Vol. 36, No. 7
ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, July 1992, p. 1478-1482
0066-4804/92/071478-05$02.00/0 Copyright ©) 1992, American Society for Microbiology
In Vitro Activity of Nonoxynol 9 on HeLa 229 Cells and Primary Monkey Cervical Epithelial Cells Infected with Chlamydia trachomatis DOROTHY L. PATTON,`* SHA-KE WANG,1 AND CHO-CHOU KUO2 Departments of Obstetrics and Gynecology' and Pathobiology, 2 University of Washington, Seattle, Washington 98195 Received 21 November 1991/Accepted 24 April 1992
Nonoxynol 9 (non-9) is the active ingredient in a wide variety of vaginal contraceptive preparations. The manufacturer recommendation for optimal contraceptive practice is repeated application every 6 h. We studied the in vitro activity of non-9 against Chlamydia trachomatis (E/UW-5/Cx) and its toxicity against HeLa 229 cells and monkey cervical epithelial cells. With a contact time of 6 h, non-9 was toxic to HeLa cells at concentrations of 50 ,ug/ml or greater and to monkey cervical cells at 100 ,ug/ml or greater. Inhibitory effects of non-9 on extracellular chiamydiae were observed at concentrations of 50 ,ug/ml or greater. Inhibition of intracellular growth of chlamydiae in monkey cervical cells was observed at a nontoxic concentration of 50 ,ug/ml. Our studies show that non-9 has antichlamydial activity. However, owing to its toxicity to cervical cells in vitro, the effects of prolonged use of non-9 in vivo should be further studied. 229 cell cultures (10). An inoculum was partially purified by one cycle of differential centrifugation, which yielded 108 to
Chlamydia trachomatis is one of the most common sexually transmitted pathogens. A contraceptive that is effective against chlamydiae would provide a means of birth control as well as prophylaxis against sexually transmitted diseases. Many in vitro studies on the antimicrobial activity of the active spermicide ingredient nonoxynol 9 (non-9) against C. trachomatis have been done in continuous cell lines. The results have been controversial. Non-9 is the active ingredient in a wide variety of vaginal contraceptive foams, creams, jellies, and suppositories. Earlier in vitro studies using non-9 have demonstrated antimicrobial activity against Neisseria gonorrhea (4, 12), Trichomonas vaginalis (4, 12), Treponema pallidum (12), Candida albicans (13), Herpes simplex virus (2, 14), and Ureaplasma urealyticum (1). Many attempts have been made to assess such activity against C. trachomatis infection in vitro. Benes and McCormack (3) and Kelly et al. (8) have demonstrated inhibition of C. trachomatis growth in McCoy cell cultures. Cytopathologic effects on McCoy cells, a mouse line, have also been described (8). In contrast, data from Kappus and Quinn (7) and Knight et al. (9) have not demonstrated inhibition of C. trachomatis growth in cultures of the same cells. To understand the effects of non-9 on C. trachomatis infection further, we examined this active spermicide ingredient under more physiological conditions by using a continuous human cell line, HeLa 229, and primary monkey cervical epithelial cells to assess the toxicity of the drug and its bactericidal activity against C. trachomatis under conditions nontoxic to host cells.
109 inclusion-forming units of C. trachomatis organisms from a 32-oz (947-ml) culture bottle. A control inoculum was prepared from uninfected cultures processed as infected cultures. Serovar E was selected because it is the most common serovar of genital isolates (11). Monkey cervical epithelial cell culture. Monkey (Macaca nemestrina) cervix tissue was obtained from the Tissue Program at the Regional Primate Research Center at the University of Washington. The tissue was cut into small pieces and rinsed in warm Hanks balanced salt solution (GIBCO Life Technologies, Inc., Santa Clara, Calif.) containing 100 ,g of streptomycin, 100 U of penicillin, and 0.25 ,ug of amphotericin B per ml. After rinsing, the tissues were digested with 0.25% pronase at 37°C for 40 min. Subsequently, thin layers of the cervical epithelium were peeled off of the underlying stroma by using fine forceps under a dissecting microscope. The epithelial layers were rinsed in serum-free Dulbecco's modified Eagle's medium (DS medium; GIBCO, Santa Clara, Calif.) and treated with 0.1% trypsin for 5 min at 37°C. The epithelium was then dissociated into a single-cell suspension by repeated pipetting through a 25-gauge hypodermic needle. The cells were washed with culture medium that consisted of modified Eagle's medium (GIBCO) supplemented with 10% new horse serum and contained 200 U of penicillin and 2.0 mg of neomycin per ml (NHS). The cells were collected by centrifugation at 800 x g for 8 min. The cells suspended in NHS were counted with a hemacytometer and plated in a Rose chamber (6) at 1.5 x 106 per chamber or a four-well multidish (Nunclon, Roskilde, Denmark) at 106 per well. The cells in the Rose chamber were incubated at 37°C, and those in the four-well multidish were incubated at 37°C in a 2% CO2 atmosphere saturated with moisture. The cell culture medium was replaced with penicillin-free NHS on day 2 or 3 after culture and regularly three times per week thereafter. Cells reached a state of confluence by 5 to 7 days in culture and remained as such for 18 days without treatment or infection (see Fig. 1A).
MATERIALS AND METHODS Contraceptive. The pure form of non-9 and the base ingredients used in this study were kindly provided by Advanced Care Products, Division of Ortho Pharmaceutical Corp., Raritan, N.J. Microorganism. The serovar E strain (UW-5/Cx) of C. trachomatis was used. The organisms were grown in HeLa *
Corresponding author. 1478
VOL. 36, 1992
TOXICITY OF NONOXYNOL 9 FOR C. TRACHOMATIS
Toxicity assay. (i) HeLa 229 cells (continuous cell line). HeLa 229 cells grown on a 12-mm-diameter coverslip in a 1-dram (4-ml) vial shell were treated with 0 to 150 p,g of non-9 per ml in chlamydial transport medium, sucrosephosphate-glutamic acid medium, for 1 h at room temperature to simulate the adsorption of C. trachomatis to HeLa cells during inoculation. The medium was then removed. The cell monolayers were either washed twice with Hanks balanced salt solution or not washed and cultured with Eagle's minimum essential medium (GIBCO) containing 10% fetal bovine serum and 100 ,g each of streptomycin and vancomycin per ml (MEM) for 72 h at 37°C. HeLa 229 cell monolayers were also incubated with 0.1 to 150 ,ug of non-9 per ml or the base (inactive ingredient) at 2 and 5% in MEM for 6, 24, or 72 h at 37°C to simulate the culture of infected cells as in the assay of non-9 for antichlamydial activity described below. (ii) Monkey cervical epithelial cells. Monolayers of monkey cervical epithelial cells were incubated with 0 to 1,000 ,ug of non-9 per ml in penicillin-free NHS for 1 or 6 h at 37°C. After 1 or 6 h of contact with non-9, the cell monolayers were washed twice, overlaid with penicillin-free NHS, and incubated for 48 h at 37°C. (iii) Evaluation of cellular integrity. Assessments of cell death were based on morphological criteria (5). The epithelial cells were in a subconfluent state for the first 4 days. By days 5 to 7, the cultures had become a confluent monolayer of epithelial cells and they remained confluent for 18 days. Multilayering and cell differentiation followed the confluence period for about 7 days. Toxicity was assessed by calculating the percentage of cell death under x400 magnification after the cells were fixed with absolute methanol and stained with Giemsa stain (Fig. 1B). Assay of antichlamydial activity. The antichlamydial activity of non-9 was evaluated in two systems: inhibition of organism growth in cell culture and toxicity to extracellular organisms. For inhibition of cell culture growth of chlamydiae, 5- to 7-day-old monkey cervical cell monolayers were inoculated with 105 infection-forming units of C trachomatis. The inoculated cells were overlaid with penicillin-free NHS containing 50 ,ug of non-9 per ml and incubated for 6 h at 37°C. The culture medium was removed. The cell monolayers were washed with Hanks balanced salt solution and overlaid with penicillin-free NHS containing no non-9 and further incubated for 48 h at 37°C. Half of each culture was stained for inclusion counts, and the other half was harvested and the yield of infectious organisms was assayed by titration in HeLa cell cultures without non-9. For passages, the culture medium was removed and the cells were harvested in 0.3 ml of sucrose-phosphate-glutamic acid medium by scraping with a Pasteur pipette. New HeLa cell monolayers were inoculated with the harvested cells. After the infected cells were fixed with absolute methanol and stained by Giemsa, the antichlamydial activity of non-9 was determined by counting (under x400 magnification) the cytoplasmic inclusions in 10 randomly chosen fields per well among eight wells per experiment. Each field contained 100 cells. For the toxicity assay, chlamydial organisms were treated with 50 p,g of non-9 per ml for 6 h at 37°C prior to inoculation of HeLa cells. Control organisms treated with buffer contained no non-9. The viability of non-9-treated or control organisms was tested by the cell culture infectivity assay as described above. TEM. The condition of primary cultures of monkey cervical cells was also assessed by transmission electron mi-
1479
FIG. 1. (A) HeLa 229 cells treated with 25 Fg of non-9 per ml in MEM. The cells were incubated for 6 h. Toxicity was 0%. Magnification, x40. (B) HeLa 229 cells treated with 125 pg of non-9 per ml in sucrose-phosphate-glutamic acid medium for 1 h. The cells were washed twice in Hanks balanced salt solution and incubated for 72 h. Toxicity was 50 to 60%. Magnification, x40.
croscopy (TEM). For TEM, the cells were fixed with Karnovsky's fixative and then osmium tetroxide. The cells were then stained with uranyl acetate, dehydrated through a graded series of alcohol washes, and embedded in Epon (Ernest F. Fullam, Schenectady, N.Y.) en bloc. Thin sections were stained with uranyl acetate and lead citrate and viewed in a CM 10 TEM (Philips Electronic Instruments,
Mahwah, N.J.).
Statistics. Data were evaluated by using a chi-square test for a two-by-two contingency table.
RESULTS Toxicity of non-9 for cultured cells. For 1 h of contact time without washing followed by incubation for 72 h without non-9, non-9 was toxic to HeLa 229 cells at concentrations of 30 to 50 jig/ml. A toxicity of 50 to 99% was observed. Concentrations of non-9 of less than 12.5 p,g/ml were nontoxic. HeLa cells remained normal. Residual drug caused toxicity in the unwashed cultures. At concentrations of less than 50 pg/ml for 1 h of incubation and two washes, no cell death was observed. In comparison, 30 to 80% cell death was observed at 100 to 150 ,g non-9 per ml for 1 h of contact time followed by removal of the residual non-9 by two washes (Fig. 1 and Table 1). The toxicity of non-9 increased with increasing concentrations and/or increasing contact times (Table 1). Concentrations of non-9 of c25 ,g/ml for 6 h of contact time did not
ANTIMICROB. AGENTS CHEMOTHER.
PATTON ET AL.
1480
TABLE 1. Toxicity of non-9 for HeLa cells Duration of drug contact (hr)
Concn of non-9
(p.g/ml)
Cell death (%)
1
50 100 150
0 30 80
6
ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, July 1992, p. 1478-1482
0066-4804/92/071478-05$02.00/0 Copyright ©) 1992, American Society for Microbiology
In Vitro Activity of Nonoxynol 9 on HeLa 229 Cells and Primary Monkey Cervical Epithelial Cells Infected with Chlamydia trachomatis DOROTHY L. PATTON,`* SHA-KE WANG,1 AND CHO-CHOU KUO2 Departments of Obstetrics and Gynecology' and Pathobiology, 2 University of Washington, Seattle, Washington 98195 Received 21 November 1991/Accepted 24 April 1992
Nonoxynol 9 (non-9) is the active ingredient in a wide variety of vaginal contraceptive preparations. The manufacturer recommendation for optimal contraceptive practice is repeated application every 6 h. We studied the in vitro activity of non-9 against Chlamydia trachomatis (E/UW-5/Cx) and its toxicity against HeLa 229 cells and monkey cervical epithelial cells. With a contact time of 6 h, non-9 was toxic to HeLa cells at concentrations of 50 ,ug/ml or greater and to monkey cervical cells at 100 ,ug/ml or greater. Inhibitory effects of non-9 on extracellular chiamydiae were observed at concentrations of 50 ,ug/ml or greater. Inhibition of intracellular growth of chlamydiae in monkey cervical cells was observed at a nontoxic concentration of 50 ,ug/ml. Our studies show that non-9 has antichlamydial activity. However, owing to its toxicity to cervical cells in vitro, the effects of prolonged use of non-9 in vivo should be further studied. 229 cell cultures (10). An inoculum was partially purified by one cycle of differential centrifugation, which yielded 108 to
Chlamydia trachomatis is one of the most common sexually transmitted pathogens. A contraceptive that is effective against chlamydiae would provide a means of birth control as well as prophylaxis against sexually transmitted diseases. Many in vitro studies on the antimicrobial activity of the active spermicide ingredient nonoxynol 9 (non-9) against C. trachomatis have been done in continuous cell lines. The results have been controversial. Non-9 is the active ingredient in a wide variety of vaginal contraceptive foams, creams, jellies, and suppositories. Earlier in vitro studies using non-9 have demonstrated antimicrobial activity against Neisseria gonorrhea (4, 12), Trichomonas vaginalis (4, 12), Treponema pallidum (12), Candida albicans (13), Herpes simplex virus (2, 14), and Ureaplasma urealyticum (1). Many attempts have been made to assess such activity against C. trachomatis infection in vitro. Benes and McCormack (3) and Kelly et al. (8) have demonstrated inhibition of C. trachomatis growth in McCoy cell cultures. Cytopathologic effects on McCoy cells, a mouse line, have also been described (8). In contrast, data from Kappus and Quinn (7) and Knight et al. (9) have not demonstrated inhibition of C. trachomatis growth in cultures of the same cells. To understand the effects of non-9 on C. trachomatis infection further, we examined this active spermicide ingredient under more physiological conditions by using a continuous human cell line, HeLa 229, and primary monkey cervical epithelial cells to assess the toxicity of the drug and its bactericidal activity against C. trachomatis under conditions nontoxic to host cells.
109 inclusion-forming units of C. trachomatis organisms from a 32-oz (947-ml) culture bottle. A control inoculum was prepared from uninfected cultures processed as infected cultures. Serovar E was selected because it is the most common serovar of genital isolates (11). Monkey cervical epithelial cell culture. Monkey (Macaca nemestrina) cervix tissue was obtained from the Tissue Program at the Regional Primate Research Center at the University of Washington. The tissue was cut into small pieces and rinsed in warm Hanks balanced salt solution (GIBCO Life Technologies, Inc., Santa Clara, Calif.) containing 100 ,g of streptomycin, 100 U of penicillin, and 0.25 ,ug of amphotericin B per ml. After rinsing, the tissues were digested with 0.25% pronase at 37°C for 40 min. Subsequently, thin layers of the cervical epithelium were peeled off of the underlying stroma by using fine forceps under a dissecting microscope. The epithelial layers were rinsed in serum-free Dulbecco's modified Eagle's medium (DS medium; GIBCO, Santa Clara, Calif.) and treated with 0.1% trypsin for 5 min at 37°C. The epithelium was then dissociated into a single-cell suspension by repeated pipetting through a 25-gauge hypodermic needle. The cells were washed with culture medium that consisted of modified Eagle's medium (GIBCO) supplemented with 10% new horse serum and contained 200 U of penicillin and 2.0 mg of neomycin per ml (NHS). The cells were collected by centrifugation at 800 x g for 8 min. The cells suspended in NHS were counted with a hemacytometer and plated in a Rose chamber (6) at 1.5 x 106 per chamber or a four-well multidish (Nunclon, Roskilde, Denmark) at 106 per well. The cells in the Rose chamber were incubated at 37°C, and those in the four-well multidish were incubated at 37°C in a 2% CO2 atmosphere saturated with moisture. The cell culture medium was replaced with penicillin-free NHS on day 2 or 3 after culture and regularly three times per week thereafter. Cells reached a state of confluence by 5 to 7 days in culture and remained as such for 18 days without treatment or infection (see Fig. 1A).
MATERIALS AND METHODS Contraceptive. The pure form of non-9 and the base ingredients used in this study were kindly provided by Advanced Care Products, Division of Ortho Pharmaceutical Corp., Raritan, N.J. Microorganism. The serovar E strain (UW-5/Cx) of C. trachomatis was used. The organisms were grown in HeLa *
Corresponding author. 1478
VOL. 36, 1992
TOXICITY OF NONOXYNOL 9 FOR C. TRACHOMATIS
Toxicity assay. (i) HeLa 229 cells (continuous cell line). HeLa 229 cells grown on a 12-mm-diameter coverslip in a 1-dram (4-ml) vial shell were treated with 0 to 150 p,g of non-9 per ml in chlamydial transport medium, sucrosephosphate-glutamic acid medium, for 1 h at room temperature to simulate the adsorption of C. trachomatis to HeLa cells during inoculation. The medium was then removed. The cell monolayers were either washed twice with Hanks balanced salt solution or not washed and cultured with Eagle's minimum essential medium (GIBCO) containing 10% fetal bovine serum and 100 ,g each of streptomycin and vancomycin per ml (MEM) for 72 h at 37°C. HeLa 229 cell monolayers were also incubated with 0.1 to 150 ,ug of non-9 per ml or the base (inactive ingredient) at 2 and 5% in MEM for 6, 24, or 72 h at 37°C to simulate the culture of infected cells as in the assay of non-9 for antichlamydial activity described below. (ii) Monkey cervical epithelial cells. Monolayers of monkey cervical epithelial cells were incubated with 0 to 1,000 ,ug of non-9 per ml in penicillin-free NHS for 1 or 6 h at 37°C. After 1 or 6 h of contact with non-9, the cell monolayers were washed twice, overlaid with penicillin-free NHS, and incubated for 48 h at 37°C. (iii) Evaluation of cellular integrity. Assessments of cell death were based on morphological criteria (5). The epithelial cells were in a subconfluent state for the first 4 days. By days 5 to 7, the cultures had become a confluent monolayer of epithelial cells and they remained confluent for 18 days. Multilayering and cell differentiation followed the confluence period for about 7 days. Toxicity was assessed by calculating the percentage of cell death under x400 magnification after the cells were fixed with absolute methanol and stained with Giemsa stain (Fig. 1B). Assay of antichlamydial activity. The antichlamydial activity of non-9 was evaluated in two systems: inhibition of organism growth in cell culture and toxicity to extracellular organisms. For inhibition of cell culture growth of chlamydiae, 5- to 7-day-old monkey cervical cell monolayers were inoculated with 105 infection-forming units of C trachomatis. The inoculated cells were overlaid with penicillin-free NHS containing 50 ,ug of non-9 per ml and incubated for 6 h at 37°C. The culture medium was removed. The cell monolayers were washed with Hanks balanced salt solution and overlaid with penicillin-free NHS containing no non-9 and further incubated for 48 h at 37°C. Half of each culture was stained for inclusion counts, and the other half was harvested and the yield of infectious organisms was assayed by titration in HeLa cell cultures without non-9. For passages, the culture medium was removed and the cells were harvested in 0.3 ml of sucrose-phosphate-glutamic acid medium by scraping with a Pasteur pipette. New HeLa cell monolayers were inoculated with the harvested cells. After the infected cells were fixed with absolute methanol and stained by Giemsa, the antichlamydial activity of non-9 was determined by counting (under x400 magnification) the cytoplasmic inclusions in 10 randomly chosen fields per well among eight wells per experiment. Each field contained 100 cells. For the toxicity assay, chlamydial organisms were treated with 50 p,g of non-9 per ml for 6 h at 37°C prior to inoculation of HeLa cells. Control organisms treated with buffer contained no non-9. The viability of non-9-treated or control organisms was tested by the cell culture infectivity assay as described above. TEM. The condition of primary cultures of monkey cervical cells was also assessed by transmission electron mi-
1479
FIG. 1. (A) HeLa 229 cells treated with 25 Fg of non-9 per ml in MEM. The cells were incubated for 6 h. Toxicity was 0%. Magnification, x40. (B) HeLa 229 cells treated with 125 pg of non-9 per ml in sucrose-phosphate-glutamic acid medium for 1 h. The cells were washed twice in Hanks balanced salt solution and incubated for 72 h. Toxicity was 50 to 60%. Magnification, x40.
croscopy (TEM). For TEM, the cells were fixed with Karnovsky's fixative and then osmium tetroxide. The cells were then stained with uranyl acetate, dehydrated through a graded series of alcohol washes, and embedded in Epon (Ernest F. Fullam, Schenectady, N.Y.) en bloc. Thin sections were stained with uranyl acetate and lead citrate and viewed in a CM 10 TEM (Philips Electronic Instruments,
Mahwah, N.J.).
Statistics. Data were evaluated by using a chi-square test for a two-by-two contingency table.
RESULTS Toxicity of non-9 for cultured cells. For 1 h of contact time without washing followed by incubation for 72 h without non-9, non-9 was toxic to HeLa 229 cells at concentrations of 30 to 50 jig/ml. A toxicity of 50 to 99% was observed. Concentrations of non-9 of less than 12.5 p,g/ml were nontoxic. HeLa cells remained normal. Residual drug caused toxicity in the unwashed cultures. At concentrations of less than 50 pg/ml for 1 h of incubation and two washes, no cell death was observed. In comparison, 30 to 80% cell death was observed at 100 to 150 ,g non-9 per ml for 1 h of contact time followed by removal of the residual non-9 by two washes (Fig. 1 and Table 1). The toxicity of non-9 increased with increasing concentrations and/or increasing contact times (Table 1). Concentrations of non-9 of c25 ,g/ml for 6 h of contact time did not
ANTIMICROB. AGENTS CHEMOTHER.
PATTON ET AL.
1480
TABLE 1. Toxicity of non-9 for HeLa cells Duration of drug contact (hr)
Concn of non-9
(p.g/ml)
Cell death (%)
1
50 100 150
0 30 80
6
