Sodium dodecyl sulfate and C31G as microbicidal alternatives to nonoxynol 9: comparative sensitivity of primary human vaginal keratinocytes

Abstract

A broad-spectrum vaginal microbicide must be effective against a variety of sexually transmitted disease pathogens and be minimally toxic to the cell types found within the vaginal epithelium, including vaginal keratinocytes. We assessed the sensitivity of primary human vaginal keratinocytes to potential topical vaginal microbicides nonoxynol-9 (N-9), C31G, and sodium dodecyl sulfate (SDS). Direct immunofluorescence and fluorescence-activated cell sorting analyses demonstrated that primary vaginal keratinocytes expressed epithelial cell-specific keratin proteins. Experiments that compared vaginal keratinocyte sensitivity to each agent during a continuous, 48-h exposure demonstrated that primary vaginal keratinocytes were almost five times more sensitive to N-9 than to either C31G or SDS. To evaluate the effect of multiple microbicide exposures on cell viability, primary vaginal keratinocytes were exposed to N-9, C31G, or SDS three times during a 78-h period. In these experiments, cells were considerably more sensitive to C31G than to N-9 or SDS at lower concentrations within the range tested. When agent concentrations were chosen to result in an endpoint of 25% viability after three daily exposures, each exposure decreased cell viability at the same constant rate. When time-dependent sensitivity during a continuous 48-h exposure was examined, exposure to C31G for 18 h resulted in losses in cell viability not caused by either N-9 or SDS until at least 24 to 48 h. Cumulatively, these results reveal important variations in time- and concentration-dependent sensitivity to N-9, C31G, or SDS within populations of primary human vaginal keratinocytes cultured in vitro. These investigations represent initial steps toward both in vitro modeling of the vaginal microenvironment and studies of factors that impact the in vivo efficacy of vaginal topical microbicides.

FIG. 1

Primary human vaginal keratinocytes express members of the keratin protein family. Primary vaginal keratinocytes were examined using flow cytometry for expression of CD4, members of the keratin protein family, or fibronectin. Analyses were conducted as described in Materials and Methods. The horizontal and vertical axes represent fluorescence intensity (log scale) and cell counts, respectively.

FIG. 2

Keratin expression in primary human vaginal keratinocytes can be identified by immunofluorescence. Primary vaginal keratinocytes were examined by direct immunofluorescence for expression of CD4 and members of the keratin protein family. Analyses were conducted as described in Materials and Methods. (A) Representative field under visible light; (B, C, and D) direct immunofluorescent micrographs of vaginal keratinocytes in the absence of antibody, labeled with CD4 antibodies, or labeled with pan cytokeratin antibodies, respectively. The arrow in panel C indicates a cell weakly positive for CD4 expression. The field of cells in panel C was selected specifically to show the cells that expressed low levels of CD4 and is not representative of a typical field; cells in most fields were devoid of any detectable CD4 expression.

FIG. 3

Primary keratinocytes are more sensitive to N-9 than to C31G or SDS during long-term exposure. Primary human vaginal keratinocytes were exposed to N-9, C31G, or SDS for 48 h and subsequently assessed for viability using MTT assays. (A) Cell survival following exposure to concentrations of 2.5 × 10⁻⁴ to 5 × 10⁻³%; (B) Cell survival following exposure to N-9 at concentrations of 2.5 × 10⁻⁵ to 2.5 × 10⁻⁴%. The arrows in panels A and B both indicate cell viability at 2.5 × 10⁻⁴%. Cell viability following microbicide exposure is expressed as the fraction of viable cells relative to the number of mock-exposed cells. The results illustrated are averages from two experiments in which triplicate wells for each concentration were assayed.

FIG. 4

Repetitive microbicide exposure affects the viability of human vaginal keratinocytes. Primary human vaginal keratinocytes were exposed to N-9, C31G, or SDS for 2 h. The cells were then washed once with PBS and cultured under new medium for 24 h. This cycle of exposure and recovery was repeated two more times. Cell survival was assessed using MTT assays following the third exposure/recovery cycle. Cell viability following microbicide exposure is expressed as the fraction of viable cells relative to the number of mock-exposed cells. The results illustrated are averages from two experiments in which triplicate wells for each concentration were assayed.

FIG. 5

Repetitive microbicide exposure results in a steady reduction in human vaginal keratinocyte viability. TC₇₅ were determined from the results illustrated in Fig. 4. Cell survival was assessed by MTT assay after each exposure and recovery cycle. The cell viability following microbicide exposure is expressed as the fraction of viable cells relative to the number of mock-exposed cells. The results illustrated are averages from two experiments in which triplicate wells for each concentration were assayed.

FIG. 6

Microbicide toxicity increases with continued exposure to equivalent concentrations of N-9, C31G, or SDS. TC₇₅ were determined from data illustrated in Fig. 3A and B. Primary human vaginal keratinocytes were exposed to N-9, C31G, or SDS for 2, 8, 16, 24, or 48 h. At the end of each exposure interval, cell survival was assessed by an MTT assay. The cell viability following microbicide exposure is expressed as the fraction of viable cells relative to the number of mock-exposed cells. The results illustrated are averages from two experiments in which triplicate wells for each concentration were assayed.

FIG. 7

Microbicide toxicity increases with continued exposure to equal concentrations of N-9, C31G, or SDS. Primary human vaginal keratinocytes were exposed to 1.25 × 10⁻³% N-9, C31G, or SDS for 2, 8, 16, 24, or 48 h. At the end of each exposure interval, cell survival was assessed by an MTT assay. Cell viability following microbicide exposure is expressed as the fraction of viable cells relative to the number of mock-exposed cells. The results illustrated are averages from two experiments in which triplicate wells for each concentration were assayed.