Carcinogenesis vol.21 no.4 pp.

811816, 2000

SHORT COMMUNICATION

Inhibitory effect of a flavonoid antioxidant silymarin on benzoyl

peroxide-induced tumor promotion, oxidative stress and
inflammatory responses in SENCAR mouse skin

Jifu Zhao1, Moushumi Lahiri-Chatterjee2,

Yogesh Sharma1 and Rajesh Agarwal1,3,4
1Center

for Cancer Causation and Prevention, AMC Cancer Research

Center, Denver, CO 80214, 2Department of Dermatology, Case Western
Reserve University, Cleveland, OH 44106 and 3University of Colorado
Cancer Center, University of Colorado Health Sciences Center, Denver,
CO 80262, USA
4To

whom correspondence should be addressed at AMC Cancer Research

Center, 1600 Pierce Street, Denver, CO 80214, USA
Email: agarwalr@amc.org

In this communication, we investigate the preventive effect

of a flavonoid antioxidant, silymarin, on free radicalgenerating skin tumor promoting agent benzoyl peroxide
(BPO)-induced tumor promotion, oxidative stress and
inflammatory responses in SENCAR mouse skin. Topical
application of silymarin at a dose of 6 mg prior to BPO
resulted in a highly significant protection against BPOinduced tumor promotion in 7,12-dimethylbenz[a]anthracene-initiated SENCAR mouse skin. The preventive effect
of silymarin was evident in terms of a 70% reduction
(P < 0.001) in tumor incidence, a 67% reduction (P < 0.001)
in tumor multiplicity and a 44% decrease (P < 0.001) in
tumor volume/tumor. In oxidative stress studies, topical
application of BPO resulted in 75, 87 and 61% depletion
in superoxide dismutase (SOD), catalase and glutathione
peroxidase (GPX) activities in mouse epidermis, respectively. These decreases in antioxidant enzyme activities were
significantly (P < 0.0050.001) reversed by pre-application
of silymarin in a dose-dependent manner. The observed
effects of silymarin were 1866, 3272 and 2067% protection against BPO-induced depletion of SOD, catalase and
GPX activity in mouse epidermis, respectively. Silymarin
pre-treatment also resulted in a dose-dependent inhibition
(3587%, P < 0.050.001) of BPO-induced lipid peroxidation in mouse epidermis. In inflammatory response studies,
silymarin showed a strong inhibition of BPO-induced skin
edema (6285% inhibition, P < 0.001), myeloperoxidase
activity (42100% inhibition, P < 0.001) and interleukin1 protein level in epidermis (3681% inhibition, P <
0.001). These results, together with our other recent studies,
suggest that silymarin could be useful in preventing a wide
range of carcinogen and tumor promoter-induced cancers.
In both experimental animals and possibly in humans, cancers
of the skin, lung, colon, breast, prostate, cervix, bladder and
esophagus arise in epithelial tissues and acquire the ability to
grow and invade through the basement membrane (1). A
Abbreviations: BPO, benzoyl peroxide; DMBA, 7,12-dimethylbenz[a]anthracene; GPX, glutathione peroxidase; IL-1, interleukin 1; MDA, malondialdehyde; MPO, myeloperoxidase; ROS, reactive oxygen species; SOD,
superoxide dismutase; TPA, 12-O-tetradecanoylphorbol 13-acetate.
Oxford University Press

common pre-neoplastic precursor lesion to all squamous cell

carcinomas arising in these epithelia is squamous dysplasia
and/or metaplasia (24). Accordingly, for more than 50 years,
the multistage model of carcinogenesis in mouse skin has
provided a conceptual framework to study the carcinogenic
process in tissues of epithelial origin (5,6). Many concepts
and mechanisms of carcinogenesis as well as cancer chemoprevention now being applied to other tissues in animal model
systems were originally derived from the mouse skin model
(5,6). It is widely recognized that carcinogenesis in mouse
skin and presumably in human skin and other tissues is a
multistage process comprised of initiation, promotion and
progression (5,6). Using experimental carcinogenesis models,
it has become well established that oxidative stress plays a
causative role during carcinogenesis, specifically in tumor
promotion (79). This is further supported by the study where
Slaga et al., for the first time, showed that benzoyl peroxide
(BPO), a free radical-generating compound, induces tumor
promotion in carcinogen-initiated mouse skin (10). Although
BPO was found to be a weak tumor promoting agent, it
was alone sufficient to induce clonal expansion of 7,12dimethylbenz[a]anthracene (DMBA)-initiated epidermal cells
into visible tumors in mouse skin (10).
Consistent with the involvement of oxidative stress in cancer
induction and its subsequent development (79), efforts are
being made to identify naturally occurring antioxidants which
could prevent, slow and/or reverse cancer induction and its
subsequent development (1114). Silymarin is a flavonoid
antioxidant isolated from the milk thistle (Silybum marianum)
and has been used clinically for almost 25 years in Europe as
an anti-hepatotoxic agent for nearly every known form of liver
disease, including cirrhosis, hepatitis and necrosis (15). These
effects of silymarin could be explained by alterations in liver
cell membrane structure, blocking the absorption of toxins
into the cells, increasing the intracellular concentration of
glutathione and the antioxidant potential (1620). Silymarin is
non-toxic and there is no known LD50 for silymarin (2123).
Several short-term studies have suggested that silymarin
may be a potent anti-carcinogenic agent (ref. 24 and references
therein). Initially, we found that silymarin affords significant
inhibition of phorbol ester and other skin tumor promoter
(including BPO)-induced induction of ornithine decarboxylase
activity and mRNA expression in mouse epidermis (24). More
recently, we also showed that silymarin exerts an exceptionally
high protective effect against UVB radiation-, 12-O-tetradecanoylphorbol 13-acetate (TPA)- and okadaic acid-induced
tumor promotion in mouse skin and that the anti-tumor
promoting effects of silymarin are primarily targeted at stage
I tumor promotion (2527). Based on the results of these
studies, and a strong antioxidant activity of silymarin (1620),
here we have performed additional studies to investigate the
preventive effect of silymarin against BPO-induced (i) tumor
811

J.Zhao et al.

promotion, (ii) oxidative stress and (iii) inflammatory responses

in mouse skin.
Six-week-old female SENCAR mice were purchased from
Harlan Sprague-Dawley (Indianapolis, IN) and maintained in
our animal facility under standard conditions. The dorsal skin
of the mice was shaved using electric clippers and mice in the
resting phase of the hair cycle were used in all studies. For
the tumor studies, the mice were randomly divided into three
groups of 20 animals each. The mice in group I were left
untreated and served as negative controls. Mice in groups II
and III were topically applied on the dorsal shaved skin with
a single 2.5 g dose of DMBA in 0.2 ml of acetone/mouse as
tumor initiator. One week later, animals in group II were
treated topically with 0.2 ml of acetone and in group III with
6 mg of silymarin in 0.2 ml of acetone/mouse/application.
Thirty minutes following these treatments, animals in both
groups II and III were applied topically with 20 mg of BPO
in 0.2 ml of acetone/mouse/application. The BPO alone (group
II) or silymarin plus BPO treatments (group III) were repeated
twice per week up to termination of the experiment at 40
weeks from the start of DMBA. We did not include a DMBA
silymarin group in this study as we have previously shown
that a 12 mg topical application of silymarin twice a week to
DMBA-initiated SENCAR mouse skin does not have a tumor
promoting effect (27). The selection of 6 mg of silymarin in
the present study was based on a recent study by us showing
that this dose causes exceptionally high anti-tumor promoting
effect against both TPA- and okadaic acid-induced skin tumor
promotion (26). Animals in all the groups were watched for
food and water consumption and any apparent signs of toxicity
such as weight loss or mortality during the entire period of
the study. Skin tumor formation was recorded weekly and
tumors 1 mm in diameter were included in the cumulative
total if they persisted for 2 weeks or more. Latent periods for
the onset of tumors in groups II and III were computed and
at the termination of the experiment, the tumor volume on
the back of each mouse was also computed. The statistical
significance of differences between tumor incidence in the
silymarin-treated and untreated groups was determined by twotailed Fishers exact test (25,27). For tumor multiplicity and
tumor volume/tumor, two sample Wilcoxon rank sum and
Students t-tests, respectively, were used (25,27).
For antioxidant enzymes and other studies, all treatments
were done on a 4 cm2 shaved area. The animals were divided
into six groups with four mice in each group and were treated
topically on the shaved area with 0.2 ml of acetone (group I)
and 0 (group II), 3 (group III), 6 (group IV) or 9 mg (group
V) of silymarin/mouse each in 0.2 ml of acetone followed
30 min later by 20 mg of BPO in 0.2 ml of acetone except
for group I. In group VI, the mice were treated topically with
6 mg of silymarin alone in 0.2 ml of acetone. These treatments
were performed once. To assess the extent of BPO-induced
edema in SENCAR mouse skin and the inhibitory effect of
pre-application of different doses of silymarin, 24 h after BPO
treatment a dial caliper was used to measure the skin bi-fold
thickness. Edema formation was expressed as net increase in
skin bi-fold thickness between BPO- and non-BPO-treated
groups. Immediately after this measurement, mice were killed
and the dorsal treated skin was removed and used for further
studies. The epidermis was separated from the whole skin and
epidermal 100 000 g supernatant and microsomal fractions
prepared as described earlier (28). Superoxide dismutase (SOD)
activity was measured by inhibition of reduction of nitroblue
812

Fig. 1. Protective effect of silymarin on BPO-induced tumor promotion in

DMBA-initiated SENCAR mouse skin. A single dose (2.5 g) of DMBA
was used as tumor initiating agent and after 1 week the mice were treated
topically either with acetone alone or silymarin (6 mg) and 30 min later
with BPO (20 mg). These treatments were performed twice weekly up to
the end of the experiment at 40 weeks. Each agent was applied in 0.2 ml of
acetone/application. The percentage of mice with tumors (A) and number of
tumors per mouse (B) were plotted as a function of number of weeks on
test. The percentages of mice with tumor data are from 20 mice/group.
Similarly, the tumors/mouse data are also means SE of 20 mice/group.
For the clarity of presentation of data, the tumor incidence and multiplicity
results are shown only for alternate weeks of study.

tetrazolium by superoxide anions produced by potassium

superoxide dissolved in dimethylsulfoxide (29). One unit of
enzyme activity is defined as the amount of enzyme causing
50% inhibition of the reduction to formazan observed in the
blank. Catalase activity was determined by measuring the
sample-catalyzed disappearance of H2O2 at 240 nm (30), and
expressed as U/mg protein (1 U OD of 1 mM H2O2).
Glutathione peroxidase (GPX) activity was measured as disappearance of NADPH at 340 nm (31), and expressed as nmol
NADPH oxidized/min/mg protein. The lipid peroxidation assay
was done as detailed recently (27). Myeloperoxidase (MPO)
activity was determined by the method of Bradley et al. (32)
as detailed earlier (33). Epidermal interleukin 1 (IL-1)
protein levels were determined as detailed recently (34) using
an ELISA kit (Endogen, Woburn, MA). For statistical analysis
of the data, Students t-test was used between the BPO and
silymarin BPO groups.
In the studies assessing the preventive effect of silymarin
on BPO-induced skin tumor promotion, as shown by the data
in Figure 1, topical application of silymarin prior to BPO
resulted in an exceptionally high preventive effect against
BPO-induced tumor promotion in SENCAR mouse skin. No
significant change was observed in the body weight gain
profile between silymarin- and non-silymarin-treated groups
of mice throughout the experiment (data not shown), which

Silymarin inhibits benzoyl peroxide effects

further supported the previous observation that silymarin

applied topically has no apparent toxicity (2527). In terms of
tumor incidence, an exceptionally high preventive effect of
silymarin against tumor promotion was evident throughout the
experiment (Figure 1A). When these data were analyzed at
the termination of the experiment at 40 weeks, compared with
the non-silymarin-treated positive controls showing 50% of
mice with skin tumors, pre-application of silymarin resulted
in only 15% of mice with tumors, giving 70% protection
(P 0.001) against tumor incidence (Figure 1A). When the
preventive effects of silymarin were analyzed for delay in
latency period of the onset of first tumor, compared with the
positive control group, a 5 week delay in the latency period
of the onset of first tumor was observed in animals treated
with silymarin prior to BPO (Figure 1A). Similar to the tumor
incidence data, pre-application of silymarin also showed a
highly significant protection against tumor multiplicity throughout the experimental protocol (Figure 1B). At termination of
the experiment at 40 weeks, compared with 2.25 0.35 (mean
SE of 20 mice) tumors/mouse in the positive control group,
silymarin treatment resulted in only 0.75 0.15 (mean SE
of 20 mice) tumors/mouse, a 67% reduction (P 0.001)
(Figure 1B). Interestingly, the inhibition of tumor multiplicity
by silymarin also correlated with its inhibitory effect on tumor
volume/tumor. Compared with the BPO alone positive group,
showing 72 13 mm3 tumor volume/tumor, animals in the
silymarin pretreated group showed a significantly (P 0.001)
smaller tumor size (41 9 mm3 tumor volume/tumor), a 44%
decrease.
Several studies have shown that topical application of skin
tumor promoters induces a strong oxidative stress in mouse
epidermis, which includes elevated levels of superoxide anions
followed by H2O2 and hydroxyl radicals and depletion of
antioxidant enzymes (e.g. SOD, catalase and GPX) that scavenge these reactive oxygen species (ROS) (59,12). This effect
of tumor promoting agents on mouse skin is possibly associated
with several other biochemical and molecular alterations
associated with inflammation and tumor promotion (59).
Consistent with this notion are studies showing that treatment
of mouse skin with ROS and free radical-generating agents
such as H2O2 and several different organic peroxides, including
BPO, is sufficient to achieve several different cellular, biochemical and molecular alterations, including tumor promotion, as
observed with TPA (510,35,36), and that treatment of mouse
skin with antioxidants protects against these oxidative conditions and tumor promotion in skin (1214). As silymarin is
also a very strong antioxidant capable of scavenging both free
radicals and ROS (1620) and shows strong protection against
BPO-induced tumor promotion, we also assessed its effect on
BPO-induced depletion of antioxidant enzyme activities in
mouse epidermis. The results for antioxidant enzyme activities
of SOD, catalase and GPX in mouse epidermis from different
groups are summarized in Figure 2. Treatment of SENCAR
mice with BPO for 24 h resulted in a strong depletion of
antioxidant enzyme activities in epidermis. The observed
reductions in enzyme activities for SOD, catalase and GPX in
mouse epidermis following a single BPO application were 75,
87 and 61% of the acetone-treated negative control, respectively
(Figure 2). In the studies assessing the effect of silymarin on
BPO-induced depletion of antioxidant enzyme activities, it
showed a highly significant dose-dependent protection (Figure
2). In the case of SOD, pre-application of silymarin at 3, 6
and 9 mg showed 18 (P 0.005), 45 (P 0.001) and 66%

Fig. 2. Inhibitory effect of silymarin on BPO-induced depletion of

antioxidant enzyme activities in SENCAR mouse epidermis. The shaved
dorsal area of mice in different groups was treated topically with 0.2 ml of
acetone alone, 6 mg of silymarin in 0.2 ml of acetone and 20 mg of BPO in
0.2 ml of acetone or 3, 6 or 9 mg of silymarin followed 30 min later by
20 mg of BPO each in 0.2 ml of acetone. Twenty-four hours after these
treatments, mice were killed, the treated area of the skin removed and
epidermal cytosolic fractions prepared. The SOD (A), catalase (B) and GPX
(C) enzyme activities were measured. Each data bar represents the mean
SD of four mice; each sample was assayed in triplicate. The up arrow
indicates a percentage increase in BPO-induced depletion of antioxidant
enzyme activity in mouse epidermis.

(P 0.001) protection against BPO-induced depletion of

SOD activity in mouse epidermis, respectively (Figure 2A).
Similarly, BPO-induced depletion of epidermal catalase activity
also recovered by 32 (P 0.001), 57 (P 0.001) and 72%
(P 0.001) following pre-treatment with 3, 6 and 9 mg of
silymarin, respectively (Figure 2B). In the case of GPX activity,
3, 6 and 9 mg of silymarin showed 20 (P 0.005), 57
(P 0.001) and 67% (P 0.001) protection, respectively
(Figure 2C). In contrast to these inhibitory effects of silymarin
on BPO-induced depletion of antioxidant enzyme activities,
treatment of mouse skin with 6 mg of silymarin alone did not
result in any change in the epidermal antioxidant enzyme
activities examined (Figure 2).
Under oxidative conditions, the superoxide anion is the first
ROS formed, which if not scavenged, ultimately leads to a
highly reactive species, the hydroxyl radical, which attacks
nucleic acid, protein and lipid-rich membranes causing severe
cell damage leading to genetic alterations (37,38). In this
regard, the most studied markers of oxidative damage are lipid
813

J.Zhao et al.

Fig. 3. Inhibitory effect of silymarin on BPO-induced epidermal lipid

peroxidation in SENCAR mice. The treatments on the shaved dorsal area of
mice in different groups were performed as described in Figure 2. Twentyfour hours after these treatments, mice were killed, the treated area of the
skin removed and epidermal microsomal fractions prepared. The epidermal
lipid peroxidation activity was measured. Each data bar represents the mean
SD of four mice; each sample was assayed in triplicate. The down arrow
indicates a percentage decrease in BPO-induced epidermal lipid
peroxidation in mice.

peroxidation [malondialdehyde (MDA) formation] and DNA

damage (8-hydroxydeoxyguanosine formation) (37,38). In
recent studies we have shown that in vitro addition of silymarin
to epidermal microsomal incubations inhibits lipid peroxidation
in a concentration-dependent manner (26). Based on this study
and the data shown in Figure 2, we also assessed the effect of
pre-treatment with silymarin on BPO-induced lipid peroxidation in mouse epidermis. As shown by the data in Figure 3,
compared with the acetone-treated control, a single topical
application of BPO resulted in strong induction of epidermal
lipid peroxidation, as measured by MDA formation. However,
pre-treatment of mouse skin with silymarin resulted in significant inhibition of BPO-induced epidermal lipid peroxidation in
a dose-dependent manner at doses of 3 and 6 mg (Figure 3).
A further increase in silymarin dose to 9 mg showed a
comparable effect to that observed at 6 mg (Figure 3). The
observed inhibitory effects of silymarin at 3, 6 and 9 mg were
35 (P 0.05), 80 (P 0.001) and 87% (P 0.001),
respectively (Figure 3). Silymarin alone, however, showed
almost comparable epidermal lipid peroxidation to that in the
acetone-treated negative control (Figure 3).
Based on the results showing that silymarin significantly
inhibits BPO-induced tumor promotion and depletion of antioxidant enzyme activities, we also assessed the effect of silymarin
on BPO-induced inflammatory responses in terms of skin
edema, neutrophil infiltration (by measuring MPO activity)
and an increase in inflammatory cytokine IL-1 in mouse
epidermis. As measured by a net increase in the skin bi-fold
thickness, compared with the acetone-treated negative controls,
a single topical application of BPO to mouse skin resulted in
a strong increase in skin bi-fold thickness (edema) in SENCAR
mice (Figure 4). However, the application of different doses
of silymarin prior to BPO treatment resulted in a highly
significant inhibition of BPO-induced skin edema (Figure 4).
The observed inhibitory effects of silymarin were dose dependent and evident as 62, 77 and 85% inhibition (P 0.001) at
3, 6 and 9 mg of silymarin, respectively (Figure 4). Similar
to other studies, silymarin alone applied topically at 6 mg
showed no skin edema (Figure 4).
Topical application of the tumor promoter TPA to mouse
814

Fig. 4. Inhibitory effect of silymarin on BPO-induced skin edema in

SENCAR mice. The treatments on the shaved dorsal area of mice in
different groups were performed as described in Figure 2. Twenty-four
hours after these treatments, a dial caliper was used to measure skin bi-fold
thickness and edema formation was calculated as net increase in skin bi-fold
thickness. Each data bar represents the mean SD of four mice; eight
measurements were made at different dorsal skin sites for each mouse. The
down arrow indicates a percentage decrease in BPO-induced skin edema.

Fig. 5. Inhibitory effect of silymarin on BPO-induced MPO activity in

SENCAR mice. The treatments on the shaved dorsal area of mice in
different groups were performed as described in Figure 2. Twenty-four
hours after these treatments, mice were killed, the treated area of the skin
removed, total MPO content extracted and MPO activity measured. Each
data bar represents the mean SD of four mice; each sample was assayed
in triplicate. The down arrow indicates a percentage decrease in BPOinduced skin MPO activity in mice.

skin has been shown to result in neutrophil infiltration that is

responsible, at least in part, for the oxidative conditions
following TPA application to mouse skin (3942). In a given
tissue, MPO is utilized as a marker to quantify the total number
of neutrophils present therein (32). Based on the data showing
that silymarin protects against BPO-induced depletion of
antioxidant enzyme activities and BPO-induced skin edema,
we next assessed its effect on BPO-induced neutrophil infiltration by measuring MPO. As shown in Figure 5, 24 h after
application of a single dose of BPO to mouse skin, total MPO
activity increased from not detectable to 53 U/mg epidermal
protein. This increase in the level of tissue MPO indicates an
influx of neutrophils into the inflamed skin. Pre-application of
silymarin before BPO, however, resulted in a significant
protection against BPO-induced infiltration of neutrophils in a
dose-dependent manner (Figure 5). Compared with the BPOtreated samples, 3 mg of silymarin showed 42% protection
(P 0.001) against BPO-induced total MPO content (Figure 5).
Much stronger effects of silymarin were observed at 6 and 9
mg, which showed 77% and complete inhibition (P 0.001)

Silymarin inhibits benzoyl peroxide effects

In this regard, the results of the present study showing that

silymarin protects against BPO-induced tumor promotion may
be of great significance in preventing any tumor promotion
risk associated with BPO exposure. Taken together, based on
our recent studies and the results of the present study, we
suggest that silymarin could be a useful cancer preventive
agent against a wide range of carcinogen and tumor promoterinduced epithelial cancers in humans.
Acknowledgment
This study was supported by USPHS grant CA64514.
Fig. 6. Inhibitory effect of silymarin on BPO-induced IL-1 protein
expression in SENCAR mouse epidermis. The treatments on the shaved
dorsal area of mice in different groups were performed as described in
Figure 2. Twenty-four hours after these treatments, mice were killed, the
treated area of the skin removed, epidermal homogenates prepared and IL1 protein expression measured. Each data bar represents the mean SD
of four mice; each sample was run in duplicate and the ELISA estimation
was made twice. The down arrow indicates a percentage decrease in BPOinduced IL-1 protein expression in mouse epidermis.

of BPO-induced neutrophil infiltration in skin, respectively

(Figure 5). Together, these data are consistent with an inhibitory
effect of silymarin on BPO-induced depletion of antioxidant
enzyme activities and skin edema and suggest that protection
against BPO-induced neutrophil infiltration by silymarin is
possibly responsible, at least in part, for its observed inhibitory
effect on BPO-induced tumor promotion.
Cytokines have been implicated in a variety of physiological
and pathological conditions, including inflammation and tumor
promotion (34,43). Several studies in recent years have shown
the involvement of IL-1 in both phorbol and non-phorbol
ester type tumor promoter (including BPO)-induced skin
inflammation and tumor promotion (34,43). We performed
studies to assess the effect of silymarin on BPO-induced IL1 protein expression in mouse epidermis. As shown in
Figure 6, compared with the acetone control, a single topical
application of BPO resulted in strong expression (6.5-fold
increase over control) of IL-1 protein in mouse epidermis.
Pre-treatment of mice with silymarin, however, showed a
strong inhibition of BPO-induced IL-1 protein expression in
mouse epidermis. The observed inhibitory effect of silymarin
was dose-dependent and evident in terms of 36 (P 0.005),
72 (P 0.001) and 81% (P 0.001) decreases in BPOinduced IL-1 protein expression in mouse epidermis at 3, 6
and 9 mg, respectively (Figure 6). Treatment of mice with
silymarin alone at 6 mg did not result in any change in
epidermal IL-1 protein level (Figure 6).
BPO is a free radical-generating compound and a strong
oxidizing agent to which humans are constantly exposed due
to the fact that BPO is widely used as: (i) a polymerization
initiator; (ii) a bleaching agent for flour and cheese; (iii) as an
additive in cosmetics and pharmaceutical products including
those for the treatment of acne (ref. 10 and references therein).
As BPO has been found to cause irritation of human skin,
extensive studies were performed which showed that though
not a complete carcinogen, BPO is a weak tumor promoting
agent (10). Since it can be argued that tumor initiation is
almost inevitable, as evident by a steady increase in cancer
incidence world wide (44), a prevention strategy against tumor
promotion could be a more practical approach to slow down
the clonal expansion of initiated cells in the first place (5,6).

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Received July 20, 1999; revised October 13, 1999; accepted November 8, 1999