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Antimicrobial Agents and Chemotherapy, April 1999, p. 745-751, Vol. 43, No. 4
0066-4804/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Bile Salts: Natural Detergents for the Prevention
of Sexually Transmitted Diseases
Betsy C.
Herold,1,*
Risa
Kirkpatrick,2
Daniel
Marcellino,1
Anna
Travelstead,2
Valentina
Pilipenko,2
Holly
Krasa,1
James
Bremer,3
Li Jin
Dong,4 and
Morris D.
Cooper2
Department of Pediatrics, Mount Sinai School
of Medicine, New York, New York1;
Department of Medical Microbiology and Immunology, Southern
Illinois University School of Medicine, Springfield,
Illinois2; and Departments of
Microbiology3 and Obstetrics and
Gynecology,4 Rush Medical College, Chicago,
Illinois
Received 27 July 1998/Returned for modification 23 November
1998/Accepted 4 January 1999
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ABSTRACT |
The development of new, safe, topical microbicides for intravaginal
use for the prevention of sexually transmitted diseases is imperative.
Previous studies have suggested that bile salts may inhibit human
immunodeficiency virus infection; however, their activities against
other sexually transmitted pathogens have not been reported. To further
explore the potential role of bile salts in preventing sexually
transmitted diseases, we examined the in vitro activities and
cytotoxicities of select bile salts against Chlamydia
trachomatis, herpes simplex virus (types 1 and 2),
Neisseria gonorrhoeae, and human immunodeficiency virus in
comparison to those of nonoxynol-9 and benzalkonium chloride using both
primary cells and cell lines derived from the human female genital
tract. We found that taurolithocholic acid 3-sulfate and a combination of glycocholic acid and taurolithocholic acid 3-sulfate showed excellent activity against all of the pathogens assayed. Moreover, taurolithocholic acid 3-sulfate alone or in combination was less cytotoxic than nonoxynol-9 and benzalkonium chloride. Thus,
taurolithocholic acid 3-sulfate alone or in combination warrants
further evaluation as a candidate topical microbicidal agent.
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INTRODUCTION |
Novel approaches to the prevention
of sexually transmitted diseases (STDs) are clearly warranted. An
urgency emerges from the growing pandemic of STDs, estimated at 333 million cases of curable STDs worldwide by the World Health
Organization (6, 12, 14). These diseases affect not only
sexually active individuals but also newborns, who may become infected
perinatally. Male condoms, in theory, remain a highly effective method
for the prevention of STDs and contraception, but problems with
compliance limit their efficacy. The development of topical
microbicides intended for intravaginal use offers several advantages.
Intravaginal formulations would enable women to make their own
decisions regarding STD prophylaxis. Ideally, intravaginal microbicides
would directly inactivate microbes at the portal of entry, preventing
the establishment of infection. Optimally, agents that are active
against multiple pathogens, that are nontoxic to genital epithelia and
normal flora, that are inexpensive to manufacture, and that could thus
be made available worldwide could be identified.
One class of intravaginal topical microbicides currently in use is
surface-active agents. For example, the cationic quaternary ammonium
compound benzalkonium chloride (BZC) is currently used in several
vaginal contraceptive preparations. It has been shown to inhibit the
reverse transcriptase activity of human immunodeficiency virus (HIV)
(31) and to protect mice challenged with Chlamydia trachomatis (22). However, BZC has profound adverse
effects on the normal vaginal microflora of pig-tailed macaques
(23), and concerns about the cytotoxicity of this detergent
have been raised. Similarly, the nonionic surface-active agent
nonoxynol-9 (N-9), the most commonly used spermicide currently on the
market, has been shown to be active in vitro against a wide array of
STD pathogens. These include Neisseria gonorrhoeae, HIV, and
herpes simplex virus (HSV) (5, 11, 19, 32, 33). However, N-9 is cytotoxic for most cell lines in vitro, including primary human cervical and vaginal cells (15). Importantly, frequent use
of N-9 causes epithelial disruption of the cervix and vagina and may
increase the risk for STDs (20, 34). In clinical studies, the effect of N-9 in preventing HIV transmission remains controversial. Improvement in the safety and efficacy of vaginal antimicrobial agents,
therefore, is a priority.
Recently, a class of naturally occurring anionic surface-active agents,
bile acids, has received attention as potential contraceptive agents.
Specifically, cholic acid has been shown to have strong spermicidal
activity and has been shown to inhibit sperm motility at a
concentration of 6 mM (26). Cholic acid is one of the active ingredients in F-5 gel preparations marketed for use within a vaginal
sponge (Protectaid) available in Canada (8, 26). However,
concerns have been raised regarding the cytotoxicity of cholic acid.
For example, Baba and colleagues (1) examined the effects of
a series of cholic acid derivatives on HIV infection and found that
sodium cholate (NaC) had a selectivity index of <1.0 for HIV
replication in MT-2 cells (1). Similarly, Psychoyos and
colleagues (26) found that NaC at a concentration of 2.5 mM
(~1 mg/ml) reduced the viability of normal peripheral blood lymphocytes by 45% (26). The effect of NaC or other bile
salts on STD pathogens other than HIV and the cytotoxic effects of this class of compounds on human vaginal or cervical epithelial cells have
not been reported.
Therefore, to further explore the potential role of bile salts as
intravaginal topical microbicides, we compared select bile salts to N-9
and BZC for their in vitro activities against Chlamydia trachomatis, HSV type 1 (HSV-1), HSV-2, N. gonorrhoeae,
and HIV. We also assayed the cellular toxicities of these compounds
with primary human vaginal and cervical cells. The results obtained suggest that taurolithocholic acid-3-sulfate (NaTLC-3-SO4)
alone or combined with glycocholic acid exhibits excellent activity in
vitro against all of the pathogens tested with little or no cytotoxicity for human cervical epithelial cells. Thus, this bile salt,
alone or in combination, may be superior to N-9 or BZC and warrants
further investigation.
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MATERIALS AND METHODS |
Primary and permanent cell cultures.
The human cervical
epithelial cell lines HeLa 229, and CaSki were purchased from the
American Type Culture Collection (Rockville, Md.). HeLa cells were
maintained in Eagle's minimal essential medium containing 10%
heat-inactivated fetal bovine serum (FBS), nonessential amino acids,
L-glutamine, and gentamicin (50 µg/ml). CaSki cells were
maintained in Dulbecco's modified Eagle's medium supplemented with
10% FBS. A 24th passage of the T-lymphocyte cell line MT-2 was
obtained from the AIDS Research and Reference Reagent Program of the
National Institutes of Health (Rockville, Md.). The MT-2 cells were
maintained in RPMI 1640 with glutamine containing 20% heat-inactivated
FBS and 50 µg of gentamicin per ml. Primary human endocervical,
ectocervical, and vaginal mucosal cultures were derived from specimens
obtained during hysterectomies performed for benign conditions such as
uterine leiomyomas and endometriosis as described previously (15,
30). For viral infectivity and cytotoxicity studies, most samples
had been subcultured one to two times.
Microbial pathogens.
The C. trachomatis strain
used in this study was serotype E/UW-5/CX. The E strain of C. trachomatis is well characterized and has previously been shown to
be capable of infecting explants of primate and human fallopian tubes
in culture (7, 21, 24, 25). Stocks were maintained through
passage in HeLa 229 cells for a minimum of nine passages, concentrated
to 108 inclusion-forming units per ml, and stored at
80°C in HEPES-sucrose-calcium (HSC) buffer (3). The
wild-type strain HSV-2(G) and strain HSV-1(17)(dUTPase/LAT) were used.
The latter strain has been genetically engineered and expresses the
-galactosidase gene under control of the viral immediate-early gene
promoter dUTPase (gift of E. Wagner, Stanford University)
(29). HSV-1(17)(dUTPase/LAT) behaves like wild-type
HSV-1(17) with respect to binding and infectivity (18). The
N. gonorrhoeae strain used was MS11. The strain was monitored for transparency and piliation, and inocula of transparent, piliated organisms were used. The gonococci were grown on GC agar (Difco Laboratories, Detroit, Mich.) plates, and stocks were stored in
10% skim milk (Difco Laboratories) at 80°C until use. The HIV
strain (G910-SI; zidovudine resistant) was obtained from the AIDS
Research and Reference Reagent Program of the National Institutes of
Health and is identified as a stable HIV clone which will produce syncytia in MT-2 cells. The virus was expanded by culture in MT-2 cells
maintained in RPMI 1640 with glutamine containing 10% heat-inactivated FBS and 50 µg of gentamicin per ml. The final virus stock
concentration was 6 × 103 PFU/ml.
Infectivity assays.
To measure the effects of the compounds
on C. trachomatis, infectivity assays with HeLa cells were
performed as described previously, with modifications (9).
Briefly, 24-well plates were seeded at 4 × 105 to
5 × 105 cells/ml and the cells were grown to
confluence. The compounds (0 to 10 mg/ml or 0 to 0.1% [vol/vol] for
N-9) were diluted in HSC (pH 7.35) and were mixed with a concentration
of chlamydia to yield 100 to 300 inclusions per well in untreated wells
for 4 h at 4°C. Control organisms were treated with buffer
containing no compounds. HeLa cells were then inoculated with chlamydia
for 1 h at 37°C. The medium was removed, and the infected cells
were incubated in tissue culture with Eagle's minimal essential medium supplemented with 10% fetal calf serum, 30 µM glucose, 1 µg of cycloheximide per ml, and 50 µg of gentamicin sulfate per ml for 48 h at 37°C. After 48 h of incubation, the HeLa cells were
trypsinized, pelleted by centrifugation, fixed, permeabilized, and
stained for flow cytometry (Caltag Laboratories, Burlingame, Calif.). The infected cells were stained with a
fluorescein-isothiocyanate-conjugated monoclonal antibody to chlamydial
lipopolysaccharide protein (Kallestad). Infected cells were enumerated
with a FACS Vantage flow cytometer (Becton Dickinson, San Jose,
Calif.), and data from 10,000 cells were collected in the list mode for
analysis with H-P-LYSYS II software.
The effects of bile salts, N-9, and BZC on N. gonorrhoeae
were also determined. Various concentrations of compounds were added to
GC agar and the agar was poured into plates. The GC inoculum was made
by using a 0.5 McFarland standard. Agar plates were inoculated with
serial dilutions of the gonococcal standard and were incubated for
24 h in a 7% CO2 atmosphere at 37°C. The plates
were scored for colony growth, and counts from plates containing
compound were compared to those from plates containing no compound. A
percentage of gonococcal viability was determined.
For studies with HSV-2, plaque assays were conducted (
15,
18). Nearly confluent primary human cervical or vaginal cells
in
24-well plates or confluent CaSki cells in 25-cm
2 flasks
were inoculated with virus (50 to 100 PFU/well or 200
to 500 PFU/flask)
in phosphate-buffered saline (PBS) in the presence
or absence of bile
salts (0 to 5 mg/ml). Plaques were counted
after 1 day (primary cells)
or 3 days (CaSki cells). To visualize
the plaques, an immunoassay was
performed as described previously
with monoclonal antibody 1103 (anti-HSV-1 and HSV-2 gD; Goodwin
Institute) (
16,
17). For
studies with HSV-1, viral infectivity
assays were conducted as
described previously (
15,
18). Briefly,
primary cells in
96-well dishes were inoculated with HSV-1(17)(dUTPase/LAT)
at a
multiplicity of infection of 5 PFU/cell in the absence or
presence of
bile salts or detergent. After a 5-h period at 37°C,
the
-galactosidase expression from infected cells was quantified.
To
determine if the bile salts were virucidal or cytotoxic, plaque
assays
were modified. For virucidal activity, HSV-2 at a concentration
of
about 10
9 PFU/ml was mixed with various concentrations of
bile salt or
with PBS as a control. After incubation for 1 h at
37°C, the mixtures
were diluted to yield 100 to 500 PFU/flask for
controls, with
a final concentration of bile salt of 10
6
to 10
7 mg/ml. CaSki cells were inoculated with the
mixture to determine
whether the compound had irreversible effects on
viral infectivity.
For cytotoxic activity, the cells were incubated in
the presence
of bile salts at various concentrations or PBS, as a
control,
for 1 h at 37°C and were then washed extensively. The
cells were
then inoculated with virus to determine whether the
compounds
were cytotoxic, resulting in an inability to support viral
infection.
For studies with HIV, MT-2 cells were infected with a
syncytium-inducing strain of HIV (G910-SI; zidovudine resistant) in
the
presence or absence of bile salts with a viral inoculum that
produced
approximately 30 to 50 syncytia/well by day 3 of incubation.
MT-2 cells
(5.0 × 10
5) were aliquoted into each test well, and
the wells were inoculated
with virus in 100 µl of medium containing
the appropriate concentration
of bile salt to be assayed (0 to 1 mg/ml). Cultures were incubated
in a humidified 37°C incubator
containing 5% CO
2. The cultures
were examined daily. The
number of syncytia in each well was quantified
on day 3. Each drug
concentration was tested in
quadruplicate.
Cytotoxicity assays.
The cytotoxic effects of the bile salts
on both primary cervical cells and CaSki cells were determined by
quantitating cell viability through the uptake of neutral red dye as
described previously (2, 4, 10, 27, 28) or by quantitating
the inhibition of host cell DNA synthesis. For quantitation of DNA
synthesis, cells were grown directly in glass scintillation vials to
half confluence and were then incubated overnight in medium containing serial dilutions of test compounds and 2.5 µCi of
[3H]thymidine per ml. The cells were washed extensively,
and the cell-associated radioactivity was quantitated. HeLa cell
viability was determined with propidium iodide (PI) by flow cytometry
analysis as described previously, with modifications (10).
The HeLa cells were exposed to compounds or PBS, as a control, for 20 to 24 h. Cells were trypsinized and incubated with 5 µg of PI
per ml in PBS for 5 min at room temperature prior to
fluorescence-activated cell sorter analysis. The positive controls were
cells permeabilized to damage the cell membrane and allow the DNA to be
stained with PI.
Bile salt compounds and detergents.
Sodium salts of
glycocholic acid (NaGC), NaTLC-3-SO4, taurocholic acid
(NaTC), and cholic acid were purchased from Sigma; BZC and N-9
(Tergitol, NP-9) were also purchased from Sigma. All bile salts and
detergents were dissolved in PBS to a stock concentration of 10 mg/ml
(1% [vol/vol] for N-9) prior to use.
Statistical analysis.
The data presented are means with
standard deviations, as indicated in the figure legends. Student
unpaired, two-tailed t tests were performed as indicated.
| |
RESULTS |
Effects of bile salts on STD pathogens.
On the basis of pilot
studies, we focused on four bile salts as potential topical
microbicides (NaC, NaTC, NaTLC-3-SO4, and NaGC) and
compared their in vitro activities to those of N-9 and BZC. We first
compared their activities against C. trachomatis. Chlamydia
were pretreated with various concentrations of the bile salts, BZC, or
N-9 for 4 h prior to inoculation onto HeLa cell monolayers. At a
concentration of 1 mg/ml, NaTLC-3-SO4 inhibited chlamydial
infection by 67% ± 10% (P = 0.0001) (Fig.
1a). None of the other bile salts
exhibited significant antimicrobial activity. Notably, neither N-9 nor
BZC at concentrations as high as 0.01% or 0.01 mg/ml, respectively,
inhibited chlamydial infection, and higher concentrations of either
detergent disrupted the HeLa cell monolayer, resulting in a selectivity
index of 
1 for either detergent (Fig. 1b and Table
1).
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FIG. 1.
Effects of bile salts at a concentration of 1.0 mg/ml
(a), N-9 at a concentration of 0.001% (b), or BZC at a concentration
of 0.01 mg/ml (b) against microbial infection. For chlamydia, the
results are presented as the number of inclusions formed in the
presence of compound as a percentage of the number of inclusions formed
in the absence of compound. Each point is the mean of values obtained
from at least two independent experiments performed in duplicate. For
gonococci, the results are presented as the number of viable gonococci
found in the presence of compound as a percentage of the number of
gonococci found in the absence of compound. Each point is the mean of
values obtained from two independent experiments performed in
duplicate. No viable gonococci were observed on the plates containing
0.01 mg of BZC per ml or 1 mg NaC or NaTLC-3-SO4 (*) per
ml. For HSV-2, results are presented as the number of PFU per well in
the presence of compound as a percentage of the number of PFU per well
in the absence of compound. For HSV-1, results are presented as
-galactosidase expression (absorbance at 410 nm) in the presence of
compound as a percentage of -galactosidase expression in the absence
of compound. Each point is the mean of three to five experiments
performed in duplicate with primary cells obtained from different
patients. For HIV-1, results are presented as syncytia observed on day
3 of infection in the presence of compound as a percentage of syncytia
formed in the absence of compound. Each point is the mean of two
experiments performed in quadruplicate. ***, no syncytia were
observed after NaC, NaTLC-3-SO4, or NaGC treatment at
concentrations as low as 0.1 mg/ml. The error bars indicate standard
deviations.
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To examine the microbicidal activities of the bile salts and detergents
against
N. gonorrhoeae, another major bacterial STD,
GC agar
cultures were incubated in the presence or absence of
various
concentrations of compounds, and gonococcal viability
was determined.
NaTLC-3-SO
4 and NaC were the most effective bile
salts and
at concentrations of 1 mg/ml completely inhibited gonococcal
growth
(Fig.
1a). Similarly, BZC at 0.01 mg/ml completely inhibited
gonococcal
growth (Fig.
1b). In contrast, even at cytotoxic concentrations
the
detergent N-9 only modestly inhibited gonococci (Table
1).
Next, we compared the activities of the same series of compounds
against HSV-2 and HSV-1 infection. Virus was mixed with bile
salt or
detergent immediately prior to inoculating primary or
permanent human
cervical cells. NaTLC-3-SO
4 inhibited 98% ± 1%
of HSV-2
infections as measured by the quantitation of plaque
formation and 96% ± 4% of HSV-1 infections as measured by quantitation
of
-galactosidase expression (
P < 0.001; Fig.
1a). NaC
also inhibited
77% ± 8% of HSV-2 and 98% ± 2% of HSV-1
infections. NaTC had little
or no inhibitory activity against either
serotype, and NaGC inhibited
HSV-2 plaque formation only by about 50%.
Similar results were
obtained with permanent cervical cell lines. At a
dose of 1 mg/ml,
NaTLC-3-SO
4 completely inhibited HSV-2
infections and inhibited
98% ± 1% of HSV-1 infections in CaSki cells
(data not shown).
In contrast, neither N-9 nor BZC inhibited HSV-2
plaque formation
at concentrations that did not appear to be cytotoxic
for primary
cells (0.001% and 0.01 mg/ml, respectively; Fig.
1b).
Higher concentrations
of either detergent disrupted the cellular
monolayer.
The observation that bile salts are surface active and inhibit HSV
early gene expression and plaque formation in parallel
suggests that
they may be virucidal and may directly inactivate
HSV. To further
explore this, we compared the virucidal and cytotoxic
effects of bile
salts as described in Materials and Methods. For
virucidal assays,
HSV-2(G) at a concentration of about 10
9 PFU/ml was mixed
with the bile salts at 1 mg/ml, and the mixture
was incubated for
1 h at 37°C. The virus-bile salt mixture was
then diluted to
yield 10
2 to 10
3 PFU/ml and a final
concentration of bile salt of 10
6 to 10
7
mg/ml and was plated onto plaque dishes. Conversely, for cytotoxicity
assays, CaSki cells were incubated with bile salts at 1 mg/ml
for
1 h at 37°C and were then extensively washed prior to
inoculation
of the cells with virus. The results are presented in Table
2.
The results suggest that following a
1-h exposure at a concentration
of 1 mg/ml, the bile salts are
irreversibly virucidal but not
cytotoxic.
Because previous studies with bile salts and HIV infection have yielded
variable results, we also examined the activities
of the bile salts
against HIV using MT-2 cells. The results from
three different
experiments conducted in quadruplicate are summarized
in Fig.
1a and
Table
1. NaC, NaTLC-3-SO
4, and NaGC at concentrations
of
0.1 mg/ml completely inhibited syncytium formation, with limited
inhibitory effects at lower concentrations. NaTC had little or
no
inhibitory
effect.
Cytotoxicities of bile salts.
To further assess the potential
role of bile salts as topical microbicides, we examined their
cytotoxicities using several different cell lines (HeLa, CaSki, or
primary human cervical cells) and several different types of assays
(neutral red dye uptake, PI uptake, or inhibition of host cellular DNA
synthesis). The results are summarized in Tables 1 and
3. Several observations are noteworthy.
First, although the trends were parallel, cytotoxicity was dependent on
both cell type and assay conditions. For example, bile salts and
detergents exhibited more cytotoxicity for primary human cervical cells
than for permanent cell lines. Moreover, cytotoxicity became more
evident when quantitation of cellular DNA synthesis was compared to
cell viability. For example, none of the bile salts would be considered
cytotoxic if one measured cell viability with CaSki cells, but some
cytotoxicity was evident when cellular DNA synthesis was quantitated.
The duration of exposure to compounds also appeared to influence their
cytotoxicity. To explore this further, we exposed CaSki
cells to
compounds at a single concentration, one that inhibited
HSV infection,
for increasing time periods and quantitated cell
viability through the
uptake of neutral red dye. The results are
shown in Fig.
2. Little or no cytotoxicity was observed
for up
to 4 h for the bile salts; however, cytotoxicity increased
following
prolonged exposure. In contrast, both N-9 and BZC were
cytotoxic
within 1 h of exposure.
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FIG. 2.
Cytotoxic effects of bile salts, BZC, or N-9 on CaSki
cells following exposure for increasing lengths of time. CaSki cells
were incubated in medium containing the compound at the indicated
concentrations for from 1 to 16 h. At each time point, the number
of viable cells was quantitated by exclusion of neutral red dye.
Results are presented as the number of viable cells in the presence of
compound as a percentage of the number of viable cells in the absence
of compound. Each point is the mean of two experiments performed in
duplicate. The error bars indicate standard deviations.
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Combinations of bile salts.
The observation that at 1 mg/ml
NaTLC-3-SO4 inhibited all of the STD pathogens but
exhibited some cytotoxicity for primary cells prompted us to explore
the possibility that a combination of bile salts that would retain
antimicrobial activity with less cytotoxicity might be identified.
Therefore, we compared the antimicrobial activities of various
combinations of NaC, NaGC, and NaTLC-3-SO4 and their
cytotoxic effects. The results for select combinations are summarized
in Fig. 3. The addition of 0.1 mg of NaC
per ml, 0.1 mg of NaGC per ml, or 1.0 mg of NaGC per ml to 0.1 mg of
NaTLC-3-SO4 per ml (combinations A, B, and C, respectively)
markedly enhanced the anti-HSV activities of the bile salts and
produced little or no cytotoxicity. However, no additive effect was
observed for chlamydia or gonococci. The combination of 1.0 mg of NaGC
per ml with 1.0 mg of NaTLC-3-SO4 per ml (combination D)
completely inhibited HSV infection and gonococcal viability and
markedly reduced chlamydial infectivity. This combination was not
cytotoxic for HeLa cells; 95% ± 0.1% of HeLa cells were viable
following a 24-h exposure to the bile salt combination, as determined
with PI by flow cytometry. However, this combination did inhibit
cellular DNA synthesis to 53% ± 25% of that for the controls
following 16 h of exposure for primary human cervical cells.
Notably, the concentrations of bile salts in this combination are well
above the 50% effective dose for HIV infection of MT-2 cells. Thus, either alone or in combination, NaTLC-3-SO4 appears to be
superior to either N-9 or BZC because of its broad range of anti-STD
activity with limited cytotoxicity.
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FIG. 3.
Antimicrobial effects of combinations of bile salts.
Chlamydial organisms were preincubated with 0.1 mg of NaC per ml plus
0.1 mg of NaTLC-3-SO4 per ml (A), 0.1 mg of NaGC per ml
plus 0.1 mg of NaTLC-3-SO4 per ml (B), 1.0 mg of NaGC per
ml plus 0.1 mg of NaTLC-3-SO4 per ml (C), or 1.0 mg of NaGC
per ml plus 1.0 mg of NaTLC-3-SO4 per ml (D) prior to
infection of HeLa cells. Results are presented as the number of
inclusions formed in the presence of the combination as a percentage of
the number of inclusions formed in the absence of compound. Each value
is the mean of values obtained from at least two independent
experiments performed in duplicate. For HSV-2 infection, primary human
cervical cells were inoculated with ~100 PFU of HSV-2(G) per well in
the absence or presence of the indicated combination, and the number of
PFU was quantitated at 24 h. Results are presented as the number
of PFU formed in the presence of the combination as a percentage of the
number of PFU formed in the presence of PBS as a control. Each value is
the mean of values obtained from three independent experiments
performed in duplicate with cells from different patients. For N. gonorrhoeae, combinations of bile salts were added to GC agar; and
the mixture was poured into plates, inoculated with serial dilutions of
the gonococcal standard, and incubated for 24 h. Results are means
of two experiments performed in duplicate. The error bars indicate
standard deviations. IFU, inclusion-forming units.
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DISCUSSION |
The studies described here were designed to explore the potential
role of select bile salts as topical microbicides for the prevention of
several STDs. We found that NaTLC-3-SO4 was highly effective against HSV-1, HSV-2, HIV, N. gonorrhoeae, and
C. trachomatis. In contrast, although BZC inhibited
gonococcal viability, it failed to inhibit HSV or chlamydial infection
except at concentrations that were toxic and that completely disrupted
the cellular monolayer. Moreover, N-9 also exhibited little or no
activity except at cytotoxic concentrations against any of the
pathogens assayed. Thus, in vitro, NaTLC-3-SO4 exhibits a
more favorable selectivity index than N-9 or BZC for multiple STD pathogens.
The optimal in vitro assay for predicting the in vivo cytotoxicities of
candidate topical microbicides is not known. The results of our studies
demonstrate that primary cells tend to be more susceptible than cell
lines to cytotoxic effects. Bile salts and detergents inhibit cellular
DNA synthesis at concentrations lower than those required to kill
cells. Presumably, this reflects the fact that any change in the
cellular membrane or cellular architecture results in a reduction in
cellular DNA synthesis (13). Cytotoxicity is also dependent
on the duration of exposure. Many of the bile salts exhibited little or
no cytotoxicity when primary or permanent cells were exposed for up to
4 h but did begin to exhibit cytotoxicity 8 to 16 h following
exposure. In contrast, BZC and N-9 were cytotoxic within 1 h of
exposure at the concentrations that inhibited HSV or chlamydial infection.
Brief exposure (1 h) of HSV to bile salts was sufficient to induce
irreversible, virucidal effects. This suggests that the viral envelope
is more susceptible than the cellular plasma membrane to the damaging
effects of bile salts. Similarly, gonococcal growth is markedly reduced
in the presence of bile salts at concentrations that fail to disrupt
the human cervical cell membranes. This suggests that the gonococcal
cell wall is also more susceptible than human cellular plasma membranes
to the damaging effects of bile salts.
Although NaTLC-3-SO4 was also microbicidal for C. trachomatis at noncytotoxic concentrations, of all the pathogens
assayed chlamydia were the most resistant to any of the surface-active agents. For example, neither N-9 nor BZC at noncytotoxic concentrations inhibited chlamydial infection. While NaTLC-3-SO4 was the
most effective of the compounds assayed, its inhibitory effects were dependent on time of exposure. Following a 4-h incubation of elementary bodies with 1 mg of NaTLC-3-SO4 per ml, chlamydial
infectivity was reduced by 67% ± 10%, whereas following a 1-h
incubation, infectivity was reduced by 50% ± 4% (data not shown).
Presumably, challenge of the vaginal mucosa by STD pathogens will occur
continuously and simultaneously with exposure to the topical
antimicrobial agent. Whether the preincubation time required in vitro
will affect the efficacy of this compound in vivo remains to be
determined. The differences in susceptibility between C. trachomatis and N. gonorrhoeae, the other bacterial
pathogen assayed, may reflect differences between chlamydia and other
gram-negative bacteria with respect to cell wall composition. For
example, the chlamydial cell wall lacks peptidoglycan. The mechanism of
microbicidal activity for surface-active detergents, at a molecular
level, has not been determined.
Taken together, the results of these studies suggest that
NaTLC-3-SO4 alone or in combination with NaGC exhibits
excellent activity in vitro against multiple STD pathogens. It appears
to be microbicidal and thus would directly inactivate STD microbes at
their portal of entry and prevent the establishment of any infection.
Moreover, NaTLC-3-SO4 alone or in combination exhibits little or no cytotoxicity for permanent cervical cells and is less
cytotoxic than N-9 or BZC for primary human cervical cells. Thus, this
bile salt may be superior to N-9 or BZC and warrants further
investigation as a candidate topical intravaginal microbicide for the
prevention of the transmission of STDs. Future studies will be directed
at formulating bile salts for intravaginal use and determining their
spermicidal activities and effects on normal vaginal flora. Studies
with animal models are in progress.
| |
ACKNOWLEDGMENTS |
We are grateful to Ed Wagner for providing viral strain
HSV-1(17)dUTPase/LAT. We thank Ching-yu Sun, Alicia Siston, and Ernest Winkfield for technical support and Larry Stanberry and George Wilbanks
for advice.
This work was supported by Public Health Service grant AI37940.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Pediatric
Infectious Diseases, Mount Sinai School of Medicine, 1 Gustave L. Levy
Place, New York, NY 10029. Phone: (212) 241-6930. Fax: (212) 426-4813. E-mail: betsy_herold{at}smtplink.mssm.edu.
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Antimicrobial Agents and Chemotherapy, April 1999, p. 745-751, Vol. 43, No. 4
0066-4804/99/$04.00+0
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Abstract
Introduction
