis nidulans algae, MNNG, and 3-MC were
purchased from Sigma Chemical Co. (St. Louis, MO). Cell culture media, other chemi-
cals and reagents for mutagenicity, and cytotoxicity assays were obtained from Fisher
Scientific (Pittsburgh, PA). Chlorophyll a was the source for the preparation of vari-
ous chlorophyll derivatives.
Pheophytin a was produced from a diethyl ether solution of the chlorophyll a
by acidification with 13% aqueous HCL. HCL was then removed by washing with
distilled water. The diethyl ether solution was dried over anhydrous Na2SO4 and evapo-
rated in vacuo [15]. Pyropheophytin a was prepared by heating a pyridine solution
of pheophytin a in a sealed tube at 100°C. After 48 h, the pyridine was removed by
evaporation in vacuo and the pyropheophytin a was obtained as a residue [16].
Pheophorbide a was prepared by treating a diethyl ether solution of the chlorophyll
a with 30% (w/w) aqueous HCL in the dark for 1 h at room temperature. The mix-

Chlorophyll Derivatives and Mutagenesis
315
Fig. 1. Structures of chlorophyll derivatives.
ture was diluted with distilled water and pheophorbide a in the ether phase was
washed with distilled water to remove HCL. The ether solution was dried over anhy-
drous Na2SO4 and the solvent was removed by evaporation in vacuo [17].
Mutagenicity Studies
Salmonella typhimurium strain TA100 was employed as a test system following
the procedure described previously [10]. All chlorophyll derivatives were initially
dissolved in dimethylsulfoxide and added, at concentrations as noted, to the media
containing the bacterial cells and the appropriate mutagen. The amount of the indi-
rect-acting mutagen, 3-MC (50 µg/plate), to cause 1,320 His+ revertants, or the di-
rect-acting mutagen, MNNG (5 µg/plate), to cause 478 His+ revertants, were employed
as standard conditions. The counts were corrected for spontaneous background re-
vertants.
Chlorophyll Derivative/Mutagen Interaction
The absorption spectrum of pheophytin a and pheophorbide a alone or mixed
with mutagens (at chlorophyll derivative/mutagen molar ratios of 1:10) in dimethyl-
sulfoxide were obtained in a range between 350–700 nm by using a computer con-
trolled Perkin-Elmer Lambda 3B UV-VIS spectrophotometer.

316
Chernomorsky et al.
Cell Cultures, Cytotoxicity, and Uptake Studies
Tumor cells (mouse myeloma line P3X63Ag8), an azaguanine resistant P3 clone
derived from MOPC-21, were cultured in Dulbecco’s modified Eagle’s medium with
10% fetal bovine serum, 2 mM L-glutamine, 0.1 mM non-essential amino acids, 50
IU/ml penicillin, and 50 µg/ml streptomycin (Gibco Laboratories, Grand Island, NY),
at 37°C in an environment of 10% CO2/90% air. Cells suspended in fresh culture
medium to give 3 × 105 cells/ml were incubated with pheophytin a and pheophorbide
a as described previously [10]. Splenocytes, obtained from the C3H mice, were sus-
pended in Hanks’ balanced salts solution, centrifuged, and resuspended in 1 ml of
distilled water for 15 s to lyse erythrocytes [18]. After dilution with Hanks’ balanced
salts solution, cells were pelleted by centrifugation, washed, and resuspended to 106
cells/ml of the same solution. Ethanolic solutions of chlorophyll derivatives were
mixed with the cells. The concentration of ethanol did not exceed 10% of the total
volume and was found to be non-toxic to cells at this concentration.
After an incubation period of 48 h with the chlorophyll derivatives, viability of
myeloma cells was determined by trypan blue exclusion. Multiplicity of myeloma
cells was assessed as a ratio of the total number of cells after exposure to pheophytin
a or pheophorbide a to that in control cultures [10]. For cellular uptake studies, cells
were incubated for 24 h with each chlorophyll derivative (230 µM). Following cen-
trifugation (500g) for 30 min, the supernate was reserved and cells were washed three
times with fresh phosphate-buffered saline. The combined medium and cell washings
were extracted with a mixture of acetone and diethyl ether (1.4:1). The organic layer
was removed, washed with water, dried over anhydrous Na2SO4, and analyzed spec-
trophotometrically for the respective chlorophyll derivatives. The amount of com-
pound recovered was compared with that added to the cells prior to incubation.
In addition, the fluorescence of cells incubated with the chlorophyll derivatives
was detected by using a Leitz Diavert microscope equipped with epi-illumination
and an MPV compact photometer (excitation 350–460 nm, emission 515 nm).
RESULTS
Antimutagenic Activity
Figure 2 demonstrates that all of chlorophyll derivatives tested are antimutagenic
against 3-MC. They exhibit similar dose-response patterns, reaching a level of ED50
and ED95 (effective doses required to afford 50 or 95% decrease in the number of
bacterial cells) at the 0.13 and 0.89 µM, respectively. Quite different responses are
evident with MNNG. As seen in Fig. 3, the 50 and 95% inhibition of His+ revertants
requires much higher concentrations for pheophorbide a (24.0 and 38.5 µM, respec-
tively) compared to pheophytin a and pyropheophytin a (3.9 and 6.9 µM, respectively).
The antimutagenic effect of pheophorbide a or pheophytin a, as reported here,
is supported in separate experiments by others, though with different indirect-acting
mutagens. The activity of these chlorophyll derivatives, however, was not determined
at identical concentrations of the mutagens and accordingly the dose-response curves
are difficult to compare [19,20].
Spectral Changes of Chlorophyll Derivative/Mutagen
Evidence suggests that chlorophyll and CHL are involved in molecular com-
plexation with a number of conjugated polycyclic aromatic mutagens [21–23], thereby

Chlorophyll Derivatives and Mutagenesis
317
Fig. 2. Antimutagenic activity of pheophytin a (closed circle), pyropheophytin a (open triangle) and
pheophorbide a (open circle) against 3-MC.
reducing or totally eliminating their bioavailability. In these studies, difference spec-
tra of chlorophyll or CHL mixed with mutagen were obtained. The spectral changes
were indicative of the complex formation. We compared the visible absorption spec-
tra of MNNG or 3-MC mixed with pheophytin a or pheophorbide a against that of
the appropriate mutagen and chlorophyll derivative alone (Table I). The results re-
vealed that the position of the Soret and Q bands for pheophytin a or pheophorbide a
remained unchanged in the presence of either MNNG or 3-MC. However, the ratios
of the absorption for these bands are different depending on the mutagen used. The
ratios for chlorophyll derivatives mixed with MNNG are the same as for those com-
pounds alone. In contrast, 3-MC added to pheophytin a or pheophorbide a decreases
the ratios of the Soret/Q bands by 20% and 57%, respectively. This pattern also is
observed in studies with CHL and benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide [22].
Cytotoxic Effect and Cellular Uptake
The dose-effect profiles of chlorophyll derivatives with respect to growth prop-
erties of myeloma cells are shown in Figure 4. Pheophorbide a is approximately 55–
57% more effective as an inhibitor of cell multiplicity than pheophytin a. It can be
seen that pheophorbide a requires concentrations of only 0.36 and 0.70 mM for ED50
and ED95, respectively. Concentrations of 0.56 and 1.10 mM are needed for pheophytin

318
Chernomorsky et al.
Fig. 3. Antimutagenic activity of pheophytin a and pyropheophytin a (closed circle) and pheophorbide
a (open circle) against MNNG.
a to cause similar effects. The impact of both chlorophyll derivatives on cell viabil-
ity, however, is identical (0.66 and 1.30 mM for ED50 and ED95, respectively).
In order to further understand the possible mechanism(s) involved in the cyto-
toxic/cytostatic activities of these chlorophyll derivatives, the distribution of each
compound between the cells and medium was determined. Table II demonstrates that
pheophorbide a which lacks the phytyl ester group and possesses a free carboxylate
moiety as compared to pheophytin a exhibits a 5-fold greater cellular uptake than the
latter. The cell/medium distribution of pheophorbide a in non-tumor cells, murine
splenocytes, was found to be significantly less (by 25 times) than that in myeloma
TABLE I. Spectral Characteristics of Chlorophyll Deirvatives and Their Mixture With Mutagens
Compounds
Soret banda
Q banda
Soret/Qb
Pheophytin a alone
413
413
413
413
413
413
666
666
666
666
666
666
2.9
2.3
2.9
3.2
1.4
3.2
Pheophytin a + 3-MC
Pheophytin a + MNNG
Pheophorbide a alone
Pheophorbide a + 3-MC
Pheophorbide a + MNNG
aWavelength of maximum absorption, nm.
bRatio of the absorbances at the maxima of the Soret and Q bands.

Chlorophyll Derivatives and Mutagenesis
319
Fig. 4. Effect of pheophytin a (closed circle) and pheophorbide a (open circle) on the growth (M-
multiplicity, V-viability) of murine myeloma cells.
cells. These observations are supported by the determination of the cell bound chlo-
rophyll derivatives detected by cellular fluorescence. The fluorescence method, how-
ever, indicated a much greater difference in uptake values of the chlorophyll
derivatives than those from the spectrophotometric measurements.
DISCUSSION
Clearly, pheophytin a, pyropheophytin a, and pheophorbide a each exhibit
antimutagenicity towards 3-MC or MNNG. Interestingly, the three chlorophyll de-
rivatives demonstrate identical activities against 3-MC but differ in regard to
TABLE II. Uptake of Chlorophyll Derivatives by Tumor and Non-Tumor Cells
Compounds
Cells
Distributiona
Relative fluorescence/cell
Pheophytin a
Pheophorbide a
Pheophorbide a
Myeloma cells
Myeloma cells
Murine splenocytes
0.10
0.50
0.02
2
264
4
aRatio of the concentrations of chlorophyll derivatives in cells and medium.

320
Chernomorsky et al.
antimutagenicity towards MNNG. In particular, the structural differences relating to
the presence or absence of the phytyl or methyl groups, which influence hydropho-
bic properties (pyropheophytin a > pheophytin a > pheophorbide a), did not affect
their antimutagenic efficacy against 3-MC. Such results suggest that the antimutagenic
properties of these compounds towards the indirect-acting mutagen, 3-MC, may in-
volve the porphyrin nucleus and is, for the most part, independent of the configura-
tion of the side chains. Tachino et al. [22] have suggested that a possible mechanism
for the antimutagenic activity of chlorophyll derivatives involves complex formation
with the mutagen through strong π-π-interactions between their planar unsaturated
cyclic rings. This may be the case with 3-MC. Difference spectral analysis in the
visible region, capable of detecting perturbations of the electron distribution of the
porphyrin nucleus in the presence of a large molar excess of 3-MC, indicates that
interaction between pheophytin a or pheophorbide a and mutagen does occur. This is
most evident by changes of absorbencies in the Soret and Q bands (Soret/Q ratio).
The impact of complexation on the principal absorption peaks of the chlorophyll
derivatives allows for a sensitive detection of interaction. This type of analysis pos-
sesses an internal control of experimental variables since it is based upon a ratio of
absorbencies at two different wavelengths of the same sample. The Soret/Q ratio
may be especially valuable in detecting the direct interaction of chlorophyll deriva-
tives and mutagens when the complexation is of non-covalent nature and relatively
weak, as found for some mutagens [24].
It should be noted that inhibition of the mutagenicity of 3-MC by a complex
formation does not exclude that pheophytin a, pyropheophytin a, and pheophorbide
a may also inactivate the enzymatic transformation of this mutagen. It is possible
that two different mechanisms may be involved simultaneously in desmutagenic ac-
tivity of chlorophyll derivatives against indirect-acting mutagens. Thus, the results
from the study by Yun et al. [25] indicate that CHL may inhibit the mutagenicity of
2-amino-3-methylimidazo[4,5-f]quinoline through inactivation of cytochrome P450
in addition to complexation with this mutagen.
In contrast to the results seen with 3-MC, the mutagenicity of MNNG is inhib-
ited differently by pheophytin a, pyropheophytin a, or pheophorbide a, suggesting
the important role of the phytol chain in such an activity. The unchanged Soret/Q
ratios imply that the complexation of MNNG with any of the chlorophyll derivatives
does not occur. This is consistent with the fact that MNNG lacks an extended planar,
π-electron cloud required for interaction with the π-system of porphyrin type com-
pounds. Romert et al. [26] suggested that a possible mechanism of inactivation in-
volves the decomposition of MNNG mediated by chlorophyll derivatives similar to
that for thiols and amines. In particular, CHL may facilitate the formation of highly
mutagenic intermediates derived from MNNG that are immediately inactivated as a
result of the reaction with water outside the cell.
The potential of chlorophyll derivatives as antimutagens raises questions about
their affect on normal and pathological tissue. Porphyrin-related compounds, and
chlorophyll derivatives in particular, have a high affinity for tumor tissue as com-
pared to normal tissue [27]. There is also evidence that individual chlorophyll de-
rivatives, as well as commercial preparations derived from chlorophyll, have cytostatic
and cytotoxic activities against tumor cells. Tissue culture studies showed that
pheophytin is cytostatic against hepatoma cells [28] and along with chlorin e6 and
CHL, has a cytotoxic effect on myeloma cells [10,11]. The chlorophyll derivatives

Chlorophyll Derivatives and Mutagenesis
321
studied here are cytostatic and at higher concentrations are cytotoxic to murine my-
eloma cells. One factor, however, that plays an important role in such activities is the
ability of the porphyrins to become cell associated. The approaches employed in our
study utilizing cell bound fluorescence or chemical extraction of chlorophyll deriva-
tives not associated with cells exhibit similar patterns for cellular uptake of pheophytin
a and pheophorbide a. Both procedures demonstrate pheophorbide a accumulates to
a higher molar concentration than pheophytin a when myeloma cells are exposed to
equimolar doses of each compound. Although the fluorescence procedure indicates
much greater differences than the chemical extraction method in the ability of these
compounds to become cell associated, the same trends are observed. Aggregation
and/or formation of metal complexes may occur within the cell, thereby affecting the
fluorescence yield of the cell bound chlorophyll derivatives [29,30]. In turn, these
factors may impact pheophytin a and pheophorbide a differently. Accordingly, the
chemical extraction approach is taken as more reliable in quantifying difference be-
tween these compounds. Pheophorbide a exhibits a significantly higher cytostatic/
cytotoxic effect than pheophytin a when calculated on the amount added to the incu-
bation medium. However, when such values are adjusted for the amount of cell asso-
ciated chlorophyll derivatives, pheophytin a is more cytostatic and cytotoxic for the
myeloma cells than pheophorbide a (3.2 and 5.0 times more active for multiplicity
and viability, respectively). The greater cellular uptake of pheophorbide a in my-
eloma cells as compared to murine splenocytes found in this study is in agreement
with the data employing MGH U1 cells, a line established from a human transitional
cell carcinoma of the bladder, and IMR-90, a non-neoplastic line established from
human fibroblasts [31].
Given the importance of antimutagenic/anticarcinogenic activities coupled with se-
lective accumulation in tumor tissue and cytostatic/cytotoxic effects against tumor cells,
chlorophyll derivatives might be a significant tool in future management of cancer.
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